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Exploring the biological and socio-economic potential of new/emerging candidate fish species for the expansion of the European aquaculture industry

Final Report Summary - DIVERSIFY (Exploring the biological and socio-economic potential of new/emerging candidate fish species for the expansion of the European aquaculture industry)

Executive Summary:
The European Union (EU) is the largest importer of fisheries and aquaculture products in the world. However, aquaculture provides only 20% of the seafood produced in the EU, while the worldwide contribution of aquaculture towards seafood production is already >50%. An efficient, sustainable and market-oriented expansion of the EU aquaculture sector based on new fish species and products will reduce the dependence of the EU on imports, reduce the pressure on over-exploited fisheries and explore new segments and tailor-made products for the EU market. This was the objective of DIVERSIFY, a consortium of a multidisciplinary group of 40 partners from 12 European countries, made up of research and academic institutions, as well as nine small or medium-sized enterprises (SMEs), three large enterprises, five professional associations and one consumer non-governmental organization (NGO). DIVERSIFY had a total budget of 11,8 million € and lasted for 5 years (2013 – 2018).

DIVERSIFY has identified six new/emerging finfish species, with a great potential for the expansion of EU aquaculture. These species are fast growing and/or large finfishes, marketed at a large size and can be processed into a range of products to provide the consumer with both a greater diversity of fish species and new value-added products. The selected fishes were meagre (Argyrosomus regius) and greater amberjack (Seriola dumerili) for warm-water marine cage culture, wreckfish (Polyprion americanus) for cool-water marine cage culture, Atlantic halibut (Hippoglossus hippoglossus) for marine cold-water culture, grey mullet (Mugil cephalus) a euryhaline omnivore for pond/extensive culture, and pikeperch (Sander lucioperca) for freshwater intensive culture using recirculating aquaculture systems (RAS). These species were selected based both on their biological and economical potential, and to cover the entire European geographic area and stimulate different aquaculture types. Research was carried out in the scientific disciplines of Reproduction and Genetics, Nutrition, Larval and Grow out husbandry, Fish health, Final product quality and Socioeconomics.

After 5 years of research, DIVERSIFY has completed successfully almost 150 scientific deliverables. The project produced broodstock management methods for the control of reproduction in captivity and production of high-quality eggs. The nutritional requirements of different life stages (larvae, juveniles and/or breeders) of the studied fishes were determined resulting in the production of species-specific diets. Larval husbandry methods were developed based on the ontogeny of development of the major organs responsible for feeding and digestion, by modifying fish larval rearing protocols that exist for other species, thus allowing the production of large numbers of juveniles adequate for commercial production. Grow out husbandry methods were developed for RAS, sea cages or earthen ponds, depending on the species studied, providing information on the required environmental parameters (density, depth, light, temperature, etc.) and feeding schemes. Significant knowledge of the major diseases and health-related issues of some of the studied species was also acquired. Technical Production Manuals for potential farmers have been made freely available for all six species, while Fish Health Manuals have been produced for greater amberjack and meagre.

Market research identified market potential in cross-cultural consumer segments, with increased-to-strong interest in new products in the main EU fish markets. Greater amberjack shows the most promising market opportunities, given its large size, processing potential and superior sensory characteristics. Grey mullet is a very interesting species due to the higher sustainability of its production methods, and the marketing of its roe (bottarga). Wreckfish has very firm flesh that discriminates it readily from other fish. The remaining species (Atlantic halibut, pikeperch and meagre) have certain advantages due to their biological and physical characteristics and are of interest to specific regions in Europe.

The acquired knowledge and developed methods of DIVERSIFY will enhance the production of the selected emerging species by the European aquaculture and will enable the incorporation of some new species, such as the grey mullet and greater amberjack.

Project Context and Objectives:
The European Union (EU) is the largest importer of fisheries and aquaculture products in the world. Of the seafood produced in the EU, aquaculture provides only 20% and capture fisheries provide the rest (Eurostat 2018), while the worldwide contribution of aquaculture towards seafood consumption is already >50%. This situation can be attributed partially to a lack of diversity of aquaculture products in Europe, since European demand increases for a diverse range of fish products, especially for fish fillets and other processed products. Nevertheless, aquaculture is undertaken in all EU states, and plays an important role in the supply of high-quality seafood to the European consumer. The EU aquaculture is a modern industry providing direct employment for 85,000 people, producing 1.3 million tons worth €4 billion ( Many world-class researchers and facilities exist in research centers and universities throughout Europe, while the private sector employs highly skilled and educated personnel, with modern production facilities. Therefore, the sector is well positioned to become the world leader in the efficient and sustainable production of safe seafood of the highest quality and nutritional value, considering consumer preferences and lifestyles, and the immense diversity of aquatic products from the wild, to which the consumer is accustomed.

Even though some 35 aquatic species are cultured in Europe, fin-fish aquaculture production is dominated both in volume and value by a handful of species --such as Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio), European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata)-- that in turn limit the number of aquaculture processed products available in the market. In fact, the 10 most common species account for up to 90% of the production and 87% of its value (Eurostat 2018). An efficient, sustainable and market-oriented expansion of the EU aquaculture sector based on new species and products will reduce the dependence of the EU consumer on imports from countries of questionable production, health, environmental and social standards, and it will reduce the pressure on over-exploited fisheries in the EU.

The objective of DIVERSIFY was to support the EU aquaculture industry in diversifying its production with new/emerging species with important advantages over the ones cultured currently, such as fast growth, large size or low requirement in fish-meal and oil. In addition, the project identified the drivers for market acceptance of the new food prototypes in order to position the EU aquaculture sector in relation to imports from outside the EU. Although the emphasis of DIVERSIFY was on Mediterranean cage-culture, fish species suitable for cold-water, pond/extensive and fresh water aquaculture have been included as well. The fish species studied were meagre (Argyrosomus regius) and greater amberjack (Seriola dumerili) for warm-water marine cage culture, wreckfish (Polyprion americanus) for warm- and cool-water marine cage culture, Atlantic halibut (Hippoglossus hippoglossus) for marine cold-water culture, grey mullet (Mugil cephalus) a euryhaline herbivore for pond/extensive culture and pikeperch (Sander lucioperca) for freshwater intensive culture using Recirculating Aquaculture Systems (RAS).

A strong socioeconomic component was included in DIVERSIFY, in order to address important bottlenecks in aquaculture development, beyond biological/production issues. The socioeconomic part of the project had a science based applied market development approach, with a lot of components. These included the perception of aquaculture products in general and processed products specifically, market potential and demand factors, consumer and professional buyer preferences, new product development, creating added value in relation to raw products and market development. An important limitation in aquaculture consumption is that in many countries and/or segments of the EU market, aquaculture fish have a weaker image than wild fish. Parallel to technological improvement of production methods for the new species, expansion opportunities for the EU aquaculture sector have been identified.

The combination of biological, technological and socioeconomic research activities developed in DIVERSIFY are expected to support the diversification of the EU aquaculture industry and help in expanding production, increasing aquaculture products and development of new markets.

Project Results:
The fish species that were included in DIVERSIFY were selected based both on their biological and economical potential, and to cover the entire European geographic area and stimulate different aquaculture types. In collaboration with 12 SMEs and large enterprises, DIVERSIFY has built on recent/current national initiatives for species diversification in aquaculture, in order to overcome a series of documented bottlenecks in the production of these species and to identify possibilities to improve the competitiveness of the EU aquaculture sector. Therefore, research was carried out in different scientific disciplines, including Reproduction and Genetics, Nutrition, Larval and Grow out husbandry, Fish health, Socioeconomics and Final product quality. The project was comprised of a total of 25 Work packages (WP) each focusing on a species-scientific discipline combination, as well as four WPs on Socioeconomic issues. The presentation of Science and Technology results/foregrounds that follows is thus organized accordingly.

1.1 Reproduction
1.1.1 Meagre
The industrial bottleneck to implement genetic breeding programs for meagre was addressed by genetically sampling over 435 breeders from broodstocks in 13 breeding centres and 7 countries using 18 microsatellite markers. The broodstocks originated from a limited number of families from three wild populations or groups. Although broodstocks appeared to have sufficient variation for breeding program(s), the majority required an increase in the number of families. Both genetic tools and protocols to control reproduction for breeding programs were developed and provided. The muscle and liver transcriptome were determined and the first genetic linkage map for meagre was constructed using the ddRAD methodology, which identified 731 markers organized in 27 linkage groups. The model mapping identified five quantitative trait locus (QTLs) on two linkage groups, which exhibited significant evidence of linkage at the genome level and multiple QTLs were related to differences in body weight and length. Protocols for the induction of tank spawning in paired crossing had a 76% efficacy of spawning pairs with male rotation and produced a total of 61 families (full and half-sib) that had >200,000 eggs with >80% fertilization success. However, a decline in spawning success that was observed with repeated induced spawning with male rotation was a possible drawback that was highlighted. Protocols were also developed for in vitro fertilization for planned crosses. Meagre sperm had a mean sperm density of 3.21·10x10 ± 1.18 spzoa/mL, motility duration was 1:43± 0:18 min, mean percentage of initial motility of spermatozoa was 48.17 ± 2.80 and the mean initial spermatozoa velocity (VAP) was 90.69 ± 5.76 µm/ s. Different sperm storage methods and cryopreservation techniques were modified to provide protocols for meagre sperm. The optimal period for stripping eggs was 38-39 hours after the application of gonadotropin releasing hormone agonist (GnRHa) and a ratio of 150,000 motile spermatozoa to egg was recommended. The results of DIVERSIFY provided the technology required to implement industrial breeding programs and scientific advances in the reproductive control of marine fish in general, as well as sperm characterisation and genetic resources for meagre and related species.

1.1.2 Greater amberjack
In order to facilitate the broodstock management of greater amberjack in aquaculture, important life history traits of wild fish were first determined. Fish are 35-40 cm in length (fork length, FL) and 1 kg in weight (body weight, BW) at age 1; 60-70 cm FL and 3-5 kg BW at age 2; 80-90 cm FL and 7-10 kg BW at age 3. Male greater amberjack are reproductively active at the age of 3 years and females reach the first sexual maturity at 3-4 years of age. The spawning season of the wild greater amberjack population from the western Mediterranean is extended from late May to early July. When greater amberjack reared in sea cages in the Mediterranean were handled as other captive species, they exhibited poor gonadal development, low pituitary gonadotropin gene expression, low gonadotropin and sex steroid plasma concentrations, atresia of vitellogenic follicles, reduced proliferation and increased apoptosis of male germ cell. As a consequence of the spermatogenesis impairment, greater amberjack confined in captivity showed low sperm quality, in terms of sperm density and motility and velocity as well as ATP content and membrane integrity. The observed reproductive impairments are likely related to the handling stress, the non-optimal conditions required for reproductive maturation and/or to nutritional unbalances caused by the lack of specific broodstock diet for the species. In fact, gonads of captive-reared greater amberjack had different lipid and fatty acid contents compared to wild individuals. An overall improvement of rearing technology, particularly as it relates to husbandry operations (e.g. fish handling and transferring) together with a better formulation of dietary ingredients is suggested to overcome the observed dysfunctions and improve greater amberjack reproductive performance.

Reproductive dysfunctions occurring in greater amberjack reared in sea cages in the Mediterranean were successfully treated with GnRHa administration through sustained-release polymer implants and injections. Treatments with GnRHa implants at the dose of ≈50 μg kg-1 body weight were more effective that injections in promoting the proper endocrine pathways leading to multiple cycles of oocyte maturation, ovulation and spawning and allowed producing more eggs without altering their quality in terms of fertilization, embryo survival, hatching and larval survival. This method helped minimizing the handling stress (i.e. one handling every two weeks as opposed to one handling every week) and was very effective in females maintained in cages during gametogenesis and moved to tanks after the administration of the hormonal therapy.

Greater amberjack caught from the wild in the eastern Atlantic (southwester coast of Gran Canaria, Spain) when they were 3-5 kg in body weight and reared for two years in indoor tanks under appropriate environmental and nutritional conditions, were able to undergo normal gametogenesis, and spontaneously spawned large quantities of high quality eggs. In the same stock, hatchery-produced F1 greater amberjack (15-30 kg body weight) reared in outdoor tanks in Tenerife (Spain) underwent normal gametogenesis and were successfully induced to undergo maturation, ovulation and spawning through the administration of polymer implants containing GnRHa at the doses of 50 and 75 μg kg-1 body weight. The repeated administration of GnRHa implants resulted in multiple spawns of high quality fertilized and viable eggs for an extended period lasting from May to September. Consistent egg production is now available for this species, and has enabled the further development of larval rearing methods within the project.

Therefore, thanks to the experimental work carried out within DIVERSIFY, a set of tools to reproduce greater amberjack reared under different conditions in the Mediterranean Sea and in the eastern Atlantic is now available and this represents a fundamental step towards the large-scale aquaculture production of this species.

1.1.3 Pikeperch
The primary objective for this species was to use genetic markers (microsatellite loci) in order to evaluate the genetic indices of captive broodstock in commercial recirculation aquaculture system (RAS) farms around Europe and compare them to those of some wild pikeperch populations. Thirteen cultured and eight wild populations with more than 950 fish in total were analyzed for a final set of 10 microsatellite genetic markers. Results showed that on average, and in contrary to what we theoretically expected, the thirteen domesticated populations exhibited a slightly higher number of alleles compared to the wild ones, whereas unbiased expected heterozygosity estimates were slightly higher in wild populations. The above, in conjunction with the FIS values, indicate that in general domesticated samples do not suffer from inbreeding, with the exception of few broodstocks with either small sample size or known past of stocks under selection.

Interestingly, in DIVERSIFY we provided evidence and confirmed results generated in previous studies indicating that pikeperch populations in Europe are part of at least two genetically differentiated groups: the first one is found in northern Europe from the Netherlands/Denmark to the West and Poland (at least) to the East, to the North of Finland and a second group which comprises all remaining populations in Central Europe to as south as Tunisia (and probably Spain, Italy and Northern Greece). These nuclear (microsatellite) data results seem to be confirmed by further mitochondrial data produced beyond the project’s deliverables. In this second group, the Hungarian populations are having a key-position being different from those found geographically near, e.g. from Czech Republic and Germany. Taking the above two groups into account, the aquaculture broodstocks analyzed seem to contain in general fish of a single origin with only few exceptions.

1.1.4 Atlantic halibut
Wild-caught females were predictable spawners that produced eggs consistently of very high quality (>85% fertilization). Farmed females also produced eggs of high quality when their ovulatory cycles were identified and stripping was carried out close to ovulation. For commercial production, as well as breeding purposes, it is not practical to rely on wild-caught females. However, relatively few farmed females produced eggs consistently with fertilization rates >80-85%. As a consequence, it may be necessary to include wild-caught broodstock also in future breeding groups in order to ensure a broad enough genetic material.

Plasma concentrations of sex steroids in farmed breeders were similar to what has been reported previously in Atlantic halibut, with annual profiles following ovarian growth and maturation. Highest 17β-estradiol (E2) levels were recorded just prior to spawning, in the beginning of February, while both E2 and testosterone (T) remained elevated through the spawning period. No differences in average concentrations were seen between wild-caught and farmed females. Plasma concentrations of the gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) were documented for the first time in Atlantic halibut. Mean Fsh concentrations were relatively stable during vitellogenesis, from October to early February, consistent with a constitutive release of FSH from the pituitary. Plasma FSH decreased to low levels during spawning, but increased again after spawning was completed. Plasma LH concentrations showed large individual variations through the reproductive cycle, but high levels were detected during spawning. This is consistent with previously reported results in other teleosts, including a number of flatfishes.

