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European eel, Anguilla anguilla, larval culture faces a bottleneck during the transition to exogenous feeding. To stimulate gut-priming, in the present study, prebiotics (AgriMOS, mannan-oligosaccharides, and beta-(1,3 and 1,6)-poly-D-glucose), probiotics (Bactocell, Pediococcus acidilactici), and synbiotics (AgriMOS + Bactocell) were administered to European eel larvae during the endogenous prefeeding stage. Eel larvae were reared in 2 L incubators with an initial stocking density of similar to 200 larvae/L. Each treatment (pre-, pro-, synbiotics, and control), represented by 3 replicated incubators, was connected to a separate recirculating aquaculture system. The gut-priming agents were introduced directly into the rearing water. Results revealed increased mortality when larvae were introduced to synbiotics and impaired growth in connection to probiotics and synbiotics. Larvae receiving prebiotics showed similar survival and growth to larvae reared without gut-priming agents. The immune gene expression revealed a lag phase between maternally inherited protection (c3, igm, and il10) and the gradual buildup of the larvae's own immune system (il1 beta, irf7). The lack of treatment-related immune (c3, igm, il10, il1 beta, and irf7) and stress/repair (hsp70, hsp90) responses revealed an immature immuno-readiness. Digestion (try, ctra, ctrb, tgl, and amyl), food intake (cck), and appetite (ghrl)-related genes were expressed at basal levels already on 4 days post-hatch, which combined with phenotypic plasticity of the appetite-regulating ghrelin (ghrl), indicated a prospective adaptive capability towards earlier maturation of the larval digestive capacity. Overall, we contemplate that the application of gut-priming agents in water has merit; however, as no beneficial effect was observed, we conclude that the regimen applied is not recommendable in the present form and needs to be customized for future eel larval culture. As such, water management strategies and rearing options need to be further explored to establish prefeeding and feeding regimens, targeting optimized culture conditions, and the production of healthy eel offspring. Grant agreement nos. 7076-00125B (ITSEEL) and the ENV-Fund info:eu-repo/semantics/publishedVersion

[EN] Aquaculture production relies on controlled management of gametogenesis, especially in species where assisted reproduction is needed for obtaining gametes in captivity. The present study used human chorionic gonadotropin (hCG) treatments to induce and sustain spermatogenesis in European eel (Anguilla anguilla). The aim was to evaluate effects of strip-spawning timing (12 vs. 24 hr) after weekly administration of hCG and the necessity of a primer dose (in addition to weekly hormonal treatment) prior to strip spawning (primer vs. no-primer) on sperm quality parameters. Sperm parameters included milt production (weight), density and sperm kinematics at Week 9, 11 and 13 after onset of treatment. Spermiation commenced in 11.5% of males in Week 5 and by Week 9, and all males produced milt. Male weight, milt production, sperm density and spermatocrit did not differ among hormonal treatments during the experimental period. Overall, male weight decreased from 106.3 to 93.0 g, milt weight increased from 3.5 to 5.4 g, sperm density counts decreased from 11.7 x 10(9) to 10.5 x 10(9) cells/ml, and spermatocrit decreased from 46.5% to 40.5%. Furthermore, spermatocrit was positively related to haemocytometer counts (R-2 = .86, p < .001), providing a reliable indicator of sperm density. Differences in sperm kinematics were observed depending on strip-spawning timing after hormonal injection (12 vs. 24 hr) but with no consistent pattern. These sperm quality parameters also did not consistently differ between the no-primer and primer treatments. Considering that each male may be stripped 4-5 times over the 2-3 months spawning season, omitting the primer would reduce animal handling, material costs and labour intensity, while sustaining high-quality sperm production., Innovation Fund Denmark, Grant/Award Number: 7076-00125B; Programa Juan de la Cierva-Incorporacion, Grant/Award Number: IJCI-2017-34200

Egg biochemical composition is among the main factors affecting offspring quality and survival during the yolk-sac stage, when larvae depend exclusively on yolk nutrients. These nutrients are primarily embedded in the developing oocytes during vitellogenesis. In aquaculture, assisted reproduction procedures may be applied enabling gamete production. For the European eel (Anguilla anguilla), reproductive treatment involves administration of pituitary extracts from carp (CPE) or salmon (SPE) to induce and sustain vitellogenesis. In the present study, we compared the influence of CPE and SPE treatments on offspring quality and composition as well as nutrient utilization during the yolk-sac stage. Thus, dry weight, proximal composition (total lipid, total protein), free amino acids, and fatty acids were assessed in eggs and larvae throughout the yolk-sac stage, where body and oil-droplet area were measured to estimate growth rate, oil-droplet utilization, and oil-droplet utilization efficiency. The results showed that CPE females spawned eggs with higher lipid and free amino acid contents. However, SPE females produced more buoyant eggs with higher fertilization rate as well as larger larvae with more energy reserves (estimated as oil-droplet area). Overall, general patterns of nutrient utilization were detected, such as the amount of total lipid and monounsaturated fatty acids decreasing from the egg stage and throughout the yolk-sac larval stage. On the contrary, essential fatty acids and free amino acids were retained. Notably, towards the end of the yolk-sac stage, the proximal composition and biometry of surviving larvae, from both treatments, were similar.

Temperature is a major factor that modulates the development and reactivity of the immune system. Only limited knowledge exists regarding the immune system of the catadromous European eel, Anguilla anguilla, especially during the oceanic early life history stages. Thus, a new molecular toolbox was developed, involving tissue specific characterisation of 3 housekeeping genes, 9 genes from the innate and 3 genes from the adaptive immune system of this species. The spatial pattern of immune genes reflected their function, e.g. complement component c3 was mainly produced in liver and il10 in the head kidney. Subsequently, the ontogeny of the immune system was studied in larvae reared from hatch to first-feeding at four temperatures, spanning their thermal tolerance range (16, 18, 20, and 22 °C). Expression of some genes (c3 and igm) declined post hatch, whilst expression of most other genes (mhc2, tlr2, il1β, irf3, irf7) increased with larval age. At the optimal temperature, 18 °C, this pattern of immune-gene expression revealed an immunocompromised phase between hatch (0 dph) and teeth-development (8 dph). The expression of two of the studied genes (mhc2, lysc) was temperature dependent, leading to increased mRNA levels at 22 °C. Additionally, at the lower end of the thermal spectrum (16 °C) immune competency appeared reduced, whilst close to the upper thermal limit (22 °C) larvae showed signs of thermal stress. Thus, protection against pathogens is probably impaired at temperatures close to the critical thermal maximum (CTmax), impacting survival and productivity in hatcheries and natural recruitment.

