Search

Four specimens of mallard (Anas platyrhynchos) shot by local hunters (December 2020 to January 2021 along the eastern coastline of the island of Bornholm in the Baltic Sea) were diagnosed with a heavy load of sarcocysts in the musculature. Morphometric and molecular diagnosis based on rDNA (18 S, ITS1, 28 S) of parasites recovered from two of the birds revealed the causative pathogen to be Sarcocystis rileyi. We further present novel sequences for the entire 5.8 S and ITS2 for this species. Elongate cysts (mean length 5.25 (SD 0.6) mm, width 1.37 (SD 0.2) mm) were recorded in all parts of the striated skeletal musculature of the birds. The main part (72%) of the 2585 cysts in one female mallard was located in the outer superficial pectoral musculature, with 11% in the inner pectoral musculature. Minor but significant parts were found in the dorsal, ventral abdominal, neck and head, legs, hand and arm (wing) musculature. No cysts were found in the smooth musculature. Each cyst contained a median of 3.2 mio bradyzoites indicating that more than 8 billion bradyzoites are available for infection of one or more predators/scavengers ingesting the bird. Bradyzoites (median length 13.5 μm (range 12.1–14.5) and median width 2.66 μm (range 2.1–3.3)) were highly resistant to proteinase treatment, which secures the passage through the stomach of the predator to its intestine where wall penetration takes place. One of the birds was ringed (tagged) in Sweden Island Øland in the Baltic Sea two years before being shot. This is documenting immigration of mallards from northern locations. The parasite species was originally described in North America in 1893 and was commonly reported in this region during the 20th century but not in Europe. Recent cases from Norway, Finland, Lithuania, Poland, UK and Hungary suggest that the species may be spreading geographically. Experienced duck hunters with a 40 years record of hunting on the island reported that this type of infection unprecedented. The final host is reported to be canines (fox, raccoon dog), skunk and mustelids, including ermines and American mink. Presence of these hosts in Europe may allow establishment of the life cycle and further colonization of the local duck populations which calls for implementation of a survey program in Europe., Graphical abstract Image 1, Highlights • Heavy loads of the apicomplexan Sarcocystis riley are described from mallards. • Sarcocysts occurred in all parts of the striated musculature. • Sarcocysts dominated in the superficial pectoral muscle. • The number of bradyzoites in one single mallard was estimated to more than 10 billion. • Sarcocystis rileyi is a new species record for Denmark.

Marine and freshwater spawning environments present fish sperm with unique challenges, but for both, gametes often signal prior to contact via biochemical interactions through maternally-derived co...

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.

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.

Several aspects of the biology of European eel (Anguilla anguilla) larvae are still unknown; particularly, information about their functional development and feeding is sparse. In the present study, we histologically characterize the digestive system of wild caught specimens of European eel leptocephalus larvae. The aim was to provide more understanding about how food may be ingested and mechanically processed in the leptocephalus larvae, and to discuss this in the context of its hypothesized feeding strategy. Larvae were caught in the Sargasso Sea during the “Danish Eel Expedition 2014” with the Danish research vessel Dana. The larval sizes ranged from 7.0 to 23.3 mm standard length (SL) at catch. We found that the mouth/pharynx, especially the anterior esophagus, was surrounded by a multi-layered striated muscle tissue and that the epithelium in the mouth/pharynx had a rough filamentous surface, followed by epithelial columnar cells with multiple cilia in the anterior esophagus. This suggests an expandable pharynx/esophagus, well-suited for the transportation of ingested food and likely with a food-crushing or grinding function. The digestive tract of the larvae consisted of a straight esophagus and intestine ventrally aligned within the larval body, and its length was linearly correlated to the larval length (SL). The length of the intestinal part constituted up to 63% of the total length of the digestive tract. The intestinal epithelium had a typical absorptive epithelium structure, with a brush border and a well-developed villi structure. Some cilia were observed in the intestine, but any surrounding muscularis was not observed. The liver was observed along the posterior part of the esophagus, and pancreatic tissue was located anterior to the intestine. Our findings support the hypothesis that the eel leptocephalus may ingest easily digestible gelatinous plankton and/or marine snow aggregates. The muscular esophagus and the ciliated epithelium appear sufficient to ensure nutrient transport and absorption of the ingested food through the digestive tract.

