Final Report Summary - LIFECYCLE (Building a biological knowledge-base on fish lifecycles for competitive, sustainable European aquaculture)
Executive Summary:
In summarizing the 54-month long LIFECYCLE project, it can first be stated that the project has been hugely successful in fulfilling its main objective, which has been to build a biological knowledge-base on fish lifecycles that will contribute to competitive sustainable European aquaculture. This biological knowledge-base, to greatest extent focusing on the physiological and molecular level, can already be found in the 77 scientific LIFECYCLE papers published so far (this number is likely to double over the next two years), in large amounts of genomic (transcriptomic) data deposited in public data-bases, and in over 150 presentations at scientific and aquaculture conferences.
The building of a biological knowledge-base is essentially a task of fundamental scientific endeavor, but the selection of the specific research areas for which LIFECYCLE would attempt to significantly advance the state-of-the-art were relevant to major current aquaculture production bottlenecks. Further choices were made in order to make the knowledge-base as relevant as possible for European aquaculture. Thus, the research was largely carried out on the four major aquaculture species; Atlantic salmon, rainbow trout, sea bass and sea bream, and to lesser extent on Atlantic halibut and Atlantic cod, and then in areas where production problems remain.
Many of the major biological production bottlenecks of European aquaculture production are directly related to life-stage transitions of the fish. These transitions from embryo to larvae, from larvae to juvenile, from juvenile to pubertal adult and then to a sexually mature fish, all represent highly vulnerable phases of the production cycle. Bad egg quality, larval-juvenile mortalities and/or abnormalities, poor on-growth, premature puberty and sexual maturation are all well-known production problems linked to the lifecycles of the various aquaculture species. Therefore, LIFECYCLE research was focused on the areas (WP1) Development and Growth, (WP2) Adaptation and Homeostasis, (WP3) Immunity, and (WP4) Sex differentiation and Puberty. In carrying out the research within each of the 15 well-defined sub-topics (subWPs) of the LIFECYCLE project, one or more relevant aquaculture species were chosen, and in elucidating molecular mechanisms, the model fish species zebrafish and medaka have provided valuable research approaches.
From the onset, the LIFECYCLE consortium was composed of 14 partners from 9 European countries. Most of the groups, placed at universities and research institutes, represent internationally leading research teams in their given areas. Several of the LIFECYCLE partners also have top-tier, large-scale aquaculture research facilities, which are now a part of the AQUAEXCEL EU infrastructure. Highly illustrative of the production challenges still facing the European aquaculture industry, the only industrial SME partner in the project, a producer of Atlantic halibut juveniles, went bankrupt during the project, having been a world-leading producer only a few years earlier. This was also the cause of the only major change in the LIFECYCLE research plans, and was the main reason for a 6-month extension of the project in order to allow for alternative research plans to be put in motion.
As pledged, the LIFECYCLE consortium directed a huge amount of nationally funded person-months to the project, almost doubling the person-months covered by the EU contribution. This emphasizes the great commitment of the whole LIFECYCLE consortium to the project objectives, and the research has been carried out in the spirit of DEDICATION, ENTHUSIASM, COLLABORATION and SCIENTIFIC and TECHNICAL EXCELLENCE. Through this, many of the teams have forged strong collaborative ties generating a network, which will strengthen the European research area and will favour initiatives for years to come.
Apart from significantly advancing the scientific state-of-the-art in all its research areas as detailed below, the LIFECYCLE consortium also trained 28 PhD students and is particularly proud of gender equality actions taken in the project, and the fact that equal numbers of male and female experienced researchers (PhD holders; 46 of each gender) as well as PhD students (14 of each gender) have participated in the project.
The project progress has been detailed in period report 1 (135 pages), period report 2 (196 pages), period report 3 (243 pages and period report 4 (340 pages) submitted to the EU Commission.
The completion of the project deliverables has been reported in 157 deliverable reports submitted to the EU Commission
The scientific research advances have been described in detail in 77 scientific papers so far published in international peer-reviewed journals (ca 1000 pages).
Project Context and Objectives:
Fisheries and aquaculture provide an important source of nutritious food and animal protein for much of the world’s population. In addition, the sector provides livelihoods and income, both directly and indirectly, for a significant share of the world’s population. Increased aquaculture production is seen globally as the single most important way of securing food for the growing population. While fisheries have reached a limit world-wide, aquaculture continues to grow rapidly. In 2011, according to FAO, aquaculture produced 63.6 million tons representing half of all aquatic food destined for human consumption worldwide.
However, while aquaculture is fasted growing food sector globally, this is not the case in Europe.
In 1994, 60% of European seafood needs were provided by European fisheries and aquaculture, whereas today 65% of seafood consumed in Europe is imported. Europe is therefore increasing its reliance on seafood imports, which results in lost revenue and employment in direct and indirect jobs, but also weakens the EU food security position.
The European Union has therefore sought ways of stimulating the European aquaculture industry in numerous ways; e.g. by policy measures, industry-driven projects aimed at supporting innovations and production small and medium sized enterprises, as well as large, scientific research projects focused on various aspects of aquaculture technology and biology. LIFECYCLE is one such large, scientific research project, bringing together 13 research groups from many of Europe’s leading universities and research institutes and 9 different European countries, in order to work on key questions which cannot be efficiently answered by research at the national level.
As an EU project, the context and objectives of the LIFECYCLE project have been broadly dictated by the original call text where the EU Commission has outlined the direction and the scope of the project. In accordance with this call text, the overall objective of LIFECYCLE has been to improve our biological knowledge about some of the most important aquacultured fish species in Europe. Why is this important objective? It is because aquaculture is the youngest branch of our animal food production sector and our current knowledge about the fish species produced in aquaculture is very limited in comparison with our knowledge of other farm animals such as chicken, pigs and cows. Furthermore, a diverse range of fish species (e.g. sea bass, Atlantic salmon) is used for aquaculture in different parts of Europe and require different production systems unlike land-farm animals. These have been bred and domesticated for farm production for hundreds or even thousands of years, whereas intensive fish farming on an industrial scale started in Europe with large-scale salmon aquaculture as late as in the 1970’s, and culture of many key marine species has only been carried out for 10-15 years.
To successfully raise and breed an animal for food production requires good understanding of the biology of the animal from conception to slaughter, so that the farmer can see to it that the animal thrives and grows and finally can be turned into a valuable product. For fish, there are still many serious bottlenecks in the aquaculture production which result in lower productivity in terms of quantity and/or quality, which in turn results in decreased economic sustainability and competitiveness of the aquaculture industry. Such bottlenecks can occur at many different stages in the life of the animal and differ depending on the species in questions. Thus, the context and objectives of LIFECYCLE has been to deliver a knowledge-base designed to improve the competitiveness and sustainability of the European aquaculture industry.
Having a defined funding base (6M€ EC contribution) and duration (4 years), a single project such as LIFECYCLE could not address all known problems and questions for all aquacultured species, so choices had to be made in terms of what questions should be addressed and for which fish species.
The project was intended to create a knowledge-base about the biology of the target species, which would benefit future production-orientated projects focusing on practical areas such as aquaculture stress control, disease prevention and control, new breeding technologies, better environmental performance and diversification into new species (see call text), the research areas where chosen based on known bottlenecks. However, it’s important to understand that the role of LIFECYCLE was not primarily to solve applied, industrial production problems, but rather to provide increased biological knowledge in areas of production bottlenecks, in order to facilitate future, knowledge-based solutions. This is in line with the EC policy to create a “knowledge-based bio-economy” through measures of which the LIFECYCLE project is a part.
Therefore, the LIFECYCLE project was designed through a combination of question-driven and problem-driven approaches. It aimed at providing novel discoveries which in turn could offer solutions to current “bottle-neck” problems. The knowledge-base created in LIFECYCLE provides a solid foundation for European aquaculture to develop ways of improved larval rearing practices, feed utilization and muscle quality, to gain better control over sexual differentiation and puberty, improve stress control and disease prevention, establish new breeding selection parameters and technologies, improve environmental performance, and support diversification into new species.
In defining the scope of the LIFECYCLE project, the first focus area was on the early life stages, encompassing biological processes from fertilization of the egg to juvenile growth. This area was chosen as the production of sufficient quantities of high-quality fish larvae is still a major problem in the culture of several species, especially the marine fish species. Understanding the importance of gene trans¬fer from the mother to the egg during early development, the mechanisms of sex differentiation into males and females, the development of the immune system, the development of the muscles and the skeleton during larval growth, the roles of the larval skin and gut, and how genes and hormones regulate larval growth and development, are all questions of key importance to solve a large number of current production bottlenecks. These production problems are not only directly related to growth and survival at the larval stage, but are also indirectly related to many problems manifested at later life stages such as disease resistance, ability to cope with changing environment, and reproduction.
The second focus was placed on the growth and environmental adaptation of the juvenile-adult “ongrowing” life stages, addressing questions relevant to production problems related to poor growth. There are many possible reasons for poor growth; lack of appetite and thus low feed intake of the fish, poor utilization of the energy of the food resulting. This is often linked to environmental maladaptation where the fish are using energy to cope with the stress of the rearing environment rather than for growth. Inappropriate rearing environments can also have a negative influence on the flesh quality. With rising feed-costs, slow growth is becoming an ever more important issue for the competitiveness of European aquaculture. Therefore, the LIFECYCLE project was aimed at obtaining new knowledge on basic biological issues such as the hormonal regulation of appetite and growth of the fish, the relationship between metabolism, growth and fattiness, and the relationship between environmental conditions such as salinity, and how this affects energy balance and growth.
The third focus area of LIFECYCLE was on important issues linked to key life-stage transitions. These are complex biological changes that take place during development and growth, and are often associated with major losses in aquaculture production. One such biological transition is the so-called metamorphosis of flatfish larvae, when they change from free-swimming symmetrical larvae to bottom-living asymmetrical juvenile flatfish.
Another crucial life-stage transition is the so-called smoltification of Atlantic salmon during which the freshwater-living parr becomes seawater tolerant smolt and can be moved into marine net-pens for ongrowth. Last but not least, the onset of puberty has a major impact on aquaculture production. In many species, puberty and the ensuing sexual maturation occurs too early in the production cycle, impacting negatively on both growth and flesh quality. These life-stage transitions are extremely demanding for the organism and as already stated, major production problems are linked to these transitional periods. This includes decreased disease resistance, increased mortalities and decline of growth performance. Therefore, a better understanding of immune function during life-stage transitions is of key importance, and improved know¬ledge leading to more effective ways of regulating the timing of puberty may greatly improve the competitiveness of European aquaculture.
In order to address these lifecycle-encompassing topics, the LIFECYCLE consortium was established and brought together a critical mass of leading European research groups dedicated to significantly advance current knowledge on mechanisms governing essential biological functions in fish across species and all major life stages, with focus on important aquaculture species and current production bottlenecks. This has been achieved by applying the tools of endocrinology, biochemistry and functional genomics to state-of-the-art physiological research.
No single life stage, nor indeed a single life process, is independent of others, and great synergies can therefore be obtained by integrating the research through “cross-talk”. A major emphasis has been placed on this in designing the many experimental studies of the LIFECYCLE project.
LIFECYCLE focused on the most important commercial aquaculture species in Europe, specifically sea bass (Dicentrarchus labrax), sea bream (Sparus auratus), Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Other species such as Atlantic halibut (Hippoglossus hippoglossus) and Atlantic cod (Gadus morhua) were included in the project due to their importance for studying specific processes and/or their potential as aquaculture species. Moreover, the inclusion of both ancient (salmoniformes), and modern teleost fish (perciformes) used for aquaculture and when appropriate, model species such as zebrafish and medaka, for the elucidation of basic biological mechanisms, has made it likely that the project results can be applied to other newly emerging aquaculture species.
A key element of research is the dissemination of the research outcome to stakeholders this includes other scientists, the aquaculture sector, companies, politicians and the public. The LIFECYCLE consortium has been committed to informing all stakeholders about the project results in as active and effective way as possible, through various channels aimed at reaching different groups. An important way of openly informing society about the project has been the project website http://lifecycle-fp7.eu/ which has explained the purpose of the project, the partnership, the species, the research areas and the project results. Validation of the experimental results and their dissemination has been through interaction with the scientific community at large through publication of a large number of peer-reviewed publications in scientific journals and deposition of large datasets in public databases. Discussion and debate about the diversity of studies carried out in LIFECYCLE have been possible through presentation of numerous posters and oral presentations at national and international conferences and also at specialised workshops. An important dissemination target and the end users of the research output, the aquaculture sector has been reached through presentations and posters at aquaculture-related conferences, not least those organized by the European and national aquaculture societies but most importantly for the European sector by personal, direct discussions by many of the LIFECYCLE partners with local fish farmers or their associations. More global dissemination of LIFECYCLE to raise awareness of basic research progress of relevance for European aquaculture was pursued through presentations of the project in the trade journals. Dissemination to the general public has come directly and indirectly through popular news articles and interviews with LIFECYCLE scientists. A more difficult dissemination activity to quantify is the multiplying effect of the numerous scientists who have participated in LIFECYCLE and are engaged in outreach activities and the direct education and training of undergraduates and graduates. Inevitably, lectures and seminars given about fish and fish biology or hands-on training activities have been enriched by the outcomes of the LIFECYCLE project in a diversity of subject areas. All the various quantifiable dissemination activities are presented in Tables A1 and A2 of this report.
Project Results:
The scientific research of the LIFECYCLE project was carried out in four major workpackages, each divided into several sub-workpackages, and each subWP was further subdivided into two or more tasks. All in all, 58 significant research tasks were defined in the project, and their successful completion has now resulted in 139 scientific deliverables having been reported to the EU Commission.
In parallel to reporting the project progress regularly to the EU Commission over the 4½ years of the LIFECYCLE project, the results have been continuously published in the scientific literature, with about seventy-five scientific papers having been published to date, and over twenty more manuscripts are currently in different stages of being published. At the same time, the LIFECYCLE data have been presented in various other ways, including over 150 oral and poster presentations at a scientific and aquaculture conferences and workshops. Perhaps the most important dissemination route, which is the hardest one to quantify, is the direct contact and personal dialogue by a large number of LIFECYCLE scientists with local fish farmers, a dialogue enriched by the new insights brought forth by the LIFECYCLE project.
The new knowledge generated by the LIFECYCLE project on the aquaculture-related biology of our most important aquaculture finfish species has thus to date been broadly disseminated through the academic publishing of about one thousand pages in top quality scientific journals. In addition, the presently submitted fourth periodic report, which is about 350 pages in length, contains largely unpublished data, which will generate a significant amount of new papers, which will be published over the next 2-3 years.
It is therefore no exaggeration to say that the following summary of the main scientific and technological results of the project is a very brief summary indeed.
A major challenge in aquaculture is the production of an abundant supply of good quality eggs to secure the production cycle by generating an abundance of good quality larvae for the subsequent grow-out phase. In aquaculture, the traditional method to evaluate egg quality and embryo viability has been empirical and based upon assessment of morphological criteria such as follicle size and embryo viability. An alternative quality assessment may be hidden within the large pool of maternal RNA which accumulates in the egg during its maturation and which is essential for early development. Thus, the LIFECYCLE project has focused on research into MATERNAL TRANSCRIPTS AND IMPACT OF BROODSTOCK STRESS. The work carried out has yielded new insight into the maternal contribution to early developmental success in fish. To achieve this goal, both important European aquaculture species such as Atlantic salmon, Atlantic cod, and rainbow trout, and also a model species, the zebrafish, have been used. The identity of maternal gene products, present in the egg and inherited from the female and used by the embryo during early development was thoroughly characterized during cod development. The crucial role of specific maternally-inherited messenger RNA (i.e. maternal mRNAs) in early developmental success was shown. In parallel, the dynamics of gene expression and post-transcriptional modifications, such as changes in the poly A tail of mRNAs, has been monitored throughout egg maturation for several genes known to be important for egg/embryo development. The importance of ancient families of genes, shared by most vertebrates, in the mechanisms defining embryonic developmental success has been highlighted. Genomic tools to investigate the mechanisms defining egg quality in fish under natural and experimental conditions revealed that elevated levels of stress hormones in eggs and embryos affect factors involved in cell division rate. This result together with recent studies in rainbow trout implies that broodstock stress can increase developmental rate of embryos. Another aspect of the project was to compare all expressed genes in eggs from different fish species. Comparison of overall gene expression repertoires in salmon and cod the revealed salmon eggs have a higher representation of immune factors and calcium balance factors compared to cod. While cod had a higher representation of genes related to transcription and metabolism. The differing pathways identified between these species may be linked to for example, life history traits or adequacy of aquaculture regimes and thereby represents a novel way to understand egg quality parameters in aquaculture fish.
Production of good quality muscle and healthy fish is a key objective of fish aquaculture. The muscle and skeleton develop from the same precursor tissue, the embryonic mesoderm, and evidence suggests that mismatch between the development of these tissues may contribute to poor larval quality, reduced muscle growth and an increased incidence of skeletal abnormalities in aquaculture. Thus, LIFECYCLE has focused research on THE MUSCULOSKELETAL SYSTEM. Genes and gene transcription determine the fundamental characteristics of organisms and mean that gene sequences are an important resource for investigating transcript expression. Transcript expression can be modified by diet, environmental factors and ontogeny and may be associated with changes in muscle or skeletal development. Gene sequences also permit discovery of population level variation associated with improved production characteristics that can be used in marker assisted selection. LIFECYCLE has dramatically increased the sequences available for genes expressed in muscle, bone and cartilage in rainbow trout and gilthead sea bream. Laser microdissection was successfully used to define a sub-set of genes (transcription factors, growth factors, and signalling molecules) that are upregulated in regions of the myotome where new muscle fibres are being formed. The function of several of these novel myogenic genes was investigated using a combination of cell culture and the zebrafish model system. STAC3 a signal adaptor molecule was found to have multiple functions and be required for normal muscle differentiation. Bony fish underwent a whole genome duplication (WGD) (2R) around 550 Mya effectively doubling the number of genes relative to human. Around 80% of the duplicated genes, called paralogues, were subsequently lost and the remainder either acquired new functions or novel patterns of regulation. Salmonid fish (salmon, trout, and charr) underwent a second WGD (4R) around 88 Mya and 50% of the duplicated genes are still present in extant species. Although the duplicates can have very similar sequences (>95% sequence identity) they can also have distinct functions. It is therefore essential to characterise the sequences of all the paralogues in each gene family in order to produce a consistent nomenclature and to design genetic assays that are specific to a single paralogue. The growth of peripheral tissues including the muscoskeletal system is controlled by the insulin-like growth (IGF) system which comprises hormones, receptors and binding proteins which regulate the effective concentration of the hormone according to tissue type and physiological status. Using phylogenomic approaches, the complete IGFBP repertoire of 4R-derived salmonid fishes (19 genes; 13 more than human) was resolved and their evolutionary relationships/nomenclature established with respect to WGDs. Traits central to IGFBP action were determined for all genes, including atomic interactions in IGFBP–IGF1/IGF2 complexes regulating IGF–IGF1R binding. Using statistical methods, we demonstrate that attributes of these protein interfaces are overwhelming a product of 2R IGFBP family membership, explain 49–68% of variation in IGFBP mRNA concentration in several different tissues, and strongly predict the strength and direction of IGFBP transcriptional regulation under differing nutritional states. The expression of IGF-system genes with feeding regimes designed to initiate increased protein synthesis and compensatory growth in muscle; bone and cartilage were described in detail for the gilthead sea bream and Atlantic salmon. We took advantage of the small size and short generation time of a wild zebrafish population to investigate the effects of artificial selection for body size at age on the IGF-signalling system during the domestication process. Replicate populations were either unselected (U-lineage) or subjected to four generations of experimental selection for small (S-lineage) or large (L-lineage) body size at 90 days post-fertilization. Body mass was on average 16.3% and 41.0% higher in the L- than in the U- and S-lineages, respectively. Transcript abundance for four genes (igf1a, igf2r, igfbp1a and igfbp1b) showed either regulated or constitutive differences between the S- and L-lineages. For example, igf2 receptor transcript abundance was higher and igbp1a/b transcript abundance was lower in the L- than in the S-lineage, indicating an effect of selection on insulin-like growth factor signalling. This research has identified several candidates for marker assisted selection for fast growth which are currently being investigated in aquaculture species including Atlantic salmon. Lighting regimes are commonly used in aquaculture to regulate growth and maturation and it is important to understand the genetic mechanisms. The zebrafish also provided a convenient model to fully characterise the repertoire and function of “clock genes” in fish skeletal muscle and to investigate circadian rhythms in the expression of IGF-system components independently of feeding. Two IGF-binding protein genes, igfbp3, igfbp5b, and the myogenic regulatory factor (MRF) myf6 showed circadian patterns and were expressed in phase with the clock genes clock1a/1b changing 2-fold from peak to trough. These finding points to an important physiological role of the clock mechanism in regulating muscle mass through integration with the IGF pathway and MRFs.
LIFECYCLE also contributed significant advances to the understanding of the phenomena of thermal imprinting. Studies on the gilthead sea bream showed that low temperatures (LT) prior to metamorphosis resulted in 20% more muscle fibres of smaller average diameter than for higher rearing temperatures (HT). Moreover, in young adults the number of vertebra in the HT group (n = 24) was higher than the LT group (n = 23) and the HT group had a lower (7%) incidence of malformations compared to the LT group (25%). Early rearing temperature had a persistent effect on the mineral content of bone and the bone density was lower in HT than LT. Furthermore it was shown that gene expression patterns during growth were not fixed, but could be modified by embryonic temperature. Altering the nutritional input to the muscle to stimulate growth revealed cryptic changes in the expression of genes involved in the correct folding of myosin filaments during sarcomere assembly including UNC45 and Hsp90α with higher transcript abundance in the LT than HT groups. The effects of temperature were not limited to muscle and genes important for skeletal homeostasis were also modified. The transcript for bone gla protein, an indicator of bone formation/mineralization, was much higher in HT compared to LT fish. The effects of thermal imprinting on other traits linked to growth were conveniently studied in the zebrafish model. We showed that embryonic temperature had dramatic and persistent effects on thermal acclimation capacity at multiple levels of biological organization. Zebrafish embryos were incubated until hatching at control temperature (TE = 27 °C) or near the extremes for normal development (TE = 22 °C or 32 °C) and were then raised to adulthood under common conditions at 27 °C. Short-term temperature challenge affected aerobic exercise performance, but each TE group had reduced thermal sensitivity at its respective TE. In contrast, unexpected differences arose after long-term acclimation to 16 °C, when performance in the cold was ∼20% higher in both 32 °C and 22 °C TE groups compared with 27 °C TE controls. Differences in performance after acclimation to cold or warm (34 °C) temperatures were partially explained by variation in fibre type composition in the swimming muscle. Cold acclimation changed the abundance of 3,452 of 19,712 unique and unambiguously identified transcripts detected in the fast muscle using RNA-Seq. Principal components analysis differentiated the general transcriptional responses to cold of the 27 °C and 32 °C TE groups. Differences in expression were observed for individual genes involved in energy metabolism, angiogenesis, cell stress, muscle contraction and remodelling, and apoptosis. Therefore, thermal acclimation capacity is not fixed and can be modified by temperature during early development. Developmental plasticity may thus help some aquaculture species cope with the more variable temperatures that are expected under future climate-change scenarios. The research on thermal imprinting carried out within LIFECYCLE stresses the importance for defining the optimal temperature conditions for rearing eggs and larvae in the hatchery in order to enhance growth, muscle texture and ensure skeletal quality in adult fish.
The ability to regulate energy balance is essential for normal development and growth under various conditions and life stages and is of major importance to aquaculture. Muscle and skeletal growth depend on food intake, extraction and use/allocation of this energy. Metabolism and energy balance also include the mobilization of energy stores in liver and adipose tissue for energy-demanding life-stage transitions such as metamorphosis, smoltification and puberty. In mammals, ghrelin and leptin are multifunctional hormones with key roles in energy homeostasis, although , fundamental understanding of how energy balance is regulated in fish and the role of hormones is still very fragmented. For this reason priority was given in LIFECYCLE to the topic METABOLISM AND ENERGY BALANCE: EXPLORING ACTIONS OF LEPTIN AND GHRELIN. This work has shown that leptin reduces and ghrelin increases appetite in Atlantic salmon, thus they may have opposing effects on energy balance. In addition, these hormones seem to be important in lipid metabolism and the development of adiposity (fattiness) in fish, as well as to have functions that help the fish cope with fasting or to respond to changes in body energy stores. The studies also revealed that the IGF-I system is likely central for adipose (fat) tissue functions, adipose cell development and glucose metabolism. In addition, methodological protocols were established that can be used for future research on the biology of adipose cells and the roles of specific genes during adipogenesis (the development of fat tissue). The project has established knowledge that is important to understand the processes that lead to fat accumulation, which may affect the health, growth, time of puberty and reproduction of aquaculture fish and in this way impact welfare issues and affect product quality. The major findings are summarized below. The results provide evidence that leptin serves various roles at different life stages of salmonid fish, e.g. regulating embryogenesis and development, growth and appetite.
The regulatory system is complex and in Atlantic salmon four evolutionary conserved leptin paralogue genes were identified and most likely act via two leptin receptor genes. Leptin genes are expressed differently between tissues and life stages. The response of the hypothalamus, which is the brain centre of appetite control, to leptin revealed it is an important target in fish. This was demonstrated by detailed anatomical mapping of leptin receptors in the hypothalamus and revealed a symmetrical pattern of clusters of neurons expressing one of the leptin receptor subtypes in the Atlantic salmon. Leptin treatment reduced food intake and growth rates in Atlantic salmon, and exerted this effect by modulating neuropeptides expressed in the hypothalamus and known to inhibit appetite, e.g. POMC. Direct central injections of leptin into the brain did not influence short term appetite, and suggests according to its location in the body, local, central or peripheral, it may have different modes of action.