Implantation with GnRHa did not advance spawning time significantly in Atlantic halibut females, but an apparent synchronization in spawning time between individuals was seen, as treated females had completed spawning 1 month before control fish were spent. In commercial production, synchronization between individuals can be an advantage as staff efforts can be concentrated to a relatively short period. Atlantic halibut breeders need to be monitored for ovulation and stripped on a regular basis, and eggs are fertilized in vitro. Therefore, the use of GnRHa implantation offers a logistic advantage to the commercial broodstock management of the species, by reducing the spawning season.

1.1.5 Wreckfish
The main objective in terms of wreckfish reproduction was to describe the reproductive cycle of this species in captivity and obtain fertilized eggs. DIVERSIFY efforts were focused at first to thoroughly understand the physiology and reproductive cycle of this species. Once this challenge was achieved, various methods to control reproduction in aquaculture situations were evaluated, such as in vitro fertilization by stripping, induction of maturation by hormones and tank spawning, and spontaneous spawning in the broodstock tanks without any manipulation. The work completed on in vitro fertilization demonstrated that this method is complicated to apply to large broodstock and that the manipulation has negative effects and caused some mortality. In addition, to successfully apply in vitro techniques it is necessary that the females reach advanced stages of maturity. In the initial stages of the project very few females have reached advanced stages of maturation and this has complicated the work with in vitro fertilization. Later in the project more females reached advanced stages of maturation and this coincided with these females spawning naturally and spontaneously. The overall aim of the project for the reproduction of wreckfish was to achieve large quantities of fertilized eggs and this was achieved with spontaneous spawning.

In terms of gamete quality, the results indicated a high fecundity of wreckfish females with multiple spawns during the season, while males were shown to remain in spermiating condition throughout the year. A computer assisted sperm analysis (CASA) method was developed specifically for wreckfish sperm and can be used to optimize sperm management and fertility potential in future spawning induction experiments. The analyses also demonstrated that sperm of captive wreckfish shares a common pattern of motility with both marine and freshwater fish. Finally, the incubation methodology was adapted to the special characteristics of these, achieving high percentages of hatching (80%).

The result of this intense work with four broodstocks from different facilities has been very rewarding, since, in addition to taking a step forward in the knowledge of biology, behavior and reproductive cycle, it has been possible to obtain fertilized eggs with a high quality regards fertilization and hatching.

1.1.6 Grey mullet
Lacking the natural spawning environment, captive grey mullet fail to reproduce spontaneously, largely due to a failure to undergo complete gametogenesis. In this respect, and within the framework of DIVERSIFY, considerable progress was made by optimizing hormonal treatments for alleviating reproductive dysfunctions among captive grey mullet broodstocks. Bio-potent yeast (Pichia pastoris) produced recombinant gonadotropins (r-FSH and r-LH) that were used as therapeutic agents in a series of in vitro and in vivo assays. The best performing treatment consisted of r-FSH and a dopamine antagonist (metoclopramide) that were co-injected during the onset of the reproductive season. The latter treatment demonstrated synchronized gonadal development within and between sexes, giving rise to stimulated spermatogenesis in males and follicle growth and maturation in females. Further spawning induction trials that timed the administration of GnRHa and metoclopramide with advanced stages of gamete maturation were relatively successful. A basic breeding unit, comprising a single female and three males, was found to facilitate synchronization and in turn increase fertilization rate. Nevertheless, our results highlight an episodic fertilization rate ranging between 0 to 98% and point to a future need to fine tune and optimize the hormone-based breeding protocol for captive grey mullet. Broodstock diet containing fish oil (FO), which is relatively rich in n-3 long chain polyunsaturated fatty acids (LC-PUFA), positively affected hatching success and larvae survival. The established breeding protocol for captive grey mullet could be effectively applied during natural as well as artificially shifted spawning seasons. Over several consecutive spawning seasons, tens of millions of high-quality eggs were produced giving rise to mass production of robust fingerlings. A shipping protocol for grey mullet eggs was also established specifying the optimized conditions including egg developmental stage (gastrula) and packing density for short term (≤ 11 h) and long term (26 h) shipments.

The assessment of the effects of captivity on first sexual maturity of wild-caught and hatchery-produced grey mullet indicated that: (1) the rearing conditions established allow for a growth rate equivalent to that of wild grey mullet from the Mediterranean Sea; (2) the reduction of the rearing density from 90 to 45 fish per m3 has no effect on grey mullet growth and sexual maturity; (3) hatchery-produced grey mullet have a good potential to develop ovaries spontaneously up to a condition useful for bottarga production. Furthermore, the effects of fish origin (wild vs. domesticated) and culture conditions on advanced and spontaneous development of gonads comprising the required criteria for the production of high quality bottarga (i.e. minimal size of 100 g, bright yellowish color and chewy texture) were assessed and indicated that (1) traditional grey mullet farming procedure in freshwater ponds could be applicable, and also an advantage, for roe production; (2) Domestication appears to have a favorable effect on the spontaneous development of mullet ovaries up to a condition useful for bottarga production and (3) Pigment-enriched diets can enhance the roe coloration to meet the criteria for high quality bottarga (roe). However, two stumbling blocks that may impair the profitability of grey mullet farming for bottarga production are (1) extended grow out to a minimum of 3 years and (2) relatively low percentages (20-50%) of females developing ovaries at the appropriate size (≥ 100 g). Future studies, therefore, should focus on genetic improvement programs giving rise to advanced sexual maturity and spontaneous ovarian development in captive grey mullet females.

1.2 Nutrition
1.2.1 Meagre
Despite the interest of meagre for aquaculture diversification in the last decade, there is a lack of information on nutrition during larval development. The importance of highly unsaturated fatty acids (HUFA) and the antioxidants vitamin E and vitamin C has not been investigated in this species, despite the fact that the oxidative risk is particularly high in fast growing larvae. Dietary HUFA levels of 3% improved larval growth and lipid absorption and deposition. Besides, among fish fed 3% HUFA, increases in vitamin E and vitamin C improved significantly body weight, as well as lipid, 22:6n-3 and n-3 fatty acids contents in the larvae. Thus, weaning diets for meagre must be optimized increasing HUFA levels up to 3% and vitamins E and C >1500 and 1800 mg kg-1, respectively, in order to spare these essential fatty acids from oxidation. A 0.4% dietary HUFA is not enough to cover the essential fatty acid requirements of larval meagre. It is also important to supplement meagre weaning diets with 2.4 mg/kg vitamin K, since the absence of this vitamin reduced markedly larval survival. Meagre seemed to be very sensitive to hypervitaminosis D and only mildly sensitive to hypervitaminosis A, since supplementation with these vitamins lead to a growth reduction. Taurine supplementation did not have any effect in meagre larvae performance.

The nutritional requirements and optimum levels of n-3 Long Chain (LC)-PUFA for meagre fingerlings were examined, evaluating its effects on survival, growth performance, feed utilization and fish composition. Meagre showed the ability to selectively conserve key fatty acids (FA), particularly Docosahexaenoic acid (DHA; 22:6n-3) and Arachidonic acid (ARA; 20:4n-6) over other FA, in response to essential FA-deficiency. Furthermore, meagre seems to have active Δ6 desaturases and Elovl5, but their activities were insufficient to produce DHA and Eicosapentaenoic acid (EPA; 20:5n-3) from PUFA precursors to sustain fast growth. The EFA deficient meagre in the present study also showed a higher incidence of granulomas than fish fed ≥2% n-3 HUFA. Based on our results DIVERSIFY showed that the requirement for n-3 LC-PUFA for meagre fingerlings is at least 2.0% Dry Matter (DM) in diets containing 16.5% DM lipids, a ratio of 0.9 EPA/DHA and 0.4% ARA of total FA content.

1.2.2. Greater amberjack
To improve larval enrichment products for greater amberjack, the optimum levels and ratios of essential fatty acids and combined PUFA and carotenoids in greater amberjack enrichment products were determined. The highest greater amberjack growth was obtained when larvae (17-35 days after hatching, dah) were fed Artemia containing DHA in a range of 5-8% Total Fatty Acid (TFA), with a maximum around 7% (1.5 g 100 g-1 DHA DM). The essential FA (EFA) requirements are similar during rotifer and Artemia feeding, as reported for larvae of other marine fish species. Requirements of amberjack larvae for DHA (1.5 g.100 g-1 DHA DM) were higher than those found in other marine fish species and similar to those for other fast-growing species. Increases in DHA tend to improve larval resistance to handling. Even the highest DHA levels in the enrichment emulsion (70% DHA in TFA) resulted in reduced incorporation of DHA into Artemia lipids (11% DHA in TFA). Despite that EPA levels in Artemia increased from 0.87 to 6.81 % TFA, EPA levels in greater amberjack larvae were only increased up to 5.2% TFA, denoting a saturation process that could be associated with the fulfillment of the EPA requirements. On the contrary, DHA levels in greater amberjack larvae showed a linear increase. Dietary DHA was linearly related to skull anomalies, dietary DHA levels over 2 g per 100 g-1 inducing a higher incidence of skeletal malformations, particularly those related with skull development.

It is well known that raising the ratio of Phospholipids (PL) to Total Lipids (TL) in larval feeds may enhance growth. Rotifers enriched with the marine lecithin (E1), displayed a fast incorporation of polar lipids particularly rich in DHA. Although the role of carotenoids in the embryonic development is not very well established, there is evidence that the presence of carotenoids mitigates deleterious oxidative damage to the developing embryo. Larvae fed diets with astaxanthin below 5.3 ppm were found to have marginal growth, whereas those fed levels above 5.3 ppm had a better performance and significantly higher lipid levels. Rotifers enriched with polar rich emulsion containing a marine natural lecithin LC60 combined with 10 ppm of Naturose also resulted in a significant advantage in larval growth, survival and welfare compared to rotifers enriched with other emulsions. Thus, DIVERSIFY established the following recommendations for enrichment products for greater amberjack larvae culture: DHA in enrichment products for Artemia 10-17% TFA, EPA 14-20% TFA, and DHA/EPA ratio 1-5. For rotifers, DHA in enrichment products 14% TFA, EPA 6% TFA, and DHA/EPA ratio 2.3. Carotenoids levels in enrichment products must be around 10 ppm.

Regarding greater amberjack juveniles, the dietary lysine requirements, based on the Broken-line model, which can support maximum weight gain of greater amberjack juveniles fed on a diet based mainly on plant ingredients, containing 45% protein, 18% lipid and 25% fish meal inclusion, was 2.11% of diet.

In broodstock diets, the requirements of essential fatty acids were determined to obtain improved spawning quality. Broodstock fed a diet containing 1.57% EPA+DHA showed high fertilization and egg viability, higher number of eggs per spawn and kg of female, with the highest percent of fertilization, egg viability, hatching rate and larval survival. Egg fatty acid composition was shown to be influenced by broodstock diets. A diet containing 14-15 % EPA+DHA of total fatty acids (corresponding to 2.5-3 % in a dry diet) resulted in the best spawning performance in greater amberjack broodstock. Increasing dietary EPA+DHA contents did not improve spawning performance. Histidine contents in broodstock diets from 1 to 1.5% and Taurine in broodstock diets increased the reproductive performance of greater amberjack.

1.2.3 Pikeperch
The experiments conducted within DIVERSIFY have gained new insight into the nutritional requirements of pikeperch larvae until early juvenility. The studies performed focused primarily on the importance of PL and FA composition and dietary requirement of Ca/P, as well as their interactions. Lack of knowledge on these nutritional parameters has been considered as a bottleneck in commercial farming with large implications for survival and growth. In addition, rearing larvae at low salinities was examined, as it was hypothesized to have a possible positive influence on larval FA requirements, metabolism and physiology. Results confirmed the importance of high dietary PL levels for optimal performance of pikeperch, as well as reduced skeletal deformities by supplementation with DHA+ EPA in the form of concentrated Triacylglycerol (TAG) in otherwise identical formulated diets. It was observed that pikeperch larvae have a limited ability to elongate and metabolize shorter chain PUFAs to LC-PUFAs (essential FAs), while saline rearing conditions had no obvious positive effect on larval performance or development, but may cause improved FA metabolism and uptake and change eicosanoid (hormonal) activity). Digestive enzymatic activity seemed to be related to ontogenetic development more than nutritional composition, while live proteomic expression of some important proteins are clearly related to the availability and presence of LC – PUFAs especially DHA. The Ca/P, FA and their interaction seem to be key nutritional factors influencing pikeperch larval development. In addition, it appears that total P % content should be considered in determining the optimal Ca/P level. Lack of sufficient dietary LC- PUFAs from early larval ontogenetic development have severe effect on long-term stress sensitivity and may impair learning abilities and behaviour. Therefore, it must be included in early diets to ensure both short- and long-term fish robustness.

Based on the above, a final test at commercial farm conditions showed that it was possible to formulate a diet that was superior to the commercial diet “Otohime” in terms of larval growth until 52 days post hatching (dph). Thus, diets with high levels of PL and sufficient LC-PUFAs are recommended to obtain good performance and lower degree of malformations, and nutrients can be supplemented as vegetable lecithin and in the form of TAG.

1.2.4 Atlantic halibut
For the development of a protocol for early weaning of Atlantic halibut larvae, we found a large difference regarding the larvae’s feed intake on 3 different commercial diets at 28 days post first feeding (dpff). Larvae fed “Otohime» had full guts after 5 days of feeding. This diet was used in an experiment aimed to find the earliest time of weaning at 15, 22 and 28 dpff. Weaning at 15 dpff gave almost 100% mortality, at 22 dpff approximately 30% mortality and at 28 dpff, almost 0% mortality. The conclusion is that diet characteristics are important to ensure feed intake in Atlantic halibut larvae and that the larvae are ready to feed on a formulated feed only at 28 dpff. Further experiments are needed to evaluate if the early larvae grow and develop on these diets. Also, a protocol for production of on-grown Artemia was developed and the nutrient composition was analyzed. Artemia grown for 3 days on “Oriculture” and enriched with “Multigain” obtained an improved nutrient profile in many aspects. The protein, free amino acid and taurine contents increased, lipid and glycogen decreased, while the ratio of PL to TL increased. The FA composition improved at one experiment, but not at the one carried out at the commercial partner. The micronutrient profiles were not negatively affected by culture of Artemia on “Oriculture”.

Since previous research had found that larvae fed on-grown Artemia developed into juveniles with better quality, larvae were fed these Artemia compared to conventional Artemia nauplii in DIVERSIFY. There were no differences in growth; pigmentation and eye migration between the two groups and the nutrient composition of the larvae after 3 weeks of feeding was very similar. The conclusion was that Artemia nauplii produced with modern methods have sufficient nutrient levels to cover the requirements of Atlantic halibut larvae. Also, the hypothesis that larvae reared in RAS would have another micro flora in the gut and therefor have different uptake of nutrients was examined. However, except for higher levels of the vitamin K derivative MK6, we found no differences in nutrient utilization between larvae reared in RAS or flow through systems. Finally, Atlantic halibut juveniles (1 g bodyweight) were fed diets with 5 PL levels varying from 9 to 32% of TL. There were no effects of PL levels on growth or lipid composition in intestine, liver and muscle, 24 hours after feeding. However, time after the meal affected the lipid composition of the intestinal tissue, with higher levels of neutral lipids 1 and 4 hours post-prandial, and higher levels of polar lipids, cholesterol esters and ceramide at 24 hours post-prandial, reflecting absorption of the lipids early after the meal. It appears that Atlantic halibut juveniles regulate their lipid species composition to be independent of the diet when a range of PL/TAG is applied, as in the present study.