The future of European eel aquaculture depends on closing the life cycle in captivity. Present focus is on developing suitable larval rearing technology. This study explored new salinity reduction applications to elucidate performance thresholds of European eel larvae produced under realistic hatchery conditions, using Kreisel tanks and recirculating aquaculture systems for larval culture. The study links eel larval survival and biometrics to expression of genes related to underlying molecular mechanisms by taking parental effects into account. Larvae from different families were reared either at constant salinity of 36 psu (Control) or subjected to salinity reduction (36 to 18 psu) initiated 3 days post hatch (dph) and at a rate of 4 psu/day, occurring either within 1 h (Fast) or 24 h (Slow). An extreme scenario, reducing salinity directly from 36 to 18 psu within 1 h on 6 dph (Drastic) was also tested. Early and gradual salinity reduction (Slow or Fast) led to increased growth rate and larger larvae, while influencing the expression of dio3 (deiodination mechanism and thyroid endocrine system). Expression of atp6 and cox1 (energy metabolism) was constant, indicating that energy metabolism was stable and independent of salinity, while expression of nkcc1a (ion regulation) was upregulated in the Control, suggesting an upregulation of active Na+, K+, and Cl− transport and thus increased cellular energy consumption. This explained that eel larvae experiencing an early and progressive salinity reduction, used their energy reserves more efficiently, leading to improved growth and survival. However, salinity reduction caused heart edema. Expression patterns of 12 genes [stress/repair (hsp90), immune response (mhc2), neurogenesis (neurod4), deiodination (dio2), thyroid metabolism (thαa, thαb, thβb), energy metabolism (atp6), skeletogenesis (bmp2b, bmp5), growth (igf2b), ion regulation (nkcc2b)] on 6 dph and 5 genes [water transport (aqp3), immune response (il1β), thyroid metabolism (thβb), skeletogenesis (bmp5), heart development (nppb)] on 12 dph were driven by genotype (family) × environment (salinity) interactions, revealing batch specific phenotypic plasticity and describing a genetic programming of molecular mechanisms and intrinsic sensitivity to environmental drivers that need to be considered in future eel aquaculture. In conclusion, early and progressive salinity reduction (Fast or Slow) benefits larval eel growth and survival, but emerging implications regarding heart edema need to be addressed in future studies. On the other hand, we show that biotechnical difficulties for introducing salinity reductions, can be circumvented by directly moving larvae from seawater to isoosmotic conditions, but suited application timing needs to be explored.

BONUS XWEBS Deliverable D2.1

Hormones and mRNA transcripts of maternal origin deposited in the egg may affect early embryonic development in oviparous species. These hormones include steroids, such as estradiol-17β (E2), testosterone (T), 11-ketotestosterone (11-kt), 17α,20ß-dihydroxy-4-pregnen-3-one (DHP), and cortisol, which also play an important role in fish reproduction. In European eel, Anguilla anguilla, which does not reproduce naturally in captivity, vitellogenesis in female broodstock is commonly induced by administration of salmon or carp pituitary extract (PE) as an exogenous source of gonadotropins, while follicular maturation is stimulated by a priming dose of PE followed by provision of DHP as a maturation inducing hormone. In this regard, the main purpose of the present study was to evaluate effects of induced follicular maturation on reproductive success in European eel, focusing on maternal transfer and dynamics of steroids and mRNA transcripts of growth- and development-related genes throughout embryogenesis. The results showed that maternal blood plasma concentrations of E2, T and DHP were reflected in the unfertilized eggs. Moreover, a negative relationship between concentrations of E2 and DHP in eggs and embryos and quality parameters measured as fertilization success, cleavage abnormalities, embryonic survival, and hatch success was found. Concomitant mRNA transcript abundance analysis including genes involved in stress response (hsp70, hsp90), somatotropic axis (gh, igf1, igf2a, igf2b), lipid (cpt1a, cpt1b, pigf5) and thyroid metabolism (dio1, dio2, dio3, thrαb, thrβa, thrβb) varied among unfertilized egg batches. For the majority of genes, mRNA abundance increased during the maternal-to-zygotic transition in connection to activation of the transcription of the embryos own genome. mRNA abundance of dio1, cpt1a and cpt1b throughout embryogenesis was related to embryonic developmental competence. Notably, mRNA abundance of dio3 was positively associated with E2 concentrations, while the mRNA abundance of thrαb was negatively related to T concentrations in the unfertilized eggs, which may suggest an interaction between the thyroid and steroid hormone systems. Altogether, maternal plasma concentrations of E2 and DHP were reflected in the eggs, with high concentrations of these steroids in the eggs being negatively associated with embryonic developmental competence. Additionally, high transcript levels of two of the investigated genes (dio1, cpt1b) were positively associated with embryonic developmental competence. This study reveals maternal transfer of steroids and mRNA transcripts to the eggs, which may be significant contributors to the variability in embryonic survival observed in European eel captive reproduction.

Highlights: • Egg production methods have been used for assessing the Eastern Baltic cod stock. • The analysis contributed to the re-establishment of an analytical assessment for the stock. • Annual and daily egg production methods gave similar results. • Results confirmed trends from trawl surveys, questioned earlier because of potential change in catchability. • Several biological processes impact on absolute spawning stock estimates and research needs are identified to improve these. Abstract: Egg production methods (EPM) provide fishery independent estimates of spawning stock sizes and dynamics of fish populations. Such methods are commonly used for short-lived pelagic species, less so for demersal fish such as cod. In this paper, we apply EPMs on cod in the eastern Baltic Sea, using a long time series of ichthyoplankton data. Stock assessment of Eastern Baltic cod has been challenged due to changing productivity of the stock invalidating some of the standard procedures, e.g. age determination and input variables, e.g. natural mortality. We demonstrate that EPMs, based on other data and assumptions than standard stock assessments, provide useful information on stock status and dynamics. We apply both the annual and daily egg production methods, which yielded similar results and were in line with stock trends derived from bottom trawl surveys. However, the EPM based spawning stock estimates were consistently lower compared to results from the latest analytical stock assessment. We identified processes introducing uncertainties in EPM estimates and their effects on the resulting estimates, and conclude that they mainly affect the absolute estimates but less the relative trends in stock dynamics. Therefore, we consider that EPMs are useful for providing relative indices for stock assessment purposes, with the Eastern Baltic cod being the first case where such indices are included in an official stock assessment of a demersal gadoid species. We also identify knowledge gaps in order to be able to derive absolute stock size estimates from EPMs in the future.

