Sune Riis Sørensen

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.

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.

Assessing pre- and post-zygotic barriers between North Atlantic eels ( Anguilla anguilla and A. rostrata )

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.

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, thrβB)]. 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 thrβB. 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.