Ghrelin stimulated appetite and body weight gain in Atlantic salmon. The effect of ghrelin, as well as GH, on appetite and growth was not influenced by water salinity and thus the role of these hormones in mediating the effect of salinity on growth is still unclear. Leptin plasma concentrations were influenced by the amount of energy stored in the body, suggesting that leptin acts as an adiposity signal in fish. However, in contrast to mammals, fasted fish that had lost body weight and body fat stores, with fat cells almost emptied of their lipid content, had elevated leptin levels in the blood. In line with this, lean fish treated with GH had higher leptin plasma levels and leptin mRNA expression compared with fat untreated fish. An explanation for the impact of body composition on the response to GH may be explained by considering wild fish. Wild fish experience periods of glut and fast and have to adjust to fasting and loss of energy stores by reducing appetite and foraging activity to decrease energy-demanding activities. The studies also revealed that the energy status of the fish modulates the action of leptin: fish fed a high fat diet may become leptin resistant (unresponsive) at the adipose tissue level. Further, the secretion of leptin by adipocytes increases as they mature. This secretion is under hormonal and nutritional control, e.g. both insulin and ghrelin stimulate the release of leptin while the amino acid leucine decreased leptin release. This project shows for the first time in fish that leptin and ghrelin have direct effects on the lipid metabolism of adipocytes. In particular, ghrelin may promote lipid synthesis and tissue lipid uptake, and hence promote the development of adiposity. In addition, the complete IGF-I system was characterized and seems to have important roles in adipose tissue development and glucose metabolism.
All major life processes such as larval development, metamorphosis, smoltification, growth, metabolism, puberty and reproduction are under complex endocrine control. Built into the endocrine system is the capacity to “sense” change in the internal and external “milieu” and stimulate an appropriate physiological response. Most hormones studied have multiple functions and their action is dependent on the responsiveness of the target tissue which is in part determined by enzymes (e.g. activation/inactivation), plasma binding proteins, specific tissue receptors and other accessory proteins. Current knowledge is primarily related to characterization of hormone function, and to some extent, information about receptor expression and distribution, since the interaction of hormone and receptor bring about the cellular response. However, recently the importance of binding proteins that regulate circulating hormone concentrations and their availability for receptor binding has been appreciated. Therefore, LIFECYCLE research was directed to MODULATION OF HORMONE ACTION by hormone binding proteins for some key hormones regulating growth, hydromineral balance, metabolism and life-stage transitions in fish such as metamorphosis and smoltification. The hormone systems which the LIFECYCLE research has focused in relation to binding proteins are the growth hormone (GH), leptin (Lep) and thyroid hormones (TH) systems. These systems regulate important production traits in aquaculture, including growth, energy balance and metabolism and the capacity of salmonids to adapt to change in salinity during smoltification. The goal was to study the interaction of these hormones with their receptors, binding proteins and transporters. To achieve this goal it was first essential to develop unique analytical tools with which to carry out the study. Considerable effort was put into the establishment of tools to measure GH and leptin binding proteins (GHBP and LepBP, respectively) and into complimentary molecular biology studies. The research has revealed that the GHBP have the same amino acid sequence as the extracellular part of the GHR and are probably shed from the cell surface in fish as occurs in most mammals. For GHBP, the work included the isolation, purification and characterization of the binding proteins from plasma, production of recombinant binding proteins and of specific antibodies. A specific analytical assay was developed and used to quantify GHBP in salmonids under several experimental situations that simulate situations that can occur during aquaculture production. Fasted rainbow trout, salmon in which plasma GH or ghrelin was increased, or salmon that were kept at different salinities did not have modified plasma GHBP,, although circulating GH levels and the abundance of GH receptors in the liver increased. However, acute physiological changes, like re-feeding after a fasting period or a sudden change in salinity, resulted in clear changes in plasma GHBP levels. Thus, GHBP may act to buffer sudden oscillations in GH.
Multiple LepBPs were characterized and cloned in rainbow trout . LepBPs are alternatively spliced Leptin receptor transcripts and consist of the extracellular segment. The different LepBP are differently expressed in different tissue of the fish. This indicates that the physiological action of Lep in fish can be modulated, at the level of tissue by modulating the expression of multiple LepBPs. LepBP were isolated from plasma and their binding specificity was assessed in a competitive binding assay. Quantitative assays revealed that plasma LepBP declines in rainbow trout after fasting. In maturing Atlantic salmon males and females no seasonal variation in LepBP in males because of large individual variation, but in females light and feeding regimes modified LepBP. Accordingly, LepBP may change transiently in response to environmental changes.
Thyroid hormones, their transporters and receptors are key-element in regulation of fish early development and metamorphosis and disregulation may explain poor larval or juvenile quality in aquaculture production. Thyroid hormones (THs) have to enter cells to bring about their biological action and recently a family of TH transporters located in cell membranes have been found to regulate this process. Once inside the cell, activation of THs occurs and the precursor hormone, thyroxine (T4), is converted into tri-iodothyronine (T3) by deiodinase enzymes and it then binds to thyroid hormone receptors (TR) and activates gene transcription. The role in development of TH transporters and receptors was studied in Atlantic halibut larvae and zebrafish larvae. In zebrafish the fundamental importance of THs in fish early development was demonstrated by ablating the TH transporter, MCT8, and blocking T3 uptake from the yolk by the embryo. The resulting larvae has abnormal brain development and the heart and GI-tract were also affected. The study reveals that an adequate supply of maternal THs in the egg is important for normal development and highlight the need for appropriate broodstock nutrition. Furthermore, TH cell transporters, deiodinase enzymes and TH receptors appear to be important to ensure normal embryonic and larval development. In a later developmental stage in Atlantic halibut when it transforms from a symmetric larva to an asymmetric juvenile TH transporters, (MCT8 and MCT10) gene transcript abundance increases from the start of metamorphosis until metamorphic climax and declines after metamorphosis, which is similar to THs, TR and deiodinase enzymes and further emphasises the fundamental importance of THs in this process. From a production perspective since metamorphosis impacts the quality and robustness of juveniles used for subsequent grow-out phases problems can severely affect sustainability.
Flatfish such as Atlantic halibut, turbot and sole are highly prized by consumers for their firm flesh and delicate flavour. Consumer demand for flatfish far outstrips supply of both wild fish from fisheries and from aquaculture production meaning that these fish command high market prices. The failure of aquaculture to meet the strong consumer demand for flatfish is a result of specific bottlenecks in their production cycle that threaten sustainability and check expansion of the sector. The flatfish offer a unique production challenge as unlike other commonly exploited finfish they shift from plankton feeding larva, that swim in the water column, to bottom dwelling juveniles with both eyes located on the same side of the head. The transformation from a symmetric larva to an asymmetric juvenile is called metamorphosis and represents a crucial moment in the lifecycle as failures in this process have a significant impact on viability and quality of the resulting juveniles. The role of thyroid hormones (THs) in orchestrating flatfish metamorphosis is well accepted, but the way in which they do this is far from understood. Therefore, METAMOPHOSIS was selected as one of the top-priority research areas of the LIFECYCLE project. It was not possible in LIFECYCLE to study all commercial flatfish species and the work focussed on Atlantic halibut for logistical and practical reasons. Nevertheless, because metamorphosis is an event that is TH driven and is highly conserved in vertebrates including the flatfish species the knowledge developed in the Atlantic halibut will be transferrable.
The challenges of flatfish larval production were evident during the project when the progress of the work was severely challenged by repeated and unexplained “collapse” of Atlantic halibut larval cultures at the commercial partners. For the project this was inconvenient and the problem was eventually overcome but for the industrial partner such unpredictable and uncontrollable events threatened sustainability. This problem highlights the urgent need for better knowledge about other aspects of early larval rearing not studied in the present project. The overall aim of research was to identify developmental programs, mechanisms and functional modifications associated with TH driven organ maturation during metamorphosis. The partners took advantage of next generation sequencing (NGS), a cost effective way to look in detail at the genes being expressed in any tissue. The absence of consolidated molecular resources for the halibut makes identification of gene transcripts difficult and this was overcome by generating an Atlantic halibut transcriptome by fusing the data from the project with existing publicly available data and this generated 17,000 unique halibut transcripts. Micro-dissection of tissues known to respond to THs followed by sequencing generated tissue specific Atlantic halibut transcriptomes and tissue specific development pathways and also general and tissue specific TH-responsive genes were identified. Taking advantage of the well-established staging scheme for Atlantic halibut the transcriptome of individual halibut larvae was determined and by comparison between them used to identify the principal molecular differences between development stages; early larvae, pre-metamorphosis, metamorphosis, climax, post-climax and juvenile. Highly differentially expressed transcripts between stages were associated with maturation of muscle, the cardiovascular system, skeletal system, skin and the GI-tract. Overall, tissue-specific gene networks activated or repressed during normal metamorphosis and TH responsive transcripts were identified. Molecular studies were linked to studies of tissue organisation using histology and immunohistochemistry and permitted identification of the main maturational changes occurring during metamorphosis. Overall, the histological picture together with specific molecular tools associated with Atlantic halibut metamorphosis could be used in future projects to predict and assess metamorphic success in response to modified culture regimes.
The importance of correct metamorphosis for function has been revealed for the GI-tract and the skin and insight into none metamorphic maturational processes have also been gained. At the level of the GI tract, a 3D model of its ontogeny was established and revealed increasing complexity is correlated with changes in function. In vivo pH analysis and quantitative PCR (qPCR) of α and β subunits of the gastric proton pump (H+/K+-ATPase) and pepsinogen A2 indicate that gastric proteolytic capacity is established during the climax of metamorphosis. Moreover, it was revealed that the stomach’s function in appetite regulation is interdependent with establishment of gastric function. A process independent of metamorphosis was the putative osmoregulatory capacity of the GI-tract already at first feeding in the Atlantic halibut and further exploration of events independent of metamorphosis should be fruitful. A better understanding of GI tract function and maturation will contribute to improved management of feeding. The skin that is an important innate immune barrier changed from a simple epithelia to a multi-layered tissue, which was less leaky and this change was pronounced from climax onwards. The reduction in skin keratin (that makes human skin impermeable) from metamorphic climax onwards coincided with the appearance of goblet cells that were presumably produced protective mucous. A better understanding of skin and its maturation has contributed knowledge for improving larval rearing conditions as it provides the basis for understanding osmoregulation, gas exchange and innate immunity. Studies to block metamorphosis by inhibiting TH production or to assess how stress impacts on metamorphosis confirmed the importance of THs and revealed a role for stress. A surprisingly strong “self-righting” mechanism exists in the Atlantic halibut and attempts to block metamorphosis caused a strong activation of the TH axis so that only 25% of fish were affected. Overall, the results of the LIFECYCLE work on metamorphosis demonstrated that direct disruption of the thyroid axis or cortisol axis modifies tissue specific developmental programs but it has not been proven yet if this directly underlies the overt changes that occur during arrested metamorphosis.
Ability to adapt to various environmental conditions is vital for fish and they rely on the development of osmoregulatory mechanisms at the level of specific organs such as the gills. Such adaptive processes occur during early development (embryo-larvae transition) when osmoregulatory functions develop and during smoltification when juvenile fish have to adapt to salinity changes. These situations are critical bottlenecks in salmonid aquaculture. For the preceding reason the LIFECYCLE project focused on DIFFERENTIATION AND PLASTICITY OF TRANSPORTING EPITHELIAL CELLS. The aim was to decipher the major molecular mechanisms regulating differentiation of osmoregulatory gill cells, a key process for development of osmoregulatory processes that give the fish the ability to withstand environmental changes. To analyse gill cell differentiation processes, descriptive studies of candidate gene expression in salmonid was combined with functional validation by transgenic approaches in a model fish species, medaka.
Several specific markers of differentiated gill cells (ionocytes and pavement cells) involved in osmoregulation were identified using microarray analysis. This work was carried out in trout by; 1) analysis of the gill transcriptome in fish transferred from freshwater to seawater; 2) by comparing gill to others organs; 3) by analysing dissociated and purified gill cells; and 4) from literature searches. Overall, this approach led to validation of 1 gene specific for freshwater ionocytes and 3 genes which are significantly expressed in all differentiated epithelial cells (pavement cells and in ionocytes) in both freshwater and seawater.
Key genes controlling epithelial gill cell differentiation were identified using 1) a survey of the literature; 2) a gill transcriptome analysis after freshwater-seawater transfer in trout; and 3) a genetic screen based on systematic in situ hybridizations. Candidate genes analysed further were selected based on their specific expression in the gill presumptive domain of the medaka embryo. Overall, these studies identified 20 candidate key genes potentially involved in epithelial cell differentiation.
The functional role of 3 candidate genes (gcm2, sgk2, GR2) controlling epithelial cell differentiation was validated in medaka during embryogenesis, using gene inactivation approaches based on morpholino injections. The inactivation of gcm2 and sgk2 led to significant modifications in ionocyte number in the embryonic skin, suggesting they have a key role in the ionocyte differentiation. Similarly, we also showed that maternal GR2 (glucocorticoid receptor form 2) is a major pathway in the ionocyte biogenesis in medaka, during early development.
Overall, during the LIFECYCLE project specific markers for differentiated epithelial gill cell and also key genes which are involved in epithelial cell differentiation were identified. The candidate genes were used in other LIFECYCLE tasks aimed at determining the effect of sexual maturation and androgens on the osmoregulatory system in Atlantic salmon. Recent findings suggest that GR2 also plays a key role in the modulation of the gill ion-transporting epithelium in salmonids during freshwater/seawater adaptation.
WP2.2 The intestine is the key organ for uptake of nutrients as well as for the uptake of water, through ion-coupled absorption mechanisms that are of key interest for aquaculture. Thus, LIFECYCLE has focused research on the MULTIFUNCTIONALITY OF THE GUT. Fish are continuously exposed to osmotic forces across all epithelia. In fresh water (FW), active absorption of ions and excretion of the excess amounts of water diffusing into the fish is crucial. In seawater (SW), on the other hand, uptake of water by the intestine in combination with secretion of ions is needed. The driving force for both the fluid uptake and the uptake of nutrients is the electrochemical gradient for Na+ ions, created by the Na+,K+-ATPase (NKA) and directed into the intestinal epithelial cells. The Na+-gradient brings along either other ions, like K+ and Cl- or nutrients, like amino acids and glucose. Thus, the utilization of the same electrochemical gradient, at the cellular level, may lead to a competition between absorbing ions for osmoregulation or nutrients for growth and explain growth check in a production setting.
Some active ion-transporters suggested for stenohaline SW fish (do not tolerate change in salinity) are present also in anadromous salmonids, but the transporters and proteins involved in intestinal water uptake is incomplete. Gene transcript (qPCR), and protein expression (immunohistochemistry) together with physiological measurements of ion- water- and nutrient transport, have revealed the developmental changes occurring in ion transport mechanisms as well as epithelial permeability during the parr smolt transformation (PST) and the subsequent transfer to SW. Understanding this process is important as it can lead to substantial losses if it fails to occur correctly in aquaculture.
The pre-adaptation of the intestine to SW includes mobilization of ion transporters that in concert work to build up a high osmolality (mainly through high Na+, Cl- and K+ levels) in the space between the cells (the lateral intercellular space; LIS) to drive fluid uptake. However, during the parr-smolt transformation, when the fish is still in FW, it would be counterproductive to start absorbing water and thereby add to the osmotic problems experienced in FW. The model for intestinal fluid transport in the smoltifying salmon developed from the results of studies in LIFECYCLE. Three, concomitantly acting mechanisms, including increased expression of the necessary ion transporters and mechanisms that can un-coupled their activity build up high osmolality in the LIS while the fish is still in FW. Thus, even though the NKA activity is gradually up-regulated throughout the time of the PST the enzymes are distributed within the cell membrane so that they don’t exert there action until the fish has entered SW. Similarly an increase in paracellular permeability of the intestinal epithelium, during the latter part of the smoltification, will prevent high ion concentrations in the LIS by allowing a leakage of especially positive ions back to the intestinal lumen. Thirdly, the proton excreting V-ATPase decreased in mRNA expression at the end of the PST when the expression of the NKA and other main transporters increased. As V-ATPase activity decreases proton excretion from the cells instead occur through a sodium-proton-exchanger protein (NHE) and leading Na+ back from the LIS and into the cell. Thus, the decreased V-ATPase activity may constitute a third way to un-couple the creation of a high osmolality in the LIS and thereby prevent fluid uptake.
In aquaculture both an efficient nutrient absorption and a maintained osmotic homeostasis is crucial for optimization of fish welfare and growth. This has led to the hypothesis that fish kept in an iso-osmotic environment, minimize energy use and utilize the available Na+-gradient for osmoregulatory mechanisms and should grow better than fish in SW. To investigate this Atlantic salmon were reared at full strength (34 ppt), 2/3 (22 ppt) and 1/3 (12 ppt) of SW in order to resolve the question of how intermediate salinities affect feed intake (FI), growth (SGR) and food conversion efficiency (FCE) and how this is related to energy requirement for osmoregulation. Fish reared in the highest (34 ppt) and lowest (12 ppt) salinity performed worse than at intermediate salinity (22 ppt). In the 12 ppt group the nutrient uptake across the intestinal mucosa was low and with low FCE and high FI indicates poor digestion and food utilization. The fish could however maintain a high specific growth rate (SGR), probably due to the low energy cost of osmoregulation at close to iso-osmotic conditions. At the other extreme, 34 ppt, the fish grew less FCE and intestinal nutrient transports were low and the results clearly suggest an allocation of the Na+-gradient towards ion transport, which then favours fluid absorption rather than nutrient uptake. Together the results suggest that the high nutrient transport in the 22 ppt group compared to the 34 ppt group is the result of allocation of the Na+-gradient from osmoregulation towards nutrient transport.
The loss of seawater tolerance related to sexual maturation is a production- as well as a welfare-problem in Atlantic salmon aquaculture in seawater net-pens, if fish start maturing prior to slaughter. Therefore, the LIFECYCLE project placed research emphasis on SEXUAL MATURATION AND OSMOREGULATORY FUNCTION. Early puberty is a problem that is most pronounced in males, as males normally reach puberty at a lower age and smaller size than females. Hence, a variable proportion of the individuals become sexually mature before harvest. In a study aimed at resolving the actions of androgens and cortisol in vivo, groups of immature Atlantic salmon post-smolts were maintained in seawater and treated with 11-keto androstenedione (OA), a precursor for the major androgen in fish, 11-ketotestosterone (11-KT), cortisol (F) only, or a combination of cortisol and OA (F+OA).
The key to seawater tolerance, is the activity of the ion pump called Na-K-ATPase (NKA), mainly situated in the fish gill. Gill NKA activity was significantly influenced by hormones and salinity. In seawater, NKA activity in the F(OA) group was reduced to about 50% of control levels, and this group also experienced mortality in seawater. Further, the interactive effect of hormones and salinity was again observed in the F and F+OA groups, which showed significantly reduced NKA activity in fresh water.
Parallel to the changes in NKA activity there was a significant increase in the freshwater isoform NKA α1a mRNA levels. An increase in expression levels by 100-fold or more was seen when control and OA fish were transferred to fresh water. A significant increase was also seen among F and F+OA groups. It is important to note that even in seawater, the F(OA) group showed a highly significant increase in NKA α1a expression levels, a freshwater form of the ion pump, despite the fact that these fish were still in seawater. Looking at the NKA α1b expression levels, a pattern emerges which shows a highly significant, and dramatic reduction in F(OA), down to levels which are characteristic of the freshwater condition. Taken together, these findings clearly demonstrate the reduction in seawater adaptive mechanisms in the F(OA) group, and points to the establishment of a freshwater adaptive situation from the combined exposure to OA followed by F.
In another experiment, the function of the intestine in mature and immature fish in SW was studied. Sexual maturation was associated with a substantial loss of intestinal barrier function, observed in both proximal and distal intestine. A decrease in intestinal barrier function is evident from increased leakage of Na+-ions through the tight junctions. This has a major impact on the fluid absorption of fish in SW as a tight epithelium towards Na+- ions is essential for creating a large osmotic gradient in the inter-cellular spaces used for fluid absorption. To compensate for this, the fish must increase the number and/or activity of the NKA, both at the level of the intestine and in the gill. Indeed, this was observed in the sexually mature fish. Although the fish is able to absorb fluid, it occurs at a higher energy cost due to the reduced barrier function. The intestinal barrier to smaller molecules is also impaired, suggesting that luminal antigens and harmful substances can more easily penetrate the intestine, suggesting that sexually mature fish are more susceptible to diseases.
This work has focused on the effect of sex steroids on critical ion transporting proteins in the gill and intestine during natural sexual maturation of Atlantic salmon. Our hypothesis proposes that the effect of sex steroids in salmon would stimulate a freshwater condition in the gill, and this is indeed what we have found. Taken together, our data show major changes in gill and intestinal function during sexual maturation of Atlantic salmon. Maturing salmon in seawater suffer osmotic stress, and critical elements of their osmoregulatory physiology are directed to a freshwater condition.
The early stages of development of farmed fish are highly susceptible to infection. At these early stages fish cannot be vaccinated as their immune system is not fully developed. A major under explored area of defence mechanisms in farmed fish is maternal influence on fry survival in terms of vertical transmission of immune gene transcripts and the protection this may provide the egg and post hatch stages of the fish. Whilst there is some evidence that protection to certain diseases may be passed on, the extent of this is unknown. Therefore, LIFECYCLE research efforts were directed against the area of OVA AND LARVAL IMMUNE GENE EXPRESSION, in particular to explore how the immune system of developing salmonid fish (rainbow trout and Atlantic salmon) cope with infections. We also examined eggs to determine the immune relevant mRNA molecules that are directly derived from the mother, termed maternal RNAs.
To assess the immune gene component of maternal RNAs a microarray approach was taken using RNA isolated from early stage zygotes before the embryonic transcription was initiated. Hence all RNAs would be maternally derived. We examined the genes present that hybridized to an Atlantic salmon custom Agilent microarray. Here, the criterion for gene selection was based on their expression threshold. The selected gene set (18499 genes) was examined by text filtering for key groups of immune related transcripts. Interestingly, we found many cytokine receptor mRNAs, enhancer binding protein transcripts and transcription factors, suggesting that the eggs are in a state of readiness to respond to immune stimulating signals. Both Interleukin enhancer binding factors 2 and 3 and interleukin receptors (IL-1, Il-4, Il-6, IL-9 and IL-10 receptors) were highly expressed in unfertilized eggs. Similarly for genes related to viral defences where interferon regulatory factors (IRFs 1, 2, 3, 5, 6, 7 and 8) were present, with IRF1, 2 and 7 being the most abundant. A number of immunoglobulins and immunoglobulin binding protein mRNAs are found which potentially relates to maternal immune protection. This research has given unique insights to early stage immune gene expression in salmonids.
During early stages of development, juvenile salmonid fish must rely on their innate immune system to defend against pathogens. At these early stages the immune system is immature and is unlikely to express the full repertoire of genes that control immune defences. Although vulnerable, these larval fish can still fight off infections indicating that there are active immune defence mechanisms. To examine later stages of development from eyed eggs to 3 weeks post first feeding, we challenged four key developmental stages with either bacterial or viral infections. These were eyed eggs, hatchling, first feeding, and 3 weeks post first feeding. Microarray analysis revealed new information about how the immune response increases dramatically during development. Early stages exhibited only innate responses, but at both first feeding and post first feeding, we observed a large number of immune genes that are regarded as part of the acquired immune response. These findings suggest that the transcripts (and probably the proteins) of the acquired immune system are present, and indicates the potential for earlier vaccination, but leads to new questions about how well developed the T cells are and the capacity for immunological memory. Hence, this work has led to new information that is highly relevant to fish health knowledge.
The final goal was to address the early growth rate of the fish. For this we placed first feeding fish on a high and low food ration. Each group was challenged with bacteria, virus or with mock infection. We examined a number of key immune responsive genes, although limited significant effect of growth rate on the response of the immune genes was observed. We extended this study to examine genes known to be involved in control of protein deposition / growth, with the aim to determine if infection altered the energy control balance differently in fast and slow growing fish. The two E3 ubiquitin ligases (these are responsible for targeting proteins for degradation) analysed showed a similar pattern of expression with a general increase in response to immune stimulation. TNFα was also the only cytokine gene to show a significant difference between fast and slow growing trout, with a greater response in the slow growing bacterially stimulated trout compared to fast growing ones.
Overall this part of the LIFECYCLE research effort has generated novel data and expanded considerably our knowledge of the ontogeny of the immune response in salmonid fish and has demonstrated that there may also be a relationship between rate of growth and immune function.
The Atlantic halibut is a marine, cold-water flatfish with a high market value and demand. The species is under development for aquaculture and is a promising candidate to complement and diversify the marine cold-water aquaculture industry.
As mentioned earlier, flatfish like the halibut, have an early unique life stage transition, metamorphosis, during which they transform from pelagic free swimming, symmetric larvae to benthic, asymmetric juveniles. Metamorphosis is common for all flatfish but the large size of the Atlantic halibut larvae as well as its potential as a future important aquaculture species, makes it suitable for studies of physiological, anatomical and immunological events during metamorphosis. One industrial bottleneck in the production cycle of many marine, cold-water species is the high mortality of the larvae after they hatch as they are relatively small and underdeveloped. For production of marine flatfish, this period is even more crucial as it also involves the complex process of metamorphosis. To address these problems, LIFECYCLE research was directed towards the PRIMARY BARRIERS AND DISEASE RESISTANCE with the overall aim to understand the functional characteristics and importance of the skin primary barrier during Atlantic halibut larval development and metamorphosis.
