Molecular detection of the swim bladder parasite Anguillicola crassus (Nematoda) in fecal samples of the endangered European eel Anguilla anguilla.

The European eel Anguilla anguilla is listed as critically endangered by the IUCN. Among many threats, the introduced parasitic nematode Anguillicola crassus is suspected to alter the eels' swim bladder and jeopardize their reproductive oceanic migration. To date, gaining knowledge about the distribution and prevalence of A. crassus requires individual sacrifice (over 50,000 eels were sacrificed for epidemiology studies since 2010). This paper describes a non-lethal molecular protocol for identifying prevalence of A. crassus in A. anguilla, based on searching for A. crassus DNA in the feces of eels. Tests using three DNA microsatellite markers specific to the nematode showed that molecular detection provided similar results to visual examination of the swim bladder in up to 80% of the cases, and allowed for comparison of prevalence among sites. Easy to implement, this non-lethal protocol for detecting A. crassus could be valuable for management plans of this endangered species.

Assessment of the quality of European silver eels and tentative approach to trace the origin of contaminants - A European overview.

The European eel is critically endangered. Although the quality of silver eels is essential for their reproduction, little is known about the effects of multiple contaminants on the spawning migration and the European eel management plan does not take this into account. To address this knowledge gap, we sampled 482 silver eels from 12 catchments across Europe and developed methods to assess three aspects of eel quality: muscular lipid content (N = 169 eels), infection with Anguillicola crassus (N = 482), and contamination by persistent organic pollutants (POPs, N = 169) and trace elements (TEs, N = 75). We developed a standardized eel quality risks index (EQR) using these aspects for the subsample of 75 female eels. Among 169 eels, 33% seem to have enough muscular lipids content to reach the Sargasso Sea to reproduce. Among 482 silver eels, 93% were infected by A. crassus at least once during their lifetime. All contaminants were above the limit of quantification, except the 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), Ag and V. The contamination by POPs was heterogeneous between catchments while TEs were relatively homogeneous, suggesting a multi-scale adaptation of management plans. The EQR revealed that eels from Warwickshire were most impacted by brominated flame-retardants and agricultural contaminants, those from Scheldt were most impacted by agricultural and construction activities, PCBs, coal burning, and land use, while Frémur eels were best characterized by lower lipid contents and high parasitic and BTBPE levels. There was a positive correlation between EQR and a human footprint index highlighting the capacity of silver eels for biomonitoring human activities and the potential impact on the suitability of the aquatic environment for eel population health. EQR therefore represents a step forward in the standardization and mapping of eel quality risks, which will help identify priorities and strategies for restocking freshwater ecosystems.

Empirical observations of the spawning migration of European eels: The long and dangerous road to the Sargasso Sea.

The spawning migration of the European eel (Anguilla anguilla L.) to the Sargasso Sea is one of the greatest animal migrations. However, the duration and route of the migration remain uncertain. Using fishery data from 20 rivers across Europe, we show that most eels begin their oceanic migration between August and December. We used electronic tagging techniques to map the oceanic migration from eels released from four regions in Europe. Of 707 eels tagged, we received 206 data sets. Many migrations ended soon after release because of predation events, but we were able to reconstruct in detail the migration routes of >80 eels. The route extended from western mainland Europe to the Azores region, more than 5000 km toward the Sargasso Sea. All eels exhibited diel vertical migrations, moving from deeper water during the day into shallower water at night. The range of migration speeds was 3 to 47 km day-1. Using data from larval surveys in the Sargasso Sea, we show that spawning likely begins in December and peaks in February. Synthesizing these results, we show that the timing of autumn escapement and the rate of migration are inconsistent with the century-long held assumption that eels spawn as a single reproductive cohort in the springtime following their escapement. Instead, we suggest that European eels adopt a mixed migratory strategy, with some individuals able to achieve a rapid migration, whereas others arrive only in time for the following spawning season. Our results have consequences for eel management.

Sexually dimorphic gene expressions in eels: useful markers for early sex assessment in a conservation context.

