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.

Sarcocystis rileyi (Apicomplexa) in Anas platyrhynchos in Europe with a potential for spread.

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.

Spawning by the European eel across 2000 km of the Sargasso Sea.

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.

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.

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.