Low egg quality and embryonic survival are critical challenges in aquaculture, where assisted reproduction procedures and other factors may impact egg quality. This includes European eel (Anguilla anguilla), where pituitary extract from carp (CPE) or salmon (SPE) is applied to override a dopaminergic inhibition of the neuroendocrine system, preventing gonadotropin secretion and gonadal development. The present study used either CPE or SPE to induce vitellogenesis in female European eel and compared impacts on egg quality and offspring developmental competence with emphasis on the maternal-to-zygotic transition (MZT). Females treated with SPE produced significantly higher proportions of floating eggs with fewer cleavage abnormalities and higher embryonic survival. These findings related successful embryogenesis to higher abundance of mRNA transcripts of genes involved in cell adhesion, activation of MZT, and immune response (dcbld1, epcam, oct4, igm) throughout embryonic development. The abundance of mRNA transcripts of cldnd, foxr1, cea, ccna1, ccnb1, ccnb2, zar1, oct4, and npm2 was relatively stable during the first eight hours, followed by a drop during MZT and low levels thereafter, indicating transfer and subsequent clearance of maternal mRNA. mRNA abundance of zar1, epcam, and dicer1 was associated with cleavage abnormalities, while mRNA abundance of zar1, sox2, foxr1, cldnd, phb2, neurod4, and neurog1 (before MZT) was associated with subsequent embryonic survival. In a second pattern, low initial mRNA abundance with an increase during MZT and higher levels persisting thereafter indicating the activation of zygotic transcription. mRNA abundance of ccna1, npm2, oct4, neurod4, and neurog1 during later embryonic development was associated with hatch success. A deviating pattern was observed for dcbld1, which mRNA levels followed the maternal-effect gene pattern but only for embryos from SPE treated females. Together, the differences in offspring production and performance reported in this study show that PE composition impacts egg quality and embryogenesis and in particular, the transition from initial maternal transcripts to zygotic transcription.

[EN] Little is known about the role of dietary amino acids on male reproductive performance and gamete quality in fishes. Thus, the objective of this study was to investigate how "enhanced" feeds (EH-4, EH-5, EH-6), with modified amino acid composition, and the standard on-growing diet (DAN-EX) impact body composition, milt biochemistry, and sperm performance in male European eel, Anguilla anguilla. The fatty acid composition of EH4, EH-5, and EH-6 was similar but differed to that in DAN-EX, while amino acid composition varied between all four diets. Diet did not influence organ-somatic indices (e.g. HSI, GSI), while males fed EH-4 were heavier than other groups. Arginine, alanine, and lysine were the most abundant amino acids in milt (>11%), followed by glycine, aspartic acid, valine, glutamic acid, and leucine ( >5.66%). Diet impacted milt arginine, serine, proline, methionine, and histidine levels. Specifically, males fed DAN-EX, EH-4, and EH-5 had the highest percentages of arginine, while males fed EH-4 to EH-6 had higher percentages of serine. Proline was most abundant in males fed DAN-EX, EH-5, and EH-6. Both methionine and histidine were detected at low percentages ( < 2%), and were impacted by diet, where males fed EH-4 and EH-5 had higher percentages of methionine, and males fed DAN-EX, EH-4, and EH-6 had the highest percentage of histidine. Milt production increased over time, where eels fed EH4 and EH-6 showed the highest probability of producing suited milt volumes ( > 0.5 mL) for fertilization procedures. Spermatocrit (43.1 +/- 1.80%) did not differ between the diets (ranged from 37.57 to 47.21%). Dietary regime had an impact on sperm motility, such that eels fed EH-5 and EH-6 had the greatest percentage of motile cells. In addition, fish fed EH-5 and EH-6 (or DAN-EX) had the fastest swimming sperm. Spermatogenic maturity index of hormonally treated eels varied within groups but did not differ between dietary treatment groups after 9 weeks of injections (ranged from 0.54 to 0.80). The most interesting amino acids to scrutinize from PCA plots were proline, histidine, and valine as well as lysine and arginine. Here, eels with highly motile sperm had milt with high relative proportions of proline, histidine, and valine, but were particularly low in lysine and arginine. Together, our findings add evidence that certain amino acids regulate milt biochemistry, and that male ejaculate traits may be promoted by amino acid intake. Further studies to evaluate effects of supplemented amino acid diets on fertilization ability and inter-linked early developmental stages are required., This study was funded by the Innovation Fund Denmark under grant agreements no. 5184-00093B (EEL-HATCH) and 7076-00125B (ITSEEL). Butts IAE, was also supported by the USDA National Institute of Food and Agriculture, Hatch project 1013854. Gallego V has a post-doc grant from the MICIU (Juan de la Cierva-Incorporacion; IJCI-201734200).

The life cycle of European eel (Anguilla anguilla), a catadromous species, is complex and enigmatic. In nature, during the silvering process prior to their long spawning migration, reproductive development is arrested, and they cease feeding. In studies of reproduction using hormonal induction, eels are equivalently not feed. Therefore, in female eels that undergo vitellogenesis, the liver plays different, essential roles being involved both in vitellogenins synthesis and in reallocating resources for the maintenance of vital functions, performing the transoceanic reproductive migration and completing reproductive development. The present work aimed at unravelling the major transcriptomic changes that occur in the liver during induced vitellogenesis in female eels. mRNA-Seq data from 16 animals (eight before induced vitellogenesis and eight after nine weeks of hormonal treatment) were generated and differential expression analysis was performed comparing the two groups. This analysis detected 1,328 upregulated and 1,490 downregulated transcripts. Overrepresentation analysis of the upregulated genes included biological processes related to biosynthesis, response to estrogens, mitochondrial activity and localization, while downregulated genes were enriched in processes related to morphogenesis and development of several organs and tissues, including liver and immune system. Among key genes, the upregulated ones included vitellogenin genes (VTG1 and VTG2) that are central in vitellogenesis, together with ESR1 and two novel genes not previously investigated in European eel (LMAN1 and NUPR1), which have been linked with reproduction in other species. Moreover, several upregulated genes, such as CYC1, ELOVL5, KARS and ACSS1, are involved in the management of the effect of fasting and NOTCH, VEGFA and NCOR are linked with development, autophagy and liver maintenance in other species. These results increase the understanding of the molecular changes that occur in the liver during vitellogenesis in this complex and distinctive fish species, bringing new insights on European eel reproduction and broodstock management.

Establishment of European eel (Anguilla anguilla) hatchery production will rely on selectively bred individuals that produce progeny with the best traits in successive generations. As such, this study used a quantitative genetic breeding design, between four females and nine males (four wild‐caught and five cultured), to investigate the effect of paternal origin (wild‐caught vs. cultured) and quantify the relative importance of parental effects, including genetic compatibility, on early life history (ELH) performance traits (i.e. fertilization success, embryonic survival at 32 hr postfertilization, hatch success and larval deformities at 2 days post‐hatch) of European eel. Wild‐caught males had higher (56%) spermatocrit values than cultured males (45%), while fertilization success, embryonic survival, hatch success and larval deformities were not significantly impacted by paternal origin. This demonstrates that short‐term domestication of male eels does not negatively affect offspring quality and enables the consideration of cultured male broodstock in future breeding programmes.Moreover, paternity significantly explained 9.5% of the variability in embryonicsurvival, providing further evidence that paternal effects need to be takeninto consideration in assisted reproduction protocols. Furthermore, maternity significantly explained 54.8% of the variation for fertilization success, 61.7% for embryonic survival, 88.1% for hatching success and 62.8% for larval deformities, validating that maternity is a major factor influencing these “critical” ELH traits. At last, the parental interaction explained 12.8% of the variation for fertilization success, 8.3% for embryonic survival, 4.5% for hatch success and 20.5% for larval deformities. Thus, we conclude that eggs of one female can develop more successfully when crossed with a compatible male, highlighting the importance of mate choice for successful propagation of high‐quality offspring. Together, this knowledge will improve early offspring performance, leading to future breeding programmes for this critically endangered and economically important species