Environmental sex determination (ESD) has been detected in a range of vertebrate reptile and fish species. Eels are characterized by an ESD that occurs relatively late, since sex cannot be histologically determined before individuals reach 28 cm. Because several eel species are at risk of extinction, assessing sex at the earliest stage is a crucial management issue. Based on preliminary results of RNA sequencing, we targeted genes susceptible to be differentially expressed between ovaries and testis at different stages of development. Using qPCR, we detected testis-specific expressions of dmrt1, amh, gsdf and pre-miR202 and ovary-specific expressions were obtained for zar1, zp3 and foxn5. We showed that gene expressions in the gonad of intersexual eels were quite similar to those of males, supporting the idea that intersexual eels represent a transitional stage towards testicular differentiation. To assess whether these genes would be effective early molecular markers, we sampled juvenile eels in two locations with highly skewed sex ratios. The combined expression of six of these genes allowed the discrimination of groups according to their potential future sex and thus this appears to be a useful tool to estimate sex ratios of undifferentiated juvenile eels.

First evidence of European eels exiting the Mediterranean Sea during their spawning migration.

The migration route and the spawning site of the European eel Anguilla anguilla are still uncertain. It has been suggested that the Mediterranean eel stock does not contribute to spawning because there is no evidence of eels leaving the Mediterranean Sea. To test this hypothesis, we equipped eight female silver eels from the south of France with pop-up satellite tags during escapement from coastal waters. Once in deeper water, the eels quickly established diel vertical migration (DVM) between the upper and lower mesopelagic zone. Five tagged eels were taken by predators within the Mediterranean, but two eels reached the Atlantic Ocean after six months and at distances greater than 2000 km from release. These eels ceased their DVM while they negotiated the Gibraltar Strait, and remained in deep water until they reached the Atlantic Ocean, when they recommenced DVM. Our results are the first to show that eels from Mediterranean can cross the Strait of Gibraltar and continue their migration into the Atlantic Ocean. This finding suggests that Mediterranean countries, as for other EU states, have an important role to play in contributing to conservation efforts for the recovery of the European eel stock.

Bages-Sigean and Canet-St Nazaire lagoons (France): physico-chemical characteristics and contaminant concentrations (Cu, Cd, PCBs and PBDEs) as environmental quality of water and sediment.

Environmental characteristics in water and sediments of two contrasted coastal Mediterranean lagoons, Bages-Sigean and Canet-St Nazaire, were measured over a three season survey. The urban pollution (treatment plant discharges) is very important in Canet-St Nazaire lagoon reflecting untreated sewages, while in Bages-Sigean, the northern part appears more impacted due to larger anthropogenic inputs. Dissolved Cd concentrations are on the whole similar in both lagoons, whereas Cu concentrations are by far higher in lagoon Canet-St Nazaire. Cu concentrations appear to be highly dependent on dissolved organic carbon whereas salinity seems to control Cd variations. Concerning the sediments, the confined northern part of lagoon Bages-Sigean shows organic carbon and total nitrogen enrichment whereas lipid concentrations are much higher in the Canet-St Nazaire lagoon. Cu complexation seems to be strongly related to organic matter as evidenced by the two significant positive relationships, on one hand between Cu and organic carbon, and on the other hand, between Cu and lipids. On the contrary, Cd concentrations appear to be mainly controlled by carbonates. PCBs and PBDEs were detected only in sediments and show relatively low concentrations compared to similar lagoon environments. Regarding the sediment quality guidelines, Cd, Cu and PCBs in both lagoons did not exceed any Probable Effect Concentration (PEC).

Influence of introduced vs. native parasites on the body condition of migrant silver eels.