1.2.5 Wreckfish
Nutrition studies performed in DIVERSIFY for wreckfish were focused mainly on the development of adequate live prey enrichments for larvae and the production of broodstock feeds for enhancing fecundity and gamete quality. These are the first steps for the development of proper nutrition and culture of this species. Enrichment products for live prey (rotifers and Artemia) were produced with two levels of ARA, being the nutrient less effective in Artemia compared to rotifers. No differences were found in FA composition of wreckfish larvae fed with the prey enriched with the two enrichment products. Enriched live prey exhibited a good FA profile and larvae of wreckfish exhibited, in general, a good acceptance of them. The FA profile of wreckfish larvae along larval development was described, showing large amounts of PUFA specially DHA, EPA and ARA.

Results obtained on the tissue composition of wild wreckfish showed that muscle and gonad have a large amount of proteins and low levels of lipids. These results and those obtained from eggs and larvae were very useful for the formulation of a specific dry food for wreckfish broodstock. The comparisons between wild and reared wreckfish composition showed that fish from intensive culture have more lipids in muscle and liver than those obtained in the wild. In contrast, protein content is higher in the muscle of wild wreckfish than in reared fish and some differences were also observed in the FA profile with higher values of PUFA and n-3 PUFA in wild than in reared wreckfish. Gonads from females of wild wreckfish have a high level of ARA (7-10%TFA) and a ratio of EPA/ARA of nearly 1. Regarding wreckfish broodstock feeding regimes, most of commercial dry food has too much fat for wreckfish and a clear relationship between FA profile of broodstock diets and FA profile of oocytes and eggs was found. A new dry food was specifically formulated for wreckfish broodstock with a high amount of protein, low level of lipids, a high amount of n-3 PUFA and an EPA/ARA ratio similar to the one obtained in wild female gonads. Finally, the first data of FA profile of sperm from wreckfish males of different broodstock were obtained. A relationship was found between broodstock diets and fecundity and number of spawnings of the females. Relative fecundity (nº of eggs/Kg of female) and number of spawns per female increased in females fed with dry feed over the duration of the DIVERSIFY, from 2015 to 2018.

1.2.6 Grey mullet
The results suggest that grey mullet >89 dph grown in low salinity (15‰) have the capability to synthesize DHA from shorter carbon chain precursors while there is little or no biosynthesis of LC-PUFA in fish exposed to high salinity (40‰). This follows as grey mullet juveniles in nature would be moving to the lower salinity waters of river mouths and estuaries, which are characterized by an environment less rich in LC-PUFA and more abundant in smaller chain PUFA precursors. Low salinity upregulated the gene expression of the rate-limiting enzyme of LC-PUFA biosynthesis (∆6 desaturase) but was independent of DHA dietary level. On the other hand, both low salinity and DHA level upregulated the gene expression of elongase. The two transcription factors, sterol regulatory element binding protein (SREBP1) and peroxisome proliferator activated receptors (PPAR) are involved in the regulation of fatty acid biosynthesis. Although both SREBP1 and PPAR expression were highest in 15‰ water, PPAR expression was inversely regulated by dietary DHA at both salinities, while SREBP1 was inversely regulated by DHA only in the low salinity. These findings suggest that dietary levels of DHA can be decreased when feeding older juvenile mullet, provided that the salinity is reduced to levels found in estuarine waters. This would translate to a significant savings for farmers as the purchase of feed for the grow-out of fish to market weight can represent 60% of production costs and DHA is costly as a feed ingredient.

Also, regarding the cysteine sulfinic acid decarboxylase (CSD), a key enzyme in taurine synthesis pathway, DIVERSIFY found that it is active in the absence of dietary taurine and that the expression of this key gene increases with increased levels of dietary taurine until 1% where CSD expression decreases rapidly possibly due to a negative feedback mechanism. The increased taurine in the blood circulation of the liver, due to higher dietary taurine, may stimulate increased endogenous synthesis within liver cells to reduce osmotic pressure across the membrane and prevent cell shrinkage and changes in intracellular hydro-mineral balance. Cholesterol 7 alpha-hydroxylase (CYP7a1) is the key enzyme in the synthesis of bile salts and was not affected by increased levels of dietary taurine. This suggests that endogenous taurine synthesis was sufficient for bile salt synthesis. Taken together, it appears that grey mullet juveniles have the capacity for endogenous taurine synthesis that may be sufficient for cell volume homeostasis and bile salt production, but may fall short in optimizing skeletal muscle function and growth, thereby requiring a minimum of 0.5% of taurine in the diet.

In grey mullet broodstock the mobilization of energy reserves in terms of lipids and proteins was quite similar between wild and captive mature females. Moreover, in fatty acids and fatty acid groups, there were no conspicuous differences, independent of age, between female gonads from domesticated and wild captive broodstock fed fish oil-based diets or broodstock fed soybean oil-based diets. This suggests a gonadal biosynthetic capability for biosynthesis of LC-PUFA from shorter chain precursors. Nevertheless, when comparing the FA and lipid class profiles between female and male gonads, there were highly marked differences. In female gonads, the TA, TAG, wax and sterol esters were higher compared to male gonads while the male gonads had higher quantities of the PL phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine, as well as cholesterol compared to female gonads. Noteworthy were also the very high levels of DHA in the male gonads compared to the female gonads. Interestingly, the male gonads from the soybean-fed group were higher in DHA than the fish oil group despite the fact that soybean oil does not contain this essential fatty acid. The fish oil diet resulted in better egg hatchability, as well as larval tolerance of food deprivation and improved swim bladder inflation. These benefits may be due to another fish oil component, possibly carotenoids.

Fish acceptability of the developed DIVERSIFY grey mullet diet appeared enhanced by replacing poultry meal with fish meal, suggesting that the inclusion of other nutrients may be necessary in order to maintain a fish meal free diet. The fatty acid profiles of the tissues generally resembled those of the diets. Feeding the developed diet resulted in fish displaying a more balanced lipid profile than fish fed the commercial carp diet. For instance, the fillets from the DIVERSIFY diet were poorer in 18:2n-6, but also exhibited a higher absolute content of n-3 LC PUFA (EPA+DHA). On the other hand, the female gonads, unlike the flesh, displayed a selective retention of the essential fatty acids EPA, DHA and ARA independent of dietary regime, which was also demonstrated in another deliverable. The surprisingly high levels of ARA in the tissues compared to the poor amount supplied by the diets highlights the physiological relevance of this FA in this species’ reproductive performance and suggests the potential capacity for its endogenous production from the 18:2n-6 precursor. The sensorial analysis found no differences in selected sensory categories between the carp diet and the DIVERSIFY diet.

The use of excessive levels of soybean in fish diets can cause inflammatory responses in the distal intestinal epithelium, which affects fish health, reduces intestinal nutrient absorption and somatic growth. Inflammation is frequently associated with oxidative stress and the up regulation of the genes involved in the innate anti-oxidation system. In the DIVERSIFY studies, there was no indication of inflammation. In fact, digestive tract samples from all fish exhibited healthy tissue with no signs of disease and presumably oxidation stress. Although there was a significant improvement in the performance of fish fed the diet that included poultry meal instead of increased soybean meal, it was likely due to a taurine deficiency. Taken together, the results suggest that there is a significant improvement in grey mullet juvenile performance when using animal-based proteins, such as poultry meal, at about 13% DW diet. On the other hand, this advantage may be modulated by the supplementation of essential amino acids such as methionine and taurine.

1.3 Larval husbandry
1.3.1 Meagre
The main task for meagre larvae research in DIVERSIFY was to provide the industry an early weaning protocol for this new species, that included co-feeding live prey with artificial micro diets. It is well documented that the cost and quality of live feed, especially Artemia, is very high and fluctuates over time because it is dependent on the worldwide aquaculture demand and the weather patterns affecting the pelagic fish harvesting areas. Furthermore, the percentage of hatchery costs attributed to Artemia is substantial and for quite a long time researchers and feed producers have been looking for alternative feeding strategies and/or new formulations to reduce larval production costs. The standard method for meagre larval production is to start weaning the larvae around 20 dph. In the trials carried out in DIVERSIFY we showed that weaning time can be advance successfully to 15 and 12 dph using a commercial micro diet with a gradual transfer from live prey to the artificial diet over a minimum period of 5 days. However, larval survival was low due to cannibalism, a major problem in the culture of many marine fish larvae. Size variation is the primary cause of cannibalism in larval fish, together with factors such as food availability, larval density, feeding frequency, light intensity, water turbidity and shelter. This project demonstrated that meagre larvae have the same capacity to digest live prey and micro diets and they can be weaned earlier reducing the production costs if some measures to reduce cannibalism are in place. These measures include increasing feeding frequency, removing dominant individuals and keeping the larvae in the dark when the food was unavailable or in short supply.

1.3.2 Greater amberjack
The objectives of this WP were to study the (1) Effects of different feeding strategies on larval performance in intensive systems, and (2) Develop feedings protocols and rearing methodologies in mesocosm semi-intensive systems for the industrial production of greater amberjack. Starting with the ontogeny of the digestion and the vision system to acquire the basic biological information, our studies focused on the prey enriching diet and feeding regime and finally to critical parameters (tank type-shape, duration of the photo phase, tank background color and light conditions, stocking density) of the rearing process. The results were evaluated in terms of growth, survival, skeletal deformities, biochemical composition, stress and larval condition.

The results of DIVERSIFY indicated that larval rearing in large tanks and low initial stocking of eggs-larvae improves the growth performance and survival of greater amberjack. Egg stocking densities > 25 eggs l-1 affects negatively the results. The recommended photo phase is 24 L:00 D from 1 to 20 dph and 18 L: 06 D between 21 and 30 dph, with light intensities of 800, 1200, 1000 and 500 lux at 3, 6, 12, and 20 dph, respectively. A renewal of filtered seawater (5 μm) at an increasing rate ranging from 15-40% day-1 at 1 dph, 30-40% at 10 dph, 100-120% at 20 dph, and 200-240% at 30 dph ensures a good quality of the rearing environment. Dissolved oxygen ranged between 4.9 and 8.2 mg l-1, but must be preferably > 6.0 mg l-1, salinity between 35 and 40 psu, pH between 7.8 and 8.5 and temperature between 23.5 and 25.0ºC. The feeding protocols used have to be coordinated with the rearing conditions and the larval development. The larva has to be able to see, ingest and digest the food, and therefore needs the coordinated development of vision and digestive system. Larval rearing developed under conditions that allow faster growth have to consider the time of beginning and duration of the feeding periods with the different food items. In general, the addition of live microalgae at 150-300 x 103 cell ml-1 from 1 dph, enriched rotifers (Brachionus sp.) two times a day, from 3 to 25 dph, at densities between 3 and 10 rot ml-1, Artemia nauplii at 12 dph, during 5-7 days and enriched Artemia EG 1-day at 14-18 dph, and weaning diet (200-800 μm) from 18 dph can be a good sequence. Moreover, the enriched emulsions of prey supplemented with PL, carotenoids, ARA and immune modulators such as Echium oil and black cumin oil improved the larval rearing of greater amberjack, so enriching that tend to have these characteristics would give better results in the larval performance of greater amberjack.

During larval rearing, and especially following 20 dph, high size variability occurred in all rearing systems tested until today. This high variability is managed until now with early sorting of the reared groups to appropriate size classes. Applying standard methods and equipment available in all hatcheries, the sorting procedure resulted in significantly higher survival compared to unsorted groups. Unsorted groups between 20 and 30 dph had >90% mortality, while for the sorted groups it was limited to approximately 10%. During sorting, transport of the individuals is also a requisite. Individuals of less than 15 mm do not tolerate netting and transfer should be performed with care and avoid air exposure of the larvae. After reaching 20 mm in total length individuals can be netted normally. Husbandry practice with larger individuals (>0.5-1 gr) is easier although in some cases light anesthesia may help.

1.3.3 Pikeperch
The overall aims of this WP were to establish an optimal combination of factors that could give the best performance of pikeperch larval populations, and to develop an industrial protocol for pikeperch larval rearing. The protocol should reduce the high mortality caused mainly by cannibalism, the high rate of deformities and the large size heterogeneity between larvae cohorts. Using a pilot scale RAS (10 x 700-L tanks) and multifactorial designs, three successive experiments were conducted to study the effects of twelve environmental, feeding and population factors (four factors per category). For each experiment, the choice of factors was a trade-off between data available in the literature and the constraints of the SME partners of the project for this species. From each experiment, according to results obtained, the most influential factors and modalities were conserved and integrated in the following experiment in order to optimize progressively the protocol. At the end of this first step, an optimal combination of factors was identified and proposed to improve pikeperch larval rearing. The combination was mainly based on an initial larvae density (100 larvae L-1), no size grading, the use of larvae from large females, a discontinuous feeding, a constant light intensity (50 lux) with a fixed photoperiod (L:D 12:12), a weaning applied at day 16 with a duration of 9 days (progressive feeding transition from live preys to artificial diet), a fixed water renewal rate (tank volume per hour) and tank current direction (bottom to top) and finally a tank cleaning period during morning. This optimal combination of factors allowed the production of 0.8 g juveniles in 52 days with a swim bladder inflation >90%, a survival between 14 and 19%, a final biomass of 9.5 kg per tank, a food conversion rate of 0.6 and a production cost of 0.2 euro per juvenile. A fourth experiment was done to confirm the efficiency of the combination (7 repetitions), the results obtained were very homogeneous and validated the reliability of the protocol proposed.

1.3.4 Atlantic halibut
A protocol for on growing of Artemia nauplii was developed and described. Use of on-grown Artemia during the critical period of metamorphosis in Atlantic halibut larva did not differ from use of Artemia nauplii with regard to growth, mortality and fry quality. In addition, the production of on-grown Artemia is labor-intensive, and high personnel costs may be prohibitive in implementation of this live feed source in commercial larviculture.

The commercial production of Atlantic halibut fry is currently carried out in flow through systems (FT), while there is a growing consensus that a RAS would offer more stable environmental and chemical water parameters that would lead to improved larval performance. Production protocols for yolk sac and first feeding larvae in RAS were developed in DIVERSIFY. No differences in survival were detected between RAS and FT rearing during yolk sac incubation. When systems were primed for one month, larval growth was significantly higher in the RAS group during first feeding. High mortality occurred in one of the FT tanks. Taken together, results suggested that with adequate conditioning of the RAS, a stable system is established where growth and survival of larvae is as good as, or better, than in FT systems with optimal conditions. The RAS was a more stable rearing system for Atlantic halibut larvae compared to the FT system.

Metagenomic characterisation of the bacterial communities in rearing water and larvae revealed that at least 300-400 different bacterial genera were present in the rearing systems. Significant differences were detected in the micro biota composition of the RAS and FT systems: both in silos and tanks, and in the water and the larvae. No obvious correlation was seen between the micro biota in the water and the micro biota of the larvae. Characterization of the micro biota composition provides important information for development of probiotic treatment of Atlantic halibut larvae.