As pathogens are present in water, both in the natural and in the aquaculture environment, the presence of a functional primary barrier pivotal for good larval survival. In the small, newly-hatched yolk-sac larvae, the intestine and gills are poorly developed. Therefore, and due to the high surface to volume ratio in small individuals, the skin constitute the most important primary barrier, in other words the first line of defence, against pathogens in the environment.
Two main technical and methodological advances have been achieved during the LIFECYCLE project. An in vitro system for assessment of Atlantic halibut skin barrier integrity and properties was successfully developed, and four antimicrobial peptides, so-called pleurocidins, were identified in Atlantic halibut, characterised and specific antibodies against these proteins produced. These methodological advances have made it possible to better understand the development of the physical skin primary barrier during halibut larval development and metamorphosis as well as to understand the relative importance of the extrinsic barrier, i.e. the secretion of mucus and antimicrobial peptides during this period.
The skin develops from one cell-layer epithelia found in the early larval stage 6 to a complex and stratified epithelia in the juvenile fish. The supporting connective tissue in the skin, the basement membrane and the collagen fibres, increases both in thickness and complexity during the larval development and metamorphosis. These morphological changes are clearly reflected also in the functionality of the skin barrier. The skin from stage 7 larvae was leaky while stage 9 larvae had twice the resistance to passive flow of water and soluble molecules and stage 8 had an intermediate resistance. Interestingly, the early developmental stages with low and leaky physical barrier also showed a lower number of mucus producing Goblet cells. Thus, the early larval stages seem to be exposed to strong passive, diffusional forces due to a poorly developed physical as well as secretory barrier. The changes in skin barrier properties are gradual except between stage 8 and 9A, when all the developmental skin parameters measured changed dramatically. This indicates that the larvae become much more robust after passing over to stage 9A.
The development of the larvae from symmetric free-swimming fish to a bottom-living asymmetric fish can also be seen in the development of the skin barrier properties. The increased complexity and thickness of both the multi-layered epithelial cells and connective tissue were more pronounced on the top side. This was also the case for specific cell types like melanophores (pigment cells, only present on the top side, and the Goblet cells, which were more numerous on the top side). This suggests that diffusional and invasive forces from the surrounding water require a larger degree of protection than the more mechanical challenge from the underlying substrate.
The finding of four different antimicrobial pleurocidins (PLCs) in the halibut transcriptome clearly suggests an important role for these substances in resistance against infection and disease. Three of the pleurocidins (PLC2, PLC3 and PLC4) were found in Goblet cells in the different epithelia, clearly indicating that these innate defence molecules are secreted with the mucus in order to prevent invasion of pathogens into the body of the larvae. The pattern of distribution of PLCs 2-4 in the different epithelia as well as larval stages were slightly different for the three pleurocidins, but PLC3 was abundant in all epithelia and all stages of larvae from stage 7 and onwards. Thus, this anti-microbial peptide seems to be a very important component in the defence against pathogens in the earliest and most vulnerable stages of Atlantic halibut, when the physical barrier is still weak and leaky.
Life stage transitions often involve major changes in phenotype and physiological functions. Such life-stage transitions represent a particularly vulnerable period in aquaculture, and in many cases, high mortalities are seen. To meet the challenges of the new life stage, changes in form and function of epithelia occur. Gill and intestinal epithelia constitute an interface between the environment and the internal milieu and have unique roles as both primary defence barriers and selectively transporting tissues. Developmental and environmental driven changes in form and function of these transporting epithelia will affect the primary barrier functions and potentially the epithelial immune system and vice versa. Therefore, the LIFECYCLE project concentrated research on the IMMUNE-ENDOCRINE-PHYSIOLOGICAL CROSS-TALK AT THE PRIMARY BARRIER AND SYSTEMIC LEVELS, in order to study the impact of key life history changes in salmonids on their ability to resist diseases. A particular interest was the cross-talk between immune function and hormones known to be at high levels during the transition from the fresh water parr stage to seawater adapted salmon (termed smoltification or parr-smolt transformation), or during sexual maturation. In addition, host immune molecules (cytokines and free radicals) that are elicited during inflammation were also studied for their impact on physiological functioning of the fish, at sites that act as primary barriers to infection but that also have important roles in ion/water/gas transport (e.g. gut and gills), to see if these functions are affected/compromised.
Both the parr-smolt transformation (PST) and the subsequent seawater (SW) migration are important life stage transitions for Atlantic salmon. At the level of the intestine, the physiological pre-adaptations to SW include de novo synthesis of ion transporters and other epithelial proteins to create a mechanism for fluid absorption. In addition to the intestinal epithelial permeability, this tissue also constitutes a physical barrier against pathogens in fish.
The barrier function, assessed using electrophysiology on live intestinal epithelia, changed during the PST. The paracellular permeability increased, rendering a decreased barrier, towards the peak of smoltification when the fish are still in FW. Directly after SW transfer, a dramatic decrease in permeability occurred, indicating a significant strengthening of the physical primary barrier. A regional difference in tightness of the paracellular pathway was also seen, with the proximal intestine being leakier than the distal intestine. Gene expression measurements as well as immunohistochemistry showed expression differences may be governed by the barrier forming claudin isoform, claudin 25b. Upregulation of two other major tight junction proteins, occluding and ZO-1, were each shown to contribute to the tightening of the physical barrier after SW transfer and the tighter barrier in the distal intestine, respectively. The extrinsic barrier, i.e. the secretion of a protective mucus layer covering the intestinal epithelia had a similar pattern of change during PST. The mucus secretion was reduced towards the peak of smoltification, but increased to levels above those of parr in smolts after SW transfer. Thus the physical and extrinsic barriers together create an enforced primary barrier in the salmon intestine after SW entry and start of drinking. This will protect the epithelium from exposure to pathogens in the ingested water.
However, the results from the LIFECYCLE project also demonstrate that several factors interfere with these developmental changes in the primary barrier. Both acute and long term stress, through the stress hormone cortisol, negatively affected the physical barrier. It widened the paracellular pathway probably by changing the expression of tight junction proteins. An increased leakiness will induce the underlying epithelial immune barrier to start secretion of pro inflammatory cytokines. Electrophysiological studies in combination with perfusion studies, using recombinant trout cytokines, and IHC showed that some of these cytokines in turn affect the physical barrier. rIL-1β and rIL-6 increased the paracellular permeability by decreasing the expression of claudin 25b and caused a cellular re-distribution of ZO-1. Such processes may contribute to the increased susceptibility to infection seen in Atlantic salmon smolts directly after SW transfer.
The effect of free radicals and their precursors, produced during pathogen infection, on gill epithelium was also studied. Incubation of gill cell cultures with different doses of hydrogen peroxide showed a toxic effect using higher levels (4mM), where a disrupted epithelium was seen, and gill permeability and gene expression were decreased. At 2mM only gene expression was affected, and below this dose no toxic effects were apparent. Curiously, a gene that encodes a tight junction protein had increased expression at low doses of hydrogen peroxide, suggesting it may have an important role in maintenance of epithelium integrity in the presence of free radicals.
The impact of PST on the expression of a number of genes involved in anti-viral defences was examined, with a focus on interferons, since viral infection is a known risk factor in smolts post transfer. Parr and smolts were stimulated with a mimic (poly I:C) of viral infection at 5 time points during the parr smolt transformation, with subsequent analysis of the immune genes in immune organs. The results show that during the critical smolting period the immune response is reduced, especially at the time of transfer to SW, and highlight that this is a vulnerable stage during the development of salmon.
Experiments were also designed to examine the impact of sexual maturation on the antiviral immune response. Initially we tried to use sexually maturing male fish (during 2011) but there were insufficient numbers of fish, and so the approach was changed and sexual maturation was induced by injection of the androgen 11 ketoandrostenedione. Three weeks later, the fish were exposed to poly I:C to induce antiviral responses and tissue samples collected 24h later for gene expression analysis. This experiment showed that up-regulation of antiviral genes by poly I:C treatment occurred as expected, with a downward trend in gene expression observed in the androgen treated fish, although no significant changes were observed.
As a key hormone in the PST process and stress responses in general, cortisol was also investigated in vivo to examine the impact of this hormone on the antiviral response. Cortisol was administered to fish as a slow release implant. Six days later the fish were exposed to poly I:C for 24h and immune tissues collected for antiviral gene expression and blood cortisol analysis. Plasma cortisol was upregulated. In the gills, poly I:C treatment provoked upregulation of the genes studied. However, there was no clear impact of cortisol treatment vs. control fish in the presence or absence of poly I:C challenge. Cortisol treatment stimulated expression of sodium/potassium ATPase genes, consistent with previous studies, illustrating the importance of this hormone in ion regulation. The only significant effect of cortisol treatment was to decrease expression of the pro-inflammatory gene IL-1β. Up-regulation of antiviral genes by poly I:C treatment was weaker in spleen tissue, where again no modulation of gene expression was attributed to cortisol treatment.
To further examine potential cross-talk between the endocrine and immune systems, a panel of hormones; cortisol, IGF-1, T3 and growth hormone (GH) were used to examine their effects on a variety of isolated immune cell types. We examined macrophages, CD4+ cells (expected to be mainly a subpopulation of T cells), B cells and neutrophils. Incubation of macrophages for 3 or 6 h with cortisol decreased expression of two cytokines (IL-6 and IL-8) involved in inflammation. Co-stimulation of cortisol with inflammatory agents resulted in a general suppression of genes related to the proinflammatory response. In contrast, cortisol in combination with pro-inflammatory agents had a synergistic effect on IL-10 expression, and anti-inflammatory molecule. However, no impact of incubation with T3 or IGF-1 was seen.
Using a trout anti-CD4 antibody we identified a CD4+ cell line (RTHK15) developed by exposing trout leucocytes to IL-15-containing medium. The results from the cell line were in sharp contrast to our previous findings of cortisol suppression of immune gene expression in macrophages. Cortisol exhibited a stimulatory phenotype particularly in the presence of a T cell stimulant (PHA). A significant synergistic effect was seen between cortisol and PHA in the case of IL-2, IL-4-like, IL-8, IL-17C2, IL-22, and TNF-3. Indeed, five genes ( IL-1β, IL-2, IL-22, RORγ, and TGFβ1.2) were significantly upregulated in the presence of cortisol alone. Interestingly, a third group of genes (IL-15 and γ-IP) were not influenced by cortisol treatment. These findings suggest the presence of cortisol responsive and cortisol unresponsive immune cell populations in trout.
Monoclonal antibodies to trout B cells and neutrophils were used to isolate these cells types, by magnetic-activated cell sorting. No significant changes were seen after incubation of B cells with the hormones, when analysing a selection of B cell markers, but some trends were found. For instance, the relative expression of the B cell receptor (IgM) and a surface expressed co-stimulatory molecule (CD40) showed a small elevation in expression after incubation with GH in the presence of LPS. Purified neutrophils also showed no statistically significant changes after stimulation with the hormones but again a trend for induction in some genes was seen. In this case GCSF expression was increased after incubation with cortisol and PMA, and IL-8 expression was increased after incubation with GH.
Overall, the CD4+ cells and macrophages were the most sensitive to hormonal treatment, especially with respect to cortisol treatment. Nevertheless, it is clear that different immune cell types in trout do respond differently to physiological levels of the hormones used
Lastly, the regulatory regions of the genes most modulated by cortisol in the macrophage experiments above were analysed to determine the presence of transcription factors known to be responsive to hormones/stress. For this we used the Atlantic salmon genome database to retrieve the for six immune genes, IL-6A, IL-6B, IL-8, IL-10, γIP and COX-2. Four elements were searched for, namely the glucocorticoid receptor responsive element, estrogen receptor-responsive element, the thyroid hormone receptor-responsive element and the cAMP responsive element. Variation in the number of elements was apparent between the genes, with IP having the fewest (10) and COX-2 the most (28), allowing predictions about their relative sensitivity to hormone exposure.
Specific immunity is crucial for long-term disease resistance. Previous studies on early expression of Ig in B cells or TCR genes in T cells are limited and cannot predict the effectiveness of a response to infection or vaccination during the larval stages. Very little is known about the impact of reproduction on the diversity of the immune repertoire and on the response effectiveness. It is important to distinguish between the non-specific effects due to stimulation of innate immunity, and the specific and long lasting protection due to antibody and T cell responses. Thus, DNA vaccination induces both specific and non-specific responses in adult fish, and is considered to afford protection to fry only through innate mechanisms such as interferon production. However, the mechanisms and the dynamics of the transition between the different responses are still unknown in the fish. Therefore, the LIFECYCLE project included research into the area of MATURATION AND SPECIFIC IMMUNE RESPONSES. Trout immune repertoires were analysed to get insight into the diversity of immunoglobulins (Igs) and T cell receptors (TCR) in fish. Upon infection Ag-specific B and T lymphocytes develop protective responses against the intruding pathogen, and after disease resolution they maintain an increased resistance against the eliciting agent through persistence of memory cells, which is the basis of vaccination.
In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT are unknown. A comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout was undertaken before and after challenge with an important fish rhabdovirus (the VHSV) using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, distinct repertoires for IgM, IgD and IgT, respectively were observed, with a few IgM- and IgT-secreting cells in the spleen. In infected animals, complex and highly diverse IgM responses were detected dominated by a few large public and private clones. A lower number of robust clonal responses involving only a few VH were detected for the mucosal isotype IgT, indicating that both IgM+ and IgT+ spleen B cells responded to systemic infection but at different degrees. In contrast, the IgD response to the infection was faint. This study is the first attempt to obtain a comprehensive description of the Ig response after vaccination in fish, and provides a basis for further characterization of responses induced by vaccination in aquaculture.
Regarding T cells in fish, the diversity of TCR expressed by CD8+(CD4-) and CD8-(CD4+)T cells remains poorly understood. This is an important issue since fish CD8+ T cells are cytotoxic while CD8- T cells appears to be the counterparts of T helper cells, which are critical to promote strong protective Ab responses. The complexity of TCR beta was compared between FACS sorted CD8+ and CD8- T cells from spleen and pronephros of rainbow trout. It was found that the repertoire of CD8+ T cells was highly diverse and polyclonal while it appeared to be very different in CD8- lymphocytes with dominant clones even in naive fish. After infection with a systemic virus, CD8+ T cells mounted a typical response; the virus also induced significant modifications of the CD8- TCR beta repertoire, but targeting different V/J combinations. These results suggest that the diversity of TRB expressed by CD8+ and CD8- alpha-beta T cells may be subject to different regulatory patterns in fish and in mammals.
In fish as in mammals, two lineages of T cells have been identified, expressing either the alpha-beta or the gamma-delta TCR. In mammals the latter type of T cells recognizes unconventional antigens and is especially important for mucosa protection. The LIFECYCLE research has discovered and characterized the TCR gamma of sea bass. Gamma-delta TCR mRNA expression was detected in basal conditions both in thymus and intestine, while in kidney and gut leukocytes it was up-regulated by poly I:C in vitro. This work provides tools to study the biology of gamma-delta T cells in an important aquaculture species in Europe.
European sea bass and salmonid aquaculture both require production of all-female populations for optimizing growth and to avoid the problems related to sexual maturation by producing all-female triploids. The available sex control methods are time consuming and not fully efficient in sea bass while androgen treatment is used in salmonids provoking environmental and consumer concern. Knowledge about key molecular regulators of sex determination/differentiation can support a rational choice of new techniques for sex control. The LIFECYCLE research programme aimed at improving this knowledge by focusing research on BRAIN AND GONAD SEX DIFFERENTIATION. It was already known that sea bass has a polygenic sex determination system which is influenced by temperature, and that salmonids have a more simple, genotypic sex determination system. Thus, gene expression was extensively analysed in larvae before and during sex differentiation of the gonads by comparing genes expression either in larvae reared at low (15°C) or high (20°C) temperature in sea bass or by comparing genetic females and males in rainbow trout (mono-sex populations). Different cutting-edge laboratory techniques have been used for identifying and characterizing gene transcripts and dozens have been found that are involved in the molecular cascade that controls gonad sex differentiation.
Firstly, it has been confirmed there is no unique molecular model for sex determination and differentiation in fish. The genes involved in sex differentiation in other vertebrates do not always have a conserved role in all vertebrates. In the sea bass some of the genes involved in the molecular cascades leading to sex determination have a role in chromatin remodelling and epigenetic regulation, i.e. not involving a change in the nucleotide sequence. Genes up-regulated in male-biased populations of sea bass during the sex differentiation phase were associated with migration, proliferation and differentiation of germ cells.. A specific sex-determining gene was not identified although a suite of genes associated with males were identified and could be used for selection of strains giving a high rate of males by rearing larvae at high temperature.
In rainbow trout, it was found that not only the gonads but the brain as well is sexualized early, exhibiting a clear sexual dimorphism with respect to the expression of several genes. Interestingly, they include the key enzyme in oestrogen synthesis (aromatase B) as well as of certain enzymes and factors involved in the synthesis of other steroids and of estrogens receptors. Complementary studies performed in zebrafish, provide the first demonstration that the brain of fish can produce true neurosteroids, notably in radial glial cells, potentially neuronal precursor cells. Thus, a dialogue between gonads and brain should be taken into account in the future for the full understanding of regulation of first stages regulating sexual differentiation. In gonads, the kinetics and cellular localization of a group of genes potentially involved in the molecular cascade of sex differentiation were defined. This group includes genes known in other vertebrates (e.g. tbx1, amh, gsdf1, dmrt1, pax2 in male; fst, wnt, foxl2, inha in female), but also new genes (e.g. irk8, pgh2, gata2, gremlin). Experiments involving steroid administration to larvae provided additional information on the steroid sensitivity of the expression of these genes. Finally, a major outcome is the discovery that a new gene called sdY (for “sexually dimorphic on the Y-chromosome”) is the major sex determinant gene in salmonids. This gene has been functionally characterized and its spatio-temporal expression during natural sex-differentiation was defined. The sdY gene sequence has been used by international biotechnology services for genotyping in salmonids, and effectively accelerating the monosexing process in trout. It will be very useful to explore the reason of male occurrence in industrial all-female population and to develop new monosexing methods.
One of the key life-stage transitions which needs to be controlled in commercial fish farming is the onset of puberty, i.e. when the fish start the process of sexual maturation. LIFECYCLE conducted research into PUBERTY CONTROL by assessing the impact of physiological conditions (growth, adiposity) and external signals (photoperiod, behaviour) and molecular factors underlying the onset of puberty in salmon, trout and sea bass. A major breakthrough was the characterization of the Kiss-system and its relation to the previously known Gnrh system, including information on the possibilities for the feedback regulation of the Kiss/Gnrh systems by sex steroids. Decisive for this success was the strategy to first establish the basis in zebrafish, before studying the considerably larger sea bass and salmonid brains in a targeted manner. Also functioning in the brain/pituitary system and representing another, very important discovery is the identification of the in salmonid fish long-sought receptor mediating reproduction-related Gnrh effects in the pituitary. An attempt to generate transgenic medaka overexpressing Kiss peptide did not succeed. However, studying the effects of Kiss injections compensated for this and had the advantage of allowing experiments with sea bass directly. It was demonstrated that in fish, similar to land vertebrates, the Kiss and Gnrh systems regulate the pituitary gonadotropins. However, different from the higher vertebrates, where Kiss seems to work exclusively via the Gnrh system, Kiss also has direct effects on the pituitary in sea bass. Altogether, this objective has been fully achieved, maybe even above the initial expectation, considering that the work dealt successfully with an unexpected genetic complexity of the Kiss/Gnrh system in particular in salmonid fish.
The chosen experimental conditions (feeding level, photoperiod regime) have resulted in the expected, clear-cut effects on the incidence of puberty in sea bass and salmon. Investigations on the key components of the above-discussed regulatory systems operating in brain and pituitary, were complemented by quantifying plasma hormone levels and by analysing morphologically the start of spermatogenesis. This allowed another breakthrough, which was also facilitated by taking some of the crucial steps using trout and zebrafish as additional experimental models. Work in all species consistently demonstrated that the initiation of pubertal testis maturation involves the pituitary hormone Fsh. Fsh then activates testicular androgen production and modulates growth factor expression, and androgen signalling and changes in the growth factor “cocktail” then initiate spermatogenesis. For the salmon, the data moreover indicate that the newly discovered Gnrh receptor form (see point 1 above) is involved in mediating the activation of the pituitary.
The relationship between experimentally (feeding and/or photoperiod) induced changes in puberty and the Kiss/Gnrh system, however, requires more work and is not clear yet. Next to technical considerations (e.g. size of brain samples used for gene expression analysis), one of the reasons, at least in the salmon, might be related to the fact that the period during which external and internal factors affect the decision to initiate puberty, apparently act as early as 12-15 months before the first spawning season, i.e. earlier than anticipated in most of the experimental set-up used here (however, see trace-back studies in point 3). The work carried out in sea bass on the possible impact of social interactions and chemical communication provided no evidence for effects of behavioural cues or pheromonal signals on the timing of puberty.
Since restricted feeding was known to reduce the incidence of puberty, we hypothesized that hormones involved in regulating growth/adiposity (leptin, ghrelin, Gh, Igf1) play a role in the recruitment into puberty. This was examined by studying aspects of growth and reproduction in terminally sampled fish during the period December – June when recruitment into puberty becomes obvious, and by repeatedly life-sampling fish from September – June which were scored for maturity June, in order to trace back the relation between hormone plasma levels and growth to the maturational state reached in June. Moreover, we examined testicular gene expression patterns in fish at the brink of puberty (December and January) that were subjected to normal versus restricted feeding.
The study on terminally sampled fish revealed no clear evidence for significant correlations between metabolic hormones and pubertal development, while unexpected correlations were found for, e.g. effects of photoperiod regimes on plasma leptin levels. The trace-back approach, on the other hand, provided clear evidence for an early predisposition of maturing males. As soon as December, i.e. 9 months before spawning, higher plasma androgen levels were found in maturing males, and even earlier already in September, these males also showed a higher body weight. These observations warrant further studies to unravel the relation between these early differences in body weight, and the subsequent differences in plasma androgen levels that herald future maturation.
In association with work spermatogenesis, feeding-related differences in testicular gene expression patterns were investigated with the view to develop hypotheses on how growth and energy stores interfere with the brain-pituitary-testis axis to influence the decision to mature. Based on parameters reflecting testicular activity (germ cell proliferation, plasma androgen levels), 36 males were selected for large-scale testicular gene expression profiling. An important observation comparing maturing males is that under normal feeding conditions, growth factor expression gradually changed in salmon testis just starting to mature, while a sharper contrast with abrupt changes in the expression of the same genes occurred in animals kept under reduced feeding conditions. This suggests that under conditions of restricted feeding, clearer signals are required to achieve activation of testis maturation.
The search for genes associated with the physiological readiness to precocious puberty in sea bass was delayed repeatedly, first due to technical problems with the chosen sequencing approach, then as a consequence of the move to another institute of the scientist leading the sequencing activities. Accordingly, the sequencing results became available rather late for the group receiving the data, so that completion of the necessary analyses was not possible within the remaining time frame of the project. Still, the data set is available now and initial evaluation of the brain samples suggests that the experiment was successful per se.
Male puberty involves the activation of steroidogenesis and spermatogenesis, and better understanding of this process is of great importance for aquaculture production. Therefore, LIFECYCLE focused research on the PARACRINE SIGNALING AND TESTICULAR GENE EXPRESSION DURING THE INITIATION OF SPERMATOGENESIS. The aim was to characterize the gonadal molecular responses to FSH and androgens (the two most important hormones regulating testis maturation) that could result in a promotion or inhibition of testis maturation. In vivo effects of recombinant Fsh on spermatogenesis were investigated in prepubertal sea bass and demonstrated for the first time that recombinant Fsh can initiate testis development in immature fish. We also confirmed that androgen production was involved in the initiation of fish testis maturation. Further characterization of regulatory pathways used by hormones to initiate testis maturation was achieved in trout by large scale gene expression changes during natural testis maturation and spermatogenetic development. Androgen-dependent genes were also searched for in prepubertal rainbow trout treated with physiological doses of androgens, while Fsh-regulated genes were identified from trout testicular explants cultured in the presence of this hormone. Orthologues of these hormone-dependent trout genes were identified in several vertebrates for which the genome sequences were available, and candidate genes were annotated for functions demonstrated in other species. These analyses provided us with a knowledge base containing around 2000 genes, essential for the interpretation of androgen and Fsh action in fish. Overall it was considered that candidate genes should be hormonally regulated, evolutionary conserved, act as paracrine factors (i.e. functionally with “growth factor” or “cell fate regulation”), and most likely expressed in the testicular somatic cells.
Based on these hypotheses, four candidates (Amh, Gsdf, Igf1b, Insl3) were studied in a zebrafish testis tissue culture. Amh, Gsdf, Igf1b are expressed in zebrafish Sertoli cells and intra-tubular somatic cells that are known to support spermatogenesis. Anti-Müllerian hormone (Amh), has inhibitory effects on androgen release and, independently, also inhibitory effects on germ cell proliferation and differentiation. The Igf1b has stimulatory effects on germ cell proliferation and differentiation, but does not modulate steroidogenesis. The third candidate, Gsdf stimulated the slow proliferation of undifferentiated spermatogonia type A in zebrafish testis tissue cultures. Finally, the fourth factor identified most recently, another insulin like factor, Insl3, is produced by the interstitial Leydig cells. A model was developed for their role in the initiation of the spermatogenic development.