Because parasitism is among the reasons invoked to explain the collapse of Anguilla anguilla, we evaluated the parasitic constraint on body condition (BC) of migrant silver eels as a proxy of fitness with inter-site comparisons. Metazoan parasites were studied in 149 silver eels from five sites (northern Europe). In total, 89% were infected by 13 species including Myxozoa, Monogenea, Cestoda, Nematoda, and Acanthocephala. Anguillicoloides crassus was most common (56%), then Acanthocephalus clavula (30%), and Pseudodactylogyrus sp. (17%). BC, calculated for 58 females, was negatively correlated by abundance of the introduced Pseudodactylogyrus sp. but not by other parasite taxa. Nevertheless, the introduced A. crassus was considered as a severe pathogen based on previous data, whereas the native A. clavula was supposed to have limited impact. Parasite component communities and BC were different between sites. Silver eels from Stockholm Archipelago (Sweden) were the least parasitized (40% vs. 90-95% for other sites) with no parasites on the gills. Burrishoole (Ireland) differed by the absence of A. crassus and high prevalence of A. clavula (84%) but without consequences on BC. Gudenaa (Denmark), Corrib (Ireland), and Frémur (France) were close due to high prevalence of A. crassus (89-93%). Gudenaa and Corrib were the most similar because Pseudodactylogyrus sp. was also highly prevalent (respectively 71% and 60%) whereas absent in Frémur. Our results suggest that the fitness loss induced by the introduced parasites could affect the spawning success of migrant silver eels from Gudenaa and Corrib, and to a lesser extent from Frémur, but probably not those from Stockholm Archipelago and Burrishoole.

Individual and combined effects of copper and parasitism on osmoregulation in the European eel Anguilla anguilla.

The European eel (Anguilla anguilla), a catadromous species, breeds in the sea and migrates to estuarine, lagoon or freshwater habitats for growth and development. Yellow eels, exposed to low or fluctuating salinities, are also exposed to multiple other stressors as pollution, over-fishing and parasitism, which contribute to the dramatic decrease of eel populations in several European countries. The objective of this study was to evaluate the single and combined effects of waterborne copper and experimental infestation of eels with the nematode Anguillicoloides crassus after a salinity challenge from nearly isotonic (18ppt) to hypo- (5ppt) and hypertonic (29ppt) conditions, in order to investigate the osmoregulatory capacity of eels exposed to these stressors. In a nearly isotonic condition (18ppt), blood osmolality remained constant over the 6 weeks contamination to Cu(2+) and Anguillicoloides crassus. In fish exposed to a salinity challenge of 29ppt for 2 weeks, no significant effect was recorded in blood osmolality, Na(+)/K(+)-ATPase (NKA) activity, Na(+) and Cl(-) concentrations. After 2 weeks at 5ppt however, a significant blood osmolality decrease was detected in fish exposed to Anguillicoloides crassus infestation with or without Cu(2+) addition. This decrease may originate from lower Cl(-) levels measured in eels exposed to both stressors. Blood Na(+) levels remained relatively stable in all tested animals, but gill NKA activities were lower in eels exposed to combined stress. No apparent branchial lesions were detected following the different treatments and immunolocalization of NKA revealed well-differentiated ionocytes. Thus, the 5ppt challenge in eels exposed to copper and Anguillicoloides crassus seems to clearly enhance iono/osmoregulatory disturbances. Funded by ANR CES/CIEL 2008-12.

Complete genomic sequence and taxonomic position of eel virus European X (EVEX), a rhabdovirus of European eel.

Eel virus European X (EVEX) was first isolated from diseased European eel Anguilla anguilla in Japan at the end of seventies. The virus was tentatively classified into the Rhabdoviridae family on the basis of morphology and serological cross reactivity. This family of viruses is organized into six genera and currently comprises approximately 200 members, many of which are still unassigned because of the lack of molecular data. This work presents the morphological, biochemical and genetic characterizations of EVEX, and proposes a taxonomic classification for this virus. We provide its complete genome sequence, plus a comprehensive sequence comparison between isolates from different geographical origins. The genome encodes the five classical structural proteins plus an overlapping open reading frame in the phosphoprotein gene, coding for a putative C protein. Phylogenic relationship with other rhabdoviruses indicates that EVEX is most closely related to the Vesiculovirus genus and shares the highest identity with trout rhabdovirus 903/87.