Effect of Food Amounts on Larval Performance, Bacteriome and Molecular Immunologic Development during First-Feeding Culture of European Eel.

Production of European eel offspring has become a reality, but liquid diets during larval culture hold new challenges. This study focused on increasing food amounts without compromising well-being or healthy larvae-bacteria interactions. First-feeding larvae were fed two food amounts (Low = 0.5 mL food/L water vs. High = 1.5 mL food/L water) until 30 days post-hatch (dph). Results indicated that ~75% of larvae ingested the diet in both treatments, but upregulation of a stress/repair-related gene (hsp90) on 25 and 30 dph indicated nutritional inadequacy. Larvae fed a High amount of food were 3.68% bigger, while larvae in the Low-food group showed 45.2% lower gut fullness and upregulated expression of the gene encoding the "hunger hormone" ghrelin (ghrl), indicating signs of starvation. The High-food group larvae exhibited a healthier bacteriome with a higher abundance of potentially beneficial orders (Lactobacillales and Bacillales), whereas the Low-food group showed more potentially harmful orders (Vibrionales, Rhodobacterales, and Alteromonadales). While survival was initially lower in the High-food group, both treatments had comparable survival by the end of the experiment. In conclusion, feeding European eel larvae with High food amounts seemed beneficial, supported by increased gut fullness, reduced ghrl expression (no starvation), enhanced growth, and the presence of a healthier bacteriome.

Ultrasonic imaging as a means of monitoring gonadal development in lumpfish (Cyclopterus lumpus).

The commercial farming of juvenile lumpfish requires monitoring of gonadal development to achieve synchronized production. Conventional methods such as gonadosomatic index (GSI), sex hormone analyses, gonadal histology, endoscopy, and gene expression analyses are costly, invasive, and often involve sacrificing the fish. We assessed the efficiency of ultrasound as a non-invasive method for monitoring gonadal development in lumpfish. Based on ultrasound observations, we categorized the fish into six stages; F0 to F5 for females and M0 to M5 for males, that represented maturity levels from immature to spent. Importantly, the ultrasound gonadal stages aligned with histological gonadal stages. Additionally, ultrasound stages aligned with profiles of GSI, testosterone (T), 11-ketotestosterone, and 17ß-estradiol throughout gonadal development including the spawning period. Moreover, these parameters exhibited significant positive correlations with each other reflecting their parallel trends during gonadal development. To minimize the frequency of ultrasound usage and fish handling, we established F3 and M3/M4 as arbitrary thresholds for identifying ripe females and males, respectively. By using these thresholds, the need for regular ultrasound monitoring could be reduced during most of the rearing period. Ultrasound proves to be useful and reliable for monitoring gonadal development in lumpfish, enabling synchronized production of juvenile fish.

Exploring bacterial community composition and immune gene expression of European eel larvae (Anguilla anguilla) in relation to first-feeding diets.

European eel (Anguilla anguilla) is a commercially important species for fisheries and aquaculture in Europe and the attempt to close the lifecycle in captivity is still at pioneering stage. The first feeding stage of this species is characterized by a critical period between 20 to 24 days post hatch (dph), which is associated with mortalities, indicating the point of no return. We hypothesized that this critical period might also be associated with larvae-bacterial interactions and the larval immune status. To test this, bacterial community composition and expression of immune and stress-related genes of hatchery-produced larvae were explored from the end of endogenous feeding (9 dph) until 28 dph, in response to three experimental first-feeding diets (Diet 1, Diet 2 and Diet 3). Changes in the water bacterial community composition were also followed. Results revealed that the larval stress/repair mechanism was activated during this critical period, marked by an upregulated expression of the hsp90 gene, independent of the diet fed. At the same time, a shift towards a potentially detrimental larval bacterial community was observed in all dietary groups. Here, a significant reduction in evenness of the larval bacterial community was observed, and several amplicon sequence variants belonging to potentially harmful bacterial genera were more abundant. This indicates that detrimental larvae-bacteria interactions were likely involved in the mortality observed. Beyond the critical period, the highest survival was registered for larvae fed Diet 3. Interestingly, genes encoding for pathogen recognition receptor TLR18 and complement component C1QC were upregulated in this group, potentially indicating a higher immunocompetency that facilitated a more successful handling of the harmful bacteria that dominated the bacterial community of larvae on 22 dph, ultimately leading to better survival, compared to the other two groups.