The biological significance of selected factors was to be explored also in vivo in transgenic zebrafish, genetically modified to over-express these factors. Experiments did not result in significant overexpression of the factors and could not induce a noticeable reproductive phenotype. It was concluded that loss-of-function approaches should be evaluated as alternative routes to study the biological significance of selected paracrine factors in vivo.
To elucidate further the molecular mechanisms underlying the hormonal regulation of the factors characterized above, potential hormone-responsive elements (HREs) located within the proximal promoter region were highlighted in the genome sequences. Then using in vitro analysis according to established procedures, two regulatory elements have been successfully confirmed that suggest that Fsh regulates the transcription of the targeted igf1b gene through the stimulation of the cAMP signalling pathways.
In can be concluded that understanding the physiology of puberty at the testicular level is necessary before new procedures to reduce early maturation in aquaculture can be established. Based on complementary biological models and on primordial original competencies, the LIFECYCE work has provided significant progress in our understanding of hormonal actions during puberty: 1) FSH induces pubertal maturation and androgen treatments modify the immature germ cell towards the profile of differentiated germ cells; 2) Transcriptome analyses provided us with a knowledge base essential for understanding hormone mechanisms of action in fish and a short-list of genes known to be regulated by hormones, evolutionary conserved from fish to mammals, expressed in testicular somatic cells, and possibly acting as paracrine factors on germ cell fate were identified; and 3) three growth factors were shown to have a role in gonad maturation and could be interesting targets in procedures to control of puberty.
I wish to thank the large number of scientists from eight European countries, representing all the thirteen LIFECYCLE partners, who have contributed to this brief summary in a continued effort to disseminate the massive biological knowledge-base, so successfully generated by the whole LIFECYCLE research consortium.
Gothenburg, September 2013
Björn Thrandur Björnsson
LIFECYCLE coordinator
Potential Impact:
LIFECYCLE built on recent major advances in marine genomic research and resources developed for key finfish aquaculture species in Europe including rainbow trout, Atlantic salmon sea bass, sea bream, Atlantic cod and Atlantic halibut, in addition to the knowledge generation by the use of model species like zebrafish and medaka. The project has utilized such new knowledge and research tools in a cross-cutting approach by parallel studies on some of the most relevant farmed fish species in Europe, in combination with model fish studies to generate basic biological knowledge of relevance to solve bottle-necks in the production cycles in European aquaculture.
LIFECYCLE has applied in silico search for relevant genes using recently available genome data in combination with various techniques to study gene expression, either on single genes or the entire transcriptome in various developmental processes along the life-cycle in farmed fish. This has been combined with more traditional physiological, endocrine, immunological, and morphological techniques applied to experimental fish studies at different life stages from egg to adult fish. To some extent this has been combined with studies on gene function in the model species zebrafish and medaka.
The approach has greatly extended our knowledge on e.g. the transcriptome of developing oocytes and eggs with relevance of broodstock rearing and egg quality in farmed fish, sex determination and differentiation in early development, metamorphosis, growth processes and adiposity, links between growth and puberty, development of the immune system and its modulation during life-stage transitions like smoltification and puberty, and various cross-talks between these processes. Finally the project has also studied in detail the impact of various environmental factors on some of these processes.
Although the main impact of the project is new basic knowledge on developmental processes in critical life stages of farmed fish in terms of scientific publications and other dissemination along with a substantial extension of genomic resources and research tools for these species, parts of the knowledge-base generated are of direct importance to farming such as new understanding on how smoltification modulates the immune response of Atlantic salmon, and how factors such as feeding and photoperiod modulate age at puberty in sea bass and salmon.
POTENTIAL IMPACT
Contribution to establishing the knowledge-base for the improved competitiveness of the EU aquaculture industry:
Introduction: The LIFECYCLE project emerged in response to the project call KBBE-2007-1-2-16: Essential biological functions related to the most relevant stages of aquaculture fish life-history Call: FP7-KBBE-2007-2A and it took close to two years to build the project consortium and proposal. When funded, a formal contract was signed between the project consortium and the European Commission and the appendix to the contract, the “Document of Work”, in essence specified the work to be performed during the project and also foresaw the outcome. The present description of the potential impact of LIFECYCLE is therefore circumscribed by the impact expected by the Commission (see below) and forms the basis for the present analysis of the actual project impact that was determined. In addition to foreseen impacts, the project has had a number of other impacts that were not defined in the formal “call text” and they will also be highlighted in the following description and include more general aspects such as, stimulating innovation, contributing to European excellence and competitiveness, structuring the European research area, stimulating international collaborations, and contributing to build a strong skill base in science and aquaculture through training as well as actions favouring gender equality. An important point to bear in mind is that the impact of the project will not terminate with its completion, but will continue for many years to come. Nevertheless, the LIFECYCLE project will have an enduring impact in a number of important areas of aquaculture production, which will be made evident in the following short report.
Expected impact foreseen in the call: The project will deliver improved competitiveness of the EU aquaculture industry by establishing for commercially important species the knowledge-base required for improved stress control, disease prevention and control, new breeding technologies, better environmental performance and also diversification into new species.
Impact in the context of Policy: The aquaculture industry is part of the “bio-economy”, which includes all industries and economic sectors that produce, manage and otherwise exploit biological resources. The biological resources targeted by the LIFECYCLE project were fish, and more specifically, species important for the European aquaculture sector; sea bass, sea bream, Atlantic salmon and rainbow trout, as well as Atlantic halibut and cod to lesser extent. The project was directed at advancing knowledge and stimulating innovations in order to promote and support the sustainable management, production and use of fish, a biological resource used in commercial aquaculture. The project generated a wealth of knowledge that has improved the understanding of fish biology and physiology and when translated to industry should help to overcome key production bottlenecks in the areas of egg and larval production, muscle growth, reproduction and disease resistance, all of which currently challenge productivity. A better understanding of the biology of fishes will make it possible to define sensible strategies that improve European aquaculture productivity and make it more competitive, but not at the cost of animal welfare or the environment. Improved productivity and competitiveness in the bio-economy is expected to generate jobs, and since aquaculture tends to be in coastal regions that have seen a decline in jobs with the loss of traditional fisheries this makes an important contribution to social welfare and well-being. LIFECYCLE has generated a knowledge base for the development of a more competitive and productive aquaculture industry and as such supports the Common Fisheries Policy reform that aims to provide sustainable development of fishing and aquaculture.
Establishing the knowledge-base for commercially important aquaculture species: The section above has given an overview of the main S&T results/foregrounds and scientific outcome of LIFECYCLE. This section will therefore, give a general consideration of the impact and some specific research highlights that have resolved hypotheses or shifted paradigms. Immediate practical impacts are presented under the sub-section innovations.
The first major impact of LIFECYCLE is the significant increase in biological knowledge available for commercial species, that has arisen from the “cross-cutting approach” deployed to study fish developmental stages. This was achieved by bringing together internationally acknowledged research groups studying individual organs/functions, and the interactions and cross-talks between different biological systems/processes were examined throughout the whole fish life-cycle from gametes to reproductive individuals. Since commercial producers may be involved in the productions of several life stages or concentrating on a specific process e.g. grow-out that may cover several biological stages, the LIFECYCLE approach has direct practical interest for producers.
The second major impact of LIFECYCLE has been to advance understanding of fish biology through the utilization of existing molecular and biochemical tools, but also through the generation of additional molecular and biochemical tools. The molecular information generated by LIFECYCLE has been released into the public domain. In this way LIFECYCLE has had a knowledge spreading effect that has benefited the scientific community in general and given added value to the project outcomes.
The third major impact of LIFECYCLE has come from studies of tractable model species in which integrated molecular, genomic and functional information from the zebrafish and medaka has been used to advance the state-of-the-art and then applied to aquaculture species. This approach was novel for aquaculture research, but its successful application in LIFECYCLE has given “proof of concept” and reveals the approach may be a rapid and cost-effective way to obtain knowledge useful to solve commercial problems. LIFECYCLE took advantage of the benefits of the model organisms zebrafish and medaka to establish and study basic biological mechanisms and since they are well conserved between teleosts it was possible to transfer knowledge to the major commercially reared fish species. Moreover, knowledge transfer between zebrafish and the commercially reared fish was facilitated by the generation of further molecular data about the transcriptomes of sea bream, sea bass, halibut and cod under a range of conditions using a next generation sequencing (NGS) approach.
The results coming from LIFECYCLE will underpin future generations of basic science and applied projects for both consortium members, and other scientists, and the impact will be long-lasting towards the horizon of 2020.
Innovation: LIFECYCLE has substantially advanced current knowledge beyond the state-of-the-art and has impacted on egg and larval production, growth and energy balance, broodstock and reproduction, fish immunity and disease resistance and osmoregulation. The diverse and complementary nature of the consortium has allowed key basic questions to be addressed at specific life stages or across the life cycle. Research has encompassed the gene, cells, proteins, tissue and the organisms and when it was possible to integrate information has consolidated understanding of specific biological processes and has also given novel insights. Through conducting high quality research LIFECYCLE has contributed to European excellence and competitiveness. Below, examples are given of some of the major advances and outputs achieved by the LIFECYCLE project:
Regulation of maternal mRNAs: It has been demonstrated in LIFECYCLE that maternally-inherited gene products play a crucial role in defining the eggs ability to support early development, once fertilized. Improper broodstock conditions and stress modify the maternal RNA content and this alters the cell division processes. Teleost eggs contain an abundance of maternally derived hormones that are assumed to be important for egg development. The importance of thyroid hormones (THs) for egg viability and larval quality was demonstrated during LIFECYCLE. It was revealed that TH cellular transporters are present in developing zebrafish embryos and their ablation modifies TH availability and caused increased mortality and the surviving larvae had a severely modified nervous system, heart, muscular and gastrointestinal tract.
Fish immunity: The very early stages of the immune response were examined in terms of antibacterial and antiviral responses. LIFECYCLE showed that the complexity of the immune response increases dramatically from the eyed egg stage to first feeding. Many of the elements of the acquired immune system were also found to be present. This suggests the cellular machinery for immunological memory is ready at stages earlier than previously thought. Relating to immunological memory, a strategy to gain a comprehensive description of the antibody diversity of rainbow trout was established. This was used it to analyse the response to VHSV, a serious viral pathogen of salmonids. The research showed that secondary infection induced a massive IgM response against the virus in the spleen. The mucosal isotype IgT also produced a significant response. This provides a frame for further characterization and monitoring of responses induced by vaccination in aquaculture.
Sex determination is governed by a ‘genetic sex determination’ (GSD) system in the rainbow trout while it is governed by a GSD with thermal effects (GSD+TE) system in the European sea bass. LIFECYCLE identified a major sex determinant linked to the Y chromosome (sdy) in the rainbow trout, whereas sea bass sex differentiation is consistent with a polygenic model not yet identified explicitly. From the point of view of basic sciences, the trout example shows that apparently unrelated genes can become recruited for the process of sex determination. From a translational point of view, it is important emphasize the strong interest of the salmonid aquaculture biotechnology industry in this finding, as it opens the possibility for having a means for secure and early differentiation between sexes.
Puberty control: A major breakthrough has been the neuro-anatomical and functional characterization of the Kiss/Gpr54 system in zebrafish and sea bass, indicating that this signalling system as very relevant for controlling puberty and reproduction of fish in aquaculture. Also, testicular growth factors mediating in a paracrine manner the stimulatory role of Fsh have been identified, and anti-Müllerian hormone has been identified as mediating inhibitory effects on spermato- and steroidogenesis.
Research towards resolving current and emerging production bottlenecks: LIFECYCLE focussed a comprehensive research effort at major current aquaculture production bottlenecks related to fish biology. The knowledge generated in LIFECYCLE about broodstock stress, maternal effects, development and growth, adaptation and homeostasis, the immune system, sex differentiation and puberty provide knowledge that now needs to be translated through applied projects to industry. The LIFECYCLE outcomes point to solutions for alleviating serious and common production problems linked to abnormal larval development, skeletal deformities, poor growth and energy utilization along with excessive fat deposition, mortalities related to life stage transitions, poor environmental performance, and unwanted sexual maturation. The knowledge should contribute to make the European aquaculture industry more efficient and competitive through less problematic production of better quality fish, both in terms of flesh and welfare.
The research programme of LIFECYCLE has led to increased understanding of mechanisms of essential biological functions which can be exploited for current commercial species but also for new species. By including in the research programme phylogenetically diverse species belonging to Gadiformes, Salmoniformes, Pleuronectiformes and Perciformes complimented by a strong comparative analytical approach at various levels (gene, protein, biochemistry and function) identification of “essential biological functions” which are conserved and “specializations” and “adaptations” that are species specific have been identified. In keeping with the “call text” and the LIFECYCLE research contract with the EU commission, a solid scientific knowledge-base was generated and delivered rather than applied empirical solutions. An added value from the project was the significant scientific outcomes of some research areas that have direct practical implications and these have been highlighted under “innovations”.
Research outcomes with a potential application in aquaculture have been generated in the majority of WPs. These will not be detailed as they are highlighted in the description of the main S&T results/foregrounds, but include:
• Improved knowledge about factors that impact on egg quality
• How hatchery conditions can influence developmental plasticity and generate biological variation in aquaculture production.
• Knowledge about genes and physiological mechanisms regulating key aqua¬culture traits (e.g. growth, flesh quality, early puberty) that is relevant to genetic selection.
• Knowledge about lipid metabolism, adiposity and energy balance.
• Disease resistance: The influence of developmental stage and sexual maturation on the capacity to mount specific immune responses.
• Adaptation and homeostasis: knowledge about physiological as well as molecular mechanisms of adaptation and symptoms associated with failed adaptation.
• Molecular mechanisms regulating gill and intestine epithelial cell differentiation that are important for survival and quality of juvenile transitions in aquaculture.
• Global molecular genomics approaches and classical endocrinology have identified genes and mechanisms underlying puberty and precocious puberty.
• Improved understanding of the regulation of reproduction and control of gamete production and quality.
• Improved knowledge about maintenance of homeostasis and normal physiological and behavioural functions that contribute to define fish welfare.
• Knowledge about cross-talk between systems/processes (e.g. homeostasis, muscle and skeletal development, metamorphosis, smoltification, immune function, energy balance, maturation and reproduction) at different lifecycle stages.
• Baseline data on immune function during critical life stages.
• A knowledge base for the development and validation of welfare indicators.
The European dimension: LIFECYCLE has truly been an European enterprise that has generated a network of twelve European laboratories in eight European countries with long expertise in fish biology and aquaculture research. This contributed to structuring the European research area in fish biology applied to aquaculture and elements of the project network will be maintained through national project funding or further EU funding. There has been a high level of interaction among partners, which has resulted in mobility and knowledge spreading and sharing within the EU. LIFECYCLE created a critical mass that has delivered cost-effective cutting edge research. An aspect that has increased cohesion of the European research area and added value to LIFECYCLE has been the unique national resources for fish research that were made available to partners and included large-scale “life cycle” research facilities at Matre (IMR, salmon) and IRTA (sea bream), Torre de la Sal (CSIC; sea bass), Jouy en Josas (INRA, facilities for infec¬tious diseases and immunology, trout) and Drennec (INRA; trout), state-of-the-art genomic/-transcriptomic and bioinformatics facilities in Rennes (INRA) and Berlin (MPG), as well as the Scottish Fish Immunology Research Center (UNIABD).
Added value and cost effectiveness: The impact of the knowledge-base delivered by LIFECYCLE represents not only the projects outcome, but also integrates that of concluded EU projects in which the partners participated, including GUTINTEGRITY, ARRDE, PUBERTIMING, AVINSI, PROBASS, STRESSGENES, SEAFOODplus, AQUAFIRST, WEALTH, FPPARS, FISHCAL and BRIDGEMAP, as well as nationally funded projects on fish biology, physiology and genomics. Thus, the success of the LIFE¬CYCLE project was in part secured by previous investments on EU and national scale, and ensures that the state-of-the-art as defined by these projects was advanced even further and has contributed to increase the competitiveness of European aquaculture.
Building competencies and skills through training: Although training was not an explicit objective of LIFECYCLE, a considerable number of training opportunities have been generated by the project. A large number of young scientists have undertaken masters and doctoral studies in the different research areas of the project. The impact of training has benefited by the opportunities for technology transfer that arose through the exchange of young scientists who frequently carried out their training in the laboratories of several LIFECYCLE partners. This has been extremely beneficial as it has exposed young scientists to a variety of different work environments allowed them to contact with well-established and well-recognised scientists and to build up their professional networks for their future. The emphasis given to promoting young scientists is evident in the project indicators; in addition to their key role in knowledge generation and production of scientific manuscripts, they have been active participants in the annual LIFECYCLE meetings and frequently were given the opportunity to present their work. Special measure were also taken to promote doctoral and young post-doctoral researchers through offering direct support for participation in conferences in which the LIFECYCLE project organised a specific session. In addition to Masters and doctoral students, a number of other junior scientists and undergraduates, as well as a large number of technical personnel have also been involved in LIFECYCLE and have benefited from the experience through specific “on–the-job training”. Overall, there has been a high level of interchange between the project participants and transfer of knowledge and skills between scientists and young scientists and among the participating groups. Through training, LIFECYCLE has contributed to the generation of a skilled workforce to meet future challenges and has also contributed to the transfer and spreading of knowledge within Europe. The network of young scientists trained in LIFECYCLE is an important resource for Europe and by training in a pan-European network of researchers will certainly contribute to future European cohesion.
Favouring gender equality: The LIFECYCLE project was built upon the concept of gender equality to ensure that the talent, perspectives and insight assured by increasing diversity in the science and technological workforce was harnessed for the project’s benefit.
An action plan was established at the planning stage ad implemented during the project. A gender-issues workgroup was established within LIFECYCLE and defined actions for implementation and also oversaw them. The budget available permitted a number of specific measures to be carried out. At the kick-off meeting of the project, an invited lecture was given by an expert on gender issues in order to stimulate awareness within the consortium of measures that should be taken to ensure gender equality. Several of the key leadership roles in LIFECYCLE were occupied by women and 3 of the 7 steering committee members were women. In addition both senior and junior scientists that were women were encouraged to take on co-ordinating responsibility for sub-workpackages (6 out of 15 subWPs were led by women). By supporting LIFECYCLE leadership roles of female researchers in grade B positions this strengthens their merits for promotion.
The visibility of female researches in grade A and B positions was encouraged by offering funding for attendance of conferences and presentation of data at the LIFECYCLE meetings. However, it was considered important to encourage all young researchers to attend meetings so funding was open to all junior scientists in LIFECYCLE, although women were given priority when a choice had to be made between applicants. One of the LIFECYCLE conference sessions (26th Conference of Comparative Endocrinology, Zurich 2012) was organised by two of the senior female scientists of LIFECYCLE and “showcased” the results of a number of young women from the LIFECYCLE consortium. Similarly, a special LIFECYCLE session at the Aquaculture Europe 2012 conference in Prague, made it possible for several young, female scientists from the LIFECYCLE consortium to give oral presentations at a major, international conference.
The collated workforce statistics show that LIFECYCLE has been particularly successful in gender equality actions, as that the same number of male and female senior scientists, as well as the same number of male and female PhD students, have carried out research in the project.
DISSEMINATION
Effective and correct dissemination is a vital key to the overall impact of any activity and LIFECYCLE started with a clear plan for the dissemination of its activities aimed at reaching all stakeholders and maximising its impact.
As a major European research project, dissemination occurred at several levels; within the project consortium to ensure execution of the project plan. The successful attainment of the project objectives is a measure of the effective communication and dissemination of information to all partners. Communication occurred with the European Commission to report project progress principally through project reports, which were detailed and completed within the planned timescale. Moreover, the scientific officer from the European Commission designated with the task of overseeing the good functioning of the LIFECYCLE project was invited to be present at all project meetings to maximise opportunities for informal exchange about the project outcomes.
Dissemination of LIFECYCLE results and outcomes to the scientific community and to the aquaculture sector occurred through several different mechanisms. To ensure effective communication and dissemination a specific WP5 was dedicated to the task and a “Dissemination and Intellectual Property Workgroup” (DIPW) was formed. The LIFECYCLE project straddles the spheres of “Basic Biologic Research” and “Academic Applied Aquaculture Research”. The outputs from LIFECYCLE have contributed significantly to the research literature and the aquaculture literature. Presently, seventy-seven papers having been published in peer-reviewed journals, both in top scientific journals such as Endocrinology and Proceedings of the US Academy of Sciences, as well as top aquaculture journals such as Aquaculture, Reviews in Aquaculture and Fish and Shellfish Immunology. The new knowledge generated by the LIFECYCLE project on the aquaculture-related biology of our most important aquaculture finfish species has thus to date been broadly disseminated through the academic publishing of about one thousand pages in top quality scientific journals. In order to enhance the accessibility of the findings to applied researchers and end users, the LIFECYCLE consortium has published when possible in OPEN ACCESS journals. Adding to the seventy-seven scientific papers already published, over twenty more manuscripts are currently in various stages of being published. Given the large amount of still unpublished data summarized in the fourth periodic report which has just been submitted, it is clear that the LIFECYCLE project will continue to disseminate novel data to the scientific and aquaculture communities for several more years. The same can be predicted for continued conference dissemination of LIFECYCLE data. This dissemination route has been a key activity of all of the participants of the LIFECYCLE project, and at present, LIFECYCLE data have been disseminated through over 150 oral and poster presentations at a scientific and aquaculture conferences and workshops. However, perhaps the most important dissemination route, although also the hardest to quantify, is the direct contact and personal dialogue by a large number of LIFECYCLE scientists with local fish farmers, a dialogue enriched by the new insights brought forth by the LIFECYCLE project.
In addition to publishing genomic/transcriptomic data in the scientific literature, LIFECYCLE scientists have adopted an “open” approach by the release of data to appropriate public domains (NCBI etc.) as early as possible to maximise benefits for research in this area. This approach will has given added value as it favours the rapid spreading and dissemination of basic molecular information which may be of use in other research projects where work is carried out on basic as well as aquaculture-related fish biology.
While the project website has been a major web-based dissemination instrument for the project, the emergence of the professional “social network” LinkedIn was used increasingly during the project life-time as it allows direct communication between members. Currently, there are over 1100 members of the LIFECYCLE project LinkedIn group, representing the full spectrum of stakeholders in fisheries and aquaculture from all over the world.
EXPLOITATION
A broad spectrum of important exploitable intellectual assets have been created and generated in LIFECYCLE, and about forty discrete items of EXPLOITABLE FOREGROUND is given in Table A2.
From a fundamental, scientific point of view for the researchers of the LIFECYCLE consortium, the knowledge generated presents one of the most valuable exploitable assets. Although all the major scientific advances of LIFECYCLE are being published, at present count 77 LIFECYCLE publications in peer reviewed scientific journals, and that these advances are thus not patentable, the data generated during the research enhance the knowledge base of the researchers involved and thereby their own qualifications and competence. This is exploited every time a researcher, a research group or a larger research consortium applies for further research funding from private or public sources. The LIFECYCLE data will undoubtedly generate a substantial number of spin-off research projects, both basic and applied, and already, it’s clear that a substantial number of on-going national research projects have been established by exploiting LIFECYCLE generated data.
In a similar fashion, a large number of methodological advances have been made in LIFECYCLE, in everything from experimental protocols, to research equipment to highly specific measuring assays for localization and/or quantification of both transcribed and translated gene products (mRNAs and proteins). Such experimental and analytical protocols are highly exploitable within the research community in a similar fashion as explained for the scientific knowledge base above.
In addition, many aspects of such methodological development can potentially be exploited commercially to various degrees. To exemplify, analysis of specific proteins such as many hormones is carried out using immunological methods. A part of establishing these methods, specific antibodies need to be generated. Such antibodies have an obvious commercial potential as indicated by the large number of biotech companies selling antibodies to the research community. However, another way of exploiting methodological advances can be for scientists to take on contractual research where they are paid for analysing samples with their unique analytical methods.
Exploitable foreground is more often than not the result of long-term research efforts, rather than being a result of a single project. LIFECYCLE has in varying degree contributed to the exploitation of such long-term efforts by LIFECYCLE researchers.
One of the academic laboratories in LIFECYCLE launched an aquaculture genetics service company in February 2013. Xelect Ltd (www.xelect.co.uk) is a company registered in Scotland No.SC438223. Xelect represents the commercialisation of over 30 years of basic research on the physiology and genetics of fish muscle growth and flesh quality and was made possible by continuous support from the UK Research Councils and the European Commission, the later through SEAFOODplus (FP6) and LIFECYCLE (FP7). The core business of Xelect is the developing of genetic markers for brood stock selection. Xelect has licensed genetic markers for superior meat yield in Atlantic salmon to SalmoBreed A/s and Landcatch Natural selection and several other license opportunities for this and other traits are currently under negotiation. In addition, Xelect offers a range of genetic services to the aquaculture industry including sex determination (using an innovation from LIFECYCLE), genotyping, ploidy testing, and disease identification. The company is trading profitably and has a growing list of global customers.
The research group at the University of Bergen, Norway (LIFECYCLE partner 7) have recently made significant advances in establishing an analytical method termed “Smoltprobe”. The Smoltprobe has been established as a predictive tool in the assessment of smolt quality and the transfer of smolts from fresh water to seawater. The work has involved two of the major international salmon producing companies, Lerøy Seafood A/S and Marine Harvest A/S, and the Smoltprobe has been tested and evaluated in two commercial smolt producing operations. The Smoltprobe holds promise both as a tool for predicting the correct timing of seawater transfers, and a tool to pick up problems arising from poor freshwater quality and desmoltification (loss of smolt status). Evaluations are on-going, and a national proposal has been submitted in September 2013 in order to bring the Smoltprobe to a fully commercially available test tool for smolt quality.