Winter flounder, Pseudopleuronectes americanus, has emerged as a promising candidate flatfish for cold‐water aquaculture and restocking. Here, juveniles were reared for 8 weeks at three temperatures: 10, 15, and 20°C under 24‐hr light. All fish were imaged at stocking and at 2‐week intervals, where growth was measured as changes in standard length (SL) and body area (BA). By week 2, fish reared at 15 and 20°C were larger than those grown at 10°C. At weeks 4 to 6, fish at 15°C were larger than fish at 20°C. Linear regressions were used to model growth dynamics over time at each temperature. Highly significant linear growth trajectories were detected over time for SL and BA. SL and BA regressions also showed a significant difference among the slopes across temperatures, where comparing slopes showed the best temperature to rear the flounder was 15°C. Weights of fish held at 15°C and 20°C were greater than at 10°C at the termination of the experiment. Within each temperature, the growth rate of malpigmented fish was not different from that of the normally pigmented fish. Overall, growth of winter flounder was comparable to that of other commercially produced flatfish species, providing strong evidence for this flatfish species as a potential species for aquaculture.

Rearing winter flounder (Pseudopleuronectes americanus) in captivity for aquaculture and stock enhancement is a viable option to relieve fishing pressure and aid in ongoing recovery strategies. The objective of this study was to determine the effect of photoperiod on growth and survival of young-of-the-year juvenile winter flounder. Juveniles were reared for 12 weeks at three photoperiods: 24 light/0 dark, 18 light/6 dark, and 12 light/12 dark. Twenty fish were stocked into each of 18 enclosures with six enclosures per treatment. Fish in all treatments were fed to satiation every four hours. All fish were imaged at stocking and at three-week intervals. Growth was measured as changes in standard length (SL) and body area (BA). As early as Week 3, fish in all treatments were significantly different in terms of SL and BA. In general, fish grown under continuous 24 light were the largest and fish in the 12 light/12 dark regime were the smallest. Maximum specific growth rates of 0.41 % per day for SL and 0.83 % per day for BA were achieved in the 24 light/0 dark photoperiod. Average mortality rate over the course of the experiment was 9.17 % and there were no significant differences in the number of dead fish among the treatments. In conclusion, this study demonstrates that a longer day coupled with available food will increase growth rate for winter flounder juveniles, without affecting survival. By extending the photoperiod, growth in young-of-the year winter flounder is accelerated, bringing the growth rate of winter flounder close to that of other commercially produced flatfish.

Levantine scraper, Capoeta damascina is a candidate species for future stock assessments, conservation studies, and hatchery efforts. Herein, we documented embryonic and early larval development, from egg activation to the exogenous feeding period, using morphological and histological landmarks. Embryos were obtained by in vitro fertilization from hormonally induced wild-caught broodstock, and subsequent development was monitored at temperatures coinciding with native conditions. Embryonic development from fertilization to hatch lasted ~105-110 hr. Larvae emerged with unpigmented eyes and body morphology, as well as an undifferentiated digestive tract. The mouth was closed at hatch by the oropharyngeal membrane and opened by the early endogenous feeding period. Trabeculae cartilage, quadrate bone, and Meckel's cartilage of the endoskeleton were present during the endogenous feeding period. During this period, the larvae underwent considerable changes in craniofacial morphology, locomotion, and organogenesis of the digestive tract. The cartilaginous floor of the neurocranium developed and the first four ceratobranchials appeared simultaneously at the end of endogenous feeding period. The digestive tract was differentiated into buccopharynx, esophagus, and small intestine during the endogenous feeding period. The intestinal valve and numerous longitudinal folds at the posterior region of the intestine formed together by the endo-exogenous feeding period. Major developmental events in retinogenesis occurred during the endogenous feeding period. When larvae entered exogenous feeding the mouth was fully-functional. Additionally, liver size and eye diameter increased. Our analysis of embryonic and early larval development in Levantine scraper aligned with other freshwater fishes.