Exploring first-feeding diets for European eel larval culture: Insights at morphological, nutritional, and molecular levels.

Closing the life cycle of European eel (Anguilla anguilla) in captivity is targeted to provide a sustainable, year-round supply of juveniles for aquaculture. Present focus is on the nutritional requirements during the larval first-feeding period. In this study, three experimental diets were tested on hatchery-produced European eel larvae from the onset of the first-feeding stage commencing 10 days post hatch (dph) until 28 dph. Larval mortality was recorded daily, while sampling was conducted at regular intervals to record larval biometrics and analyze the expression of genes related to digestion, appetite, feed intake and growth. Two periods of high mortality were identified: the first appeared shortly after introduction of feeds (10-12 dph), while the second occurred 20-24 dph, indicating the "point of no return". This interpretation was supported at the molecular level by the expression of the gene encoding the "hunger hormone" ghrelin (ghrl) that peaked at 22 dph in all dietary trials, suggesting that most larvae were fasting. However, in larvae fed diet 3, ghrl expression was downregulated beyond 22 dph, which indicated that those larvae were no longer starving at this stage, while upregulation of genes encoding the major digestive enzymes (try, tgl, and amyl2a) advocated their healthy development. Moreover, for larvae fed diet 3, the expression of those genes as well as genes for feed intake (pomca) and growth (gh) continued to increase towards 28 dph. These results together with the registered highest survival, largest dry weight increase, and enhanced biometrics (length and body area) pointed to diet 3 as the best-performing. As a whole, this first-feeding study represents a landmark being the first to document European eel larval growth and survival beyond the point of no return, providing novel insights into the molecular development of digestive functions during the first feeding stage.

Blood-based gene expression as non-lethal tool for inferring salinity-habitat history of European eel (Anguilla anguilla).

The European eel is a facultative catadromous species, meaning that it can skip the freshwater phase or move between marine and freshwater habitats during its continental life stage. Otolith microchemistry, used to determine the habitat use of eel or its salinity history, requires the sacrifice of animals. In this context, blood-based gene expression may represent a non-lethal alternative. In this work, we tested the ability of blood transcriptional profiling to identify the different salinity-habitat histories of European eel. Eels collected from different locations in Norway were classified through otolith microchemistry as freshwater residents (FWR), seawater residents (SWR) or inter-habitat shifters (IHS). We detected 3451 differentially expressed genes from blood by comparing FWR and SWR groups, and then used that subset of genes in a machine learning approach (i.e., random forest) to the extended FWR, SWR, and IHS group. Random forest correctly classified 100% of FWR and SWR and 83% of the IHS using a minimum of 30 genes. The implementation of this non-lethal approach may replace otolith-based microchemistry analysis for the general assessment of life-history tactics in European eels. Overall, this approach is promising for the replacement or reduction of other lethal analyses in determining certain fish traits.

CO2 induced seawater acidification impacts survival and development of European eel embryos.