List of Websites:
http://lifecycle-fp7.eu/
In summarizing the 54-month long LIFECYCLE project, it can first be stated that the project has been hugely successful in fulfilling its main objective, which has been to build a biological knowledge-base on fish lifecycles that will contribute to competitive sustainable European aquaculture. This biological knowledge-base, to greatest extent focusing on the physiological and molecular level, can already be found in the 77 scientific LIFECYCLE papers published so far (this number is likely to double over the next two years), in large amounts of genomic (transcriptomic) data deposited in public data-bases, and in over 150 presentations at scientific and aquaculture conferences.
The building of a biological knowledge-base is essentially a task of fundamental scientific endeavor, but the selection of the specific research areas for which LIFECYCLE would attempt to significantly advance the state-of-the-art were relevant to major current aquaculture production bottlenecks. Further choices were made in order to make the knowledge-base as relevant as possible for European aquaculture. Thus, the research was largely carried out on the four major aquaculture species; Atlantic salmon, rainbow trout, sea bass and sea bream, and to lesser extent on Atlantic halibut and Atlantic cod, and then in areas where production problems remain.
Many of the major biological production bottlenecks of European aquaculture production are directly related to life-stage transitions of the fish. These transitions from embryo to larvae, from larvae to juvenile, from juvenile to pubertal adult and then to a sexually mature fish, all represent highly vulnerable phases of the production cycle. Bad egg quality, larval-juvenile mortalities and/or abnormalities, poor on-growth, premature puberty and sexual maturation are all well-known production problems linked to the lifecycles of the various aquaculture species. Therefore, LIFECYCLE research was focused on the areas (WP1) Development and Growth, (WP2) Adaptation and Homeostasis, (WP3) Immunity, and (WP4) Sex differentiation and Puberty. In carrying out the research within each of the 15 well-defined sub-topics (subWPs) of the LIFECYCLE project, one or more relevant aquaculture species were chosen, and in elucidating molecular mechanisms, the model fish species zebrafish and medaka have provided valuable research approaches.
From the onset, the LIFECYCLE consortium was composed of 14 partners from 9 European countries. Most of the groups, placed at universities and research institutes, represent internationally leading research teams in their given areas. Several of the LIFECYCLE partners also have top-tier, large-scale aquaculture research facilities, which are now a part of the AQUAEXCEL EU infrastructure. Highly illustrative of the production challenges still facing the European aquaculture industry, the only industrial SME partner in the project, a producer of Atlantic halibut juveniles, went bankrupt during the project, having been a world-leading producer only a few years earlier. This was also the cause of the only major change in the LIFECYCLE research plans, and was the main reason for a 6-month extension of the project in order to allow for alternative research plans to be put in motion.
As pledged, the LIFECYCLE consortium directed a huge amount of nationally funded person-months to the project, almost doubling the person-months covered by the EU contribution. This emphasizes the great commitment of the whole LIFECYCLE consortium to the project objectives, and the research has been carried out in the spirit of DEDICATION, ENTHUSIASM, COLLABORATION and SCIENTIFIC and TECHNICAL EXCELLENCE. Through this, many of the teams have forged strong collaborative ties generating a network, which will strengthen the European research area and will favour initiatives for years to come.
Apart from significantly advancing the scientific state-of-the-art in all its research areas as detailed below, the LIFECYCLE consortium also trained 28 PhD students and is particularly proud of gender equality actions taken in the project, and the fact that equal numbers of male and female experienced researchers (PhD holders; 46 of each gender) as well as PhD students (14 of each gender) have participated in the project.
The project progress has been detailed in period report 1 (135 pages), period report 2 (196 pages), period report 3 (243 pages and period report 4 (340 pages) submitted to the EU Commission.
The completion of the project deliverables has been reported in 157 deliverable reports submitted to the EU Commission
The scientific research advances have been described in detail in 77 scientific papers so far published in international peer-reviewed journals (ca 1000 pages).
Project Context and Objectives:
Fisheries and aquaculture provide an important source of nutritious food and animal protein for much of the world’s population. In addition, the sector provides livelihoods and income, both directly and indirectly, for a significant share of the world’s population. Increased aquaculture production is seen globally as the single most important way of securing food for the growing population. While fisheries have reached a limit world-wide, aquaculture continues to grow rapidly. In 2011, according to FAO, aquaculture produced 63.6 million tons representing half of all aquatic food destined for human consumption worldwide.
However, while aquaculture is fasted growing food sector globally, this is not the case in Europe.
In 1994, 60% of European seafood needs were provided by European fisheries and aquaculture, whereas today 65% of seafood consumed in Europe is imported. Europe is therefore increasing its reliance on seafood imports, which results in lost revenue and employment in direct and indirect jobs, but also weakens the EU food security position.
The European Union has therefore sought ways of stimulating the European aquaculture industry in numerous ways; e.g. by policy measures, industry-driven projects aimed at supporting innovations and production small and medium sized enterprises, as well as large, scientific research projects focused on various aspects of aquaculture technology and biology. LIFECYCLE is one such large, scientific research project, bringing together 13 research groups from many of Europe’s leading universities and research institutes and 9 different European countries, in order to work on key questions which cannot be efficiently answered by research at the national level.
As an EU project, the context and objectives of the LIFECYCLE project have been broadly dictated by the original call text where the EU Commission has outlined the direction and the scope of the project. In accordance with this call text, the overall objective of LIFECYCLE has been to improve our biological knowledge about some of the most important aquacultured fish species in Europe. Why is this important objective? It is because aquaculture is the youngest branch of our animal food production sector and our current knowledge about the fish species produced in aquaculture is very limited in comparison with our knowledge of other farm animals such as chicken, pigs and cows. Furthermore, a diverse range of fish species (e.g. sea bass, Atlantic salmon) is used for aquaculture in different parts of Europe and require different production systems unlike land-farm animals. These have been bred and domesticated for farm production for hundreds or even thousands of years, whereas intensive fish farming on an industrial scale started in Europe with large-scale salmon aquaculture as late as in the 1970’s, and culture of many key marine species has only been carried out for 10-15 years.
To successfully raise and breed an animal for food production requires good understanding of the biology of the animal from conception to slaughter, so that the farmer can see to it that the animal thrives and grows and finally can be turned into a valuable product. For fish, there are still many serious bottlenecks in the aquaculture production which result in lower productivity in terms of quantity and/or quality, which in turn results in decreased economic sustainability and competitiveness of the aquaculture industry. Such bottlenecks can occur at many different stages in the life of the animal and differ depending on the species in questions. Thus, the context and objectives of LIFECYCLE has been to deliver a knowledge-base designed to improve the competitiveness and sustainability of the European aquaculture industry.
Having a defined funding base (6M€ EC contribution) and duration (4 years), a single project such as LIFECYCLE could not address all known problems and questions for all aquacultured species, so choices had to be made in terms of what questions should be addressed and for which fish species.
The project was intended to create a knowledge-base about the biology of the target species, which would benefit future production-orientated projects focusing on practical areas such as aquaculture stress control, disease prevention and control, new breeding technologies, better environmental performance and diversification into new species (see call text), the research areas where chosen based on known bottlenecks. However, it’s important to understand that the role of LIFECYCLE was not primarily to solve applied, industrial production problems, but rather to provide increased biological knowledge in areas of production bottlenecks, in order to facilitate future, knowledge-based solutions. This is in line with the EC policy to create a “knowledge-based bio-economy” through measures of which the LIFECYCLE project is a part.
Therefore, the LIFECYCLE project was designed through a combination of question-driven and problem-driven approaches. It aimed at providing novel discoveries which in turn could offer solutions to current “bottle-neck” problems. The knowledge-base created in LIFECYCLE provides a solid foundation for European aquaculture to develop ways of improved larval rearing practices, feed utilization and muscle quality, to gain better control over sexual differentiation and puberty, improve stress control and disease prevention, establish new breeding selection parameters and technologies, improve environmental performance, and support diversification into new species.
In defining the scope of the LIFECYCLE project, the first focus area was on the early life stages, encompassing biological processes from fertilization of the egg to juvenile growth. This area was chosen as the production of sufficient quantities of high-quality fish larvae is still a major problem in the culture of several species, especially the marine fish species. Understanding the importance of gene trans¬fer from the mother to the egg during early development, the mechanisms of sex differentiation into males and females, the development of the immune system, the development of the muscles and the skeleton during larval growth, the roles of the larval skin and gut, and how genes and hormones regulate larval growth and development, are all questions of key importance to solve a large number of current production bottlenecks. These production problems are not only directly related to growth and survival at the larval stage, but are also indirectly related to many problems manifested at later life stages such as disease resistance, ability to cope with changing environment, and reproduction.
The second focus was placed on the growth and environmental adaptation of the juvenile-adult “ongrowing” life stages, addressing questions relevant to production problems related to poor growth. There are many possible reasons for poor growth; lack of appetite and thus low feed intake of the fish, poor utilization of the energy of the food resulting. This is often linked to environmental maladaptation where the fish are using energy to cope with the stress of the rearing environment rather than for growth. Inappropriate rearing environments can also have a negative influence on the flesh quality. With rising feed-costs, slow growth is becoming an ever more important issue for the competitiveness of European aquaculture. Therefore, the LIFECYCLE project was aimed at obtaining new knowledge on basic biological issues such as the hormonal regulation of appetite and growth of the fish, the relationship between metabolism, growth and fattiness, and the relationship between environmental conditions such as salinity, and how this affects energy balance and growth.
The third focus area of LIFECYCLE was on important issues linked to key life-stage transitions. These are complex biological changes that take place during development and growth, and are often associated with major losses in aquaculture production. One such biological transition is the so-called metamorphosis of flatfish larvae, when they change from free-swimming symmetrical larvae to bottom-living asymmetrical juvenile flatfish.
Another crucial life-stage transition is the so-called smoltification of Atlantic salmon during which the freshwater-living parr becomes seawater tolerant smolt and can be moved into marine net-pens for ongrowth. Last but not least, the onset of puberty has a major impact on aquaculture production. In many species, puberty and the ensuing sexual maturation occurs too early in the production cycle, impacting negatively on both growth and flesh quality. These life-stage transitions are extremely demanding for the organism and as already stated, major production problems are linked to these transitional periods. This includes decreased disease resistance, increased mortalities and decline of growth performance. Therefore, a better understanding of immune function during life-stage transitions is of key importance, and improved know¬ledge leading to more effective ways of regulating the timing of puberty may greatly improve the competitiveness of European aquaculture.
In order to address these lifecycle-encompassing topics, the LIFECYCLE consortium was established and brought together a critical mass of leading European research groups dedicated to significantly advance current knowledge on mechanisms governing essential biological functions in fish across species and all major life stages, with focus on important aquaculture species and current production bottlenecks. This has been achieved by applying the tools of endocrinology, biochemistry and functional genomics to state-of-the-art physiological research.
No single life stage, nor indeed a single life process, is independent of others, and great synergies can therefore be obtained by integrating the research through “cross-talk”. A major emphasis has been placed on this in designing the many experimental studies of the LIFECYCLE project.
LIFECYCLE focused on the most important commercial aquaculture species in Europe, specifically sea bass (Dicentrarchus labrax), sea bream (Sparus auratus), Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Other species such as Atlantic halibut (Hippoglossus hippoglossus) and Atlantic cod (Gadus morhua) were included in the project due to their importance for studying specific processes and/or their potential as aquaculture species. Moreover, the inclusion of both ancient (salmoniformes), and modern teleost fish (perciformes) used for aquaculture and when appropriate, model species such as zebrafish and medaka, for the elucidation of basic biological mechanisms, has made it likely that the project results can be applied to other newly emerging aquaculture species.
A key element of research is the dissemination of the research outcome to stakeholders this includes other scientists, the aquaculture sector, companies, politicians and the public. The LIFECYCLE consortium has been committed to informing all stakeholders about the project results in as active and effective way as possible, through various channels aimed at reaching different groups. An important way of openly informing society about the project has been the project website http://lifecycle-fp7.eu/ which has explained the purpose of the project, the partnership, the species, the research areas and the project results. Validation of the experimental results and their dissemination has been through interaction with the scientific community at large through publication of a large number of peer-reviewed publications in scientific journals and deposition of large datasets in public databases. Discussion and debate about the diversity of studies carried out in LIFECYCLE have been possible through presentation of numerous posters and oral presentations at national and international conferences and also at specialised workshops. An important dissemination target and the end users of the research output, the aquaculture sector has been reached through presentations and posters at aquaculture-related conferences, not least those organized by the European and national aquaculture societies but most importantly for the European sector by personal, direct discussions by many of the LIFECYCLE partners with local fish farmers or their associations. More global dissemination of LIFECYCLE to raise awareness of basic research progress of relevance for European aquaculture was pursued through presentations of the project in the trade journals. Dissemination to the general public has come directly and indirectly through popular news articles and interviews with LIFECYCLE scientists. A more difficult dissemination activity to quantify is the multiplying effect of the numerous scientists who have participated in LIFECYCLE and are engaged in outreach activities and the direct education and training of undergraduates and graduates. Inevitably, lectures and seminars given about fish and fish biology or hands-on training activities have been enriched by the outcomes of the LIFECYCLE project in a diversity of subject areas. All the various quantifiable dissemination activities are presented in Tables A1 and A2 of this report.
Project Results:
The scientific research of the LIFECYCLE project was carried out in four major workpackages, each divided into several sub-workpackages, and each subWP was further subdivided into two or more tasks. All in all, 58 significant research tasks were defined in the project, and their successful completion has now resulted in 139 scientific deliverables having been reported to the EU Commission.
In parallel to reporting the project progress regularly to the EU Commission over the 4½ years of the LIFECYCLE project, the results have been continuously published in the scientific literature, with about seventy-five scientific papers having been published to date, and over twenty more manuscripts are currently in different stages of being published. At the same time, the LIFECYCLE data have been presented in various other ways, including over 150 oral and poster presentations at a scientific and aquaculture conferences and workshops. Perhaps the most important dissemination route, which is the hardest one to quantify, is the direct contact and personal dialogue by a large number of LIFECYCLE scientists with local fish farmers, a dialogue enriched by the new insights brought forth by the LIFECYCLE project.
The new knowledge generated by the LIFECYCLE project on the aquaculture-related biology of our most important aquaculture finfish species has thus to date been broadly disseminated through the academic publishing of about one thousand pages in top quality scientific journals. In addition, the presently submitted fourth periodic report, which is about 350 pages in length, contains largely unpublished data, which will generate a significant amount of new papers, which will be published over the next 2-3 years.
It is therefore no exaggeration to say that the following summary of the main scientific and technological results of the project is a very brief summary indeed.
A major challenge in aquaculture is the production of an abundant supply of good quality eggs to secure the production cycle by generating an abundance of good quality larvae for the subsequent grow-out phase. In aquaculture, the traditional method to evaluate egg quality and embryo viability has been empirical and based upon assessment of morphological criteria such as follicle size and embryo viability. An alternative quality assessment may be hidden within the large pool of maternal RNA which accumulates in the egg during its maturation and which is essential for early development. Thus, the LIFECYCLE project has focused on research into MATERNAL TRANSCRIPTS AND IMPACT OF BROODSTOCK STRESS. The work carried out has yielded new insight into the maternal contribution to early developmental success in fish. To achieve this goal, both important European aquaculture species such as Atlantic salmon, Atlantic cod, and rainbow trout, and also a model species, the zebrafish, have been used. The identity of maternal gene products, present in the egg and inherited from the female and used by the embryo during early development was thoroughly characterized during cod development. The crucial role of specific maternally-inherited messenger RNA (i.e. maternal mRNAs) in early developmental success was shown. In parallel, the dynamics of gene expression and post-transcriptional modifications, such as changes in the poly A tail of mRNAs, has been monitored throughout egg maturation for several genes known to be important for egg/embryo development. The importance of ancient families of genes, shared by most vertebrates, in the mechanisms defining embryonic developmental success has been highlighted. Genomic tools to investigate the mechanisms defining egg quality in fish under natural and experimental conditions revealed that elevated levels of stress hormones in eggs and embryos affect factors involved in cell division rate. This result together with recent studies in rainbow trout implies that broodstock stress can increase developmental rate of embryos. Another aspect of the project was to compare all expressed genes in eggs from different fish species. Comparison of overall gene expression repertoires in salmon and cod the revealed salmon eggs have a higher representation of immune factors and calcium balance factors compared to cod. While cod had a higher representation of genes related to transcription and metabolism. The differing pathways identified between these species may be linked to for example, life history traits or adequacy of aquaculture regimes and thereby represents a novel way to understand egg quality parameters in aquaculture fish.
Production of good quality muscle and healthy fish is a key objective of fish aquaculture. The muscle and skeleton develop from the same precursor tissue, the embryonic mesoderm, and evidence suggests that mismatch between the development of these tissues may contribute to poor larval quality, reduced muscle growth and an increased incidence of skeletal abnormalities in aquaculture. Thus, LIFECYCLE has focused research on THE MUSCULOSKELETAL SYSTEM. Genes and gene transcription determine the fundamental characteristics of organisms and mean that gene sequences are an important resource for investigating transcript expression. Transcript expression can be modified by diet, environmental factors and ontogeny and may be associated with changes in muscle or skeletal development. Gene sequences also permit discovery of population level variation associated with improved production characteristics that can be used in marker assisted selection. LIFECYCLE has dramatically increased the sequences available for genes expressed in muscle, bone and cartilage in rainbow trout and gilthead sea bream. Laser microdissection was successfully used to define a sub-set of genes (transcription factors, growth factors, and signalling molecules) that are upregulated in regions of the myotome where new muscle fibres are being formed. The function of several of these novel myogenic genes was investigated using a combination of cell culture and the zebrafish model system. STAC3 a signal adaptor molecule was found to have multiple functions and be required for normal muscle differentiation. Bony fish underwent a whole genome duplication (WGD) (2R) around 550 Mya effectively doubling the number of genes relative to human. Around 80% of the duplicated genes, called paralogues, were subsequently lost and the remainder either acquired new functions or novel patterns of regulation. Salmonid fish (salmon, trout, and charr) underwent a second WGD (4R) around 88 Mya and 50% of the duplicated genes are still present in extant species. Although the duplicates can have very similar sequences (>95% sequence identity) they can also have distinct functions. It is therefore essential to characterise the sequences of all the paralogues in each gene family in order to produce a consistent nomenclature and to design genetic assays that are specific to a single paralogue. The growth of peripheral tissues including the muscoskeletal system is controlled by the insulin-like growth (IGF) system which comprises hormones, receptors and binding proteins which regulate the effective concentration of the hormone according to tissue type and physiological status. Using phylogenomic approaches, the complete IGFBP repertoire of 4R-derived salmonid fishes (19 genes; 13 more than human) was resolved and their evolutionary relationships/nomenclature established with respect to WGDs. Traits central to IGFBP action were determined for all genes, including atomic interactions in IGFBP–IGF1/IGF2 complexes regulating IGF–IGF1R binding. Using statistical methods, we demonstrate that attributes of these protein interfaces are overwhelming a product of 2R IGFBP family membership, explain 49–68% of variation in IGFBP mRNA concentration in several different tissues, and strongly predict the strength and direction of IGFBP transcriptional regulation under differing nutritional states. The expression of IGF-system genes with feeding regimes designed to initiate increased protein synthesis and compensatory growth in muscle; bone and cartilage were described in detail for the gilthead sea bream and Atlantic salmon. We took advantage of the small size and short generation time of a wild zebrafish population to investigate the effects of artificial selection for body size at age on the IGF-signalling system during the domestication process. Replicate populations were either unselected (U-lineage) or subjected to four generations of experimental selection for small (S-lineage) or large (L-lineage) body size at 90 days post-fertilization. Body mass was on average 16.3% and 41.0% higher in the L- than in the U- and S-lineages, respectively. Transcript abundance for four genes (igf1a, igf2r, igfbp1a and igfbp1b) showed either regulated or constitutive differences between the S- and L-lineages. For example, igf2 receptor transcript abundance was higher and igbp1a/b transcript abundance was lower in the L- than in the S-lineage, indicating an effect of selection on insulin-like growth factor signalling. This research has identified several candidates for marker assisted selection for fast growth which are currently being investigated in aquaculture species including Atlantic salmon. Lighting regimes are commonly used in aquaculture to regulate growth and maturation and it is important to understand the genetic mechanisms. The zebrafish also provided a convenient model to fully characterise the repertoire and function of “clock genes” in fish skeletal muscle and to investigate circadian rhythms in the expression of IGF-system components independently of feeding. Two IGF-binding protein genes, igfbp3, igfbp5b, and the myogenic regulatory factor (MRF) myf6 showed circadian patterns and were expressed in phase with the clock genes clock1a/1b changing 2-fold from peak to trough. These finding points to an important physiological role of the clock mechanism in regulating muscle mass through integration with the IGF pathway and MRFs.
LIFECYCLE also contributed significant advances to the understanding of the phenomena of thermal imprinting. Studies on the gilthead sea bream showed that low temperatures (LT) prior to metamorphosis resulted in 20% more muscle fibres of smaller average diameter than for higher rearing temperatures (HT). Moreover, in young adults the number of vertebra in the HT group (n = 24) was higher than the LT group (n = 23) and the HT group had a lower (7%) incidence of malformations compared to the LT group (25%). Early rearing temperature had a persistent effect on the mineral content of bone and the bone density was lower in HT than LT. Furthermore it was shown that gene expression patterns during growth were not fixed, but could be modified by embryonic temperature. Altering the nutritional input to the muscle to stimulate growth revealed cryptic changes in the expression of genes involved in the correct folding of myosin filaments during sarcomere assembly including UNC45 and Hsp90α with higher transcript abundance in the LT than HT groups. The effects of temperature were not limited to muscle and genes important for skeletal homeostasis were also modified. The transcript for bone gla protein, an indicator of bone formation/mineralization, was much higher in HT compared to LT fish. The effects of thermal imprinting on other traits linked to growth were conveniently studied in the zebrafish model. We showed that embryonic temperature had dramatic and persistent effects on thermal acclimation capacity at multiple levels of biological organization. Zebrafish embryos were incubated until hatching at control temperature (TE = 27 °C) or near the extremes for normal development (TE = 22 °C or 32 °C) and were then raised to adulthood under common conditions at 27 °C. Short-term temperature challenge affected aerobic exercise performance, but each TE group had reduced thermal sensitivity at its respective TE. In contrast, unexpected differences arose after long-term acclimation to 16 °C, when performance in the cold was ∼20% higher in both 32 °C and 22 °C TE groups compared with 27 °C TE controls. Differences in performance after acclimation to cold or warm (34 °C) temperatures were partially explained by variation in fibre type composition in the swimming muscle. Cold acclimation changed the abundance of 3,452 of 19,712 unique and unambiguously identified transcripts detected in the fast muscle using RNA-Seq. Principal components analysis differentiated the general transcriptional responses to cold of the 27 °C and 32 °C TE groups. Differences in expression were observed for individual genes involved in energy metabolism, angiogenesis, cell stress, muscle contraction and remodelling, and apoptosis. Therefore, thermal acclimation capacity is not fixed and can be modified by temperature during early development. Developmental plasticity may thus help some aquaculture species cope with the more variable temperatures that are expected under future climate-change scenarios. The research on thermal imprinting carried out within LIFECYCLE stresses the importance for defining the optimal temperature conditions for rearing eggs and larvae in the hatchery in order to enhance growth, muscle texture and ensure skeletal quality in adult fish.
The ability to regulate energy balance is essential for normal development and growth under various conditions and life stages and is of major importance to aquaculture. Muscle and skeletal growth depend on food intake, extraction and use/allocation of this energy. Metabolism and energy balance also include the mobilization of energy stores in liver and adipose tissue for energy-demanding life-stage transitions such as metamorphosis, smoltification and puberty. In mammals, ghrelin and leptin are multifunctional hormones with key roles in energy homeostasis, although , fundamental understanding of how energy balance is regulated in fish and the role of hormones is still very fragmented. For this reason priority was given in LIFECYCLE to the topic METABOLISM AND ENERGY BALANCE: EXPLORING ACTIONS OF LEPTIN AND GHRELIN. This work has shown that leptin reduces and ghrelin increases appetite in Atlantic salmon, thus they may have opposing effects on energy balance. In addition, these hormones seem to be important in lipid metabolism and the development of adiposity (fattiness) in fish, as well as to have functions that help the fish cope with fasting or to respond to changes in body energy stores. The studies also revealed that the IGF-I system is likely central for adipose (fat) tissue functions, adipose cell development and glucose metabolism. In addition, methodological protocols were established that can be used for future research on the biology of adipose cells and the roles of specific genes during adipogenesis (the development of fat tissue). The project has established knowledge that is important to understand the processes that lead to fat accumulation, which may affect the health, growth, time of puberty and reproduction of aquaculture fish and in this way impact welfare issues and affect product quality. The major findings are summarized below. The results provide evidence that leptin serves various roles at different life stages of salmonid fish, e.g. regulating embryogenesis and development, growth and appetite.
The regulatory system is complex and in Atlantic salmon four evolutionary conserved leptin paralogue genes were identified and most likely act via two leptin receptor genes. Leptin genes are expressed differently between tissues and life stages. The response of the hypothalamus, which is the brain centre of appetite control, to leptin revealed it is an important target in fish. This was demonstrated by detailed anatomical mapping of leptin receptors in the hypothalamus and revealed a symmetrical pattern of clusters of neurons expressing one of the leptin receptor subtypes in the Atlantic salmon. Leptin treatment reduced food intake and growth rates in Atlantic salmon, and exerted this effect by modulating neuropeptides expressed in the hypothalamus and known to inhibit appetite, e.g. POMC. Direct central injections of leptin into the brain did not influence short term appetite, and suggests according to its location in the body, local, central or peripheral, it may have different modes of action.