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

Factors such as gamete quality can profoundly affect fertility, but the spawning micro-environment that surrounds the spermatozoa and eggs during gamete contact has largely been neglected. In fishes, understanding these gametic interactions is crucial because each female creates a unique spawning environment by simultaneously expelling her distinct ovarian fluid (OF) along with an egg batch. In turn, OF has been shown to influence spermatozoa performance traits by modifying spermatozoa behaviors and fertilization outcomes. Here, we shed light on these gametic interactions by overviewing literature on OF and how it impacts spermatozoa performance traits. Fish OF is clear or has slight coloration and can constitute ≤10–30% of egg mass. Viscosity of the OF is ∼2- to 3-fold higher than water and its pH ranges 6.2 to 8.8. Osmolality of the OF is lower in freshwater (190–322 mOsmol/kg) than marine species (289–514 mOsmol/kg). Na+ (98.3–213.7 mmol/L) and Cl− (89.8–172.7 mmol/L) are predominant ions in OF, while K+ (1.7–19.3 mmol/L), Mg2+ (0.4–8.1 mmol/L), and Ca2+(0.5–9.7 mmol/L) ions are detected at lower concentrations. Protein levels can be high in OF and exhibit intra- and inter-species variation (54–826 mg/100 mL). Fish OF also contains a series of organic components and substances that enhance and/or attract sperm towards the vicinity of an egg. OF can also differentially impact sperm based on genetic relatedness of mates, male phenotype (i.e. alternative reproductive tactics), or geographic origin. To conclude, when testing further reproductive paradigms, we suggest a shift from classic spermatozoa activation medium (water only) to more natural spawning media, which encompass OF-spermatozoa interactions.

Background Gamete cryopreservation causes cellular damage and death. This study develops cryopreservation techniques for Levantine scraper, and deciphers how early offspring development is affected when eggs are sired with fresh and frozen-thawed sperm. Results Cryopreserved sperm did not affect embryogenesis at two- and four-cell stages, but impaired embryonic development at eight-cell stage. Embryonic viability decreased at organogenesis, where only 34-49% of embryos showed viability with frozen-thawed sperm. Hatching success and percentage of normal hatched embryos declined when fertilized with frozen-thawed sperm. Considering only frozen-thawed cells the dimethyl sulfoxide (DMSO)-5%, methanol (METH)-5%, and METH-10% treatments yielded highest hatch, while METH-5% and propylene glycol-5% yielded the most normal hatched embryos. Larval spinal torsion was higher for fresh than frozen-thawed sperm, where larvae with spinal torsion showed vertebral fusion and shape alterations during exogenous feeding. Both fresh and cryopreserved treatments showed abnormalities in caudal skeleton, while rates of defective yolk-sacs were higher for cryopreserved sperm, where larvae with defective yolks showed oversized yolk extension. Percentage of larvae with defective heads/eyes were also higher for cryopreserved sperm. Conclusions Results show how frozen-thawed sperm impairs embryonic/larvae development and identifies frequency and position of abnormalities. Future studies should investigate how sperm DNA damage may have caused these alterations.

It has been known for about a century that European eels have a unique life history that includes offshore spawning in the Sargasso Sea about 5000–7000 km away from their juvenile and adult habitats in Europe and northern Africa. Recently hatched eel larvae were historically collected during Danish, German and American surveys in specific areas in the southern Sargasso Sea. During a 31 day period of March and April 2014, Danish and German research ships sampled for European eel larvae along 15 alternating transects of stations across the Sargasso Sea. The collection of recently hatched eel larvae (≤12 mm) from 70° W and eastward to 50° W showed that the European eel had been spawning across a 2000 km wide region of the North Atlantic Ocean. Historical collections made from 1921 to 2007 showed that small larvae had also previously been collected in this wide longitudinal zone, showing that the spatial extent of spawning has not diminished in recent decades, irrespective of the dramatic decline in recruitment. The use of such a wide spawning area may be related to variations in the onset of the silver eel spawning migration, individual differences in their long-term swimming ability, or aspects of larval drift.

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.

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.

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.

Elucidating barriers to gene flow is important for understanding the dynamics of speciation. Here we investigate pre- and post-zygotic mechanisms acting between the two hybridizing species of Atlantic eels: Anguilla anguilla and A. rostrata. Temporally varying hybridization was examined by analyzing 85 species-diagnostic single-nucleotide polymorphisms (SNPs; FST ⩾0.95) in eel larvae sampled in the spawning region in the Sargasso Sea in 2007 (N=92) and 2014 (N=460). We further investigated whether genotypes at these SNPs were nonrandomly distributed in post-F1 hybrids, indicating selection. Finally, we sequenced the mitochondrial ATP6 and nuclear ATP5c1 genes in 19 hybrids, identified using SNP and restriction site associated DNA (RAD) sequencing data, to test a previously proposed hypothesis of cytonuclear incompatibility leading to adenosine triphosphate (ATP) synthase dysfunction and selection against hybrids. No F1 hybrids but only later backcrosses were observed in the Sargasso Sea in 2007 and 2014. This suggests that interbreeding between the two species only occurs in some years, possibly controlled by environmental conditions at the spawning grounds, or that interbreeding has diminished through time as a result of a declining number of spawners. Moreover, potential selection was found at the nuclear and the cytonuclear levels. Nonetheless, one glass eel individual showed a mismatch, involving an American ATP6 haplotype and European ATP5c1 alleles. This contradicted the presence of cytonuclear incompatibility but may be explained by that (1) cytonuclear incompatibility is incomplete, (2) selection acts at a later life stage or (3) other genes are important for protein function. In total, the study demonstrates the utility of genomic data when examining pre- and post-zyotic barriers in natural hybrids.