Fish embryos may be vulnerable to seawater acidification resulting from anthropogenic carbon dioxide (CO2) emissions or from excessive biological CO2 production in aquaculture systems. This study investigated CO2 effects on embryos of the European eel (Anguilla anguilla), a catadromous fish that is considered at risk from climate change and that is targeted for hatchery production to sustain aquaculture of the species. Eel embryos were reared in three independent recirculation systems with different pH/CO2 levels representing "control" (pH 8.1, 300 µatm CO2), end-of-century climate change ("intermediate", pH 7.6, 900 µatm CO2) and "extreme" aquaculture conditions (pH 7.1, 3000 µatm CO2). Sensitivity analyses were conducted at 4, 24, and 48 hours post-fertilization (hpf) by focusing on development, survival, and expression of genes related to acute stress response (crhr1, crfr2), stress/repair response (hsp70, hsp90), water and solute transport (aqp1, aqp3), acid-base regulation (nkcc1a, ncc, car15), and inhibitory neurotransmission (GABAAα6b, Gabra1). Results revealed that embryos developing at intermediate pH showed similar survival rates to the control, but egg swelling was impaired, resulting in a reduction in egg size with decreasing pH. Embryos exposed to extreme pH had 0.6-fold decrease in survival at 24 hpf and a 0.3-fold change at 48 compared to the control. These observed effects of acidification were not reflected by changes in expression of any of the here studied genes. On the contrary, differential expression was observed along embryonic development independent of treatment, indicating that the underlying regulating systems are under development and that embryos are limited in their ability to regulate molecular responses to acidification. In conclusion, exposure to predicted end-of-century ocean pCO2 conditions may affect normal development of this species in nature during sensitive early life history stages with limited physiological response capacities, while extreme acidification will negatively influence embryonic survival and development under hatchery conditions.

Sperm production and quality in European eel (Anguilla anguilla) in relation to hormonal treatment.

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 × 109 to 10.5 × 109  cells/ml, and spermatocrit decreased from 46.5% to 40.5%. Furthermore, spermatocrit was positively related to haemocytometer counts (R2  = .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.

Sex steroid dynamics and mRNA transcript profiles of growth- and development-related genes during embryogenesis following induced follicular maturation in European eel.

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-17ß (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, thrßa, thrßb) 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.

Unravelling the changes during induced vitellogenesis in female European eel through RNA-Seq: What happens to the liver?

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.

Differential impacts of carp and salmon pituitary extracts on induced oogenesis, egg quality, molecular ontogeny and embryonic developmental competence in European eel.

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.

Molecular ontogeny of larval immunity in European eel at increasing temperatures.

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, il1ß, 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.

Molecular Ontogeny of First-Feeding European Eel Larvae.

Digestive system functionality of fish larvae relies on the onset of genetically pre-programmed and extrinsically influenced digestive functions. This study explored how algal supplementation (green-water) until 14 days post hatch (dph) and the ingestion of food [enriched rotifer (Brachionus plicatilis) paste] from 15 dph onward affects molecular maturation and functionality of European eel larval ingestion and digestion mechanisms. For this, we linked larval biometrics to expression of genes relating to appetite [ghrelin (ghrl), cholecystokinin (cck)], food intake [proopiomelanocortin (pomc)], digestion [trypsin (try), triglyceride lipase (tgl), amylase (amyl)], energy metabolism [ATP synthase F0 subunit 6 (atp6), cytochrome-c-oxidase 1 (cox1)], growth [insulin-like growth factor (igf1)] and thyroid metabolism [thyroid hormone receptors (thrαA, thrßB)]. Additionally, we estimated larval nutritional status via nucleic acid analysis during transition from endogenous and throughout the exogenous feeding stage. Results showed increased expression of ghrl and cck on 12 dph, marking the beginning of the first-feeding window, but no benefit of larviculture in green-water was observed. Moreover, expression of genes relating to protein (try) and lipid (tgl) hydrolysis revealed essential digestive processes occurring from 14 to 20 dph. On 16 dph, a molecular response to initiation of exogenous feeding was observed in the expression patterns of pomc, atp6, cox1, igf1, thrαA and thrßB. Additionally, we detected increased DNA contents, which coincided with increased RNA contents and greater body area, reflecting growth in feeding compared to non-feeding larvae. Thus, the here applied nutritional regime facilitated a short-term benefit, where feeding larvae were able to sustain growth and better condition than their non-feeding conspecifics. However, RNA:DNA ratios decreased from 12 dph onward, indicating a generally low larval nutritional condition, probably leading to the point-of-no-return and subsequent irreversible mortality due to unsuccessful utilization of exogenous feeding. In conclusion, this study molecularly identified the first-feeding window in European eel and revealed that exogenous feeding success occurs concurrently with the onset of a broad array of enzymes and hormones, which are known to regulate molecular processes in feeding physiology. This knowledge constitutes essential information to develop efficient larval feeding strategies and will hopefully provide a promising step toward sustainable aquaculture of European eel.