Ghrelin stimulated appetite and body weight gain in Atlantic salmon. The effect of ghrelin, as well as GH, on appetite and growth was not influenced by water salinity and thus the role of these hormones in mediating the effect of salinity on growth is still unclear. Leptin plasma concentrations were influenced by the amount of energy stored in the body, suggesting that leptin acts as an adiposity signal in fish. However, in contrast to mammals, fasted fish that had lost body weight and body fat stores, with fat cells almost emptied of their lipid content, had elevated leptin levels in the blood. In line with this, lean fish treated with GH had higher leptin plasma levels and leptin mRNA expression compared with fat untreated fish. An explanation for the impact of body composition on the response to GH may be explained by considering wild fish. Wild fish experience periods of glut and fast and have to adjust to fasting and loss of energy stores by reducing appetite and foraging activity to decrease energy-demanding activities. The studies also revealed that the energy status of the fish modulates the action of leptin: fish fed a high fat diet may become leptin resistant (unresponsive) at the adipose tissue level. Further, the secretion of leptin by adipocytes increases as they mature. This secretion is under hormonal and nutritional control, e.g. both insulin and ghrelin stimulate the release of leptin while the amino acid leucine decreased leptin release. This project shows for the first time in fish that leptin and ghrelin have direct effects on the lipid metabolism of adipocytes. In particular, ghrelin may promote lipid synthesis and tissue lipid uptake, and hence promote the development of adiposity. In addition, the complete IGF-I system was characterized and seems to have important roles in adipose tissue development and glucose metabolism.
All major life processes such as larval development, metamorphosis, smoltification, growth, metabolism, puberty and reproduction are under complex endocrine control. Built into the endocrine system is the capacity to “sense” change in the internal and external “milieu” and stimulate an appropriate physiological response. Most hormones studied have multiple functions and their action is dependent on the responsiveness of the target tissue which is in part determined by enzymes (e.g. activation/inactivation), plasma binding proteins, specific tissue receptors and other accessory proteins. Current knowledge is primarily related to characterization of hormone function, and to some extent, information about receptor expression and distribution, since the interaction of hormone and receptor bring about the cellular response. However, recently the importance of binding proteins that regulate circulating hormone concentrations and their availability for receptor binding has been appreciated. Therefore, LIFECYCLE research was directed to MODULATION OF HORMONE ACTION by hormone binding proteins for some key hormones regulating growth, hydromineral balance, metabolism and life-stage transitions in fish such as metamorphosis and smoltification. The hormone systems which the LIFECYCLE research has focused in relation to binding proteins are the growth hormone (GH), leptin (Lep) and thyroid hormones (TH) systems. These systems regulate important production traits in aquaculture, including growth, energy balance and metabolism and the capacity of salmonids to adapt to change in salinity during smoltification. The goal was to study the interaction of these hormones with their receptors, binding proteins and transporters. To achieve this goal it was first essential to develop unique analytical tools with which to carry out the study. Considerable effort was put into the establishment of tools to measure GH and leptin binding proteins (GHBP and LepBP, respectively) and into complimentary molecular biology studies. The research has revealed that the GHBP have the same amino acid sequence as the extracellular part of the GHR and are probably shed from the cell surface in fish as occurs in most mammals. For GHBP, the work included the isolation, purification and characterization of the binding proteins from plasma, production of recombinant binding proteins and of specific antibodies. A specific analytical assay was developed and used to quantify GHBP in salmonids under several experimental situations that simulate situations that can occur during aquaculture production. Fasted rainbow trout, salmon in which plasma GH or ghrelin was increased, or salmon that were kept at different salinities did not have modified plasma GHBP,, although circulating GH levels and the abundance of GH receptors in the liver increased. However, acute physiological changes, like re-feeding after a fasting period or a sudden change in salinity, resulted in clear changes in plasma GHBP levels. Thus, GHBP may act to buffer sudden oscillations in GH.
Multiple LepBPs were characterized and cloned in rainbow trout . LepBPs are alternatively spliced Leptin receptor transcripts and consist of the extracellular segment. The different LepBP are differently expressed in different tissue of the fish. This indicates that the physiological action of Lep in fish can be modulated, at the level of tissue by modulating the expression of multiple LepBPs. LepBP were isolated from plasma and their binding specificity was assessed in a competitive binding assay. Quantitative assays revealed that plasma LepBP declines in rainbow trout after fasting. In maturing Atlantic salmon males and females no seasonal variation in LepBP in males because of large individual variation, but in females light and feeding regimes modified LepBP. Accordingly, LepBP may change transiently in response to environmental changes.
Thyroid hormones, their transporters and receptors are key-element in regulation of fish early development and metamorphosis and disregulation may explain poor larval or juvenile quality in aquaculture production. Thyroid hormones (THs) have to enter cells to bring about their biological action and recently a family of TH transporters located in cell membranes have been found to regulate this process. Once inside the cell, activation of THs occurs and the precursor hormone, thyroxine (T4), is converted into tri-iodothyronine (T3) by deiodinase enzymes and it then binds to thyroid hormone receptors (TR) and activates gene transcription. The role in development of TH transporters and receptors was studied in Atlantic halibut larvae and zebrafish larvae. In zebrafish the fundamental importance of THs in fish early development was demonstrated by ablating the TH transporter, MCT8, and blocking T3 uptake from the yolk by the embryo. The resulting larvae has abnormal brain development and the heart and GI-tract were also affected. The study reveals that an adequate supply of maternal THs in the egg is important for normal development and highlight the need for appropriate broodstock nutrition. Furthermore, TH cell transporters, deiodinase enzymes and TH receptors appear to be important to ensure normal embryonic and larval development. In a later developmental stage in Atlantic halibut when it transforms from a symmetric larva to an asymmetric juvenile TH transporters, (MCT8 and MCT10) gene transcript abundance increases from the start of metamorphosis until metamorphic climax and declines after metamorphosis, which is similar to THs, TR and deiodinase enzymes and further emphasises the fundamental importance of THs in this process. From a production perspective since metamorphosis impacts the quality and robustness of juveniles used for subsequent grow-out phases problems can severely affect sustainability.
Flatfish such as Atlantic halibut, turbot and sole are highly prized by consumers for their firm flesh and delicate flavour. Consumer demand for flatfish far outstrips supply of both wild fish from fisheries and from aquaculture production meaning that these fish command high market prices. The failure of aquaculture to meet the strong consumer demand for flatfish is a result of specific bottlenecks in their production cycle that threaten sustainability and check expansion of the sector. The flatfish offer a unique production challenge as unlike other commonly exploited finfish they shift from plankton feeding larva, that swim in the water column, to bottom dwelling juveniles with both eyes located on the same side of the head. The transformation from a symmetric larva to an asymmetric juvenile is called metamorphosis and represents a crucial moment in the lifecycle as failures in this process have a significant impact on viability and quality of the resulting juveniles. The role of thyroid hormones (THs) in orchestrating flatfish metamorphosis is well accepted, but the way in which they do this is far from understood. Therefore, METAMOPHOSIS was selected as one of the top-priority research areas of the LIFECYCLE project. It was not possible in LIFECYCLE to study all commercial flatfish species and the work focussed on Atlantic halibut for logistical and practical reasons. Nevertheless, because metamorphosis is an event that is TH driven and is highly conserved in vertebrates including the flatfish species the knowledge developed in the Atlantic halibut will be transferrable.
The challenges of flatfish larval production were evident during the project when the progress of the work was severely challenged by repeated and unexplained “collapse” of Atlantic halibut larval cultures at the commercial partners. For the project this was inconvenient and the problem was eventually overcome but for the industrial partner such unpredictable and uncontrollable events threatened sustainability. This problem highlights the urgent need for better knowledge about other aspects of early larval rearing not studied in the present project. The overall aim of research was to identify developmental programs, mechanisms and functional modifications associated with TH driven organ maturation during metamorphosis. The partners took advantage of next generation sequencing (NGS), a cost effective way to look in detail at the genes being expressed in any tissue. The absence of consolidated molecular resources for the halibut makes identification of gene transcripts difficult and this was overcome by generating an Atlantic halibut transcriptome by fusing the data from the project with existing publicly available data and this generated 17,000 unique halibut transcripts. Micro-dissection of tissues known to respond to THs followed by sequencing generated tissue specific Atlantic halibut transcriptomes and tissue specific development pathways and also general and tissue specific TH-responsive genes were identified. Taking advantage of the well-established staging scheme for Atlantic halibut the transcriptome of individual halibut larvae was determined and by comparison between them used to identify the principal molecular differences between development stages; early larvae, pre-metamorphosis, metamorphosis, climax, post-climax and juvenile. Highly differentially expressed transcripts between stages were associated with maturation of muscle, the cardiovascular system, skeletal system, skin and the GI-tract. Overall, tissue-specific gene networks activated or repressed during normal metamorphosis and TH responsive transcripts were identified. Molecular studies were linked to studies of tissue organisation using histology and immunohistochemistry and permitted identification of the main maturational changes occurring during metamorphosis. Overall, the histological picture together with specific molecular tools associated with Atlantic halibut metamorphosis could be used in future projects to predict and assess metamorphic success in response to modified culture regimes.
The importance of correct metamorphosis for function has been revealed for the GI-tract and the skin and insight into none metamorphic maturational processes have also been gained. At the level of the GI tract, a 3D model of its ontogeny was established and revealed increasing complexity is correlated with changes in function. In vivo pH analysis and quantitative PCR (qPCR) of α and β subunits of the gastric proton pump (H+/K+-ATPase) and pepsinogen A2 indicate that gastric proteolytic capacity is established during the climax of metamorphosis. Moreover, it was revealed that the stomach’s function in appetite regulation is interdependent with establishment of gastric function. A process independent of metamorphosis was the putative osmoregulatory capacity of the GI-tract already at first feeding in the Atlantic halibut and further exploration of events independent of metamorphosis should be fruitful. A better understanding of GI tract function and maturation will contribute to improved management of feeding. The skin that is an important innate immune barrier changed from a simple epithelia to a multi-layered tissue, which was less leaky and this change was pronounced from climax onwards. The reduction in skin keratin (that makes human skin impermeable) from metamorphic climax onwards coincided with the appearance of goblet cells that were presumably produced protective mucous. A better understanding of skin and its maturation has contributed knowledge for improving larval rearing conditions as it provides the basis for understanding osmoregulation, gas exchange and innate immunity. Studies to block metamorphosis by inhibiting TH production or to assess how stress impacts on metamorphosis confirmed the importance of THs and revealed a role for stress. A surprisingly strong “self-righting” mechanism exists in the Atlantic halibut and attempts to block metamorphosis caused a strong activation of the TH axis so that only 25% of fish were affected. Overall, the results of the LIFECYCLE work on metamorphosis demonstrated that direct disruption of the thyroid axis or cortisol axis modifies tissue specific developmental programs but it has not been proven yet if this directly underlies the overt changes that occur during arrested metamorphosis.
Ability to adapt to various environmental conditions is vital for fish and they rely on the development of osmoregulatory mechanisms at the level of specific organs such as the gills. Such adaptive processes occur during early development (embryo-larvae transition) when osmoregulatory functions develop and during smoltification when juvenile fish have to adapt to salinity changes. These situations are critical bottlenecks in salmonid aquaculture. For the preceding reason the LIFECYCLE project focused on DIFFERENTIATION AND PLASTICITY OF TRANSPORTING EPITHELIAL CELLS. The aim was to decipher the major molecular mechanisms regulating differentiation of osmoregulatory gill cells, a key process for development of osmoregulatory processes that give the fish the ability to withstand environmental changes. To analyse gill cell differentiation processes, descriptive studies of candidate gene expression in salmonid was combined with functional validation by transgenic approaches in a model fish species, medaka.
Several specific markers of differentiated gill cells (ionocytes and pavement cells) involved in osmoregulation were identified using microarray analysis. This work was carried out in trout by; 1) analysis of the gill transcriptome in fish transferred from freshwater to seawater; 2) by comparing gill to others organs; 3) by analysing dissociated and purified gill cells; and 4) from literature searches. Overall, this approach led to validation of 1 gene specific for freshwater ionocytes and 3 genes which are significantly expressed in all differentiated epithelial cells (pavement cells and in ionocytes) in both freshwater and seawater.
Key genes controlling epithelial gill cell differentiation were identified using 1) a survey of the literature; 2) a gill transcriptome analysis after freshwater-seawater transfer in trout; and 3) a genetic screen based on systematic in situ hybridizations. Candidate genes analysed further were selected based on their specific expression in the gill presumptive domain of the medaka embryo. Overall, these studies identified 20 candidate key genes potentially involved in epithelial cell differentiation.
The functional role of 3 candidate genes (gcm2, sgk2, GR2) controlling epithelial cell differentiation was validated in medaka during embryogenesis, using gene inactivation approaches based on morpholino injections. The inactivation of gcm2 and sgk2 led to significant modifications in ionocyte number in the embryonic skin, suggesting they have a key role in the ionocyte differentiation. Similarly, we also showed that maternal GR2 (glucocorticoid receptor form 2) is a major pathway in the ionocyte biogenesis in medaka, during early development.
Overall, during the LIFECYCLE project specific markers for differentiated epithelial gill cell and also key genes which are involved in epithelial cell differentiation were identified. The candidate genes were used in other LIFECYCLE tasks aimed at determining the effect of sexual maturation and androgens on the osmoregulatory system in Atlantic salmon. Recent findings suggest that GR2 also plays a key role in the modulation of the gill ion-transporting epithelium in salmonids during freshwater/seawater adaptation.
WP2.2 The intestine is the key organ for uptake of nutrients as well as for the uptake of water, through ion-coupled absorption mechanisms that are of key interest for aquaculture. Thus, LIFECYCLE has focused research on the MULTIFUNCTIONALITY OF THE GUT. Fish are continuously exposed to osmotic forces across all epithelia. In fresh water (FW), active absorption of ions and excretion of the excess amounts of water diffusing into the fish is crucial. In seawater (SW), on the other hand, uptake of water by the intestine in combination with secretion of ions is needed. The driving force for both the fluid uptake and the uptake of nutrients is the electrochemical gradient for Na+ ions, created by the Na+,K+-ATPase (NKA) and directed into the intestinal epithelial cells. The Na+-gradient brings along either other ions, like K+ and Cl- or nutrients, like amino acids and glucose. Thus, the utilization of the same electrochemical gradient, at the cellular level, may lead to a competition between absorbing ions for osmoregulation or nutrients for growth and explain growth check in a production setting.
Some active ion-transporters suggested for stenohaline SW fish (do not tolerate change in salinity) are present also in anadromous salmonids, but the transporters and proteins involved in intestinal water uptake is incomplete. Gene transcript (qPCR), and protein expression (immunohistochemistry) together with physiological measurements of ion- water- and nutrient transport, have revealed the developmental changes occurring in ion transport mechanisms as well as epithelial permeability during the parr smolt transformation (PST) and the subsequent transfer to SW. Understanding this process is important as it can lead to substantial losses if it fails to occur correctly in aquaculture.
The pre-adaptation of the intestine to SW includes mobilization of ion transporters that in concert work to build up a high osmolality (mainly through high Na+, Cl- and K+ levels) in the space between the cells (the lateral intercellular space; LIS) to drive fluid uptake. However, during the parr-smolt transformation, when the fish is still in FW, it would be counterproductive to start absorbing water and thereby add to the osmotic problems experienced in FW. The model for intestinal fluid transport in the smoltifying salmon developed from the results of studies in LIFECYCLE. Three, concomitantly acting mechanisms, including increased expression of the necessary ion transporters and mechanisms that can un-coupled their activity build up high osmolality in the LIS while the fish is still in FW. Thus, even though the NKA activity is gradually up-regulated throughout the time of the PST the enzymes are distributed within the cell membrane so that they don’t exert there action until the fish has entered SW. Similarly an increase in paracellular permeability of the intestinal epithelium, during the latter part of the smoltification, will prevent high ion concentrations in the LIS by allowing a leakage of especially positive ions back to the intestinal lumen. Thirdly, the proton excreting V-ATPase decreased in mRNA expression at the end of the PST when the expression of the NKA and other main transporters increased. As V-ATPase activity decreases proton excretion from the cells instead occur through a sodium-proton-exchanger protein (NHE) and leading Na+ back from the LIS and into the cell. Thus, the decreased V-ATPase activity may constitute a third way to un-couple the creation of a high osmolality in the LIS and thereby prevent fluid uptake.
In aquaculture both an efficient nutrient absorption and a maintained osmotic homeostasis is crucial for optimization of fish welfare and growth. This has led to the hypothesis that fish kept in an iso-osmotic environment, minimize energy use and utilize the available Na+-gradient for osmoregulatory mechanisms and should grow better than fish in SW. To investigate this Atlantic salmon were reared at full strength (34 ppt), 2/3 (22 ppt) and 1/3 (12 ppt) of SW in order to resolve the question of how intermediate salinities affect feed intake (FI), growth (SGR) and food conversion efficiency (FCE) and how this is related to energy requirement for osmoregulation. Fish reared in the highest (34 ppt) and lowest (12 ppt) salinity performed worse than at intermediate salinity (22 ppt). In the 12 ppt group the nutrient uptake across the intestinal mucosa was low and with low FCE and high FI indicates poor digestion and food utilization. The fish could however maintain a high specific growth rate (SGR), probably due to the low energy cost of osmoregulation at close to iso-osmotic conditions. At the other extreme, 34 ppt, the fish grew less FCE and intestinal nutrient transports were low and the results clearly suggest an allocation of the Na+-gradient towards ion transport, which then favours fluid absorption rather than nutrient uptake. Together the results suggest that the high nutrient transport in the 22 ppt group compared to the 34 ppt group is the result of allocation of the Na+-gradient from osmoregulation towards nutrient transport.
The loss of seawater tolerance related to sexual maturation is a production- as well as a welfare-problem in Atlantic salmon aquaculture in seawater net-pens, if fish start maturing prior to slaughter. Therefore, the LIFECYCLE project placed research emphasis on SEXUAL MATURATION AND OSMOREGULATORY FUNCTION. Early puberty is a problem that is most pronounced in males, as males normally reach puberty at a lower age and smaller size than females. Hence, a variable proportion of the individuals become sexually mature before harvest. In a study aimed at resolving the actions of androgens and cortisol in vivo, groups of immature Atlantic salmon post-smolts were maintained in seawater and treated with 11-keto androstenedione (OA), a precursor for the major androgen in fish, 11-ketotestosterone (11-KT), cortisol (F) only, or a combination of cortisol and OA (F+OA).
The key to seawater tolerance, is the activity of the ion pump called Na-K-ATPase (NKA), mainly situated in the fish gill. Gill NKA activity was significantly influenced by hormones and salinity. In seawater, NKA activity in the F(OA) group was reduced to about 50% of control levels, and this group also experienced mortality in seawater. Further, the interactive effect of hormones and salinity was again observed in the F and F+OA groups, which showed significantly reduced NKA activity in fresh water.
Parallel to the changes in NKA activity there was a significant increase in the freshwater isoform NKA α1a mRNA levels. An increase in expression levels by 100-fold or more was seen when control and OA fish were transferred to fresh water. A significant increase was also seen among F and F+OA groups. It is important to note that even in seawater, the F(OA) group showed a highly significant increase in NKA α1a expression levels, a freshwater form of the ion pump, despite the fact that these fish were still in seawater. Looking at the NKA α1b expression levels, a pattern emerges which shows a highly significant, and dramatic reduction in F(OA), down to levels which are characteristic of the freshwater condition. Taken together, these findings clearly demonstrate the reduction in seawater adaptive mechanisms in the F(OA) group, and points to the establishment of a freshwater adaptive situation from the combined exposure to OA followed by F.
In another experiment, the function of the intestine in mature and immature fish in SW was studied. Sexual maturation was associated with a substantial loss of intestinal barrier function, observed in both proximal and distal intestine. A decrease in intestinal barrier function is evident from increased leakage of Na+-ions through the tight junctions. This has a major impact on the fluid absorption of fish in SW as a tight epithelium towards Na+- ions is essential for creating a large osmotic gradient in the inter-cellular spaces used for fluid absorption. To compensate for this, the fish must increase the number and/or activity of the NKA, both at the level of the intestine and in the gill. Indeed, this was observed in the sexually mature fish. Although the fish is able to absorb fluid, it occurs at a higher energy cost due to the reduced barrier function. The intestinal barrier to smaller molecules is also impaired, suggesting that luminal antigens and harmful substances can more easily penetrate the intestine, suggesting that sexually mature fish are more susceptible to diseases.
This work has focused on the effect of sex steroids on critical ion transporting proteins in the gill and intestine during natural sexual maturation of Atlantic salmon. Our hypothesis proposes that the effect of sex steroids in salmon would stimulate a freshwater condition in the gill, and this is indeed what we have found. Taken together, our data show major changes in gill and intestinal function during sexual maturation of Atlantic salmon. Maturing salmon in seawater suffer osmotic stress, and critical elements of their osmoregulatory physiology are directed to a freshwater condition.
The early stages of development of farmed fish are highly susceptible to infection. At these early stages fish cannot be vaccinated as their immune system is not fully developed. A major under explored area of defence mechanisms in farmed fish is maternal influence on fry survival in terms of vertical transmission of immune gene transcripts and the protection this may provide the egg and post hatch stages of the fish. Whilst there is some evidence that protection to certain diseases may be passed on, the extent of this is unknown. Therefore, LIFECYCLE research efforts were directed against the area of OVA AND LARVAL IMMUNE GENE EXPRESSION, in particular to explore how the immune system of developing salmonid fish (rainbow trout and Atlantic salmon) cope with infections. We also examined eggs to determine the immune relevant mRNA molecules that are directly derived from the mother, termed maternal RNAs.
To assess the immune gene component of maternal RNAs a microarray approach was taken using RNA isolated from early stage zygotes before the embryonic transcription was initiated. Hence all RNAs would be maternally derived. We examined the genes present that hybridized to an Atlantic salmon custom Agilent microarray. Here, the criterion for gene selection was based on their expression threshold. The selected gene set (18499 genes) was examined by text filtering for key groups of immune related transcripts. Interestingly, we found many cytokine receptor mRNAs, enhancer binding protein transcripts and transcription factors, suggesting that the eggs are in a state of readiness to respond to immune stimulating signals. Both Interleukin enhancer binding factors 2 and 3 and interleukin receptors (IL-1, Il-4, Il-6, IL-9 and IL-10 receptors) were highly expressed in unfertilized eggs. Similarly for genes related to viral defences where interferon regulatory factors (IRFs 1, 2, 3, 5, 6, 7 and 8) were present, with IRF1, 2 and 7 being the most abundant. A number of immunoglobulins and immunoglobulin binding protein mRNAs are found which potentially relates to maternal immune protection. This research has given unique insights to early stage immune gene expression in salmonids.
During early stages of development, juvenile salmonid fish must rely on their innate immune system to defend against pathogens. At these early stages the immune system is immature and is unlikely to express the full repertoire of genes that control immune defences. Although vulnerable, these larval fish can still fight off infections indicating that there are active immune defence mechanisms. To examine later stages of development from eyed eggs to 3 weeks post first feeding, we challenged four key developmental stages with either bacterial or viral infections. These were eyed eggs, hatchling, first feeding, and 3 weeks post first feeding. Microarray analysis revealed new information about how the immune response increases dramatically during development. Early stages exhibited only innate responses, but at both first feeding and post first feeding, we observed a large number of immune genes that are regarded as part of the acquired immune response. These findings suggest that the transcripts (and probably the proteins) of the acquired immune system are present, and indicates the potential for earlier vaccination, but leads to new questions about how well developed the T cells are and the capacity for immunological memory. Hence, this work has led to new information that is highly relevant to fish health knowledge.
The final goal was to address the early growth rate of the fish. For this we placed first feeding fish on a high and low food ration. Each group was challenged with bacteria, virus or with mock infection. We examined a number of key immune responsive genes, although limited significant effect of growth rate on the response of the immune genes was observed. We extended this study to examine genes known to be involved in control of protein deposition / growth, with the aim to determine if infection altered the energy control balance differently in fast and slow growing fish. The two E3 ubiquitin ligases (these are responsible for targeting proteins for degradation) analysed showed a similar pattern of expression with a general increase in response to immune stimulation. TNFα was also the only cytokine gene to show a significant difference between fast and slow growing trout, with a greater response in the slow growing bacterially stimulated trout compared to fast growing ones.
Overall this part of the LIFECYCLE research effort has generated novel data and expanded considerably our knowledge of the ontogeny of the immune response in salmonid fish and has demonstrated that there may also be a relationship between rate of growth and immune function.
The Atlantic halibut is a marine, cold-water flatfish with a high market value and demand. The species is under development for aquaculture and is a promising candidate to complement and diversify the marine cold-water aquaculture industry.
As mentioned earlier, flatfish like the halibut, have an early unique life stage transition, metamorphosis, during which they transform from pelagic free swimming, symmetric larvae to benthic, asymmetric juveniles. Metamorphosis is common for all flatfish but the large size of the Atlantic halibut larvae as well as its potential as a future important aquaculture species, makes it suitable for studies of physiological, anatomical and immunological events during metamorphosis. One industrial bottleneck in the production cycle of many marine, cold-water species is the high mortality of the larvae after they hatch as they are relatively small and underdeveloped. For production of marine flatfish, this period is even more crucial as it also involves the complex process of metamorphosis. To address these problems, LIFECYCLE research was directed towards the PRIMARY BARRIERS AND DISEASE RESISTANCE with the overall aim to understand the functional characteristics and importance of the skin primary barrier during Atlantic halibut larval development and metamorphosis.