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.

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.

Background: The tyrosine kinase receptor HER4 is a member of the epidermal growth factor receptor (EGFR) family. It plays diverse roles in cancer development and cancer progression and can both exert oncogenic and tumour-suppressive activities. Alternatively spliced isoforms of HER4 are critical to the different signalling possibilities of HER4. Methods: We use a splice-switching oligonucleotide (SSO) to direct the alternative splicing of HER4 from the CYT1 to the CYT2 isoform in HER4-expressing breast cancer cells. Results: Treatment with a target-specific SSO was accompanied by a decreased growth of the cells (P

[EN] Contents Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20 degrees C, or cryopreserved, following existing protocols. Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20 degrees C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few larvae (cryolarvae') were obtained 55h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success., This study was part of the project: 'Reproduction of European Eel: Towards a Self-sustained Aquaculture' (PRO-EEL) European Community's 7th Framework Programme under the Theme 2 'Food, Agriculture and Fisheries, and Biotechnology', Grant Agreement no245257. Juan F. Asturiano and Luz Perez had a grant to staying in Denmark from Programa de Apoyo a la Investigacion y Desarrollo (PAID-00-11) of the Universitat Politecnica de Valencia. We thank Christian Graver and Lars B. Tybjerg for assistance during

Standard, single-exposure alizarin complexone- (ALC) marking was conducted on early life stages of Baltic cod ( Gadus morhua L.) to examine acute and chronic effects. Embryos and yolk sac larvae were marked using different concentrations of ALC (0, 50, 100 and 200 mg l − 1 ). Experiments included control groups for treatment and handling effects. In agreement with previous studies, long-lasting, distinct otolith marks were produced by immersion of yolk sac larvae in ≥ 50 mg l − 1 ALC for 24 h. Mortality of eggs and larvae was low during the marking procedure. Hatching success of ALC marked embryos was significantly reduced and hatching was delayed with increasing ALC concentration. Growth rates of larvae through 21 dph (exogenous feeding stage) were significantly reduced in ALC marked fish compared to controls. Biochemical condition (RNA–DNA ratio) was not affected. Subtle changes in activity and metabolism were indicated by reduced first feeding success and yolk absorption rates. Our results reveal that batch marking of finfish early life stages using ALC can have not only significant, acute impacts on survival but, despite long-standing assumptions to the contrary, also sublethal effects on other vital parameters like growth. We discuss these acute and chronic effects and give recommendations for assessment of life stage- and species-specific ALC immersion marking procedures.

Standardization of artificial fertilization protocols for the European eel, Anguilla anguilla, is a prerequisite for optimizing the use of available gametes in hatchery facilities and for conserving sperm from high quality males, which is either cryopreserved or in living gene banks. The objectives of this research were to provide a rapid, accurate and precise method to quantify sperm density by examining the relationship between sperm density and absorbance by use of a spectrophotometer, determine the optimal number of sperm required to fertilize eggs in a controlled setting, and explore how long eggs are receptive to fertilization post-stripping. Mean sperm density and absorbance at 350 nm were 1.54e + 10 +/- 4.95e + 9 sperm/mL and 1.91 +/- 0.22 nm, respectively. Regression analysis demonstrated a highly significant positive relationship between sperm density and absorbance using a spectrophotometer at 350 nm(R-2 - 0.94, p < 0.001, y - 2.273e + 10x - 2.805e + 10); significant but slightly weaker relationships were also detected at 400, 500, and 600 nm (R-2 >= 0.93, p < 0.001). Fertilization success using sperm to egg ratios ranging from 1.3e + 3 to 1.0e + 6 sperm per egg increased from 37.5 to 68.1%, respectively. Spermto egg ratio had a significant effect on fertilization success (p b 0.0001), where fertilization success increased from1.3e + 3 to 2.5e + 4 sperm per egg; adding greater than 2.5e + 4 spermper egg had no significant effect. Furthermore, the duration of time post-stripping had a significant effect on egg fertilization success (p < 0.0001), such that between 0 and 10 min post-stripping 57.4 to 78.2% of the eggs were fertilized while at 15 min post-stripping a significant decrease in fertilization success was detected (47.5%). For all statistical models, the female variance component was significant for fertilization success (p < 0.0001) and explained >= 84% of the models variance. In conclusion, European eel eggs should be fertilized within 10 min post- stripping using 2.5e + 4 sperm per egg. Together, these findings will contribute to the development of European eel breeding technology and further our understanding on sperm biology and reproductive biology in fishes. (C) 2014 Elsevier B.V. All rights reserved.