1.3.5 Wreckfish
The efforts in this area were focused on the development of a methodology for larval rearing and to define the optimal conditions, both in terms of water temperature, as well as in terms of a rearing system (RAS or FT). More advances in achieving natural spawns and in larval husbandry have been done in the three Galician wreckfish stocks. This was the first time ever that a project succeeded in producing a significant number of juveniles weaned to inert food, and it signifies a milestone in the efforts to produce wreckfish under aquaculture conditions. The work implemented in DIVERSIFY acquired important data on growth and increased our knowledge about the feeding protocol and the specific behavior and metamorphosis of wreckfish larvae. Technical changes have been made in incubation and larval husbandry that can be decisive to avoid the problem of malformed larval and achieve greater survival. During the first stages of egg development, vulnerability to external conditions is higher; nowadays the incubation parameters were adjusted and the facilities and systems were optimized in order to increase the quality of embryogenesis, resulting in larvae of the best conditions, and increased survival. The work also advanced the knowledge of the optimal incubation temperature and the adequate parameters regarding aeration, water flow and form of creating an adequate water circulation. Important observations were also made about larval behavior and feeding sequence. Very important results were achieved in larval feeding sequence in RAS culture. These data could be the starting point for future experiments and a reality to propose the cultivation of wreckfish as a possibility for the diversification of aquaculture.

1.3.6 Grey mullet
The WP on grey mullet larval husbandry determined that the most effective concentration of microalgae daily added to the larval rearing tanks of grey mullet was 0.4 x 106 cells ml-1 of Nannochloropsis oculata or 0.023x106cells ml-1 of Isochrysis galbana, in terms of larval growth and survival. These microalgal concentrations, although differing between these species, both provided the same level of turbidity of 1.19 NTU. Turbidity is considered a factor that facilitates prey recognition and larval consumption by providing a contrasting background. On the other hand, further studies revealed that the dominant factor defining the benefit of algal tank supplementation was the biochemical composition of the microalgae, which contain unidentified compounds common to both Isochrysis galbana and Nannochloropsis oculata that promote larval growth and survival. Although algal supplementation to the larval rearing tanks did not affect the ontogeny of brush border and pancreatic digestive enzymes, there were dramatic changes in enzyme activity as a function of age and the transition from strictly carnivorous larvae to omnivorous juveniles. Alkaline phosphatase activity, a marker for brush border absorption, was ca. 8 times higher and α-amylase activity increased 5.3 times in 79 dph fish compared to 40 dph individuals. In addition, gut maturation occurred around 61 dph. The results suggest that aquaculture feeds at this developmental stage should include not only considerable protein but also higher levels of starch or other low cost amylolytic energetic compounds compared to starter feeds fed to younger grey mullet or the juvenile stages of carnivorous species.

From these studies, the clear benefits of microalgal addition at species-specific concentrations to the larval rearing tanks of grey mullet was shown. Further studies also highlighted that the use of lyophilized microalgae was just as effective as the use of live microalgae, in terms of tank turbidity as well as larval rotifer consumption, swim bladder inflation, growth and survival. Interestingly, the use of lyophilized microalgae enhanced the maturation of the intestine more rapidly in grey mullet fry, suggesting earlier weaning onto a dry prepared diet is possible, when using this dried alga. Taken together, the results of this study showed that using lyophilized algae would be a significant saving in time, labour and infrastructure and may have expressed a growth advantage in older fish and is recommended in the larva rearing of grey mullet. From these studies it was shown that juvenileς are producing increasing amounts of amylase at the same time that protease activity is decreasing at an age when they are migrating to lower salinity estuarine waters. This begs the question whether weaning diets should be designed for a carnivorous, herbivorous or omnivorous mode of feeding. The results showed that fish performance was best, in terms of growth, survival, feed efficiency and gut maturation when fed an omnivorous diet. Furthermore, the high amylase and maltase activity in the omnivorous diet would provide glucose as an energy substrate, which could be protein sparing resulting in improved growth. These results continue to support the use of high carbohydrate-low protein diets to wean juvenile grey mullet, which would be more economical.

The results of this WP were implemented in the development of a grey mullet larval rearing protocol, which was tested in 6 m3 semi-commercial V-tanks in Israel. In the 2017 season, 78704 juveniles were produced as a result of the production protocols. This did not include the juveniles harvested for experimental tasks that year within the framework of DIVERSIFY. This meant that the entire juvenile production for 2017 was ca. 200,000 fish and survival was 20% from egg to 60 dph, which makes commercial juvenile production of grey mullet a reality.

1.4 Grow out husbandry
1.4.1 Meagre
The industry bottlenecks of variable growth rates and the possibly related need for defined feeding methodologies during grow out were addressed. The development of size variability in populations was described and was due to different growth rates amongst individuals and was observed at all stages including early juvenile stages before cage grow out sizes were attained and when cannibalism was a problem. There was no compensatory growth of slow growing fish and it was recommended that slow growing fish were not selected for grow out as an economic analysis indicated that these fish would need six months more to attain 500 g. Genetic differences were observed between fish that grow at different growth rates and genetic markers were identified that were associated to growth (see Reproduction meagre WP). A wide range of husbandry parameters did not alter the variable growth rates and consequently did not alter the wide size distribution obtained. These included light conditions (shaded or non shaded cages), depth (8 m or 6 m cages), feeding methods (self-feeding, hand feeding or automatic feeding), time of feeding (night or day) and depth of feeding (surface or bottom of the cage). In addition, the studies indicated many aspects that can improve feeding methodologies: Mortality and feed conversion ratio (FCR) were lower in deeper cages (8 m). High light intensity from natural sunlight had negative effects on feeding behavior. The structure of the visual system indicated that meagre are a nocturnal species that prefers low light intensity environments. A total of 50% of the stomach content had been transferred to the rest of the digestive channel 8 hours after feeding. Self-feeding fish feed during the entire 24-hour period throughout the year. Feeding behavior was stimulated by both visual (light) and mechanical (aeration) cues. In conclusion, variable growth rates appeared to be only related to genetic differences, which suggested that genetic breeding programs and domestication might be the solution to this problem. The information obtained, indicated that an optimal feeding methodology should adjust to the biological characteristics of meagre by feeding when light intensity is low (dusk, dawn and night), using stimuli to ensure a good feeding response from fish that often cannot be observed and fish should be left to digest during periods of high light intensity (daytime – particularly mid-day).

1.4.2 Greater amberjack
For the grow-out tasks of greater amberjack, development of methodologies emphasized cage technology. The feeding pattern of different age classes has been studied while trials to define optimal stocking densities were implemented. Furthermore there were trials aiming to study temperature effects on growth performance of greater amberjack. Cage rearing is important for the commercial production of amberjack, but appears to be challenging. Several trials have been performed at an industrial scale and during all trials fish accepted commercial feed of appropriate composition, i.e. high protein (of fish origin) without problem. There was also no problem during the standard husbandry practices of net cleaning/changing and although the stocking density was not high, a value of ~ 5 kg m-3 is considered acceptable for a pelagic fish. Regarding the growth performance, during the first 4 months the growth was high (5g d-1) while it decreases later by 50%. Significant variations in growth were observed among individuals resulting in size variability of almost 100% a problem that requires further investigation. Another significant difficulty during grow out is the occurrence of parasitic infestations by monogenean worms. Even though treatment with peroxide was effective, the application is not easy and appropriate methodologies suitable for large cages should be developed. The species is also facing bacterial infections and in the case of Greece incidences with Vibrio harvey were reported, causing significant mortalities.

Environmental temperature was shown to affect significantly the performance of greater amberjack. Juveniles of 5 g held at 26ºC showed significantly higher body weight compared with fish held at 22ºC or 17ºC. Morphological analysis showed that the increase of temperature led to an elongated fish body, especially of the head. Also, the specimens reared at 26ºC showed significant swimming differences compared to the individuals reared at 17ºC and 22ºC, while there was no difference between the later groups. For individuals of 350 g body weight, fish held at 21ºC showed significantly higher growth compared to fish held at 26ºC, while fish held at 16ºC showed the lowest final body weight. The survival rate was higher at 16ºC, but there was no significant difference in the FCR for the whole experimental period of 3 months. Plasma Cortisol levels were analogous to temperature and showed a high inter-individual variability. Nutrient digestibility coefficients were high indicating the good quality of the diets. Although temperature is one of many parameters affecting gut transit time it did not affect energy fat, protein and dry matter digestibility of greater amberjack. Finally, fish of 500 g showed no significant differences for the temperature studied (20ºC and 23 ºC) on feed intake and growth.

Regarding stocking densities, results showed that it affects growth rates and feed intake. Fish maintained at a high density (7 kg m-3) had better specific growth rate, while the condition index presented no difference between the groups. Further to this, feed intake was significantly lower at low density (2.5 kg m-3) during the second and third months, a tendency that changed in the four month during which the feed intake decreased with the increase of density. No negative effects on growth were observed in fish of 150 g initially stocked at 3.2 kg m-3 reaching a final stocking density of 6.8 kg m-3. Results from immunological parameters suggest that greater amberjack reared at the higher stocking density are not under a stressful condition. For the feeding pattern, studies showed that greater amberjack juveniles grew less when fed at 2.5% body weight d-1 compared to fish fed 3.5% d-1 or at apparent satiation. Furthermore animals fed one meal daily showed lower growth compared to those fed 3 or 4 meals per day. Similarly, FCR was higher for fish fed 2.5% body weight d-1 or once daily compared to the other conditions tested. For larger individuals (200 g), between the feeding frequencies tested (1, 2, 3 and 7 meals d-1) the best results in growth and FCR were obtained with 7 meals daily. The absence of changes among the hematological and biochemical parameters suggests that greater amberjack juveniles were able to adapt to the different feeding frequencies under the particular culture conditions. However, results from immunological parameters reveal differences in the immune status among fish subjected to different feeding frequencies that could influence the health status of fish.

1.4.3 Pikeperch
High mortality and unpredictable growth rate during the on growing of juveniles are among the major bottlenecks for the development of pikeperch aquaculture. These may be related to high stress responsiveness, since they are often observed after procedures of aquaculture management. The multifactorial study provided relevant outputs that covered well the objective to determine major directive husbandry and environmental factors for growth and welfare of pikeperch. The main results from this multifactorial study and from the confirming experiments indicated that pikeperch juveniles are highly sensitive to aquaculture manipulations such as grading and that long-term application of grading manipulations may alter growth rate and food utilization. In addition, other environmental variables were shown to affect the welfare of pikeperch, including the light intensity and, to a lesser extent, the light spectrum. Pikeperch is sensitive to the light environment due to its specialized retina and inadequate lighting conditions induce stress and immune suppression that may alter its resistance to pathogens. The second main objective was to study whether domestication level and geographical origin affect physiological stress response and immune status of pikeperch. The results indicated that domestication increases the stress response to the net chasing stress test. Moreover, the highest stress sensitivity of domesticated populations was associated to the best immune status. These results established basic knowledge for future selection studies of pikeperch strains according to the rearing conditions of commercial fish farms.

1.4.4 Grey mullet
Fish meal substitution between 50 and 75% by a mixture of different plant protein sources (corn gluten, wheat gluten and soy protein concentrate) in wild grey mullet fry weaned onto compound diets did not affect good growth performance and survival. The proximate composition, pancreatic and intestinal enzyme activity confirm the capacity of this species to digest plant protein sources at early life stages. These results indicated that weaning diets for wild grey mullet harvested for restocking aquaculture ponds and on-growing may be formulated with a high level of fish meal replacement by alternative plant protein sources. Moreover, it seems plausible that fry of this species may accept and use satisfactorily compound diets with a complete fishmeal substitution by plant protein sources. Diets with a 50 and 75% of fish meal replacement by plant protein sources were 15.5 and 23.6% less expensive than the fish meal diet, which is very relevant considering that feed costs account for >50% of the production costs in aquaculture production.

Three separate experiments tested the effect of stocking grey mullet at different densities (4, 6, 10, 12, 29, 55 and 286 fish per m2) in a range of cement and polypropylene tanks. The results showed that increasing the fish stocking density above 6 fish per m2 can lead to decreased growth in an increasing segment of the population resulting in larger numbers of smaller fish. This may be a result of higher stress among cohorts from increasing competition for the same food sources. In future studies, the effect of increased ration size, use of extruded and not pelleted diets as well as the number of meals per day (simulate continuous feeding) will be employed. This should reduce the number of slower growing, smaller fish in the population and increase the efficiency of grow-out. The effect of different stocking densities during grow-out was tested in Greece (4 and 6 individuals per m2), Spain (0.5 and 1.0 fish per m2) and Israel (1 and 2 fish per m2). Generally, poor growth was reported in all countries with no significant effect of density or observed differences in proximate and fatty acid analyses. Spain did report a trend of improved growth and feed efficiency in the lower stocking density treatment, while this inclination was muted in the Greece and Israel trials. The generally poor performance of the grey mullet in the Greek, Spanish and Israeli trials can be due to a number of factors. Certainly attempting to grow mullet in full strength seawater (40‰), which was the case in the Israeli trial, is not going to deliver the best growth, as much energy will be channeled into osmoregulation instead of building tissue. However, a major impediment is likely the extruded diet, which remains not sufficiently attractive to the fish as they appear to prefer the detritus and primary productivity of the pond to the more nutrient dense feed. Moreover, in earthen ponds the mullet are likely using sediment to aid mashing of the plant material in the gizzard for better digestion and absorption. In order to improve the feasibility of intensive monoculture of this species, the dietary formula of the current grey mullet feed must be improved

1.5 Fish Health
1.5.1 Meagre
During the course of the DIVERSIFY project, all major diseases and health-related issues of meagre were recorded and studied. Through the various tasks, studies of key disease states, development of appropriate treatments, and a first characterization of the meagre immune system/immune responses were carried out. One of the most important bottlenecks of meagre production is Systemic Granulomatosis (SG), a pathological condition affecting the majority of farmed populations. Through various tasks we tried to identify the etiology of the disease; we have run various feeding trials to identify potential nutritional causes of SG and we monitored meagre populations farmed in various locations in order to isolate and identify Nocardia spp., or other granuloma-associated pathogens. The general conclusions from these tasks were that nocardiosis is not the cause of SG, the addition of Vitamin D3, Se and Mn did not stop the development of the disease, while high dietary content of phosphorus, vitamin E and C in a fishmeal-based diet seemed to improve the condition. From the pathological assessment performed on various fish samples, a diagnostic protocol for SG was created based on the results of visual inspection, histopathology, electron microscopy and on the assessment of selected blood biochemical parameters.
Meagre is one of the fish species that are sensitive to Chronic Ulcerative Dermatopathy (CUD). The results of the studies in DIVERSIFY indicated that CUD is induced by the use of borehole water, however neither pH nor CO2 are the factors affecting the underlying causative agents. The causative agent is still unknown, however a full description of the disease in meagre was made using histology and SEM, as well as osteoclast activity using molecular markers.
Most of the currently important diseases in meagre are of parasitic aetiology, such as the monogenean Sciaenacotyle panceri. One of the tasks was focused on the use of essential oils with vermicide properties in order to test their efficiency as parasiticide. Overall, cinnamon showed immunostimulant properties and a clear potential to treat a parasitosis with Sciaenacotyle pancerii when administered orally to juvenile meagre. Furthermore, the first report of Diplectanum scianae infecting cultured meagre and the first report associating this parasite with fish mortality was recorded within the project framework.
Regarding the other pathogens of meagre, a wide range of molecular protocols for diagnosis by PCR have been developed that can be used for the detection of Vibrio anguillarum, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio vulnificus, Photobacterium damselae subsp. piscicida and Nocardia spp. Moreover, based on the results obtained of the occurrence of different pathogens and experimental challenge test, recommended protocols have been developed with the recommendations on specific antibiotic dosages and treatment regimes. Furthermore, since meagre is a relatively new species to aquaculture it was necessary to document the immune response of specific genes under conditions of vaccination and against pathogens of significance for commercial aquaculture, such as Vibrio anguillarum as it is a pathogen with broad host-range and likely to be of concern for intensive rearing facilities of meagre. In total, 28 assays have been developed for measuring the expression of genes related to the immune function in this species. Moreover, two different vaccine preparations were evaluated in the trials performed that appeared to stimulate positively immune responses of a diverse repertoire. Further, immersion vaccination against V. anguillarum showed that it conferred protection in vaccinates when challenged with V. anguillarum. These data, and related published work from this project, show the potential to modulate immune responses in meagre in culture, such as by delivery of immuno-stimulants, to enhance particular immune pathways at a time of disease risk such as prior to transport.