As pathogens are present in water, both in the natural and in the aquaculture environment, the presence of a functional primary barrier pivotal for good larval survival. In the small, newly-hatched yolk-sac larvae, the intestine and gills are poorly developed. Therefore, and due to the high surface to volume ratio in small individuals, the skin constitute the most important primary barrier, in other words the first line of defence, against pathogens in the environment.
Two main technical and methodological advances have been achieved during the LIFECYCLE project. An in vitro system for assessment of Atlantic halibut skin barrier integrity and properties was successfully developed, and four antimicrobial peptides, so-called pleurocidins, were identified in Atlantic halibut, characterised and specific antibodies against these proteins produced. These methodological advances have made it possible to better understand the development of the physical skin primary barrier during halibut larval development and metamorphosis as well as to understand the relative importance of the extrinsic barrier, i.e. the secretion of mucus and antimicrobial peptides during this period.
The skin develops from one cell-layer epithelia found in the early larval stage 6 to a complex and stratified epithelia in the juvenile fish. The supporting connective tissue in the skin, the basement membrane and the collagen fibres, increases both in thickness and complexity during the larval development and metamorphosis. These morphological changes are clearly reflected also in the functionality of the skin barrier. The skin from stage 7 larvae was leaky while stage 9 larvae had twice the resistance to passive flow of water and soluble molecules and stage 8 had an intermediate resistance. Interestingly, the early developmental stages with low and leaky physical barrier also showed a lower number of mucus producing Goblet cells. Thus, the early larval stages seem to be exposed to strong passive, diffusional forces due to a poorly developed physical as well as secretory barrier. The changes in skin barrier properties are gradual except between stage 8 and 9A, when all the developmental skin parameters measured changed dramatically. This indicates that the larvae become much more robust after passing over to stage 9A.
The development of the larvae from symmetric free-swimming fish to a bottom-living asymmetric fish can also be seen in the development of the skin barrier properties. The increased complexity and thickness of both the multi-layered epithelial cells and connective tissue were more pronounced on the top side. This was also the case for specific cell types like melanophores (pigment cells, only present on the top side, and the Goblet cells, which were more numerous on the top side). This suggests that diffusional and invasive forces from the surrounding water require a larger degree of protection than the more mechanical challenge from the underlying substrate.
The finding of four different antimicrobial pleurocidins (PLCs) in the halibut transcriptome clearly suggests an important role for these substances in resistance against infection and disease. Three of the pleurocidins (PLC2, PLC3 and PLC4) were found in Goblet cells in the different epithelia, clearly indicating that these innate defence molecules are secreted with the mucus in order to prevent invasion of pathogens into the body of the larvae. The pattern of distribution of PLCs 2-4 in the different epithelia as well as larval stages were slightly different for the three pleurocidins, but PLC3 was abundant in all epithelia and all stages of larvae from stage 7 and onwards. Thus, this anti-microbial peptide seems to be a very important component in the defence against pathogens in the earliest and most vulnerable stages of Atlantic halibut, when the physical barrier is still weak and leaky.
Life stage transitions often involve major changes in phenotype and physiological functions. Such life-stage transitions represent a particularly vulnerable period in aquaculture, and in many cases, high mortalities are seen. To meet the challenges of the new life stage, changes in form and function of epithelia occur. Gill and intestinal epithelia constitute an interface between the environment and the internal milieu and have unique roles as both primary defence barriers and selectively transporting tissues. Developmental and environmental driven changes in form and function of these transporting epithelia will affect the primary barrier functions and potentially the epithelial immune system and vice versa. Therefore, the LIFECYCLE project concentrated research on the IMMUNE-ENDOCRINE-PHYSIOLOGICAL CROSS-TALK AT THE PRIMARY BARRIER AND SYSTEMIC LEVELS, in order to study the impact of key life history changes in salmonids on their ability to resist diseases. A particular interest was the cross-talk between immune function and hormones known to be at high levels during the transition from the fresh water parr stage to seawater adapted salmon (termed smoltification or parr-smolt transformation), or during sexual maturation. In addition, host immune molecules (cytokines and free radicals) that are elicited during inflammation were also studied for their impact on physiological functioning of the fish, at sites that act as primary barriers to infection but that also have important roles in ion/water/gas transport (e.g. gut and gills), to see if these functions are affected/compromised.
Both the parr-smolt transformation (PST) and the subsequent seawater (SW) migration are important life stage transitions for Atlantic salmon. At the level of the intestine, the physiological pre-adaptations to SW include de novo synthesis of ion transporters and other epithelial proteins to create a mechanism for fluid absorption. In addition to the intestinal epithelial permeability, this tissue also constitutes a physical barrier against pathogens in fish.
The barrier function, assessed using electrophysiology on live intestinal epithelia, changed during the PST. The paracellular permeability increased, rendering a decreased barrier, towards the peak of smoltification when the fish are still in FW. Directly after SW transfer, a dramatic decrease in permeability occurred, indicating a significant strengthening of the physical primary barrier. A regional difference in tightness of the paracellular pathway was also seen, with the proximal intestine being leakier than the distal intestine. Gene expression measurements as well as immunohistochemistry showed expression differences may be governed by the barrier forming claudin isoform, claudin 25b. Upregulation of two other major tight junction proteins, occluding and ZO-1, were each shown to contribute to the tightening of the physical barrier after SW transfer and the tighter barrier in the distal intestine, respectively. The extrinsic barrier, i.e. the secretion of a protective mucus layer covering the intestinal epithelia had a similar pattern of change during PST. The mucus secretion was reduced towards the peak of smoltification, but increased to levels above those of parr in smolts after SW transfer. Thus the physical and extrinsic barriers together create an enforced primary barrier in the salmon intestine after SW entry and start of drinking. This will protect the epithelium from exposure to pathogens in the ingested water.
However, the results from the LIFECYCLE project also demonstrate that several factors interfere with these developmental changes in the primary barrier. Both acute and long term stress, through the stress hormone cortisol, negatively affected the physical barrier. It widened the paracellular pathway probably by changing the expression of tight junction proteins. An increased leakiness will induce the underlying epithelial immune barrier to start secretion of pro inflammatory cytokines. Electrophysiological studies in combination with perfusion studies, using recombinant trout cytokines, and IHC showed that some of these cytokines in turn affect the physical barrier. rIL-1β and rIL-6 increased the paracellular permeability by decreasing the expression of claudin 25b and caused a cellular re-distribution of ZO-1. Such processes may contribute to the increased susceptibility to infection seen in Atlantic salmon smolts directly after SW transfer.
The effect of free radicals and their precursors, produced during pathogen infection, on gill epithelium was also studied. Incubation of gill cell cultures with different doses of hydrogen peroxide showed a toxic effect using higher levels (4mM), where a disrupted epithelium was seen, and gill permeability and gene expression were decreased. At 2mM only gene expression was affected, and below this dose no toxic effects were apparent. Curiously, a gene that encodes a tight junction protein had increased expression at low doses of hydrogen peroxide, suggesting it may have an important role in maintenance of epithelium integrity in the presence of free radicals.
The impact of PST on the expression of a number of genes involved in anti-viral defences was examined, with a focus on interferons, since viral infection is a known risk factor in smolts post transfer. Parr and smolts were stimulated with a mimic (poly I:C) of viral infection at 5 time points during the parr smolt transformation, with subsequent analysis of the immune genes in immune organs. The results show that during the critical smolting period the immune response is reduced, especially at the time of transfer to SW, and highlight that this is a vulnerable stage during the development of salmon.
Experiments were also designed to examine the impact of sexual maturation on the antiviral immune response. Initially we tried to use sexually maturing male fish (during 2011) but there were insufficient numbers of fish, and so the approach was changed and sexual maturation was induced by injection of the androgen 11 ketoandrostenedione. Three weeks later, the fish were exposed to poly I:C to induce antiviral responses and tissue samples collected 24h later for gene expression analysis. This experiment showed that up-regulation of antiviral genes by poly I:C treatment occurred as expected, with a downward trend in gene expression observed in the androgen treated fish, although no significant changes were observed.
As a key hormone in the PST process and stress responses in general, cortisol was also investigated in vivo to examine the impact of this hormone on the antiviral response. Cortisol was administered to fish as a slow release implant. Six days later the fish were exposed to poly I:C for 24h and immune tissues collected for antiviral gene expression and blood cortisol analysis. Plasma cortisol was upregulated. In the gills, poly I:C treatment provoked upregulation of the genes studied. However, there was no clear impact of cortisol treatment vs. control fish in the presence or absence of poly I:C challenge. Cortisol treatment stimulated expression of sodium/potassium ATPase genes, consistent with previous studies, illustrating the importance of this hormone in ion regulation. The only significant effect of cortisol treatment was to decrease expression of the pro-inflammatory gene IL-1β. Up-regulation of antiviral genes by poly I:C treatment was weaker in spleen tissue, where again no modulation of gene expression was attributed to cortisol treatment.
To further examine potential cross-talk between the endocrine and immune systems, a panel of hormones; cortisol, IGF-1, T3 and growth hormone (GH) were used to examine their effects on a variety of isolated immune cell types. We examined macrophages, CD4+ cells (expected to be mainly a subpopulation of T cells), B cells and neutrophils. Incubation of macrophages for 3 or 6 h with cortisol decreased expression of two cytokines (IL-6 and IL-8) involved in inflammation. Co-stimulation of cortisol with inflammatory agents resulted in a general suppression of genes related to the proinflammatory response. In contrast, cortisol in combination with pro-inflammatory agents had a synergistic effect on IL-10 expression, and anti-inflammatory molecule. However, no impact of incubation with T3 or IGF-1 was seen.
Using a trout anti-CD4 antibody we identified a CD4+ cell line (RTHK15) developed by exposing trout leucocytes to IL-15-containing medium. The results from the cell line were in sharp contrast to our previous findings of cortisol suppression of immune gene expression in macrophages. Cortisol exhibited a stimulatory phenotype particularly in the presence of a T cell stimulant (PHA). A significant synergistic effect was seen between cortisol and PHA in the case of IL-2, IL-4-like, IL-8, IL-17C2, IL-22, and TNF-3. Indeed, five genes ( IL-1β, IL-2, IL-22, RORγ, and TGFβ1.2) were significantly upregulated in the presence of cortisol alone. Interestingly, a third group of genes (IL-15 and γ-IP) were not influenced by cortisol treatment. These findings suggest the presence of cortisol responsive and cortisol unresponsive immune cell populations in trout.
Monoclonal antibodies to trout B cells and neutrophils were used to isolate these cells types, by magnetic-activated cell sorting. No significant changes were seen after incubation of B cells with the hormones, when analysing a selection of B cell markers, but some trends were found. For instance, the relative expression of the B cell receptor (IgM) and a surface expressed co-stimulatory molecule (CD40) showed a small elevation in expression after incubation with GH in the presence of LPS. Purified neutrophils also showed no statistically significant changes after stimulation with the hormones but again a trend for induction in some genes was seen. In this case GCSF expression was increased after incubation with cortisol and PMA, and IL-8 expression was increased after incubation with GH.
Overall, the CD4+ cells and macrophages were the most sensitive to hormonal treatment, especially with respect to cortisol treatment. Nevertheless, it is clear that different immune cell types in trout do respond differently to physiological levels of the hormones used
Lastly, the regulatory regions of the genes most modulated by cortisol in the macrophage experiments above were analysed to determine the presence of transcription factors known to be responsive to hormones/stress. For this we used the Atlantic salmon genome database to retrieve the for six immune genes, IL-6A, IL-6B, IL-8, IL-10, γIP and COX-2. Four elements were searched for, namely the glucocorticoid receptor responsive element, estrogen receptor-responsive element, the thyroid hormone receptor-responsive element and the cAMP responsive element. Variation in the number of elements was apparent between the genes, with IP having the fewest (10) and COX-2 the most (28), allowing predictions about their relative sensitivity to hormone exposure.
Specific immunity is crucial for long-term disease resistance. Previous studies on early expression of Ig in B cells or TCR genes in T cells are limited and cannot predict the effectiveness of a response to infection or vaccination during the larval stages. Very little is known about the impact of reproduction on the diversity of the immune repertoire and on the response effectiveness. It is important to distinguish between the non-specific effects due to stimulation of innate immunity, and the specific and long lasting protection due to antibody and T cell responses. Thus, DNA vaccination induces both specific and non-specific responses in adult fish, and is considered to afford protection to fry only through innate mechanisms such as interferon production. However, the mechanisms and the dynamics of the transition between the different responses are still unknown in the fish. Therefore, the LIFECYCLE project included research into the area of MATURATION AND SPECIFIC IMMUNE RESPONSES. Trout immune repertoires were analysed to get insight into the diversity of immunoglobulins (Igs) and T cell receptors (TCR) in fish. Upon infection Ag-specific B and T lymphocytes develop protective responses against the intruding pathogen, and after disease resolution they maintain an increased resistance against the eliciting agent through persistence of memory cells, which is the basis of vaccination.
In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT are unknown. A comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout was undertaken before and after challenge with an important fish rhabdovirus (the VHSV) using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, distinct repertoires for IgM, IgD and IgT, respectively were observed, with a few IgM- and IgT-secreting cells in the spleen. In infected animals, complex and highly diverse IgM responses were detected dominated by a few large public and private clones. A lower number of robust clonal responses involving only a few VH were detected for the mucosal isotype IgT, indicating that both IgM+ and IgT+ spleen B cells responded to systemic infection but at different degrees. In contrast, the IgD response to the infection was faint. This study is the first attempt to obtain a comprehensive description of the Ig response after vaccination in fish, and provides a basis for further characterization of responses induced by vaccination in aquaculture.
Regarding T cells in fish, the diversity of TCR expressed by CD8+(CD4-) and CD8-(CD4+)T cells remains poorly understood. This is an important issue since fish CD8+ T cells are cytotoxic while CD8- T cells appears to be the counterparts of T helper cells, which are critical to promote strong protective Ab responses. The complexity of TCR beta was compared between FACS sorted CD8+ and CD8- T cells from spleen and pronephros of rainbow trout. It was found that the repertoire of CD8+ T cells was highly diverse and polyclonal while it appeared to be very different in CD8- lymphocytes with dominant clones even in naive fish. After infection with a systemic virus, CD8+ T cells mounted a typical response; the virus also induced significant modifications of the CD8- TCR beta repertoire, but targeting different V/J combinations. These results suggest that the diversity of TRB expressed by CD8+ and CD8- alpha-beta T cells may be subject to different regulatory patterns in fish and in mammals.
In fish as in mammals, two lineages of T cells have been identified, expressing either the alpha-beta or the gamma-delta TCR. In mammals the latter type of T cells recognizes unconventional antigens and is especially important for mucosa protection. The LIFECYCLE research has discovered and characterized the TCR gamma of sea bass. Gamma-delta TCR mRNA expression was detected in basal conditions both in thymus and intestine, while in kidney and gut leukocytes it was up-regulated by poly I:C in vitro. This work provides tools to study the biology of gamma-delta T cells in an important aquaculture species in Europe.
European sea bass and salmonid aquaculture both require production of all-female populations for optimizing growth and to avoid the problems related to sexual maturation by producing all-female triploids. The available sex control methods are time consuming and not fully efficient in sea bass while androgen treatment is used in salmonids provoking environmental and consumer concern. Knowledge about key molecular regulators of sex determination/differentiation can support a rational choice of new techniques for sex control. The LIFECYCLE research programme aimed at improving this knowledge by focusing research on BRAIN AND GONAD SEX DIFFERENTIATION. It was already known that sea bass has a polygenic sex determination system which is influenced by temperature, and that salmonids have a more simple, genotypic sex determination system. Thus, gene expression was extensively analysed in larvae before and during sex differentiation of the gonads by comparing genes expression either in larvae reared at low (15°C) or high (20°C) temperature in sea bass or by comparing genetic females and males in rainbow trout (mono-sex populations). Different cutting-edge laboratory techniques have been used for identifying and characterizing gene transcripts and dozens have been found that are involved in the molecular cascade that controls gonad sex differentiation.
Firstly, it has been confirmed there is no unique molecular model for sex determination and differentiation in fish. The genes involved in sex differentiation in other vertebrates do not always have a conserved role in all vertebrates. In the sea bass some of the genes involved in the molecular cascades leading to sex determination have a role in chromatin remodelling and epigenetic regulation, i.e. not involving a change in the nucleotide sequence. Genes up-regulated in male-biased populations of sea bass during the sex differentiation phase were associated with migration, proliferation and differentiation of germ cells.. A specific sex-determining gene was not identified although a suite of genes associated with males were identified and could be used for selection of strains giving a high rate of males by rearing larvae at high temperature.
In rainbow trout, it was found that not only the gonads but the brain as well is sexualized early, exhibiting a clear sexual dimorphism with respect to the expression of several genes. Interestingly, they include the key enzyme in oestrogen synthesis (aromatase B) as well as of certain enzymes and factors involved in the synthesis of other steroids and of estrogens receptors. Complementary studies performed in zebrafish, provide the first demonstration that the brain of fish can produce true neurosteroids, notably in radial glial cells, potentially neuronal precursor cells. Thus, a dialogue between gonads and brain should be taken into account in the future for the full understanding of regulation of first stages regulating sexual differentiation. In gonads, the kinetics and cellular localization of a group of genes potentially involved in the molecular cascade of sex differentiation were defined. This group includes genes known in other vertebrates (e.g. tbx1, amh, gsdf1, dmrt1, pax2 in male; fst, wnt, foxl2, inha in female), but also new genes (e.g. irk8, pgh2, gata2, gremlin). Experiments involving steroid administration to larvae provided additional information on the steroid sensitivity of the expression of these genes. Finally, a major outcome is the discovery that a new gene called sdY (for “sexually dimorphic on the Y-chromosome”) is the major sex determinant gene in salmonids. This gene has been functionally characterized and its spatio-temporal expression during natural sex-differentiation was defined. The sdY gene sequence has been used by international biotechnology services for genotyping in salmonids, and effectively accelerating the monosexing process in trout. It will be very useful to explore the reason of male occurrence in industrial all-female population and to develop new monosexing methods.
One of the key life-stage transitions which needs to be controlled in commercial fish farming is the onset of puberty, i.e. when the fish start the process of sexual maturation. LIFECYCLE conducted research into PUBERTY CONTROL by assessing the impact of physiological conditions (growth, adiposity) and external signals (photoperiod, behaviour) and molecular factors underlying the onset of puberty in salmon, trout and sea bass. A major breakthrough was the characterization of the Kiss-system and its relation to the previously known Gnrh system, including information on the possibilities for the feedback regulation of the Kiss/Gnrh systems by sex steroids. Decisive for this success was the strategy to first establish the basis in zebrafish, before studying the considerably larger sea bass and salmonid brains in a targeted manner. Also functioning in the brain/pituitary system and representing another, very important discovery is the identification of the in salmonid fish long-sought receptor mediating reproduction-related Gnrh effects in the pituitary. An attempt to generate transgenic medaka overexpressing Kiss peptide did not succeed. However, studying the effects of Kiss injections compensated for this and had the advantage of allowing experiments with sea bass directly. It was demonstrated that in fish, similar to land vertebrates, the Kiss and Gnrh systems regulate the pituitary gonadotropins. However, different from the higher vertebrates, where Kiss seems to work exclusively via the Gnrh system, Kiss also has direct effects on the pituitary in sea bass. Altogether, this objective has been fully achieved, maybe even above the initial expectation, considering that the work dealt successfully with an unexpected genetic complexity of the Kiss/Gnrh system in particular in salmonid fish.
The chosen experimental conditions (feeding level, photoperiod regime) have resulted in the expected, clear-cut effects on the incidence of puberty in sea bass and salmon. Investigations on the key components of the above-discussed regulatory systems operating in brain and pituitary, were complemented by quantifying plasma hormone levels and by analysing morphologically the start of spermatogenesis. This allowed another breakthrough, which was also facilitated by taking some of the crucial steps using trout and zebrafish as additional experimental models. Work in all species consistently demonstrated that the initiation of pubertal testis maturation involves the pituitary hormone Fsh. Fsh then activates testicular androgen production and modulates growth factor expression, and androgen signalling and changes in the growth factor “cocktail” then initiate spermatogenesis. For the salmon, the data moreover indicate that the newly discovered Gnrh receptor form (see point 1 above) is involved in mediating the activation of the pituitary.
The relationship between experimentally (feeding and/or photoperiod) induced changes in puberty and the Kiss/Gnrh system, however, requires more work and is not clear yet. Next to technical considerations (e.g. size of brain samples used for gene expression analysis), one of the reasons, at least in the salmon, might be related to the fact that the period during which external and internal factors affect the decision to initiate puberty, apparently act as early as 12-15 months before the first spawning season, i.e. earlier than anticipated in most of the experimental set-up used here (however, see trace-back studies in point 3). The work carried out in sea bass on the possible impact of social interactions and chemical communication provided no evidence for effects of behavioural cues or pheromonal signals on the timing of puberty.
Since restricted feeding was known to reduce the incidence of puberty, we hypothesized that hormones involved in regulating growth/adiposity (leptin, ghrelin, Gh, Igf1) play a role in the recruitment into puberty. This was examined by studying aspects of growth and reproduction in terminally sampled fish during the period December – June when recruitment into puberty becomes obvious, and by repeatedly life-sampling fish from September – June which were scored for maturity June, in order to trace back the relation between hormone plasma levels and growth to the maturational state reached in June. Moreover, we examined testicular gene expression patterns in fish at the brink of puberty (December and January) that were subjected to normal versus restricted feeding.
The study on terminally sampled fish revealed no clear evidence for significant correlations between metabolic hormones and pubertal development, while unexpected correlations were found for, e.g. effects of photoperiod regimes on plasma leptin levels. The trace-back approach, on the other hand, provided clear evidence for an early predisposition of maturing males. As soon as December, i.e. 9 months before spawning, higher plasma androgen levels were found in maturing males, and even earlier already in September, these males also showed a higher body weight. These observations warrant further studies to unravel the relation between these early differences in body weight, and the subsequent differences in plasma androgen levels that herald future maturation.
In association with work spermatogenesis, feeding-related differences in testicular gene expression patterns were investigated with the view to develop hypotheses on how growth and energy stores interfere with the brain-pituitary-testis axis to influence the decision to mature. Based on parameters reflecting testicular activity (germ cell proliferation, plasma androgen levels), 36 males were selected for large-scale testicular gene expression profiling. An important observation comparing maturing males is that under normal feeding conditions, growth factor expression gradually changed in salmon testis just starting to mature, while a sharper contrast with abrupt changes in the expression of the same genes occurred in animals kept under reduced feeding conditions. This suggests that under conditions of restricted feeding, clearer signals are required to achieve activation of testis maturation.
The search for genes associated with the physiological readiness to precocious puberty in sea bass was delayed repeatedly, first due to technical problems with the chosen sequencing approach, then as a consequence of the move to another institute of the scientist leading the sequencing activities. Accordingly, the sequencing results became available rather late for the group receiving the data, so that completion of the necessary analyses was not possible within the remaining time frame of the project. Still, the data set is available now and initial evaluation of the brain samples suggests that the experiment was successful per se.
Male puberty involves the activation of steroidogenesis and spermatogenesis, and better understanding of this process is of great importance for aquaculture production. Therefore, LIFECYCLE focused research on the PARACRINE SIGNALING AND TESTICULAR GENE EXPRESSION DURING THE INITIATION OF SPERMATOGENESIS. The aim was to characterize the gonadal molecular responses to FSH and androgens (the two most important hormones regulating testis maturation) that could result in a promotion or inhibition of testis maturation. In vivo effects of recombinant Fsh on spermatogenesis were investigated in prepubertal sea bass and demonstrated for the first time that recombinant Fsh can initiate testis development in immature fish. We also confirmed that androgen production was involved in the initiation of fish testis maturation. Further characterization of regulatory pathways used by hormones to initiate testis maturation was achieved in trout by large scale gene expression changes during natural testis maturation and spermatogenetic development. Androgen-dependent genes were also searched for in prepubertal rainbow trout treated with physiological doses of androgens, while Fsh-regulated genes were identified from trout testicular explants cultured in the presence of this hormone. Orthologues of these hormone-dependent trout genes were identified in several vertebrates for which the genome sequences were available, and candidate genes were annotated for functions demonstrated in other species. These analyses provided us with a knowledge base containing around 2000 genes, essential for the interpretation of androgen and Fsh action in fish. Overall it was considered that candidate genes should be hormonally regulated, evolutionary conserved, act as paracrine factors (i.e. functionally with “growth factor” or “cell fate regulation”), and most likely expressed in the testicular somatic cells.
Based on these hypotheses, four candidates (Amh, Gsdf, Igf1b, Insl3) were studied in a zebrafish testis tissue culture. Amh, Gsdf, Igf1b are expressed in zebrafish Sertoli cells and intra-tubular somatic cells that are known to support spermatogenesis. Anti-Müllerian hormone (Amh), has inhibitory effects on androgen release and, independently, also inhibitory effects on germ cell proliferation and differentiation. The Igf1b has stimulatory effects on germ cell proliferation and differentiation, but does not modulate steroidogenesis. The third candidate, Gsdf stimulated the slow proliferation of undifferentiated spermatogonia type A in zebrafish testis tissue cultures. Finally, the fourth factor identified most recently, another insulin like factor, Insl3, is produced by the interstitial Leydig cells. A model was developed for their role in the initiation of the spermatogenic development.