Knowledge on dietary nutrient requirements of first-feeding European eel larvae (Anguilla anguilla) is very limited. This study provides first ever information on in vivo lipid uptake and fatty acid (FA) metabolism of European pre-leptocephalus eel larvae and advances directions for dietary lipid and FA inclusions. The in vivo capability of eel larvae to incorporate and metabolize unsaturated fatty acids was tested on larvae at different ontogenetic stages (4, 8 and 12 days post hatch, DPH). Larvae were incubated in 10 mL flat-bottom tissue culture plates, with [1-14C]-labelled FA (18:2n-6, ALA; 18:3n-3, LA; 20:4n-6, ARA and 20:5n-3, EPA) directly added to seawater. The capability of the larvae for de-acylation and re-acylation of [1-14C]arachidonic acid (ARA), initially bound to phosphatidylcholine (PC) and phosphatidylethanolamine (PE), was also investigated. In all cases, control incubations without any radiolabelled substrate were performed for further lipid analysis. The results revealed that direct incubation with 14C-labelled FA is a feasible method to investigate in vivo FA and phospholipids metabolism of pre-leptocephalus stages of the European eel. No enzymatic elongation/desaturation activity towards [1-14C]C18 or [1-14C]C20 FA was detected. Consequently, ARA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) must be considered essential FA and thus provided firstly through female broodstock and later through diet at least during the first-feeding stage. Pre-leptocephalus larvae display a high capacity to remodel dietary phospholipids with a preferential esterification of all FA substrates into PC. The unexpectedly high esterification rate of [1-14C] ARA into PC and PE is supported by the individual FA profiles of the larval phospholipids. The high levels of ARA present in the European eel larvae denotes its physiological relevance for this species. It is therefore essential to consider this FA as particularly important when designing suitable broodstock – or first-feeding diets for this species.

Breeding European eel in captivity is a complex task. However, recent advances in assisted reproduction and culturing techniques now allow mass production of high-quality gametes, embryos, and first-feeding larvae. Here, three studies were conducted to determine whether dietary regime, chemoattractants, and light intensity have an impact on the incidence of larval first-feeding, gut fullness, and behavior. Firstly, larvae at 12, 16, and 20 days post-hatch (DPH) were allowed to forage on one of five diets, rotifer paste (RP), RP + cod roe, RP + octopus juice, RP + live rotifers, or Sargasso Sea plankton for 2 h and afterwards evaluated for presence or absence of ingested diet. Secondly, light effects on feeding at 15 and 16 DPH were tested, using the following intensities: High light at 21.5 ± 3.9 μmol m− 2 s− 1; intermediate at 6.8 ± 1.4 μmol m− 2 s− 1; low at 0.6 ± 0.2 μmol m− 2 s− 1; darkness; and a control where no feed was added in darkness. Lastly, behavioral observations were recorded on larvae at first-feeding. Results showed no evidence of first-feeding at 12 DPH. At 16 DPH, 23–50% of larvae had ingested feed when offered either RP, RP + live rotifers, or RP + cod roe. At 20 DPH, the highest incidence of feeding was detected when larvae were fed RP and chemoattractants; 8–30% of larvae ingested feed. Light had a positive impact on feeding at 15 and 16 DPH, such that incidence of first-feeding and gut fullness increased at the intermediate to high light intensities, where up to 50% of larvae ingested the RP diet and had 9% gut fullness. Swimming of larvae was characterized by bouts produced by undulations of the caudal region, followed by pauses where larvae remained motionless. Duration of swimming increased from 41 to 218 s at 13–17 DPH, respectively. Larvae exposed to RP-based diets swam more than the control (no feed). Larval attacks were observed in benthic and pelagic zones of the tank, however were more frequently observed in the benthos. Here, 8–10%, 60–73% and 69–86% of larvae were observed grazing on RP-based diets at 13, 15 and 17 DPH, respectively. Typically, attacks were associated with S-bend posture and upon initiation of S-bends the larvae remained stationary before an explosive lunge. At 13–17 DPH, larvae fed the RP-based diets exhibited a higher incidence of attacks than the control. Together, this work provides benchmark diets and conditions for future feeding and growth trials. Statement of relevance First-feeding by European eel larvae in captivity.

The Eastern Baltic cod abundance started rapidly to increase in the mid-2000s as evidenced by analytical stock assessments, due to increased recruitment and declining fishing mortality. Since 2014, the analytical stock assessment is not available, leaving the present stock status unclear and casting doubts about the magnitude of the recent increase in recruitment. Earlier studies identified main factors impacting on cod reproductive success to be related to the loss of two out of three spawning areas in the 1980s caused by lack of major Baltic inflows with a concurrent reduction in salinity and oxygen. Other important factors include prey availability for first-feeding larvae, egg predation by sprat and herring and cannibalism on juveniles, all in one way or the other related to the prevailing hydrographic conditions. These factors cannot explain increased reproductive success in the last decade, as the period was characterized by an absence of large-scale Baltic inflows since 2003 and persistent anoxic conditions in the bottom water of the deep Baltic basins. This questions the perception of the increased recruitment in later years and challenges our present understanding of cod recruitment dynamics in the Baltic Sea. In this contribution, we review evidence from the recent literature supplemented by information from latest research cruises to elucidate whether cod reproductive success indeed has increased during the last decade, and we suggest the key processes responsible for the recent dynamics in cod recruitment and outline directions for future research.

OBJECTIVE To examine ultrasonographic predictors of ovarian development in European eels (Anguilla anguilla) undergoing hormonal treatment for assisted reproduction. ANIMALS 83 female European eels. PROCEDURES Eels received weekly IM injections of salmon pituitary extract (first injection = week 1). Ultrasonography of the ovaries was performed twice during hormonal treatment (weeks 7 and 11). Eels were identified on the basis of body weight as having an adequate response by weeks 14 to 20 or an inadequate response after injections for 21 weeks. Eels were euthanized at the end of the experiment and classified by use of ovarian histologic examination. Ovarian cross-sectional area and size of eel (ie, length3) were used to classify eels (fast responder, slow responder, or nonresponder) and to calculate an ultrasonographic-derived gonadosomatic index. Gray-level co-occurrence matrices were calculated from ovarian images, and 22 texture features were calculated from these matrices. RESULTS The ultrasonographic-derived gonadosomatic index differed significantly between fast responders and slow responders or nonresponders at both weeks 7 and 11. Principal component analysis revealed a pattern of separation between the groups, and partial least squares discriminant analysis revealed signals in the ovarian texture that discriminated females that responded to treatment from those that did not. CONCLUSIONS AND CLINICAL RELEVANCE Ovarian texture information in addition to morphometric variables can enhance ultrasonographic applications for assisted reproduction of eels and potentially other fish species. This was a novel, nonlethal method for classifying reproductive response of eels and the first objective texture analysis performed on ultrasonographic images of the gonads of fish.

