Periodic Reporting for period 1 - NEUROSHELL (Advancing knowledge and acquiring expertise in shellfish research by investigating environmental, neuroendocrine and endocrine control of key stages in oyster aquaculture, using a rhythmic approach) Reporting period: 2019-09-02 to 2022-09-01 Summary of the context and overall objectives of the project NEUROSHELL has made significant scientific progress, in the understanding of the environmental and endocrine control of physiology in European native oyster (Ostrea edulis), for practical application to aquaculture. Bivalve shellfish farming is rapidly expanding worldwide, it is an industry with low environmental impact, yet yields an end-product of high nutrient density. Increase in hatchery efficiency and seed availability, to support this expansion, is however being hampered by a lack of synchrony of animals, with inconsistency in broodstock conditioning and difficulties in spawning prediction and larval development. The overall objective of NEUROSHELL was to help address these issues, through focused research on the environmental/endocrine control of O. edulis physiology. Native oyster aquaculture heavily depends on hatchery seed supply not only for on-growing for the food market but for coastal restoration initiatives and for the future-proofing of oyster beds/breeding ponds vulnerable to years when there is poor natural seeding.NEUROSHELL consisted of two research sub-projects each focusing on a key life stage event: Sub-project 1) Reproduction in Adults, Sub-project 2) Early Development and Metamorphosis in Larvae. Within each sub-project, a multi-targeted approach was employed combining in vivo experiments on conditioning regimes (temperature, light), with endocrine factor gene characterisation and profiling. Significant results included the definition of a number of key endocrine players (neurohormones, neurotransmitter receptors) with potential roles in decoding environmental information into physiological effects, evidence of molecular clock and melatonergic system components in O. edulis, and the evaluation of broodstock conditioning regimes. Such results have not only increased the state of the art in the field, contributing to laying the foundations for future research studies tailored to industry needs, but may also be used directly improve hatchery production. In addition to the scientific core of NEUROSHELL, shellfish aquaculture industry integration, transfer of knowledge and training of the Research Fellow including a secondment in Spain, were key objectives of the project.In a broad sense, NEUROSHELL has built upon an essential platform for EU research and excellence: cutting-edge science for practical application in shellfish aquaculture. The advancement of the Fellow’s training and career, established position at the host institution and strong long-lasting collaboration with Spain can only contribute to strengthening this platform. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far SUB-PROJECT 1: adult Ostrea edulisObjective 1A). Two 7-month in vivo studies on reproduction: (I) Effects of daily ambient thermocycles under fixed daylength conditions of 18L:6D (light:dark hours) white light; (II) Effects of daily photocycles and spectrum under fixed constant temperature conditions. Four lighting treatments were set-up: Constant White Light at 24L:0D; Simulated Natural Photoperiod using White light; Blue Light at 18L:6D; Red Light at 18L:6D. Spawning occurred under all regimes tested, indicating the reproductive plasticity of the oysters. Nevertheless, histological analyses of the gonads revealed a poorer synchronisation of individuals under the Constant White Light and Red Light regimes, the latter also delayed reproductive development. At the endocrine level, profiles of neuropeptide and glycoprotein elevation were more pronounced for oysters maintained under ambient versus constant temperature. The Simulated Natural Photoperiod regime (at constant temperature) yielded similar trends of elevation but at a reduced scale. It is well known that temperature is the over-riding parameter affecting reproduction in oysters, but this work also demonstrates the relevance of lighting for the synchronisation of individuals.Objective 1B). 48-hour profile sampling of oysters (12 every 6 hours) maintained outside under natural light and temperature conditions. Day-night differences in the gonads in neurohormone glycoprotein levels were observed, suggesting a potential role in synchronisation. Objective 1C) successful development of molecular tools (qPCR assays for gene expression analyses) for studying the endocrine/environmental control of reproduction in O. edulis. Sex steroid (estradiol, testosterone) levels were measured using a commercial ELISA kit, optimised in-house.SUB-PROJECT 2: larval Ostrea edulis Objective 2A). Two in vivo experiments on early development: (I) Effects of daily thermocycles under a white light photoperiod of 12L:12D. Three daily temperature regimes were set-up: Constant High Temperature (20°C), Constant Low Temperature (16.5°C) and a TC cycle (thermophase-cryophase, 18-15°C); (II) Effects of daily photocycles and spectrum, under constant temperature conditions. Four lighting conditions were set-up: Constant White Light at 24L:0D, and White, Blue or Red Light at 12L:12D. Larval survival and growth were measured throughout with main results indicating that the Red Light regime significantly reduced the survival of larvae. In parallel, samples of outdoor reared larvae were obtained from a commercial grower. Neurotransmitter and hormone receptor genes were measured in these samples at three principal pre-metamorphosis stages (from 150-400 µm), a gene-dependent increase in expression was observed. Objective 2B) demonstration of the existence of clock and melatonergic system machinery in O. edulis. Gene expression of both systems was evidenced in outdoor reared larvae at all three stages measured (see Objective 2A). To investigate rhythmicity of these genes, expression was measured in adult oysters sampled under a light-dark cycle (see Objective 1B). Day-night differences were observed in clock gene expression, in mantle and gonad tissue.Objective 2C) successful development of molecular tools (qPCR assays) for studying the endocrine/environmental control of early development in O. edulis.Exploitation of results and dissemination through transfer of knowledge has ranged from industry conference attendance, discussions with the oyster industry (hatcheries, growers) about broodstock conditioning regimes/reproductive technologies, to site visits and discussions with research groups and restoration project representatives. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Progress, beyond the state of the art, and innovation outputs for O. edulis include: 1) Localisation of neurohormone and glycoprotein reproductive systems i.e. which genes are expressed in which tissues (central nervous system, peripheral), 2) qPCR assays for novel markers of reproduction (neurohormones, glycoproteins) and markers for metamorphosis (neurotransmitter receptors) , 3) qPCR assays for molecular clock and melatonergic system components, 4) Temperature and Light Conditioning Regime evaluation, for optimising reproduction, synchronisation and promoting survival.Results from NEUROSHELL may be used not only to directly improve hatchery production (e.g. conditioning regimes) but they have also contributed to laying the foundations for future research studies (e.g. functional reproductive hormone diagnostics). Such Research and Innovation is essential for supporting O. edulis and other bivalve industries. Improved larvae production efficiency and security in seed supply being a fundamental requirement for the resilience and expansion of EU shellfish aquaculture, for food and for restoration. neuroshell.jpg