The biological significance of selected factors was to be explored also in vivo in transgenic zebrafish, genetically modified to over-express these factors. Experiments did not result in significant overexpression of the factors and could not induce a noticeable reproductive phenotype. It was concluded that loss-of-function approaches should be evaluated as alternative routes to study the biological significance of selected paracrine factors in vivo.
To elucidate further the molecular mechanisms underlying the hormonal regulation of the factors characterized above, potential hormone-responsive elements (HREs) located within the proximal promoter region were highlighted in the genome sequences. Then using in vitro analysis according to established procedures, two regulatory elements have been successfully confirmed that suggest that Fsh regulates the transcription of the targeted igf1b gene through the stimulation of the cAMP signalling pathways.
In can be concluded that understanding the physiology of puberty at the testicular level is necessary before new procedures to reduce early maturation in aquaculture can be established. Based on complementary biological models and on primordial original competencies, the LIFECYCE work has provided significant progress in our understanding of hormonal actions during puberty: 1) FSH induces pubertal maturation and androgen treatments modify the immature germ cell towards the profile of differentiated germ cells; 2) Transcriptome analyses provided us with a knowledge base essential for understanding hormone mechanisms of action in fish and a short-list of genes known to be regulated by hormones, evolutionary conserved from fish to mammals, expressed in testicular somatic cells, and possibly acting as paracrine factors on germ cell fate were identified; and 3) three growth factors were shown to have a role in gonad maturation and could be interesting targets in procedures to control of puberty.
I wish to thank the large number of scientists from eight European countries, representing all the thirteen LIFECYCLE partners, who have contributed to this brief summary in a continued effort to disseminate the massive biological knowledge-base, so successfully generated by the whole LIFECYCLE research consortium.
Gothenburg, September 2013
Björn Thrandur Björnsson
LIFECYCLE coordinator
Potential Impact:
LIFECYCLE built on recent major advances in marine genomic research and resources developed for key finfish aquaculture species in Europe including rainbow trout, Atlantic salmon sea bass, sea bream, Atlantic cod and Atlantic halibut, in addition to the knowledge generation by the use of model species like zebrafish and medaka. The project has utilized such new knowledge and research tools in a cross-cutting approach by parallel studies on some of the most relevant farmed fish species in Europe, in combination with model fish studies to generate basic biological knowledge of relevance to solve bottle-necks in the production cycles in European aquaculture.
LIFECYCLE has applied in silico search for relevant genes using recently available genome data in combination with various techniques to study gene expression, either on single genes or the entire transcriptome in various developmental processes along the life-cycle in farmed fish. This has been combined with more traditional physiological, endocrine, immunological, and morphological techniques applied to experimental fish studies at different life stages from egg to adult fish. To some extent this has been combined with studies on gene function in the model species zebrafish and medaka.
The approach has greatly extended our knowledge on e.g. the transcriptome of developing oocytes and eggs with relevance of broodstock rearing and egg quality in farmed fish, sex determination and differentiation in early development, metamorphosis, growth processes and adiposity, links between growth and puberty, development of the immune system and its modulation during life-stage transitions like smoltification and puberty, and various cross-talks between these processes. Finally the project has also studied in detail the impact of various environmental factors on some of these processes.
Although the main impact of the project is new basic knowledge on developmental processes in critical life stages of farmed fish in terms of scientific publications and other dissemination along with a substantial extension of genomic resources and research tools for these species, parts of the knowledge-base generated are of direct importance to farming such as new understanding on how smoltification modulates the immune response of Atlantic salmon, and how factors such as feeding and photoperiod modulate age at puberty in sea bass and salmon.
POTENTIAL IMPACT
Contribution to establishing the knowledge-base for the improved competitiveness of the EU aquaculture industry:
Introduction: The LIFECYCLE project emerged in response to the project call KBBE-2007-1-2-16: Essential biological functions related to the most relevant stages of aquaculture fish life-history Call: FP7-KBBE-2007-2A and it took close to two years to build the project consortium and proposal. When funded, a formal contract was signed between the project consortium and the European Commission and the appendix to the contract, the “Document of Work”, in essence specified the work to be performed during the project and also foresaw the outcome. The present description of the potential impact of LIFECYCLE is therefore circumscribed by the impact expected by the Commission (see below) and forms the basis for the present analysis of the actual project impact that was determined. In addition to foreseen impacts, the project has had a number of other impacts that were not defined in the formal “call text” and they will also be highlighted in the following description and include more general aspects such as, stimulating innovation, contributing to European excellence and competitiveness, structuring the European research area, stimulating international collaborations, and contributing to build a strong skill base in science and aquaculture through training as well as actions favouring gender equality. An important point to bear in mind is that the impact of the project will not terminate with its completion, but will continue for many years to come. Nevertheless, the LIFECYCLE project will have an enduring impact in a number of important areas of aquaculture production, which will be made evident in the following short report.
Expected impact foreseen in the call: The project will deliver improved competitiveness of the EU aquaculture industry by establishing for commercially important species the knowledge-base required for improved stress control, disease prevention and control, new breeding technologies, better environmental performance and also diversification into new species.
Impact in the context of Policy: The aquaculture industry is part of the “bio-economy”, which includes all industries and economic sectors that produce, manage and otherwise exploit biological resources. The biological resources targeted by the LIFECYCLE project were fish, and more specifically, species important for the European aquaculture sector; sea bass, sea bream, Atlantic salmon and rainbow trout, as well as Atlantic halibut and cod to lesser extent. The project was directed at advancing knowledge and stimulating innovations in order to promote and support the sustainable management, production and use of fish, a biological resource used in commercial aquaculture. The project generated a wealth of knowledge that has improved the understanding of fish biology and physiology and when translated to industry should help to overcome key production bottlenecks in the areas of egg and larval production, muscle growth, reproduction and disease resistance, all of which currently challenge productivity. A better understanding of the biology of fishes will make it possible to define sensible strategies that improve European aquaculture productivity and make it more competitive, but not at the cost of animal welfare or the environment. Improved productivity and competitiveness in the bio-economy is expected to generate jobs, and since aquaculture tends to be in coastal regions that have seen a decline in jobs with the loss of traditional fisheries this makes an important contribution to social welfare and well-being. LIFECYCLE has generated a knowledge base for the development of a more competitive and productive aquaculture industry and as such supports the Common Fisheries Policy reform that aims to provide sustainable development of fishing and aquaculture.
Establishing the knowledge-base for commercially important aquaculture species: The section above has given an overview of the main S&T results/foregrounds and scientific outcome of LIFECYCLE. This section will therefore, give a general consideration of the impact and some specific research highlights that have resolved hypotheses or shifted paradigms. Immediate practical impacts are presented under the sub-section innovations.
The first major impact of LIFECYCLE is the significant increase in biological knowledge available for commercial species, that has arisen from the “cross-cutting approach” deployed to study fish developmental stages. This was achieved by bringing together internationally acknowledged research groups studying individual organs/functions, and the interactions and cross-talks between different biological systems/processes were examined throughout the whole fish life-cycle from gametes to reproductive individuals. Since commercial producers may be involved in the productions of several life stages or concentrating on a specific process e.g. grow-out that may cover several biological stages, the LIFECYCLE approach has direct practical interest for producers.
The second major impact of LIFECYCLE has been to advance understanding of fish biology through the utilization of existing molecular and biochemical tools, but also through the generation of additional molecular and biochemical tools. The molecular information generated by LIFECYCLE has been released into the public domain. In this way LIFECYCLE has had a knowledge spreading effect that has benefited the scientific community in general and given added value to the project outcomes.
The third major impact of LIFECYCLE has come from studies of tractable model species in which integrated molecular, genomic and functional information from the zebrafish and medaka has been used to advance the state-of-the-art and then applied to aquaculture species. This approach was novel for aquaculture research, but its successful application in LIFECYCLE has given “proof of concept” and reveals the approach may be a rapid and cost-effective way to obtain knowledge useful to solve commercial problems. LIFECYCLE took advantage of the benefits of the model organisms zebrafish and medaka to establish and study basic biological mechanisms and since they are well conserved between teleosts it was possible to transfer knowledge to the major commercially reared fish species. Moreover, knowledge transfer between zebrafish and the commercially reared fish was facilitated by the generation of further molecular data about the transcriptomes of sea bream, sea bass, halibut and cod under a range of conditions using a next generation sequencing (NGS) approach.
The results coming from LIFECYCLE will underpin future generations of basic science and applied projects for both consortium members, and other scientists, and the impact will be long-lasting towards the horizon of 2020.
Innovation: LIFECYCLE has substantially advanced current knowledge beyond the state-of-the-art and has impacted on egg and larval production, growth and energy balance, broodstock and reproduction, fish immunity and disease resistance and osmoregulation. The diverse and complementary nature of the consortium has allowed key basic questions to be addressed at specific life stages or across the life cycle. Research has encompassed the gene, cells, proteins, tissue and the organisms and when it was possible to integrate information has consolidated understanding of specific biological processes and has also given novel insights. Through conducting high quality research LIFECYCLE has contributed to European excellence and competitiveness. Below, examples are given of some of the major advances and outputs achieved by the LIFECYCLE project:
Regulation of maternal mRNAs: It has been demonstrated in LIFECYCLE that maternally-inherited gene products play a crucial role in defining the eggs ability to support early development, once fertilized. Improper broodstock conditions and stress modify the maternal RNA content and this alters the cell division processes. Teleost eggs contain an abundance of maternally derived hormones that are assumed to be important for egg development. The importance of thyroid hormones (THs) for egg viability and larval quality was demonstrated during LIFECYCLE. It was revealed that TH cellular transporters are present in developing zebrafish embryos and their ablation modifies TH availability and caused increased mortality and the surviving larvae had a severely modified nervous system, heart, muscular and gastrointestinal tract.
Fish immunity: The very early stages of the immune response were examined in terms of antibacterial and antiviral responses. LIFECYCLE showed that the complexity of the immune response increases dramatically from the eyed egg stage to first feeding. Many of the elements of the acquired immune system were also found to be present. This suggests the cellular machinery for immunological memory is ready at stages earlier than previously thought. Relating to immunological memory, a strategy to gain a comprehensive description of the antibody diversity of rainbow trout was established. This was used it to analyse the response to VHSV, a serious viral pathogen of salmonids. The research showed that secondary infection induced a massive IgM response against the virus in the spleen. The mucosal isotype IgT also produced a significant response. This provides a frame for further characterization and monitoring of responses induced by vaccination in aquaculture.
Sex determination is governed by a ‘genetic sex determination’ (GSD) system in the rainbow trout while it is governed by a GSD with thermal effects (GSD+TE) system in the European sea bass. LIFECYCLE identified a major sex determinant linked to the Y chromosome (sdy) in the rainbow trout, whereas sea bass sex differentiation is consistent with a polygenic model not yet identified explicitly. From the point of view of basic sciences, the trout example shows that apparently unrelated genes can become recruited for the process of sex determination. From a translational point of view, it is important emphasize the strong interest of the salmonid aquaculture biotechnology industry in this finding, as it opens the possibility for having a means for secure and early differentiation between sexes.
Puberty control: A major breakthrough has been the neuro-anatomical and functional characterization of the Kiss/Gpr54 system in zebrafish and sea bass, indicating that this signalling system as very relevant for controlling puberty and reproduction of fish in aquaculture. Also, testicular growth factors mediating in a paracrine manner the stimulatory role of Fsh have been identified, and anti-Müllerian hormone has been identified as mediating inhibitory effects on spermato- and steroidogenesis.
Research towards resolving current and emerging production bottlenecks: LIFECYCLE focussed a comprehensive research effort at major current aquaculture production bottlenecks related to fish biology. The knowledge generated in LIFECYCLE about broodstock stress, maternal effects, development and growth, adaptation and homeostasis, the immune system, sex differentiation and puberty provide knowledge that now needs to be translated through applied projects to industry. The LIFECYCLE outcomes point to solutions for alleviating serious and common production problems linked to abnormal larval development, skeletal deformities, poor growth and energy utilization along with excessive fat deposition, mortalities related to life stage transitions, poor environmental performance, and unwanted sexual maturation. The knowledge should contribute to make the European aquaculture industry more efficient and competitive through less problematic production of better quality fish, both in terms of flesh and welfare.
The research programme of LIFECYCLE has led to increased understanding of mechanisms of essential biological functions which can be exploited for current commercial species but also for new species. By including in the research programme phylogenetically diverse species belonging to Gadiformes, Salmoniformes, Pleuronectiformes and Perciformes complimented by a strong comparative analytical approach at various levels (gene, protein, biochemistry and function) identification of “essential biological functions” which are conserved and “specializations” and “adaptations” that are species specific have been identified. In keeping with the “call text” and the LIFECYCLE research contract with the EU commission, a solid scientific knowledge-base was generated and delivered rather than applied empirical solutions. An added value from the project was the significant scientific outcomes of some research areas that have direct practical implications and these have been highlighted under “innovations”.
Research outcomes with a potential application in aquaculture have been generated in the majority of WPs. These will not be detailed as they are highlighted in the description of the main S&T results/foregrounds, but include:
• Improved knowledge about factors that impact on egg quality
• How hatchery conditions can influence developmental plasticity and generate biological variation in aquaculture production.
• Knowledge about genes and physiological mechanisms regulating key aqua¬culture traits (e.g. growth, flesh quality, early puberty) that is relevant to genetic selection.
• Knowledge about lipid metabolism, adiposity and energy balance.
• Disease resistance: The influence of developmental stage and sexual maturation on the capacity to mount specific immune responses.
• Adaptation and homeostasis: knowledge about physiological as well as molecular mechanisms of adaptation and symptoms associated with failed adaptation.
• Molecular mechanisms regulating gill and intestine epithelial cell differentiation that are important for survival and quality of juvenile transitions in aquaculture.
• Global molecular genomics approaches and classical endocrinology have identified genes and mechanisms underlying puberty and precocious puberty.
• Improved understanding of the regulation of reproduction and control of gamete production and quality.
• Improved knowledge about maintenance of homeostasis and normal physiological and behavioural functions that contribute to define fish welfare.
• Knowledge about cross-talk between systems/processes (e.g. homeostasis, muscle and skeletal development, metamorphosis, smoltification, immune function, energy balance, maturation and reproduction) at different lifecycle stages.
• Baseline data on immune function during critical life stages.
• A knowledge base for the development and validation of welfare indicators.
The European dimension: LIFECYCLE has truly been an European enterprise that has generated a network of twelve European laboratories in eight European countries with long expertise in fish biology and aquaculture research. This contributed to structuring the European research area in fish biology applied to aquaculture and elements of the project network will be maintained through national project funding or further EU funding. There has been a high level of interaction among partners, which has resulted in mobility and knowledge spreading and sharing within the EU. LIFECYCLE created a critical mass that has delivered cost-effective cutting edge research. An aspect that has increased cohesion of the European research area and added value to LIFECYCLE has been the unique national resources for fish research that were made available to partners and included large-scale “life cycle” research facilities at Matre (IMR, salmon) and IRTA (sea bream), Torre de la Sal (CSIC; sea bass), Jouy en Josas (INRA, facilities for infec¬tious diseases and immunology, trout) and Drennec (INRA; trout), state-of-the-art genomic/-transcriptomic and bioinformatics facilities in Rennes (INRA) and Berlin (MPG), as well as the Scottish Fish Immunology Research Center (UNIABD).
Added value and cost effectiveness: The impact of the knowledge-base delivered by LIFECYCLE represents not only the projects outcome, but also integrates that of concluded EU projects in which the partners participated, including GUTINTEGRITY, ARRDE, PUBERTIMING, AVINSI, PROBASS, STRESSGENES, SEAFOODplus, AQUAFIRST, WEALTH, FPPARS, FISHCAL and BRIDGEMAP, as well as nationally funded projects on fish biology, physiology and genomics. Thus, the success of the LIFE¬CYCLE project was in part secured by previous investments on EU and national scale, and ensures that the state-of-the-art as defined by these projects was advanced even further and has contributed to increase the competitiveness of European aquaculture.
Building competencies and skills through training: Although training was not an explicit objective of LIFECYCLE, a considerable number of training opportunities have been generated by the project. A large number of young scientists have undertaken masters and doctoral studies in the different research areas of the project. The impact of training has benefited by the opportunities for technology transfer that arose through the exchange of young scientists who frequently carried out their training in the laboratories of several LIFECYCLE partners. This has been extremely beneficial as it has exposed young scientists to a variety of different work environments allowed them to contact with well-established and well-recognised scientists and to build up their professional networks for their future. The emphasis given to promoting young scientists is evident in the project indicators; in addition to their key role in knowledge generation and production of scientific manuscripts, they have been active participants in the annual LIFECYCLE meetings and frequently were given the opportunity to present their work. Special measure were also taken to promote doctoral and young post-doctoral researchers through offering direct support for participation in conferences in which the LIFECYCLE project organised a specific session. In addition to Masters and doctoral students, a number of other junior scientists and undergraduates, as well as a large number of technical personnel have also been involved in LIFECYCLE and have benefited from the experience through specific “on–the-job training”. Overall, there has been a high level of interchange between the project participants and transfer of knowledge and skills between scientists and young scientists and among the participating groups. Through training, LIFECYCLE has contributed to the generation of a skilled workforce to meet future challenges and has also contributed to the transfer and spreading of knowledge within Europe. The network of young scientists trained in LIFECYCLE is an important resource for Europe and by training in a pan-European network of researchers will certainly contribute to future European cohesion.
Favouring gender equality: The LIFECYCLE project was built upon the concept of gender equality to ensure that the talent, perspectives and insight assured by increasing diversity in the science and technological workforce was harnessed for the project’s benefit.
An action plan was established at the planning stage ad implemented during the project. A gender-issues workgroup was established within LIFECYCLE and defined actions for implementation and also oversaw them. The budget available permitted a number of specific measures to be carried out. At the kick-off meeting of the project, an invited lecture was given by an expert on gender issues in order to stimulate awareness within the consortium of measures that should be taken to ensure gender equality. Several of the key leadership roles in LIFECYCLE were occupied by women and 3 of the 7 steering committee members were women. In addition both senior and junior scientists that were women were encouraged to take on co-ordinating responsibility for sub-workpackages (6 out of 15 subWPs were led by women). By supporting LIFECYCLE leadership roles of female researchers in grade B positions this strengthens their merits for promotion.
The visibility of female researches in grade A and B positions was encouraged by offering funding for attendance of conferences and presentation of data at the LIFECYCLE meetings. However, it was considered important to encourage all young researchers to attend meetings so funding was open to all junior scientists in LIFECYCLE, although women were given priority when a choice had to be made between applicants. One of the LIFECYCLE conference sessions (26th Conference of Comparative Endocrinology, Zurich 2012) was organised by two of the senior female scientists of LIFECYCLE and “showcased” the results of a number of young women from the LIFECYCLE consortium. Similarly, a special LIFECYCLE session at the Aquaculture Europe 2012 conference in Prague, made it possible for several young, female scientists from the LIFECYCLE consortium to give oral presentations at a major, international conference.
The collated workforce statistics show that LIFECYCLE has been particularly successful in gender equality actions, as that the same number of male and female senior scientists, as well as the same number of male and female PhD students, have carried out research in the project.
DISSEMINATION
Effective and correct dissemination is a vital key to the overall impact of any activity and LIFECYCLE started with a clear plan for the dissemination of its activities aimed at reaching all stakeholders and maximising its impact.
As a major European research project, dissemination occurred at several levels; within the project consortium to ensure execution of the project plan. The successful attainment of the project objectives is a measure of the effective communication and dissemination of information to all partners. Communication occurred with the European Commission to report project progress principally through project reports, which were detailed and completed within the planned timescale. Moreover, the scientific officer from the European Commission designated with the task of overseeing the good functioning of the LIFECYCLE project was invited to be present at all project meetings to maximise opportunities for informal exchange about the project outcomes.
Dissemination of LIFECYCLE results and outcomes to the scientific community and to the aquaculture sector occurred through several different mechanisms. To ensure effective communication and dissemination a specific WP5 was dedicated to the task and a “Dissemination and Intellectual Property Workgroup” (DIPW) was formed. The LIFECYCLE project straddles the spheres of “Basic Biologic Research” and “Academic Applied Aquaculture Research”. The outputs from LIFECYCLE have contributed significantly to the research literature and the aquaculture literature. Presently, seventy-seven papers having been published in peer-reviewed journals, both in top scientific journals such as Endocrinology and Proceedings of the US Academy of Sciences, as well as top aquaculture journals such as Aquaculture, Reviews in Aquaculture and Fish and Shellfish Immunology. The new knowledge generated by the LIFECYCLE project on the aquaculture-related biology of our most important aquaculture finfish species has thus to date been broadly disseminated through the academic publishing of about one thousand pages in top quality scientific journals. In order to enhance the accessibility of the findings to applied researchers and end users, the LIFECYCLE consortium has published when possible in OPEN ACCESS journals. Adding to the seventy-seven scientific papers already published, over twenty more manuscripts are currently in various stages of being published. Given the large amount of still unpublished data summarized in the fourth periodic report which has just been submitted, it is clear that the LIFECYCLE project will continue to disseminate novel data to the scientific and aquaculture communities for several more years. The same can be predicted for continued conference dissemination of LIFECYCLE data. This dissemination route has been a key activity of all of the participants of the LIFECYCLE project, and at present, LIFECYCLE data have been disseminated through over 150 oral and poster presentations at a scientific and aquaculture conferences and workshops. However, perhaps the most important dissemination route, although also the hardest to quantify, is the direct contact and personal dialogue by a large number of LIFECYCLE scientists with local fish farmers, a dialogue enriched by the new insights brought forth by the LIFECYCLE project.
In addition to publishing genomic/transcriptomic data in the scientific literature, LIFECYCLE scientists have adopted an “open” approach by the release of data to appropriate public domains (NCBI etc.) as early as possible to maximise benefits for research in this area. This approach will has given added value as it favours the rapid spreading and dissemination of basic molecular information which may be of use in other research projects where work is carried out on basic as well as aquaculture-related fish biology.
While the project website has been a major web-based dissemination instrument for the project, the emergence of the professional “social network” LinkedIn was used increasingly during the project life-time as it allows direct communication between members. Currently, there are over 1100 members of the LIFECYCLE project LinkedIn group, representing the full spectrum of stakeholders in fisheries and aquaculture from all over the world.
EXPLOITATION
A broad spectrum of important exploitable intellectual assets have been created and generated in LIFECYCLE, and about forty discrete items of EXPLOITABLE FOREGROUND is given in Table A2.
From a fundamental, scientific point of view for the researchers of the LIFECYCLE consortium, the knowledge generated presents one of the most valuable exploitable assets. Although all the major scientific advances of LIFECYCLE are being published, at present count 77 LIFECYCLE publications in peer reviewed scientific journals, and that these advances are thus not patentable, the data generated during the research enhance the knowledge base of the researchers involved and thereby their own qualifications and competence. This is exploited every time a researcher, a research group or a larger research consortium applies for further research funding from private or public sources. The LIFECYCLE data will undoubtedly generate a substantial number of spin-off research projects, both basic and applied, and already, it’s clear that a substantial number of on-going national research projects have been established by exploiting LIFECYCLE generated data.
In a similar fashion, a large number of methodological advances have been made in LIFECYCLE, in everything from experimental protocols, to research equipment to highly specific measuring assays for localization and/or quantification of both transcribed and translated gene products (mRNAs and proteins). Such experimental and analytical protocols are highly exploitable within the research community in a similar fashion as explained for the scientific knowledge base above.
In addition, many aspects of such methodological development can potentially be exploited commercially to various degrees. To exemplify, analysis of specific proteins such as many hormones is carried out using immunological methods. A part of establishing these methods, specific antibodies need to be generated. Such antibodies have an obvious commercial potential as indicated by the large number of biotech companies selling antibodies to the research community. However, another way of exploiting methodological advances can be for scientists to take on contractual research where they are paid for analysing samples with their unique analytical methods.
Exploitable foreground is more often than not the result of long-term research efforts, rather than being a result of a single project. LIFECYCLE has in varying degree contributed to the exploitation of such long-term efforts by LIFECYCLE researchers.
One of the academic laboratories in LIFECYCLE launched an aquaculture genetics service company in February 2013. Xelect Ltd (www.xelect.co.uk) is a company registered in Scotland No.SC438223. Xelect represents the commercialisation of over 30 years of basic research on the physiology and genetics of fish muscle growth and flesh quality and was made possible by continuous support from the UK Research Councils and the European Commission, the later through SEAFOODplus (FP6) and LIFECYCLE (FP7). The core business of Xelect is the developing of genetic markers for brood stock selection. Xelect has licensed genetic markers for superior meat yield in Atlantic salmon to SalmoBreed A/s and Landcatch Natural selection and several other license opportunities for this and other traits are currently under negotiation. In addition, Xelect offers a range of genetic services to the aquaculture industry including sex determination (using an innovation from LIFECYCLE), genotyping, ploidy testing, and disease identification. The company is trading profitably and has a growing list of global customers.
The research group at the University of Bergen, Norway (LIFECYCLE partner 7) have recently made significant advances in establishing an analytical method termed “Smoltprobe”. The Smoltprobe has been established as a predictive tool in the assessment of smolt quality and the transfer of smolts from fresh water to seawater. The work has involved two of the major international salmon producing companies, Lerøy Seafood A/S and Marine Harvest A/S, and the Smoltprobe has been tested and evaluated in two commercial smolt producing operations. The Smoltprobe holds promise both as a tool for predicting the correct timing of seawater transfers, and a tool to pick up problems arising from poor freshwater quality and desmoltification (loss of smolt status). Evaluations are on-going, and a national proposal has been submitted in September 2013 in order to bring the Smoltprobe to a fully commercially available test tool for smolt quality.
List of Websites:
http://lifecycle-fp7.eu/