Captive breeding of European eel, Anguilla anguilla is challenged by the complex hormonal control of Anguillid eel reproduction and the distinctive ontogeny of the leptocephalus larvae that are unique to the Elopomorph superorder. Recent experimental research has succeeded in the production of viable eggs and larvae of European eel, providing the basis for studies on early life stages of this species in captivity. In this study, we describe and illustrate morphological characteristics of eggs, embryos, and larvae from fertilization to termination of the yolk sac stage and provide a comparison with additional commercially important eel species. Furthermore, we model growth during the critical first phase in larval ontogeny, i.e. the yolk sac stage, and test for maternal effects. The eggs of A. anguilla typically have numerous oil droplets that coalesce into a single large oil droplet, while the zygote forms a large perivitelline space, reaching an egg diameter of 1.45 ± 0.12 mm at 3.0 to 3.5 h post fertilization. Embryonic development from fertilization to larval hatch lasted ~ 46–48 h at 20 °C with the larvae emerging in a relatively undeveloped stage with a protuberant yolk sac. During the period of yolk and oil absorption, the larvae undertook significant changes in head and body morphology. At the completion of yolk sac absorption, the largely transparent larvae had a set of protruding teeth, pigmented eyes and tail, and a simple alimentary tract. Larvae appeared capable of feeding at ~ 12 days post hatch at 20 °C, and were able to survive another ~ 10 days without feeding. Larval length approached an asymptotic maximum of 6.8 mm around day 10 in non-fed larvae. Larval batches from different maternal origins varied in yolk sac size and the extent of yolk sac resources influenced larval size at the end of the yolk sac stage. The ontogenetic description presented here fills a gap in knowledge about the yet undiscovered early life stages of native European eel, which can provide a framework of reference for the development of hatchery technology. Such progress is urgently needed for a self-sustained aquaculture of this high-value and critically endangered species. Statement of relevance European eel is a high-value species in aquaculture, however, production is presently hampered by reliance on wild caught fry. Captive production of glass eels will reopen markets in Europe and Asia, benefiting European eel producers. The results presented here document recent progress within assisted reproduction and larval culture of this species in aquaculture and aid establishing hatchery technology of this species.

International audience; Maternal mRNA governs early embryonic development in fish and variation in abundance of maternal transcripts may contribute to variation in embryonic survival and hatch success in European eel, Anguilla anguilla. Previous studies have shown that quantities of the maternal gene products β-tubulin, insulin-like growth factor 2 (igf2), nucleoplasmin (npm2), prohibitin 2 (phb2), phosphatidylinositol glycan biosynthesis class F protein 5 (pigf5), and carnitine O-palmitoyltransferase liver isoform-like 1 (cpt1) are associated with embryonic developmental competence in other teleosts. Here, the relations between relative mRNA abundance of these genes in eggs and/or embryos and egg quality, was studied and analyzed. We compared egg quality of the two groups: i) batches with hatching and ii) batches with no hatching. Results showed no significant differences in relative mRNA abundance between the hatch and no hatching groups for any of the selected genes at 0, 2.5, and 5 HPF. However, at 30 HPF the hatch group showed significantly higher abundance of cpt1a, cpt1b, β-tubulin, phb2, and pigf5 transcripts than the no hatch group. Therefore, these results indicate that up-regulation of the transcription of these genes in European eel after the mid-blastula transition, may be needed to sustain embryonic development and hatching success.

Production of high-quality offspring from farm-raised broodstock is fundamental to establish a closed-cycle hatchery production of European eel, Anguilla anguilla. While development of larval culture technologies progresses, the present study focused on effects of essential fatty acid (EFA) composition of eggs on offspring quality. Three reproduction experiments were conducted, two of which included farm-raised broodstock fed different diets for different periods of time and one wild-caught broodstock, using size-matched females. The formulated diets varied in levels and ratios of three essential fatty acids, arachidonic acid (20:4n-6; ARA), eicosapentaenoic acid (20:5n-3; EPA), and docosahexaenoic acid (22:6n-3; DHA), while feeding periods lasted either 55 or 79 weeks. Dietary influences on egg and offspring fatty acid composition and offspring quality were evaluated and results of the most successful dietary regime was compared to those of wild-caught females. Results showed that elevated dietary levels of ARA were reflected in unfertilized eggs, with high ARA diets significantly increasing the amounts of floating eggs, total lipid content in eggs, fertilization success, and embryonic survival. Further EFA enhancements and prolonged feeding resulted in higher ARA and lower EPA levels in the unfertilized eggs, while DHA levels did not change. Females with prolonged feeding produced offspring of higher quality, i.e. higher egg dry weight and larval survival. Overall, offspring of farm-raised females showed higher EFA levels than those of wild-caught females. However, while fertilization success was comparable, offspring of farm-raised females had significantly lower embryonic survival and hatch success as well as higher proportions of cleavage abnormalities. These results identified embryonic development as the main bottleneck in offspring production from farm-raised females. Once hatched, larval survival and quality was comparable between farm-raised and wild-caught females. Notably, enhancement of essential fatty acids in female broodstock diets in combination with a long feeding period improved the production of high quality offspring.

WOS:000435090700050; International audience; European eel (Anguilla anguilla) is a euryhaline species, that has adapted to cope with both, hyper- and hypo-osmotic environments. This study investigates the effect of salinity, from a morphological and molecular point of view on European eel larvae reared from 0 to 12 days post hatch (dph). Offspring reared in 36 practical salinity units (psu; control), were compared with larvae reared in six scenarios, where salinity was decreased on 0 or 3 dph and in rates of 1, 2 or 4 psu/day, towards iso-osmotic conditions. Results showed that several genes relating to osmoregulation (nkcc2 alpha, nkcc2 beta, aqp1dup, aqpe), stress response (hsp70, hsp90), and thyroid metabolism (thr alpha A, thr alpha B, thr beta B, diol, dio2, dio3) were differentially expressed throughout larval development, while nkcc1 alpha, nkcc2 beta, aqp3, aqp 1dup, aqpe, hsp90, thr alpha A and dio3 showed lower expression in response to the salinity reduction. Moreover, larvae were able to keep energy metabolism related gene expression (atp6, cox1) at stable levels, irrespective of the salinity reduction. As such, when reducing salinity, an energy surplus associated to reduced osmoregulation demands and stress (lower nkcc, aqp and hsp expression), likely facilitated the observed increased survival, improved biometry and enhanced growth efficiency. Additionally, the salinity reduction decreased the amount of severe deformities such as spinal curvature and emaciation but also induced an edematous state of the larval heart, resulting in the most balanced mortality/deformity ratio when salinity was decreased on 3 dph and at 2 psu/day. However, the persistency of the pericardial edema and if or how it represents an obstacle in further larval development needs to be further clarified. In conclusion, this study clearly showed that salinity reduction regimes towards iso-osmotic conditions facilitated the European eel pre-leptocephalus development and revealed the existence of highly sensitive and regulated osmoregulation processes at such early life stage of this species.