1.5.2 Greater amberjack
New tools have been developed to study the immune system of greater amberjack, with the design of 20 primers relevant genes (IL-1b, IL-8, IL-10, IL-17AF, IL-17D, IL-22, Defensin, Hepcidin, Piscidin, RAG2, IgM, IgT, Mx prot, iNOS, IFN1, IFNg, TNFa, EF-1a, b-actin, MUC-2). SImilarly, the characterization of greater amberjack piscidin, as an important antimicrobial peptide (AMP) gives new insights about the function of this species’ immune system.

Neobenedenia girellae is a monogenean parasite of the skin, and causes the main health problem for Atlantic populations of greater amberjack in aquaculture. This monogenean has been described in relation with water temperature increases in sea cages of the Canary Islands. New insights about the relation of this parasite with its host shows the mechanical damage that the fixation causes, resulting in thickening of the epidermis, vacuolization of epidermal cells, disruption of cellular layers, recruitment of goblet cells, and mononuclear cell lymphocytic type mobilization to the adhesion regions. Because of this, secondary infections appear and could result in 100% mortality. New prevention strategies have been developed, such as the inclusion in the diet of mannan oligosaccharides (MOS and cMOS), which enhanced mucus production and increases the immune response, reducing the parasite load and growth.

Zeuxapa seriolae is another monogenean parasite of greater amberjack, considered the main health problem for greater amberjack culture in the Mediterranean region. This parasite gets attached to the gills, being hematophagous, producing important gill anaemia and inefficient oxygen exchange. Due to its rapid lifecycle and its increase with water temperature, it could cause the demise of the whole production. Treatments with hydrogen peroxide at 75ppm during 30 minutes have been reported to be efficient for killing the adults, always combined with repeated treatments after 150 and 30 fays, and net changes to avoid reinfection from the released eggs. Other parasites have also been described, such as the blood fluke Paradeontaxylix sp. is a blood parasite that has been observed in cultured greater amberjack in the Mediterranean. The proliferation inside the host circulatory system could produce obstruction of blood flow, resulting in ischemia and necrosis, and gill destruction when the eggs hatch. Penella one of the largest copepod parasites of fish, typically from swordfish (Xiphias gladius) and marine mammals. This parasite gets imbedded inside the skin of greater amberjack, nevertheless, it is not considered a problem for greater amberjack culture.

Epitheliocystis is a bacterial disease caused by Chlamydia, Endozoicomonas spp. and Ichthyocystis spp. The presence of multiple cysts in the gills cause respiration problems in the fish and high mortality. New tools have been developed for the early diagnosis of the disease, with molecular PCR probes.

1.5.3 Atlantic halibut
In order to develop a vaccine for Viral Neural Necrosis for Atlantic halibut larvae, the capsid protein of Nodavirus was successfully expressed recombinantly in three different systems; E. coli, Leishmania tarentolae and in tobacco plant, and as expected there was variation in the amount of expression between the systems. In addition, the recombinant capsid protein expressed in Pichia was provided from the EU project Targetfish. These four expression systems differ in the way the expressed proteins are post-translationally glycosylated. By constructing and using E.coli and Leishmania tarentolea expressing green fluorescent protein (GFP), it could be visualized by fluorescence microscope that Artemia filtered efficiently and ingested these microbes, and thereby the harboring recombinant protein. Artemia ingested recombinant Nodavirus capsid protein expressed by the various systems, which could be confirmed by immunoblotting. The recombinant capsid protein expressed by the different system was then fed to Artemia, which were fed to Atlantic halibut larvae at 100 dph. Ten weeks later the juveniles in all treatment groups were challenged by an i.p. injection with Nodavirus to check for efficacy. The challenged fish were terminated 8 weeks post challenge and tested for the presence of Nodavirus in the brain by real-time RT-PCR targeting the viral RNA2-segment. No significant difference could be seen between the different treatment groups, including the group with recombinant protein that has shown protection earlier. This indicates that the size of the fish and the need to sort fish to minimize huge variation between individuals in different phases at the time of vaccination have its inherent limitations, and should be carefully considered. In conclusion, although it has been shown that Artemia will take up and accumulate the various forms of recombinant Nodavirus capsid proteins and act as a vector for oral delivery to larvae of Atlantic halibut, the challenge experiments indicate that this strategy of antigen delivery does not induce protection against Nodavirus infection, at least under the conditions used in this study.

1.6 Socioeconomics
DIVERSIFY had a strong socioeconomic component, in order to address issues that are presently important bottlenecks in EU aquaculture development, consumption and diversification -- beyond biological/production issues. The socioeconomic part of the project had a science based applied market development approach with multiple components: the perception of aquaculture products in general and products specifically, market potential and demand factors and motives, consumer and professional buyer preferences, new product development, creating added value in relation to raw products and market development. An important bottleneck is that a large proportion of EU consumers is product-loyal in buying food and, therefore also, in fish products. Therefore, introduction of new species requires in depth market research, as it has been done in DIVERSIFY. So, parallel to the technological improvement of production methods for the new species, expansion opportunities for the EU aquaculture sector have been identified.

Market research identified market potential for aquaculture fish products in cross-cultural consumer segments, with increased-to-strong interest in new products in the main EU fish markets (France, F, Germany, D, Italy, I, Spain, ES and United Kingdom, UK). In this project further processed new products were developed and sensory and conceptually tested by consumers in the five selected countries. This resulted in a sensory positioning in regard to other species in the market and framing suggestions for marketing. Next to that all consumer research was based on the Canvas business models. Buyers and consumers would welcome new species, if they are a) sustainably farmed, ideally in domestic or EU waters; b) fresh (especially southern-EU) or mildly processed (northern-EU); c) easy to prepare and/or ready to eat; and d) competitively priced.

In Europe, greater amberjack shows the most promising market opportunities, given its large size, processing potential and superior sensory characteristics. Grey mullet is a very interesting species due to the higher sustainability of its production methods. No specific preference region has been identified for this species. Wreckfish has very firm flesh that discriminates it readily from other fish. The remaining species (Atlantic halibut, pikeperch and meagre) have certain advantages due to their biological and physical characteristic and are of interest to specific regions in Europe.

Introduction of the new species seems to have most impact if it is done country by country instead of pan-European, because in each market other buying factors and motives are important. Next to that the growing perspectives per country are different. In some of the countries early adopters make that the majority of consumers try the new species, while in France the growth model shows that early adopters try the product easily, but that growth to the majority of consumers need extra marketing efforts. In all countries, introduction of the new products with a reference product that consumers already know improves acceptance and buying from retail buyers and consumers. Price actions to penetrate the market were only successful in Spain. In all other markets price actions had a negative impact on market penetration.

1.6.1 Final product development
The main objectives of this WP were to develop new product concepts from the selected species, by incorporating consumer and expert input; to select product ideas and develop physical new products from the selected species; to monitor the quality of new products in terms of organoleptic characteristics and nutrition-rearing history; and to make a technical assessment of the products. In order to generate input in the form of a set of ideas that will be screened out and further developed into product concepts for further testing in subsequent tasks in the new product development, different focus group discussions with consumers and interviews with experts in the selected countries of the project (UK, D, ES, F, I) were performed. This research provided a list of ideas for new product development that were further tested in the experimental and quantitative research that touch upon the two main areas of the work, namely related to development and selection of new product concepts from selected fish species, and evaluation and optimization of newly developed fish products. In total, fifteen feasible ideas were generated from consumer focus groups. Interviews with experts were conducted by using a structured questionnaire in each of the five countries to explore the possibility of creating new fish products from the ideas gathered from the focus groups. Experts from different countries agreed that the created products were attractive and feasible ideas that have potential in the market. They considered that in overall these ideas could increase profits of the fish industry due to the higher diversity of choice. Generally, they stated that these ideas had a possible future if they are developed with good coordination between the fish farmers and consumers. Experts considered that some of the product concepts should be revised in collaboration with consumers and that they require product testing and sampling.

New product concepts, generated combining information of the market perceptions and the technical limitations and the economical prospect efficiencies, were submitted to a quantitative screening. From this screening, 12 concepts or ideas out 43 that acquired the highest scores were suggested for product development. These ideas covered different options: mass market products, products targeted to specific market segments and added-value products. Before the development of the new products based on the suggested ideas, an extensive characterization of the raw material was performed. The five selected fish species (meagre, pikeperch, greater amberjack, grey mullet and wreckfish) were studied for their fillet composition, their technical yields, their fillet sensory properties and mechanical texture. Based on the results obtained, some processing recommendations (products best fitted for each species) were also suggested. Physical prototypes of new products per species of varying degree of processing in commercial format (size, packaging and presentation) were developed. The physical prototypes were designed based on the market potential of the new species, the product concepts technically and consumer driven, the consumer value perceptions and segmentation, the physicochemical characteristics of each raw material, the technical properties of the products and the process, and the availability of similar product in the market. Provided that wreckfish was not available as farmed fish, the possibility to create prototypes out of this species and conduct further consumer test was not possible. Accordingly, the effort on new products development was only made on the available fish species from aquaculture. Twelve different prototypes were elaborated based on ten selected ideas and using 4 fish species.

Meagre was used for the development of the “frozen fish fillets with different recipes”, “fish burgers shaped as fish” and “ready to eat meal: salad with fish”. Pikeperch was used for the development of “fresh fish fillet with different ‘healthy’ seasoning and marinades”, “ready-made fish tartar with additional soy sauce” and “fish spreads/pate”. Grey mullet was used for the development of “thin smoked fillets”, “ready-made fish fillets in olive oil” and “fresh fish fillet with different ‘healthy’ seasoning and marinades”. Finally, greater amberjack was used for the development of “frozen fish fillet that is seasoned or marinated”, “ready-made fish tartar with additional soy sauce” and “fresh fish steak for grilling in the pan”. The necessary information to obtain these new products, as well as a number of guidelines, processing conditions, technical specifications and troubleshooting were also described. In addition, basic information regarding the packaging of the food products, conservation conditions, preliminary product shelf life and consumer handling/cooking specifications were provided as well. Regarding the different processing conditions, these new product ideas had different degree of complexity, but in all cases it was possible to elaborate different prototypes with the selected fish species. The technical feasibility suggested that it was possible to produce these products at an industrial scale, which was corroborated by the presence of other similar products in the market.

The technical quality of the developed products was also assessed. The total proximate composition of the products (protein, lipid, moisture, inorganic and carbohydrate content), the energy contents of the selected products, the quantitative nutritional value in aspects of fatty acids and the sensory profile of each of them was determined. As expected, processing had an effect on both the proximate composition and fatty quality of the products when compared to the raw fillet tissue. However, the effect depended on the processing method used as well as the inclusion of additional materials (such as olive oil) during the product formulation. Processing generally had a negative effect on nutritional quality reducing the proportion of essential fatty acids, i.e. EPA and DHA, of the majority of products when compared to the corresponding fish fillets. Regarding sensory properties, all processed products exhibited unique sensory profiles. The processed products showed a more complex sensory profile, with more attributes than the unprocessed cooked fillet of the species. The developed characteristics of the processed products in their majority were connected to the added materials and/or the processing method.

Finally, the correlation between the fish dietary history (e.g. dietary fat and protein levels, fat sources, etc.) or other rearing parameters (e.g. rearing system, temperature, or density) and the end-product quality was evaluated. Results indicated that filleting yields and protein contents did not seem to be influenced significantly by rearing and dietary histories at grow out stage. Greater amberjack displayed the highest filleting yields and final contents of protein, fat and especially EPA+DHA. Due to its vulnerability to fat oxidation, it was suggested that commercial sizes should be 1-2 kg with a relatively lower fillet fat content than bigger fish. Meagre filleting yield and protein content were quite attractive. Its total fat contents did not seem to be highly influenced by the dietary or growing history, displaying low contents of fat even in the wild, an attractive feature for low fat dietary regimes. Grey mullet was confirmed as the best candidate for marine ingredient substitution of its diet either by terrestrial or marine origin vegetable sources. Regarding the sensory properties, diet had only an important effect on the sensory characteristics of the grey mullet, especially in aspects related to the fillet fat content and its oxidative stability.

1.6.2 Consumer value perceptions and behavioral change
The main aim of this WP was to comprehend overall value perceptions of consumers with regard to aquaculture fish in general and the DIVERSIFY fish species in particular, to undertake a value-based segmentation study, to evaluate consumer sensory perceptions of the newly developed products, and to optimize the newly developed products in terms of ideal intrinsic-extrinsic product attribute combinations.

Consumer value perceptions and segmentation: Based on a number of psychographic characteristics (i.e. category involvement, domain-specific innovativeness, subjective knowledge, suspicion of novelties and optimistic bias) three cross-cultural consumer segments have been provided that comprise the top fish markets in Europe (i.e. France, Germany, Italy, Spain, and the United Kingdom). These were named “involved traditional”, “involved innovators” and “ambiguous indifferent”. The consumer segments have been found to be uniform across investigated countries with homogeneous, converging fish-related culture, that bring opportunity to fashion new product concepts at the cross-border European level. The segmentation results further show that European aquaculture could profit from a market segmentation strategy that take full advantage of the market potential of the above segments besides nurturing consumer awareness regarding aquaculture products. The future of aquaculture seems less dependent on geography and more dependent on consumer lifestyles and their psychographic profiles.

Consumer sensory perceptions: Sensory profile of 10 newly developed products from the DIVERSIFY fish species (with low, medium and high processing level) have been tested with consumers in a cross-cultural context in all test countries, where products with a lower degree of processing and the genuine sensory properties of fish were those that generated higher overall acceptability of the consumer. On the other hand, products having a higher degree of processing were found to be more appropriate for consumers who do not like fish because of its taste, presence of bones and odour, among other attributes, where the existence of different processed product alternatives is found to be a good solution. In terms of the consumer segments previously identified, the overall acceptability and preferences across products were minimal, again showing that newly developed products from the new/emerging DIVERSIFY fish species could work well across the different consumer segments.

Optimization of intrinsic-extrinsic attribute combinations: Based on the experimentation with newly developed product mock-ups from DIVERSIFY fish species in the cross-cultural context, the optimal intrinsic-extrinsic product quality profiles have been identified. It was concluded that it is possible to create new aquaculture products targeting similar high-profile consumer segments across all big EU markets. Similar pattern in consumer choice-drivers have been observed where country of origin and price come first, followed by quality certification (i.e. ASC label), while nutrition/health claims appear to have varying effect. For the aquaculture industry, the use of ASC label in their marketing to signal consumers that their products come from a “controlled”, certified and responsible aquaculture source increases the probability of consumers considering the product as well as trusting it. For the general public, the use of nutrition and health claims would actually help EU consumer to make more informed choices, aligned with their preferences, stimulating health-related behaviour.