Recent research has significantly improved our knowledge and capabilities in the field of in vitro production of yolk sac larvae from European eel (Anguilla anguilla). Female broodstock European eels are matured by weekly administration of pituitary extract and male eels with hCG (human chorionic gonadotropin), which afford gametes for in vitro fertilization studies. The maturing process may lead to mass hatchings of up to ½ million larvae of which some survive the entire yolk sac phase. However, the rearing of larvae suffers from high larval mortalities, and water quality might be a crucial factor for larval survival in rearing systems. By applying antibiotic treatment as a research tool, it was possible to determine the extent of microbial interference in the production of high numbers of good quality larvae. By controlling microbiota during egg and larval incubation, the egg hatching success and larval longevity more than doubled. Using scanning electron microscopic analysis it was observed that microbe inhibiting treatments reduced bacterial colonization of the eggs surface, which possibly cause reduced gas and ionic exchange across chorionic membrane.These results suggest that future eel larviculture should not only focus on optimizing physical incubation conditions, but certainly also on the control over microbial interference.

International audience

[EN] Contents European eel, Anguilla anguilla, is a target species for future captive breeding, yet best methodology to estimate sperm density for application in in vitro fertilization is not established. Thus, our objectives were to evaluate methods to estimate European eel sperm density including spermatocrit, computer-assisted sperm analysis (CASA) and flow cytometry (FCM), using Neubauer Improved haemocytometer as benchmark. Initially, relationships between spermatocrit, haemocytometer counts and sperm motility were analysed, as well as the effect of sperm dilution on haemocytometer counts. Furthermore, accuracy and precision of spermatocrit, applying a range of G-forces, were tested and the best G-force used in method comparisons. We found no effect of dilution on haemocytometer sperm density estimates, whereas motility associated positively with haemocytometer counts, but not with spermatocrit. Results from all techniques, spermatocrit, CASA and FCM, showed significant positive correlations with haemocytometer counts. The best correlation between spermatocrit and haemocytometer counts was obtained at 6000xg (r=0.68). Of two CASA variants, one or three photographic fields (CASA-1 and CASA-2), CASA-2 showed a very high accuracy to haemocytometer counts (r=0.93), but low precision (CV: CASA-2=28.4%). FCM was tested with and without microfluorospheres (FCM-1 and FCM-2), and relationships to haemocytometer counts were highly accurate (FCM-1: r=0.94; FCM-2: r=0.88) and precise (CV: FCM-1=2.5; FCM-2=2.7%). Overall, CASA-2 and FCM-1 feature reliable methods for quantification of European eel sperm, but FCM-1 has a clear advantage featuring highest precision and accuracy. Together, these results provide a useful basis for gamete management in fertilization protocols., This work was conducted as part of the project 'Reproduction of European Eel: towards a Self-sustained Aquaculture' (PRO-EEL) supported financially by the European Commission's 7th Framework Programme under the Theme 2 'Food, Agriculture and Fisheries, and Biotechnology', Grant Agreement no245257. Juan F. Asturiano and Luz Perez received a grant to stay in Denmark from Programa de Apoyo a la Investigacion y Desarrollo (PAID-00-11) of the Universitat Politecnica de Valencia. Victor Gallego has a predoctoral grant from Spanish Ministry of Science and Innovation (MICINN). Fish were raised at a commercial eel farm in Jutland, Denmark (Stensgard Eel Farm A/S). We want to thank Peter Lauesen, Billund Aquaculture Service, Christian Graver, Danish Eel Producers Association and Maria K. Johnsen, Technical University of Denmark, for help and assistance in experiments. All fish were handled in accordance with the European Union regulations concerning the protection of experimental animals (Dir 86/609/EEC).