During European eel assisted reproduction, timely administration of hormones that induce oocyte maturation and ovulation is a major factor influencing subsequent egg quality. This treatment commonly comprises one injection of fish pituitary extract (PE) as a primer followed by a 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) injection. In this context, the present study aimed at optimizing timing of the dual hormone administration by applying a lipid droplet-based oocyte maturation scale, previously developed for Japanese eel to determine the maturational status of each female. Using wild-caught female eels, the potential effect of female size, egg fatty acid composition and dry weight on egg quality was also analyzed. Larval survival at 3 days post hatch was used to differentiate High- and Low-quality egg batches. Results showed that lipid droplet diameter was significantly smaller in High-quality eggs than in Low-quality egg batches, indicating that females producing High-quality eggs received the PE primer and DHP generally at an earlier developmental stage than those producing Low-quality batches. These results confirm that oocyte lipid droplet diameter is a useful indicator of female maturational status for optimization of induction of oocyte maturation and ovulation in European eel. Additional parameters, including female size, egg fatty acid composition and dry weight, were similar between high and low quality egg batches. This insight regarding the fatty acid composition of eggs obtained from wild-caught female eels may help advancing the development of tailored diets for increased reproductive success of farmed broodstock.

WOS:000426336600006; International audience; Thyroid hormones (THs) are key regulators of growth, development, and metabolism in vertebrates and influence early life development of fish. TH is produced in the thyroid gland (or thyroid follicles) mainly as T4 (thyroxine), which is metabolized to T3 (3,5,3'-triiodothyronine) and T2 (3,5-diiodothyronine) by deiodinase (DIO) enzymes in peripheral tissues. The action of these hormones is mostly exerted by binding to a specific nuclear thyroid hormone receptor (THR). In this study, we i) cloned and characterized thr sequences, ii) investigated the expression pattern of the different subtypes of thrs and dios, and iii) studied how temperature affects the expression of those genes in artificially produced early life history stages of European eel (Anguilla anguilla), reared in different thermal regimes (16, 18, 20 and 22 degrees C) from hatch until first-feeding. We identified 2 subtypes of thr (thr alpha and thr beta) with 2 isoforms each (thr alpha A, thr alpha B, thr beta A, thr beta beta) and 3 subtypes of deiodinases (dio1, dio2, dio3). All thr genes identified showed high similarity to the closely related Japanese eel (Anguilla japonica). We found that all genes investigated in this study were affected by larval age (in real time or at specific developmental stages), temperature, and/or their interaction. More specifically, the warmer the temperature the earlier the expression response of a specific target gene. In real time, the expression profiles appeared very similar and only shifted with temperature. In developmental time, gene expression of all genes differed across selected developmental stages, such as at hatch, during teeth formation or at first-feeding. Thus, we demonstrate that thrs and dios show sensitivity to temperature and are involved in and during early life development of European eel.

Being part of the elopomorph group of fishes, Anguillidae species show a leptocephalus larval stage. However, due to largely unknown spawning locations and habitats of their earliest life stages, as well as their transparency, these Anguilla larvae are rarely encountered in nature. Therefore, information regarding the early life history of these larvae, including their exogenous feeding strategy and feeding performance, is rather scarce. To better understand the structural basis and functional performance of larval feeding in captivity, the functional morphology of the cranial musculoskeletal system in pre- and first-feeding engyodontic leptocephali of the European eel (Anguilla anguilla) was studied. A 3D reconstruction of the feeding apparatus (head of the leptocephali < 1 mm) was used to visualize and describe the musculoskeletal changes throughout these stages. To analyze the ontogenetic changes in the functionality of the feeding apparatus towards the active feeding phase, 3D data of joints, levers and muscles derived from the reconstructions were used to estimate bite and joint reaction forces (JRFs). Observing a maximum estimated bite force of about 65 μN (and corresponding JRFs of 260 μN), it can be hypothesized that leptocephalus larvae are functionally constrained to feed only on soft food particles. Additionally, potential prey items are size delimited, based on the theoretically estimated average gape of these larvae of about 100 μm. This hypothesis appears to be in line with recent observations of a diet consisting of small and/or gelatinous prey items (Hydrozoa, Thaliacea, Ctenophora, Polycystenia) found in the guts of euryodontic leptocephalus larvae.

[EN] In order for European eel aquaculture to be sustainable, the life cycle should be completed in captivity. Development of broodstock diets may improve the species' reproductive success in captivity, through the production of high-quality gametes. Here, our aim was to evaluate the influence of dietary regime on muscle composition, and liver lipids prior to induced maturation, and the resulting sperm composition and performance. To accomplish this fish were reared on three "enhanced" diets and one commercial diet, each with different levels of fatty acids, arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Neutral lipids from the muscle and liver incorporated the majority of the fatty acid profile, while phospholipids incorporated only certain fatty acids. Diet had an effect on the majority of sperm fatty acids, on the total volume of extractable milt, and on the percentage of motile sperm. Here, our results suggest that the total volume of extractable milt is a DHA-dependent process, as we found the diets with the highest DHA levels induced the most milt while the diet with the lowest DHA level induced the least amount of milt The diet with the highest level of ARA induced medium milt volumes but had the highest sperm motility. EPA also seems important for sperm quality parameters since diets with higher EPA percentages had a higher volume of milt and higher sperm motility. In conclusion, dietary fatty acids had an influence on fatty acids in the tissues of male eel and this impacted sperm performance. (C) 2015 Elsevier Inc All rights reserved., This study relates to the project: Reproduction of European Eel: Towards a Self-sustained Aquaculture (PRO-EEL) funded by the European Commission 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology" (Grant Agreement no. 245257). JFA and LP had a grant to stay in Denmark from the Universitat Politecnica de Valencia (PAID-00-11). Special thanks to P. Lauesen (Billund Aquaculture Service), and C. Graver (Danish Aquaculture Organisation) for help during experimentation, and Lars Holst, BioMar A/S, who took part in sourcing and feed production. IAEB, RB, LP, JFA and JT received travel grants from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE).

SummaryImproper activation and swelling of in vitro produced eggs of European eel, Anguilla anguilla, has been shown to negatively affect embryonic development and hatching. We investigated this phenomenon by examining the effects of salinity and sea salt type on egg dimensions, cell cleavage patterns and egg buoyancy. Egg diameter after activation, using natural seawater adjusted to different salinities, varied among female eels, but no consistent pattern emerged. Activation salinities between 30–40 practical salinity unit (psu) produced higher quality eggs and generally larger egg diameters. Chorion diameters reached maximal values of 1642 ± 8 μm at 35 psu. A positive relationship was found between egg neutral buoyancy and activation salinity. Nine salt types were investigated as activation and incubation media. Five of these types induced a substantial perivitelline space (PVS), leading to large egg sizes, while the remaining four salt types resulted in smaller eggs. All salt types except NaCl treatments led to high fertilization rates and had no effect on fertilization success as well as egg neutral buoyancies at 7 h post-fertilization. The study points to the importance of considering ionic composition of the media when rearing fish eggs and further studies are encouraged.