Communication effectiveness in behavioural change: The evaluation of communication effectiveness in changing consumers attitudes and behaviour towards the newly developed product mock-ups from DIVERSIFY fish species in the cross-cultural setting shows that there is a clear need for the aquaculture industry to promote new products and persuade/engage consumers that they can constitute an excellent source of sustainable and quality food. It further shows that aquaculture products should not be promoted using generic messages that cannot stand competition and defend against bad word of mouth, but should promote unique production process by increasing their visibility as traceable, healthy but also tasty alternatives for modern diets and responsible consumption.

1.6.3 Business model and marketing strategy
Each of the DIVERSIFY species has advantages in relation to the current aquaculture fish assortment in EU stores. The selection of species has been broad and diverse. However, production challenges make production process outcomes still uncertain for some of the species (e.g. wreckfish and grey mullet) and a constant and high-quality supply is therefore still difficult. Consequently, selling to large retail chains could be difficult or impossible at this stage. This channel demands a continuous stream of production. Based on this, suppliers of the experimental species are advised to begin by selling to smaller retailers/parties and local restaurants. This strategy will help generate cash flow that can be invested to further professionalize production. For these producers, collaborating with innovative channel partners (for co-creation and co-investment) is the best bet.

The more promising business opportunities and thus business models concern greater amberjack, pikeperch, meagre, and Atlantic halibut. For these species, most bottlenecks in production have been addressed satisfactorily. The challenge now is to grow customer demand and market acceptance. The newly developed products can help give an impulse to this effort. The results showed a coherent business story for these four species, which is the first litmus test for any viable business model. The value propositions can be summarized as follows:
(i) Greater amberjack is a delicious and excellent alternative to fresh tuna. It grows fast and offers an excellent food conversion rate. Prices could be high but set slightly below tuna. Because it is a large, rapidly growing species it is interesting as a fresh product, frozen and for further processing. In promotion, the excellent flesh quality and high omega 3 can be stressed.
(ii) Pikeperch is a freshwater fish and resembles rainbow trout. Its meat is white with soft texture and a mild taste. It lends itself to many different forms in many preparations (e.g. salad). It is a good alternative for trout. An exclusive high-end positioning with fairly high price is recommended combined with an initial focus on the fish’s traditional geographical markets first.
(iii) Meagre is a white-flesh fish with an attractive fish shape that offers lean fillets with excellent texture and mild flavor. Its firm texture makes it very versatile; it is suitable for a large variety of recipes. Its rapid growth rate allows producers to farm them to larger size than many other farmed competitors (e.g. European sea bass or gilthead sea bream). It is appropriate for filleting and further processing. Promotion of this marine fish could stress high omega 3, leanness, and excellent taste. The fact that the species is known under different names in different countries requires attention.
(iv) Atlantic halibut is a large semi-fat flatfish rich in omega 3, with a characteristic flaky white meat and few bones. It has a reasonably good fillet yield. It competes on the market with turbot and sole. Building on its excellent reputation in the North European markets, firms can expand presence in retail channels with a high value/price.

While very good possibilities exist for the above production-ready four species, results did show that firms should increase their attention for marketing and relationship building with channel partners. Although farmers will benefit from enhancing their production processes to further increase quality/growth and decrease cost, investments in marketing and sales/channel management generally lag behind. This could jeopardize the chance of penetrating the market and reaping profits from their efforts. However, only with a buy-in from distribution partners and adequate marketing efforts can consumers be reached and convinced to adopt and continue purchasing these new species and products. Marketing can benefit from using country/region of origin branding and health claims (e.g. high omega 3), among others. Certification issues also need to be remedied, particularly since e.g. super markets consider this a key-purchasing requirement. Without proper certification, market development may proof difficult at best.

The final, online market test performed in DIVERSIFY focused on greater amberjack (as the most representative new species and one with the highest potential) and fillets to study acceptance in a competitive store setting. Results confirmed that consumers had a positive attitude and willingness to buy the new farmed products when instructed to buy fish for a meal for their families. However, communicating the new species’ closest neighbor (referent product, i.e. similar to tuna) was found to be important in order to help consumers categorize the new fish and its product. Results showed that Spain and Italy are the most attractive EU markets to enter first. Whether these results can be generalized to all four (or six) species remains to be seen. Since production levels even for these four species remain fickle/ uncertain, soft launches with slow ramp up scenarios are advised. By focusing on one distribution channel and country at a time, supply levels can be best managed and controlled. It can help mitigate risk related to sudden shortfall in production due to disease or other setbacks, or inability to scale up production in a timely manner. Regarding an internationalization strategy, it involves selecting a country-by-country (i.e. waterfall approach) rather than all-countries-at-once approach (i.e. the sprinkler approach). Similar to the communication tests, the fish in the online market test was sold under the DIVERSIFY label. Using such a fancy label or brand may help farmers build a more sustainable market position. A brand can help differentiate the product in the market and prevent the new product from becoming generic quickly. It can help to maintain a profitable margin for farmers and will help protect them against new entrants and followers with similar products. It will help make the farmers’ business model more resilient. Farmers may create their own brand or explore developing collaborative/cooperative structures for this purpose.

Potential Impact:
The DIVERSIFY consortium integrated a multidisciplinary group of research and academic institutions, small and medium-sized enterprises, large enterprises, five professional associations and one consumer NGO from 12 European countries. The acquired knowledge and developed methods will enhance the production of the selected emerging species (meagre, Atlantic halibut, pikeperch) by the European aquaculture and will enable the incorporation of some new species (greater amberjack and grey mullet). Wreckfish is still far from commercial production, although significant advances have been made in the area of broodstock management, control of reproduction and production of fertilized eggs, and larval rearing with the production of a small number of juveniles.

2.1 Reproduction
In the area of reproduction, DIVERSIFY has provided improved understanding of the regulation of reproduction and the dysfunctions that occur when different fishes are maintained in captivity. The project has defined optimal broodstock management conditions and has developed species-specific spawning induction protocols for the acquisition of optimal gamete quality.

2.1.1 Meagre
Given the developed methods for reproduction control, we expect meagre production to increase, the cost of meagre production would decrease and this would have positive impacts on the rural coastal communities that farm meagre. In addition, the technologies would improve the production of similar species to which similar or the same technologies can be applied. The description of the limited genetic diversity of the meagre broodstocks held across Europe has highlighted the need for the implementation of meagre genetic breeding programs. The provision of genetic tools and protocols to control reproduction to produce the families necessary for a breeding program will enable the industry to implement breeding programs, thus enhancing the industries efficiency and profitability. The implementation of breeding programs has the potential to avoid negative effects of inbreeding on the production of meagre. Genetic breeding programs have many positive effects on the production traits that are selected, for example growth rates have been improved in other species by 10-20% each generation. Improved growth rates for meagre would increase production in a given time and decrease production costs as the production cycle would be shorter. These benefits would be with each generation enabling the industry to improve continually. These advantages would stabilize the production of meagre and would have socio-economic benefits for the areas, often rural coastal communities where meagre is cultured.

2.1.2 Greater amberjack
The greater amberjack is a highly valuable commercial species and a popular game fish. The total worldwide catch of this species has increased tenfold since 1990, still only reaching 3,287 tonnes in 2009, of which about 17% was taken by United State of America and around 80 % was fished in the Mediterranean and Black Sea by European (Greece, Italy and Spain), African (Algeria and Tunisia) and Asiatic countries (Cyprus, Israel and Syria). The greater amberjack flesh is much appreciated by consumers and its market quotations are high, ranging between 10 and 20 USD per kg in Europe and reaching 20–30 (sometimes up to 50) USD per kg in Japan. In the late 1980s, a greater amberjack farming activity initiated in the Mediterranean basin, based on the grow-out of juveniles caught from the wild and cultured in tanks and cages. Greater amberjack aquaculture production has an enormous potential for expansion: the total value of the global aquaculture production was just 808,051 USD in 2007 (source: Cultured Aquatic Species Information Programme, Seriola dumerili, FAO, 2016), despite its high quality flesh and high consumers’ appreciation. Its rapid growth rate, worldwide market demand and high quotations make the greater amberjack a very promising aquaculture species. However, a proper commercial aquaculture production of the species has not developed so far, mainly due to its inconsistent and unpredictable reproduction in captivity which prevented the development of hatchery production of juveniles and the conversion of this capture-based farming activity into a true aquaculture industry. We expect that the spawning induction methods developed within DIVERSIFY will represent valuable tools towards the incorporation of this species in the aquaculture industry in Europe and its large-scale aquaculture production. Moreover, the production of a large number of artificially propagated juveniles will help reduce the fishing pressure on wild greater amberjack populations, whose state of conservation is unknown. Already within the time frame of DIVERSIFY, a number of commercial operations in Greece –both partners of DIVERSIFY and non-partners—have begun pilot production of greater amberjack using eggs and juveniles produced by the project.

2.1.3 Pikeperch
Genetic studies in pikeperch were until recently limited and information was lacking on the genetic structure of wild populations, and more importantly on that of domesticated aquaculture stocks. Understanding spatial genetic variability patterns in pikeperch is expected to have a dual major throughput and impact: the first one more theoretical, which is linked to conservation actions and translocation regulations, and the second more practical and applied having to do with farming efficiency.

Pikeperch populations in Europe seem to be part of at least two genetically differentiated groups. The first group is more widely distributed in central and northern Europe and the second one comprises few populations in Central Europe and probably southern countries. In the latter, the Hungarian populations are having a key-position being different from those found geographically near, and this might imply that we are talking for another stock associated with Hungarian lakes, as opposed to all other populations that probably dispersed through the Danube River west and southwards. This should be taken into account for conservation actions as well as translocations of samples outside the species range for aquaculture practices through trading. We already know that this may have many conservation implications, such as biological invasion, pathogen spill over, etc.

Furthermore, aiming at having a long term and sustainable breeding program, it is fundamental to i) ensure sufficient genetic variation within populations, and ii) take into account different zootechnical performances that are due to the geographic differentiations of stocks. In domesticated stocks, caution is required because the loss of genetic variability within the first generations of breeding procedures limits the potential for future genetic improvement through selection practices. Last, we should examine carefully if differences in production traits (such as growth, disease resistance and/or survival rates, aggressiveness, food conversion ratio, etc.) between populations of different origin are consistent to genetically differentiated groups in our analyses, in order to recognize populations with best performances for the aquaculture industry.

2.1.4 Atlantic halibut
Documentation of reproductive performance and endocrine cycles in wild-caught and farmed female Atlantic halibut led to the development of optimized spawning protocols with consistently high fertilization of eggs. The optimized protocols also meant that fish were subjected to less handling stress and improved fish welfare. Protocols for induction of maturation and spawning with GnRHa were tested and showed that ovulation and spawning advanced in females that were treated during late vitellogenesis, before final maturation had started. However, results were not consistent and gamete quality was highly variable in treated fish. We expect that these results will have a positive impact on output and availability of high-quality gametes from Atlantic halibut. In addition, we expect an impact on handling routines and improved welfare in breeders.

2.1.5 Wreckfish
The wreckfish is one of the most prominent species for the diversification of European aquaculture due to its fast growth, late reproductive maturation and limited fisheries landings. With a high market price, worldwide distribution, and the high potential for the production of value-added products, this is a great fish for aquaculture production. However, lack of reproduction control and larval rearing protocols were considered major bottlenecks preventing the establishment of the wreckfish aquaculture. The objectives of DIVERSIFY were to understand and control the species reproductive function and develop larval rearing protocols for the industry. The expected results have been achieved in the area of reproduction with the production of large number of eggs of good quality. However, we have not yet produced consistent results in the area of larval rearing, so the species is not yet ready to be incorporated in the aquaculture industry. Nevertheless, DIVERSIFY has acquired significant knowledge on the ontogeny of the fish in culture, and the success in larval rearing we had during the last year of the project is encouraging for the European aquaculture. These results warrant and should encourage further research efforts –both at the National and European level— for the development of working protocols for the production of juveniles by the industry.

2.1.6 Grey mullet
Grey mullet, which are fished and farmed extensively around the world for centuries, have many favorable culture characteristics such as being omnivorous, euryhaline, fast growing, disease resistant and producing not only flesh of high quality, but also a secondary product of very high value, the egg roe called “bottarga”. Currently, the supply of fingerlings for grey mullet culture is almost exclusively from the wild, which increases pressure on the dwindling wild fry populations, particularly along the Mediterranean coasts. This project enabled the establishment of a potent hormonal therapy to synchronize gonadal development in captive grey mullet females and males, and sequentially induced their spawning. In addition, by using artificial thermo- and photoperiod regimes, we have managed to shift the natural spawning season, which increases the feasibility of a year-round supply of fingerlings. The achievements accrued in this WP are expected to influence the economics of grey mullet culture and help reduce the harvesting of wild fry from the sea for stocking fish culture systems.

The roe of grey mullet is consumed either as salted individual eggs or as salted and dried whole ovaries (bottarga). The catching of grey mullet and the processing of its high value roe has a long tradition in countries on both the northern and southern coasts of the Mediterranean Sea. In this regard, the current study points to favorable effects of captive conditions on the fish’s competence to undergo and complete vitellogenesis. These results emphasize the potential of hatchery-produced grey mullet to develop their ovaries up to a condition appropriate for bottarga production (advanced vitellogenesis). Furthermore, the development of a method to enhance coloration of grey mullet roe in farmed fish through the use of natural pigments in the feed ingredients incorporated in their food is a major advantage in guaranteeing final product quality. Thus, the increase in egg roe for bottarga production, that will occur if fish are grown out to sufficient size, may increase the farming economic sustainability by accelerating the production cycle of this high-value "mullet caviar", and reduce the need for importing ovaries from third countries for the production of bottarga in Europe (Italy).

2.2. Nutrition
The cost of feeding in aquaculture production is around 40-70% of total production cost; therefore, improving feed conversion efficiency and growth rates is directly related to the profitability of the industry. New species in aquaculture are fed with available diets designed for other well-established species, which may constraint their growth performance, welfare and health. For this reason, it is important to develop species-specific feeds that consider the nutritional requirements of each species at different stages of development and that can improve their performance, quality and health condition. To achieve this goal, DIVERSIFY has established the nutritional requirements of several macro- and micronutrients for most of the species considered in the project. In addition, specific live prey enrichment products have been developed. Specific formulated feeds, live prey enrichment products and feeding protocols will result in new products that can be commercialized worldwide.

2.2.1 Meagre
The nutrient requirements for meagre larvae were determined, improving larvae growth and survival and reducing both skeleton deformities and early incidence of systemic granulomatosis. Essential fatty acid requirements have been also determined for meagre juveniles, requirements linked also with the incidence of systemic granulomatosis. The results obtained have a clear positive impact on the meagre manufactured diets, improving the formulae to produce high quality fingerlings. This is expected to improve the quality of the produced juveniles by the EU aquaculture industry, thus improving its cost efficiency.

2.2.2 Greater amberjack
Nutrient requirements for greater amberjack larvae were determined, improving larval growth and survival and reducing skeleton deformities. Recommendations for enrichment protocols for both Artemia and rotifers have been produced, improving the quality of the fingerlings produced. The lysine requirement has been determined for juveniles, improving the feed conversion and growth of juveniles in sea cages. For broodstock diets, important improvements have been done, determining the levels of essential fatty acid, histidine and Taurine to improve the spawning quality of this species. The different results obtained will be indispensable for the feed production companies that will be called to produce specialized feed for the emerging greater amberjack aquaculture industry.