Effect of parental origin on early life history traits of European eel.

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 post-fertilization, 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 embryonic survival, providing further evidence that paternal effects need to be taken into 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.

Salinity reduction benefits European eel larvae: Insights at the morphological and molecular level.

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 (nkcc2α, nkcc2ß, aqp1dup, aqpe), stress response (hsp70, hsp90), and thyroid metabolism (thrαA, thrαB, thrßB, dio1, dio2, dio3) were differentially expressed throughout larval development, while nkcc1α, nkcc2ß, aqp3, aqp1dup, aqpe, hsp90, thrαA 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.

Differential expression of gonadotropin and estrogen receptors and oocyte cytology during follicular maturation associated with egg viability in European eel (Anguilla anguilla).

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, 20ß-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.

Temperature effects on gene expression and morphological development of European eel, Anguilla anguilla larvae.

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°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°C accelerated larval development, while larval gene expression patterns (hsp70, hsp90, gh and igf-1) were delayed at cold temperatures (16°C) or accelerated at warm temperatures (20-22°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°C during the jaw/teeth formation, and the first-feeding developmental stages, while expression of hsp90 was highest at 22°C, suggesting thermal stress. Furthermore, 24°C was shown to be deleterious (resulting in 100% mortality), while 16°C and 22°C (~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°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.

Interactive effects of dietary composition and hormonal treatment on reproductive development of cultured female European eel, Anguilla anguilla.

Farmed female eels were fed two experimental diets with similar proximate composition but different n-3 polyunsaturated fatty acid (PUFA) levels. Both diets had similar levels of arachidonic acid (ARA), while levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in one diet were approximately 4.5 and 2.6 times higher compared to the other diet, respectively. After the feeding period, each diet group was divided into two and each half received one of two hormonal treatments using salmon pituitary extract (SPE) for 13 weeks: i) a constant hormone dose of 18.75mg SPE/kg initial body weight (BW) and ii) a variable hormone dosage that increased from 12.5mg SPE/kg initial BW to 25mg SPE/kg initial BW. Results showed a significant interaction between diets and hormonal treatments on gonadosomatic index (GSI), indicating that the effect of broodstock diets on ovarian development depends on both nutritional status and hormonal regime. Females fed with higher levels of n-3 series PUFAs and stimulated with the constant hormonal treatment reached higher GSIs than those receiving the variable hormonal treatment. However, when females were fed lower levels of n-3 series PUFAs there was no difference in the effect of hormonal treatments on GSI. We also found that, independent of hormonal treatment, the diet with higher levels of n-3 series PUFAs led to the most advanced stages of oocyte development, such as germinal vesicle migration. Concentration of sex steroids (E2, T, and 11-KT) in the plasma did not differ between diets and hormonal treatments, but was significantly correlated with ovarian developmental stage. In conclusion, increasing dietary levels of n-3 PUFAs seemed to promote oocyte growth, leading to a more rapid progression of ovarian development in European eel subjected to hormonal treatment.

Ultrasonographic predictors of response of European eels (Anguilla anguilla) to hormonal treatment for induction of ovarian development.

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, length (3) ) 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.

Effects of salinity and sea salt type on egg activation, fertilization, buoyancy and early embryology of European eel, Anguilla anguilla.

Improper 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.

Abundance of specific mRNA transcripts impacts hatching success in European eel, Anguilla anguilla L.

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 ß-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 5HPF. However, at 30HPF the hatch group showed significantly higher abundance of cpt1a, cpt1b, ß-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.