In captivity, oogenesis and ovarian follicle maturation in European eel can be induced experimentally using hormonal therapy. The follicle's ability to respond effectively to the induction of maturation and ovulation, resulting in viable eggs, depends on the oocyte stage at the time of induction. We hypothesized that variation in the expression of key hormone receptors in the ovary and size of oocyte lipid droplets are associated with changes in oocyte stage. Thus, we induced ovarian follicle maturation using a priming dose of fish pituitary extract followed by the administration of a 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) injection. Females were then strip-spawned, the eggs were fertilized in vitro, incubated and larval survival was recorded at 3 days post hatch (dph). The expression of gonadotropin receptors (fshr, lhcgr1 and lhcgr2) and estrogen receptors (esr1, esr2a, esr2b, gpera and gperb) was quantified and the size of oocyte lipid droplets measured. Larval survival at 3 dph was used to differentiate high- and low-quality egg batches. Results showed significantly higher abundance of lhcgr1 and esr2a at priming for high-quality egg batches whereas fshr and gperb transcripts were significantly higher at DHP injection for low-quality egg batches. Therefore, high levels of lhcgr1 and esr2a may be important for attaining follicular maturational competence, while high fshr and gperb mRNA levels may indicate inadequate maturational competence. Furthermore, lipid droplet size at DHP and in ovulated eggs was significantly smaller in high-quality egg batches than in low-quality, which indicates that droplet size may be a useful marker of follicular maturational stage. © 2018. This is the authors’ accepted and refereed manuscript to the article. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

WOS:000407548800031; International audience; Temperature is important for optimization of rearing conditions in aquaculture, especially during the critical early life history stages of fish. Here, we experimentally investigated the impact of temperature (16, 18, 20, 22 and 24 degrees C) on thermally induced phenotypic variability, from larval hatch to first-feeding, and the linked expression of targeted genes [heat shock proteins (hsp), growth hormone (gh) and insulin-like growth factors (igf)] associated to larval performance of European eel, Anguilla anguilla. Temperature effects on larval morphology and gene expression were investigated throughout early larval development (in real time from 0 to 18 days post hatch) and at specific developmental stages (hatch, jaw/teeth formation, and first-feeding). Results showed that hatch success, yolk utilization efficiency, survival, deformities, yolk utilization, and growth rates were all significantly affected by temperature. In real time, increasing temperature from 16 to 22 degrees C accelerated larval development, while larval gene expression patterns (hsp70, hsp90, gh and igf-1) were delayed at cold temperatures (16 degrees C) or accelerated at warm temperatures (20-22 degrees C). All targeted genes (hsp70, hsp90, gh, igf-1, igf-2a, igf-2b) were differentially expressed during larval development. Moreover, expression of gh was highest at 16 degrees C during the jaw/teeth formation, and the first-feeding developmental stages, while expression of hsp90 was highest at 22 degrees C, suggesting thermal stress. Furthermore, 24 degrees C was shown to be deleterious (resulting in 100% mortality), while 16 degrees C and 22 degrees C (similar to 50 and 90% deformities respectively) represent the lower and upper thermal tolerance limits. In conclusion, the high survival, lowest incidence of deformities at hatch, high yolk utilization efficiency, high gh and low hsp expression, suggest 18 degrees C as the optimal temperature for offspring of European eel. Furthermore, our results suggest that the still enigmatic early life history stages of European eel may inhabit the deeper layer of the Sargasso Sea and indicate vulnerability of this critically endangered species to increasing ocean temperature.

Cultivated Atlantic cod (Gadus morhua) entering their first year of gamete maturation were fed diets with different levels of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) for 6.5 months prior to commencement of spawning. Gravid females were stripped three times: at the beginning, peak and end of spawning. Lipid composition and egg and larval quality of 34 family crosses were investigated. Results indicated that ARA uptake into eggs from broodstock diet was highly efficient achieving proportions of ARA up to 84% higher in eggs than in the diet. EPA was 42–76% higher, and DHA was 155–173% higher in eggs than in diets. Cod fed the diet with the lowest EPA/ARA ratio had the greatest egg production. Eggs from fish on a diet with high ARA level had significantly higher fertilization and hatching success than those fed low levels of ARA. This diet produced on average 71 viable eggs g−1 female compared with 32.5 and 4 eggs in diet B and C, respectively. Furthermore, larval survival until 8 days posthatch was higher in diets with lower ARA levels. The combined results showed that ARA dietary supplementation and low EPA/ARA ratio yielded a greater number of viable larvae kg−1 female.

A presumed parasitic protozoan was found in the eggs of European eel obtained from an experiment on captive breeding of eel, Anguilla anguilla, based on silver eels from a freshwater lake in the northern part of Denmark. Gross morphology of the organism was comparable to that of early stages of Ichthyodinium, a syndinian dinoflagellate parasite found in pelagic eggs of various marine fish species. Sequences of genes coding for small subunit ribosomal RNA confirmed that the organism was an Ichthyodinium species, and molecular phylogenetic analysis demonstrated the presence of two Ichthyodinium genotypes: one occurring in the Atlantic Ocean and adjacent coastal waters and one in the Pacific Ocean area. The inclusion of several GenBank-derived environmental gene sequences, from the Caribbean Sea, revealed to represent Ichthyodinium, suggesting that this parasite genus is ubiquitous in the World's oceans.

Recent experimental research applying hormonally induced maturation in European eel has resulted in production of viable eggs and yolk-sac larvae. However, present incubation and larval rearing conditions are suboptimal and few larvae survive until onset of first feeding. The aim of this work was to investigate if high mortality during egg incubation and larval culture resulted from microbial interference. By suppressing microbial coverage and activity on fertilised eel eggs using antibiotic and disinfection treatment, egg hatching success and larval longevity were significantly improved. A new approach based on scanning electron microscopy was developed to quantify microbial coverage of eggs. Measurements of microbial coverage in combination with growth curves of egg-associated bacteria indicated that microbial activity rather than physical coverage led to reduced hatch success. In addition, an inverse relationship between microbial coverage of eggs and larval survival indicated that attachment of micro-organisms on the egg surface during the last 24 h of incubation affected later larval survival. These results suggest that microbial control through application of egg surface disinfection in combination with microbial management will be fundamental for improved post-hatch larval survival.