2.2.3 Pikeperch
Development of intensive farming of pikeperch in Europe is a new industry and tailor-made commercial feeds have yet to be formulated for several rearing stages of this species. The project DIVERSIFY focused on nutritional challenges in larval, early juvenile rearing and the specific dietary requirements of phospholipids, fatty acids and minerals (Ca, P) and the effect on their interaction during early fish ontogeny. DIVERSIFY established the requirement of these nutrients in pikeperch and proved accordingly that available existing commercial extruded feeds could be optimized. If the obtained DIVERSIFY knowledge is commercialized to marketable feeds, this will benefit both the feeding industry, which can offer new better products, as well as boosting the economy of farmers due to higher growth rate, a better feed utilization, higher survival and lower frequency of deformities. Thus, in a broader sense, the DIVERSIFY results obtained will improve performance and stimulate an increased hatchery production of pikeperch juveniles for the benefits of the EU aquaculture industry.

2.2.4 Atlantic halibut
The work on Atlantic halibut nutrition established a method for early weaning of larvae, indicated at which developmental stage larvae can be weaned and established a quantitative difference between how different feeds are accepted by the larvae. This is one step towards early weaning of Atlantic halibut larvae. The ability to use formulated feeds in larval rearing will lower costs and increase efficiency for commercial hatcheries. Also, DIVERSIFY has established that Artemia nauplii produced with modern methods have sufficient levels of nutrients to cover the requirements of Atlantic halibut larvae. Phospholipid (PL) levels did not affect growth and lipid composition of juveniles. This means that further research should concentrate on other issues than nutrition in larvae fed Artemia and PL requirements of juveniles. This will release resources for research on other topics related to Atlantic halibut farming.

2.2.5 Wreckfish
Studies on wreckfish nutrition were very scarce until the onset of DIVERSIFY and the advances in this area are very important for the development of the culture of this species. In DIVERSIFY, studies about broodstock and larval nutrition of wreckfish were performed. Two new live food enrichment products for feeding larvae were developed and these products had a good acceptance by wreckfish larvae. Advances in the knowledge of nutritional requirements of this species were obtained with data obtained from different tissues and eggs from wild fish. Based on this and regarding broodstock nutrition a new dry food was specifically formulated for this species. The development of the products obtained for wreckfish nutrition in DIVERSIFY will contribute to the improvement of feeding regimes for wreckfish broodstock and larvae leading to a higher production and a good commercialization of this species in the future. In addition, a new dry feed has been designed for the fattening phase and will be tested with the fingerlings obtained during the last year of the project.

2.2.6 Grey mullet
The findings of the nutrition WP on grey mullet have made a significant impact on our understanding of the nutritional requirements of this species, which leads to more efficient and profitable production. Specifically, the conclusion that the costly ingredient DHA, an essential fatty acid for growth and development, can be reduced under low salinity during juvenile grow-out is very important. On the other hand, DHA must be added to broodstock diets to improve the quality of bottarga, a high price added value product for the industry, and increase larval performance and robustness. Taurine is also a necessary dietary supplement at 0.5% of the diet, despite the fact that this species has some capacity for endogenous synthesis, as it improves both larval and juvenile growth. On the other hand, studies carried out in this WP have shown that the current grey mullet grow-out diet formula continues to require animal based meals to achieve satisfactory growth. However, the supplementation of taurine and selected amino acids might negate the necessity of animal based meals and provide a fish meal-free and more sustainable feed for fish farmers. Taken together, these advances in the grey mullet nutrition would translate as significant savings for farmers as the purchase of feed for the grow-out of fish to market weight can represent 60% of production costs.

2.3 Larval husbandry
A larval rearing system is a complex artificial environment, with numerous factors influencing fish development and performance, as well as behavior and survival. These factors can be environmental, nutritional, social and genetic. For species such as meagre, pikeperch, grey mullet and Atlantic halibut, improvements in fish growth and husbandry have been addressed to refine the existing protocols and facilities in order to solve existing bottlenecks. In contrast, emphasis has been given to developing new species-specific larval rearing protocols in the case of greater amberjack and wreckfish, since these were species with important knowledge gaps in these areas. The output of these tasks is the development and refining of rearing protocols for the selected species that will result in the improvement of current practices, and will provide an increase in production yields.

2.3.1 Meagre
For meagre larval rearing the most important factor to be improved was to reduce production costs derived from the use of live prey (cost of Artemia cysts and labor dedicated to live prey culture and enrichment). Thus, in the project we have provided the industry a protocol for early weaning the larvae onto artificial feeds using a co-feeding period of at least 5 days and introducing several methods to reduce cannibalism during this phase. The protocol is effective in advancing larval feeding on micro diets by 5 days compared to the standard rearing method, although it produces an increase in the incidence of larval cannibalism. Thus, more research is needed to reduce this cannibalism in the larvae and to formulate more attractive feeds for meagre larvae. Increasing the survival rate and the use of artificial feeds will contribute to increase the production of meagre juveniles for on growing.

2.3.2 Greater amberjack
As a result of DIVERSIFY, a commercial larval rearing protocol has been developed, with a considerable increase in larval survival over previous attempts. This protocol enabled the industry –both partners in DIVERSIFY, but also non-partner companies—to obtain juveniles from the research partners or to produce juveniles themselves, and then start pilot sea cage rearing. As a result, the first harvesting of food-size fish begun already before the end of the project (Dec 2017) and in the Canary Islands this species has already been included in the Regional Plan for the Management of Aquaculture (PROAC), approved on July 9, 2018. Therefore, as a result of DIVERSIFY we expect the EU production of greater amberjack to continue to increase gradually in the coming years.

2.3.3 Pikeperch
The optimal combination of factors that was developed to improve larval rearing in RAS is very important to secure the biological and economical basis for the design of further hatchery – nursery for pikeperch production. For the first time a reliable protocol is available for people who intend to invest in pikeperch culture. At that moment, only few farms produced pikeperch using RAS in Europe (including Switzerland), they have developed their own protocol, which remains confidential and not accessible for new investors or producers who want to diversify their fish production. Moreover the production cost of a juvenile was estimated, consequently that will help also the building of new farms. It must be also considered here that the protocol proposed could be still improved in the future.

2.3.4 Atlantic halibut
Infections with opportunistic bacteria are a severe problem in marine larviculture, thus limiting the production of juvenile fish for commercial fish farming or for re-stocking of natural populations. While at later life stages the frequency of bacterial infections can be reduced by preventive measures such as vaccination and good management practices, the very young larvae and small fish have an immature immune system and cannot be protected by vaccination. Very often infections in larviculture are treated by antibiotics. However, this is not a sustainable practice since bacterial antibiotic resistance will develop and antibiotic-contaminated effluents are deleterious to marine ecosystems. Therefore, alternative strategies for preventing bacterial infections in fish larvae, such as pathogen-reducing probiotic bacteria or bacteriophages are highly needed. The commercial production of Atlantic halibut fry is currently carried out in flow through systems (FT). The successful application of RAS in first feeding of Atlantic halibut in the DIVERSIFY project is an important breakthrough, as the more stable environmental and chemical water parameters led to improved larval performance. A metagenomic analysis of the bacteriological composition of water and larvae in RAS and FT systems for both yolk sac and first feeding stages has been completed, and will provide a basis for selection of candidates for probiotic bacteria for use in Atlantic halibut larviculture. We expect these results to have a major positive impact on juvenile production of Atlantic halibut, by providing a rearing system with higher stability and reduced use of antibiotics compared to the FT systems that are commonly used at present.

2.3.5 Wreckfish
This was the first time that a project succeeded in producing juveniles weaned to inert food, and it signifies a milestone in the efforts to produce wreckfish under aquaculture conditions. The work in DIVERSIFY acquired important data on growth and increased our knowledge about the feeding protocol and the specific behavior and metamorphosis of wreckfish larvae. These data could be the starting point for future experiments and a reality to propose the cultivation of wreckfish as a possibility for the diversification of EU aquaculture. The success in producing wreckfish juveniles in captivity has generated great interest among aquaculture companies interested in diversifying and innovating their production, from Spain to Greece. The general interest for scientific advances in the cultivation of wreckfish is clear, and in particular from the few companies that currently maintain broodstocks of this species, and see the possibilities of incorporating this species in their production increasing. The increase of our knowledge of this species in the larval and juvenile period are definitely very important for the encouragement of subsequent projects and studies.

2.3.6 Grey mullet
The results of the WP on larval husbandry in grey mullet improved significantly many practical aspects of the current larval rearing protocol. The most effective concentration, turbidity and type of microalgae added to the tanks, in terms of larval growth, survival and rotifer ingestion, was determined. Moreover, it was shown that lyophilized microalgae was just as effective as the use of live microalgae, in terms of tank turbidity, larval rotifer consumption, swim bladder inflation, growth and survival. The use of dry microalgae instead of live represents a considerable saving in infrastructure, labor and off-the-shelf convenience. Although the co-feeding of copepods and rotifers to larval grey mullet was not completed in this WP due to technical complications, a protocol for the ozone disinfection of copepods before feeding them to the larvae was developed. This is particularly important in controlling the red-pigmented pathogenic bacterium Pseudoaltermonas spp., which can decimate or eradicate larval populations of grey mullet and other species.

This WP determined the dramatic changes in digestive tract enzyme activity, as a function of age, during the transition from carnivorous larvae to omnivorous juveniles. Alkaline phosphatase activity, a marker for brush border absorption, was ca. 8 times higher and α-amylase activity increased 5.3 times in 79 dph fish compared to 40 dph individuals. In addition, gut maturation occurred relatively late at around 61 dph. These results suggested that fish performance would improve, in terms of growth, survival, feed efficiency and gut maturation, when fed an omnivorous diet. This was confirmed in weaning studies which demonstrated that fish fed an omnivorous weaning diet performed markedly better than cohorts fed a strictly carnivorous or herbivorous diet. These findings suggest that aquaculture feeds at this developmental stage should include not only considerable protein but also higher levels of starch or other low cost amylolytic energetic compounds compared to starter feeds fed to younger grey mullet or the juvenile stages of carnivorous species. The combined results of this WP were implemented in the development of a grey mullet larval rearing protocol, which was tested in 6 m3 semi-commercial V-tanks. This resulted in a juvenile production of ca. 200,000 fish where survival was > 20% from egg to 60 dph. This result is certainly a proof that hatchery production of grey mullet is possible, and we expect the industry to capitalize on this in the coming years.

2.4 Grow out husbandry
As with the larvae, juvenile rearing systems are artificial environments, with numerous factors influencing behavior, growth and performance. For species such as meagre and pikeperch, improvements in fish growth and husbandry have been addressed to refine the existing protocols and facilities in order to solve existing bottlenecks. In contrast, emphasis has been given to developing new species-specific rearing protocols in the case of grey mullet and greater amberjack, since these were species with important knowledge gaps in these areas. The output of these tasks is the development and refining of rearing protocols for the selected species that will result in the improvement of current practices, and will provide an increase in production yields.

2.4.1 Meagre
The information on variable growth rates and optimal feeding methodologies has clarified how to address these bottlenecks. The knowledge on the biology of meagre in the grow out systems has identified optimal feeding methodologies. Optimal feeding methodologies should improve feed utilization and reduce feed conversion ratios (FCR). Feed is the most expensive aspect in growout and even small reductions in FCR can translate to massive economic gains to reduce production costs. The work on optimal feeding methodologies also demonstrated that feeding and husbandry aspects were not related to variable growth rates. Variable growth rates were related or partially related to genetic aspects indicating that genetic breeding programs and domestication should reduce or solve this bottleneck. Therefore, the implementation of genetic breeding programs (see meagre reproduction) can in addition to improving growth rates, reduce growth variation to facilitate management of stocks and provide a uniform product. Improved growth rates and improved feed utilization for meagre would increase production and decrease production costs. These advances would enable the industry to establish management practices that can in the long term improve production and, therefore, attract investment for existing and new companies for the culture of meagre. Improved production and investment in aquaculture companies that produce meagre would have socio-economic benefits for the areas, often rural coastal communities, where meagre is cultured.

2.4.2 Greater amberjack
The results obtain in DIVERSIFY are of great importance, as several barriers for the industrial production of the species have been removed. The information accumulated during the project enriched the specific knowledge base with a range of parameters appropriate for the biological requirements of the greater amberjack (temperature ranges, feeding rates, stocking densities, innate immune status). Of particular importance was the involvement of private companies in the implementation of task and the associated direct transfer of research results in the everyday husbandry practice in the farms. Worth noting is the initiation of the species commercialization in Greece by project partners and follow up by more companies. Despite the difficulties identified related to health management and husbandry, the growout of the species is feasible. Alongside with the results obtained for the larval rearing of the species it is apparent that greater amberjack may represent a new aquaculture product. Due to its characteristics (size, market, etc.) this may result in wider socioeconomic implications that can shape the Mediterranean aquaculture.

2.4.3 Pikeperch
Due to its fast growth, high quality flesh and high economical expectation, pikeperch is one of the most promising freshwater fish species for the diversification of European inland aquaculture. However, its culture is still limited by impairment in growth rate and high mortality rate during on growing. These failures may be related to inadequate rearing conditions inducing high stress level since the pikeperch aquaculture management has not been optimized yet. It has been shown that percid fishes are more sensitive to aquaculture stressors than other species with a longer history of domestication. And since decreased welfare may lead to increased stress level and to disease outbreaks, it was essential to improve its management strategy. Our results defined key elements to improve pikeperch welfare, leading to the improvement of pikeperch culture. The results include specific environmental conditions that reduce stress and, as a consequence, the negative effects on growth, immunity and disease resistance. In addition, our results on domestication level and geographical origin establish the basic knowledge for future selection studies of pikeperch strains, which are essential for the development of pikeperch aquaculture.

2.4.4 Grey mullet
This WP found that fish meal substitution between 50 and 75% by a mixture of different plant protein sources (corn gluten, wheat gluten and soy protein concentrate) in wild grey mullet fry weaned onto compound diets, did not affect growth performance and survival. This means that weaning diets for wild grey mullet harvested for restocking aquaculture ponds and on growing may be formulated with a high level of alternative plant protein sources. Diets with a 50 and 75% of fish meal replacement by plant protein sources were 15.5 and 23.6% cheaper, respectively, than a diet containing fish meal as its protein source. This is very relevant considering that the expense of feed account for >50% of the production costs in aquaculture facilities. Separate experiments tested the effect of stocking various sizes of grey mullet at different densities in a range of cement and polypropylene tanks. The results demonstrated that increasing the fish stocking density above 6 fish per m2 can led to decreased growth in an increasing segment of the population resulting in larger numbers of smaller fish. Further studies testing the effect of different mullet stocking densities in grow-out ponds in Greece (4 and 6 individuals per m2), Spain (0.5 and 1.0 fish per m2) and Israel (1 and 2 fish per m2) resulted in generally poor growth with no significant effect of density or observed differences in proximate and fatty acid analyses. A major impediment to better fish performance is likely the extruded diet. This formulated feed remains not sufficiently attractive to the fish as they appear to prefer the detritus and primary productivity of the pond to the more nutrient dense diet. Moreover, in earthen ponds the mullet are likely using sediment to aid mashing of the plant material in the gizzard for better digestion and absorption. In order to improve the feasibility of intensive monoculture of this species, the dietary formula and feeding protocols of the current grey mullet feed must be improved. In future studies, the effect of increased ration size, use of extruded and not pelleted diets as well as the number of meals per day (simulate continuous feeding) will be tested. This should reduce the number of slower growing, smaller fish in the population, and increase the efficiency of grow-out.