International audience; To sustain eel aquaculture, development of reproduction in captivity is vital. The aim of this review is to assess our current knowledge on the nutrition of broodstock eels in order to improve the quality of broodstock under farming conditions, drawing information from wild adult eels and other marine pelagic spawners. Freshwater eels spawn marine pelagic eggs with an oil droplet (type II), and with a large perivitelline space. Compared with other marine fish eggs, eel eggs are at the extreme end of the spectrum in terms of egg composition, even within this type II group. Eel eggs contain a large amount of total lipids, and a shortage of neutral lipids has been implied a cause for reduced survival of larvae. Eel eggs have higher ARA but lower EPA and DHA levels than in other fish. Too high levels of ARA negatively affected reproduction in the Japanese eel, although high levels of 18:2n-6 in the eggs of farmed eels were not detrimental. The total free amino acid amount and profile of eel eggs appears much different from other marine pelagic spawners. Nutritional intervention to influence egg composition seems feasible, but responsiveness of farmed eels to induced maturation might also require environmental manipulation. The challenge remains to succeed in raising European eel broodstock with formulated feeds and to enable the procurement of viable eggs and larvae, once adequate protocols for induced maturation have been developed

Herring (Clupea harengus) is a capital breeder that stores energy reserves in muscle tissue. Individual potential fecundity relies on the size and weight of female fish. Poor condition during the maturation process can lead to a heavy down-regulation of fecundity through atresia and, in the extreme, cause skipped spawning. Herring in the Central Baltic Sea exist in a variable environment where food availability fluctuates substantially. Compared to other herring populations their condition is generally poor. In the present study, the oocyte dynamics and fecundity in relation to the condition of Central Baltic herring was investigated. A modern stereological method, the physical fractionator, was used to quantify the number of oocytes in previtellogenic (PG), cortical alveoli (CA) as well as successive vitellogenic (VT1 and VT2) stages in central Baltic herring during ovarian maturation. The potential fecundity, i.e. the number of VT2 oocytes, was low compared to other Atlantic stocks but the relative potential fecundity was higher. The latter decreased by 71% when comparing early-maturing individuals with CA oocytes and late-maturing individuals with VT2 oocytes, suggesting a substantial down-regulation of fecundity. Although determined as spring spawners by otolith hatch type, 15% of the randomly sampled females were characterized by oocytes in CA stage in the prespawning period, indicating skipped spawning. The condition of these females was poor, which might have resulted in skipped spawning. Ovary weight was a good predictor of potential fecundity within maturing stages of females. Combined with estimates of skipped spawning, this ovary weight could be used to estimate egg production thereby improving Central Baltic herring stock-recruitment models.

Røjbek, M. C., Tomkiewicz, J., Jacobsen, C., and Støttrup, J. G. Forage fish quality: seasonal lipid dynamics of herring (Clupea harengus L.) and sprat (Sprattus sprattus L.) in the Baltic Sea. – ICES Journal of Marine Science, 71: . This study investigates lipid content and fatty acid composition of two important forage fish, sprat (Sprattus sprattus) and herring (Clupea harengus) in the Baltic Sea ecosystem. Seasonal variation in lipids was studied during three periods following the annual reproductive cycle considering potential differences relating to fish size, sex, and reproductive status. The isopod Saduria entomon, being at times an important prey for predatory fish, was included for comparison. In both sprat and herring, lipid content and absolute contents of essential polyunsaturated fatty acids (PUFAs) varied seasonally with high levels towards the end of the annual zooplankton production cycle, succeeded by a decline. Lipid content and fatty acid composition differed significantly between sprat and herring. Sprat lipid content was higher than herring, increasing with fish size and characterized by large proportions of monounsaturated fatty acids. Herring lipid content was related to the reproductive cycle and proportions of PUFAs were high compared with sprat. Levels of essential PUFAs were high in S. entomon compared with clupeids rendering it a valuable alternative prey species in the Baltic Sea ecosystem. The lipid dynamics of forage fish and benthos, combined with changes in availability and abundance, will affect growth and reproduction of their predators.

Sexual maturation patterns of 22 North Atlantic stocks of cod (Gadus morhua) were examined and related to geographical distribution area, ambient water temperature, growth and surplus production. Four patterns were identified, i.e. sexual maturation early in life at small size, early in life at large size, late in life at small size and lastly, late in life at large size. These maturation patterns were geographically clustered and associated with differences in growth and surplus production. Stocks maturing late in life at small size were characterised by slow growth and low surplus production (e.g. stocks in the Gulf of St. Lawrence, Labrador/Newfoundland). Stocks maturing early in life at large size exhibited high to intermediate growth and surplus production (e.g. Celtic Sea, North Sea). Stocks maturing late in life at large size had low to intermediate growth rates and surplus production (e.g. Iceland, North East Arctic), while stocks maturing early in life at small size generally showed intermediate growth and surplus production (e.g. Baltic stocks). Production of recruits per unit biomass showed a latitudinal trend, but appeared largely independent of maturation pattern, growth rate and surplus production. Recruit production of northernmost stocks was lowest and variability highest, mid-latitude stocks exhibited highest productivity and least variability, while stocks at the southern distribution range also showed low productivity. Thus, southern Gulf of St. Lawrence and eastern Scotian Shelf cod maturing late in life at small size with slow growth and low surplus production showed highest recruit production in the Western Atlantic, while the early maturing, fast growing and productive Icelandic and Faroese stocks showed the lowest recruitment production of all Eastern Atlantic stocks. This comparative analysis suggests that maturation patterns relate to growth potential and surplus production whereas annual production of recruits per unit biomass appears unrelated to average size at sexual maturation.

In various vertebrate species, dopamine (DA) exerts an inhibitory action on reproduction. In the European eel, DA plays a pivotal role in the inhibitory control of gonadotroph function and the blockade of puberty. In vivo studies have suggested that this effect is mediated by receptors pharmacologically related to the D2 family. In the European eel, two distinct D2 receptor (D2-R) paralogous genes have been identified (D2A-R and D2B-R) and both were shown to be expressed in the pituitary. We investigated the potential role of each paralogue in the control of gonadotroph function in this species. Eel recombinant D2A-R or D2B-R were expressed in HEK 293 cells, with a universal Gα subunit, and receptor activation was followed by inositol phosphate production. Recombinant D2-Rs exhibited a comparable affinity for DA, although they had differential affinities for mammalian D2-R agonists and antagonists, supporting subtle structure/activity differences. Furthermore, using eel pituitary cell primary cultures, the expression by gonadotroph cells of both native eel D2-R paralogues was examined by in situ hybridisation of D2A-R or D2B-R transcripts, coupled with immunofluorescence of luteinising hormone (LH)β or follicle-stimulating (FSH)β. LH and to a lesser extent, FSH cells expressed both D2-R transcripts but with a clear predominance of D2B-R. Notably, D2B-R transcripts were detected for the majority of LH cells. Accordingly, using these cultures, we showed that DA potently inhibited basal and testosterone-stimulated LHβ expression and less potently basal and activin-stimulated FSHβ expression. We also tested some D2-R antagonists, aiming to select the most adequate one to be used in innovative protocols for induction of eel sexual maturation. We identified eticlopride as the most potent inhibitor of DA action on basal and stimulated LH expression in vitro. Our data suggest a differential functionalisation of the duplicated receptor genes and demonstrate that mainly D2B-R is involved in the dopaminergic inhibitory control of eel gonadotroph function.