2.5 Fish Health
Fish health is a key aspect to be optimized in cultured fish. The effect of the developmental stage, rearing conditions and nutrition on the capacity to modulate specific immune responses will help predict vaccine responsiveness and fish health. DIVERSIFY has characterized the immune system of meagre and greater amberjack to identify key immune molecules as potential markers of immune system development, and induction of antiviral and antibacterial responses in preparation for vaccine development for disease management. In addition, potential solutions for specific bacterial infections and parasitoses have been investigated, providing means to prevent and/or minimize these issues at an industrial scale. DIVERSIFY has produced practical health manuals for meagre and greater amberjack, which are freely available and can be used immediately by the industry in order to improve their stock management.

2.5.1 Meagre
One of the requirements for the successful introduction of a new species in the aquaculture industry is the knowledge of the pathological problems that may arise. For Systemic Granulomatosis and Chronic Ulcerative Dermatopathy, which are diseases that affect 100% of meagre cultured population, diagnostics protocols with causes and solutions were produced addressing fish health specialists and aquaculture scientists and producers. The etiological factors of these two diseases are still unknown; however several factors have been eliminated and the results obtained from DIVERSIFY may lead to future investigation. In addition, in the effort to find alternative treatment for parasitoses, parasiticides obtained from natural sources such as cinnamon, which can be administered with the feed, would be tremendously convenient and safe for use from the aquaculture industry. Through the various tasks of DIVERSIFY, immune markers are now established for the innate, adaptive and inflammatory responses of the immune system of meagre. The assays that developed for genes of interest for the study of immune function in this species, will be of interest for anyone who may be studying this species outside of the DIVERSIFY consortium.

2.5.2 Greater amberjack
New markers have been described to assess changes in immune status of greater amberjack. These tools are an important advance in our ability to evaluate the health status of this species, which is highly susceptible to parasite infections. A functional diet has been formulated to increase resistance of greater amberjack to the monogenean parasite Neobenedenia girellae and could be applicable for other monogenean parasites. This diet is based on a high protein diet (required for fast growing species) and the utilization of additives with immunostimulant properties, including mannan oligosaccharides. This important milestone will provide a tool to reduce the incidence of this parasite in sea cages, reducing mortality of greater amberjack juveniles in farms. Besides, the formulation of the functional diet will have a direct impact on feed producers that can produce an effective feed for improving the amberjack quality in farms.

A Health Manual for greater amberjack describing different pathologies has been produced, that describes the general incidence and potential treatments and some tools for identification. Epitheliocystis, Zeuxapa seriolae, the blood fluke Paradeontaxylix sp. and the copepod Penella sp. has been described. The description of new or emerging pathologies for greater amberjack is of special importance for a rapid diagnosis and selection of the most efficient treatment to reduce mortalities in farms.

2.5.3 Atlantic halibut
Artemia showed to uptake and accumulate the various forms of recombinant Nodavirus capsid proteins and act as a vector for oral delivery to late larval/juvenile stage of Atlantic halibut. The challenge experiments, however, was inconclusive whether this strategy of antigen delivery does induce protection against Nodavirus infection.

2.6 Socioeconomics
The impact of the Socioeconomic work package of DIVERSIFY goes much further than marketing of the new species. Primary insights for the new species are provided, on how the product can be marketed with added value, what segments of consumers could be interesting in the selected five countries, how the product should be priced to have a good margin and where costs could or should be reduced to improve the margins. So entrepreneurs active in the species can have direct practical use of the outcomes of the project. Furthermore, the project has provided a lot of new scientific insights on consumer behavior, choice motives and sensory perception. This part of the project has provided a large number of scientific papers in peer reviewed international scientific journals. In addition to the objectives of the project, all this research has provided also new insights in choice and buying behavior of fish in the five main markets of the EU, which can be of interest for all stakeholders in the fish sector. In relation to other sectors, scientific consumer research in the fish sector is very unique, so the participants in this project have actively shared the outcomes with stakeholders in Germany, Spain, Italy, Greece and the Netherlands.

2.6.1 Final product development
The production at an industrial scale of the developed products from the DIVERSIFY fish species can be a feasible task, subject to the application of certain principles and conditions. The technical yields that can be achieved are very satisfying for all products, thus providing high profit margins. The duration of high quality life ranges depends on the product nature, but also on the optimization of processing and preservation procedures. Frozen and sterilized products (e.g. oil-preserved fish fillet and fish pate) have long high quality shelf life, spanning in months, while fresh products have a high quality shelf life of a few days, varying with the ingredients included (most sensitive ingredient is the limiting factor), the manufacturing process and the packaging type. The principles for proper production include three aspects: 1. raw materials of good quality 2. good manufacturing (processing) practices. 3. Proper traceability. Freshness of the raw materials should always be ensured. The ISO and HACCP principles should apply throughout the whole processing chain and commercialization for ensuring safety and maximum quality. Food traceability systems should be implemented in all products but also in raw materials that are incorporated during processing. These rules are necessary and sufficient condition for high quality and economic sufficient products.

Altogether, the activities included and results produced by DIVERSIFY provide a broad picture of the main quality characteristics of the raw material and the developed products, thus giving a valuable input for those interested in the commercialization of any of the studied species and/or products..

2.6.2 Consumer value perceptions and behavioral change
Economically feasible production of new aquaculture products is dependent on stable consumer demand. To be able to guarantee stable consumer demand, it is of outmost importance for the aquaculture industry to increase consumer acceptance of European aquaculture and new aquaculture products optimized to consumer preferences and eating habits. Marketing and communication strategies produced by DIVERSIFY increase consumer trust and endorse stable consumer demand.

Newly developed products from DIVERSIFY fish species have desirable nutritional characteristics and can be part of a balanced diet that contribute to consumer health. This may only be accomplished, though, if consumers develop preferences for these products, that is find claims about their nutritional and health properties reliable, and accept the underlying production method. The effort undertaken in the DIVERSIFY project proliferate the product-market fit and lead to improved consumer acceptance of the newly developed aquaculture products.

2.6.3 Business model and marketing strategy
Fish farming is still a very product- and production-driven industry. As a result, diversification efforts have focused on eco system development and value creation at the supply side, and much less on developing the eco-system at the demand side. Although farmers will benefit from continuing to work with partners to enhance their production processes for increasing quality/growth and decrease cost, it is clear that serious investments in marketing and sales/channel management are/will be needed to develop the market and create socio-economic gains of employment and healthier living. Certification is a license-to-produce for large retail chains and lack of such quality standard may cripple farmers’ business models for their new species. Only with a buy-in from distribution partners and adequate marketing efforts can retailers and consumers be reached and convinced to purchase the new species and their products. Such marketing efforts may benefit from country/region of origin branding and health claims (e.g. high Omega 3 content), among others. Such efforts will make business models for these species more resilient, resulting in a positive long lasting impact on the providers’ business and industry. The best chances exist, in the short run, for meagre, greater amberjack and pikeperch, i.e. the species that are most production ready.
Aquaculture offers important opportunities for new species of the DIVERSIFY project. However, carefully positioning the species/new products and managing the reputation of farmed fish are important to prevent bad media coverage about farming conditions at fish farms outside the EU (e.g. in Asian and African countries) from spoiling the market. Through their market strategy and promotion of their new species providers can help not only increase fish consumption, but stimulate more balanced diets and healthier living in the countries they decide to target.

2.7 Sustainability
Sustainability of aquaculture production is a major concern worldwide. DIVERSIFY has considered this issue from different points of view. For example, the acquired knowledge will support the growth and expansion of the sector based on different production systems that can be regarded as more sustainable: cage culture – no competition with land resources; RAS- ecologically friendly, with efficient use of water; extensive pond-lagoon culture, with very low environmental effects and in some cases even contributing to the restoration of ecosystems. Also, the introduction of an omnivorous fish into the aquaculture sector, such as the grey mullet, with positive influence in the environment where it is cultured and requiring low or close to no input from fish meal/oils, will contribute to the reduction of the pressure in the capture fisheries. The determination of species-specific dietary requirements, as well as feeding behavior will result in less waste of diets and nutrients into the environment. Altogether, these factors will ensure a sustainable growth and expansion of aquaculture within the EU and EEA member states.

So, overall, the main expected impact of DIVERSIFY will be the improvement of production technologies for the new/emerging species of the project. Furthermore, DIVERSIFY is expected to have also a significant impact on removing bottlenecks in markets and consumer's perception and preferences, resulting in increases in the EU consumption of aquaculture products. Such an integrated combination of biological, technological and socioeconomic activities will lead to a reduction in the dependence of the EU on imports from third countries of at times questionable production, health, environmental and social standards.

2.8 Description of the main dissemination activities
(For the Figures and Tables cited in this Section, please see the attachment “DIVERSIFY Final Report Appendix I.pdf)

During the five years of DIVERSIFY, multiple dissemination activities have been carried out to broadcast the findings and outputs of the project. The website of the project (Fig. 2.8.1) was one of the first dissemination tools to communicate the main news on the research activities and to update the general public with the latest events and achievements in the project. The website has evolved with the project and its structure has been adapted to the project evolution. Along the project life, the website has proven to be a very powerful tool also to keep the large consortium of DIVERSIFY up to date with the internal meeting organization, the legal documentation of the project, the deliverables, reports and publications.

In the first year of the project (M6), a printed folder (initial project folder, Fig. 2.8.2) with the main information of DIVERSIFY was design and distributed to all the partners and in all the meetings, conferences and events in general attended by the PC and the Dissemination leader, as well as by any project partner. An estimated 2.600 initial folders have been distributed during the project’s lifetime. A final folder has been designed in the year 5 of the project with the objective of presenting the most relevant results of the DIVERSIFY research for each of the fish species in a summarized way (Fig. 2.8.3). The folder also includes a concise resume of the socioeconomic findings and the updated list of participants. This final folder has also been distributed in different types of events, starting with the Final Coordination meeting held in Brussels, in November 2018, and will be distributed to future aquaculture event in 2019 and 2020.

During the five years of existence, DIVERSIFY has been presented in different forums and conferences involving all kind of audiences. In addition to presentations in the European Aquaculture Society and the World Aquaculture Society meetings, DIVERSIFY presentation were given to specific scientific events focused on fish biology and culture, such as LARVI; the International Symposium on Fish Reproductive Physiology, the International Symposium on Fish Nutrition and Feeding; the International Conference on Diseases of Fish and Shellfish, etc.; to meetings with Regional and National Government representatives (Regional Development Officer from Junta de Andalucía, Spain); or presentations to university students (National Aquaculture Day, University of Cádiz, Spain).

DIVERSIFY has been brought to the aquaculture stakeholders and to the society in general through more than one hundred press releases, websites, applications and articles published in the popular press along its life time. The project has been presented to a wider public and to scientific events more than 120 times (oral presentations). During the five years of the project, DIVERSIFY related scientific information has been displayed with 66 posters in conferences and aquaculture events all over Europe. A total of 15 videos have been produced to bring the results of the project to the society as audiovisual material. All the videos are available in the project web, including a 3-min professional dissemination video (Fig. 2.8.4). DIVERSIFY has been present in the social media too, in twitter and Facebook with a total number of 490 followers.

With regard to scientific publications, at the moment of writing this report, a total of 44 scientific articles have been published in peer-review scientific journals, and 6 are under review. More publications are expected to be published in the coming months since there is still a considerable amount of data and information being processed by the researchers and the project has successfully produced more than 200 Deliverables, the vast majority of them with scientific results, which will find their way in the scientific literature.

DIVERSIFY has also given special attention to the dissemination of the project’s results to the aquaculture sector and to the whole seafood value chain. Two types of activities have been developed during the last two years of the project to achieve those objectives:

Promotional Workshops were specialized one-day workshop in strategic countries to present the project’s activities and results. These events were addressed to fish farmers, feed producers, farmer’s associations, fish processors, fish retailers, hypermarkets, consumer organizations and aquaculture authorities. Four Promotional Workshops were organized, one each in Germany (Bremen), Spain (Cádiz), Italy (Verona) and Greece (Athens). Each workshop consisted of several presentations dealing with fish products, consumer issues, regulatory issues, etc., and was followed by a debate to analyze with the audience the acceptance of aquaculture products, consumer perception, value added products and trends in farmed fish markets. The Promotional Workshops had an average attendance of 30 people, which promoted constructive and highly informative debates on the real constrains of the market acceptance of new fish species.

Species Workshops were full day seminars on know-how transfer to the fish producers and researchers. Six Species Workshops (one per species) were organized in different European countries, where the species was well known and where it could have a potential to become industrially produced aquaculture species. In Table 3.1 species, locations and timing of the Species Workshops are included. The Species Workshops were designed to have a set of oral presentations from the DIVERSIFY partners working on the specific species and also presentations from other researchers and specialists in the world invited for the meeting. The event was addressed to fish farmers, feed companies, pharmaceutical companies, equipment suppliers, academia, government institutions and aquaculture media. The agenda of the event included 10 presentations of 30 minutes each to explain and present the advances and achievements of DIVERSIFY with the species of interest and the corresponding work done on the socioeconomic aspect such as the development of new/traditional product presentation and the business model developed. A final debate on the possibilities of culturing the species and on the concerns of the industry representatives present in the conferences allowed to identify the main bottlenecks for incorporating the DIVERSIFY species into the production of aquaculture companies.

As part of the exploitation of the research done in the project, six Technical Species Manuals have been produced and are freely available at the website of the project, in English, Spanish and German. Some of them have been translated to other languages as well. These Manuals provide protocols and instructions to culture each of the species of DIVERSIFY. Market studies and, especially, the business models per species have been followed closely during the presentations in the Species Seminars. Moreover, the transfer of the developed know-how in DIVERSIFY has been materialized also in the production of two Health Technical Manuals, one for greater amberjack and another one for meagre. Both documents are freely available at the DIVERSIFY website. A new device for collection and quantification of parasites in tanks has been also developed by DIVERSIFY research and it is available for the farmers.

The presence of DIVERSIFY during Y4 and Y5 in the Seafood Expo in Brussels and the establishment of close links and cooperation with the European Association of Fish Processors and the Association of International Seafood Professionals have been very helpful dissemination tools to bring the DIVERISFY message on new species to the fish processing and new fish product development sector.

Also, within the Exploitation of project outcomes, the results of the project have lead to the production of 35 Master Thesis and PhD Dissertations on greater amberjack (15), pikeperch (8), meagre (6), grey mullet (2) and on new product development (3).

List of Websites:
The website of the project is It is managed by the Project Coordinator Dr Constantinos C Mylonas of the Hellenic Centre for Marine Research (Greece) and the WP31 Dissemination leader Dr Rocio Robles from CTAQUA (Spain). Their full contact details are shown below.

Dr Constantinos C Mylonas
Director of Research
Hellenic Center for Marine Research
Institute of Marine Biology, Biotechnology and Aquaculture
POBox 2204, Heraklion 71003
Crete, Greece
tel +30 2810 337878
mob +30 6944 229754

Rocio Robles
Ctaqua Technical Consultant
Edif. Ctaqua
Muelle Comercial s/n
11500 El Puerto de Santa María

The site will remain active for the next few years (at least 2) and be updated with relevant information such as published articles (Scientific Journals and Sector magazines), and organisation of meetings and workshops.