Signatures of natural selection between life cycle stages separated by metamorphosis in European eel

Background

Species showing complex life cycles provide excellent opportunities to study the genetic associations between life cycle stages, as selective pressures may differ before and after metamorphosis. The European eel presents a complex life cycle with two metamorphoses, a first metamorphosis from larvae into glass eels (juvenile stage) and a second metamorphosis into silver eels (adult stage). We tested the hypothesis that different genes and gene pathways will be under selection at different life stages when comparing the genetic associations between glass eels and silver eels.

Results

We used two sets of markers to test for selection: first, we genotyped individuals using a panel of 80 coding-gene single nucleotide polymorphisms (SNPs) developed in American eel; second, we investigated selection at the genome level using a total of 153,423 RAD-sequencing generated SNPs widely distributed across the genome. Using the RAD approach, outlier tests identified a total of 2413 (1.57 %) potentially selected SNPs. Functional annotation analysis identified signal transduction pathways as the most over-represented group of genes, including MAPK/Erk signalling, calcium signalling and GnRH (gonadotropin-releasing hormone) signalling. Many of the over-represented pathways were related to growth, while others could result from the different conditions that eels inhabit during their life cycle.

Conclusions

The observation of different genes and gene pathways under selection when comparing glass eels vs. silver eels supports the adaptive decoupling hypothesis for the benefits of metamorphosis. Partitioning the life cycle into discrete morphological phases may be overall beneficial since it allows the different life stages to respond independently to their unique selection pressures. This might translate into a more effective use of food and niche resources and/or performance of phase-specific tasks (e.g. feeding in the case of glass eels, migrating and reproducing in the case of silver eels).

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1754-3) contains supplementary material, which is available to authorized users.     

Endurance swimming of European eel

A long‐term swim trial was performed with five female silver eels Anguilla anguilla of 0·8–1·0 kg ( c . 80 cm total length, L _T) swimming at 0·5 body lengths (BL) s⁻¹, corresponding to the mean swimming speed during spawning migration. The design of the Blazka‐type swim tunnel was significantly improved, and for the first time the flow pattern of a swim tunnel for fish was evaluated with the Laser‐Doppler method. The velocity profile over three different cross‐sections was determined. It was observed that 80% of the water velocity drop‐off occurred over a boundary layer of 20 mm. Therefore, swim velocity errors were negligible as the eels always swam outside this layer. The fish were able to swim continuously day and night during a period of 3 months in the swim tunnel through which fresh water at 19° C was passed. The oxygen consumption rates remained stable at 36·9 ± 2·9 mg O₂ kg⁻¹ h⁻¹ over the 3 months swimming period for all tested eels. The mean cost of transportation was 28·2 mg O₂ kg⁻¹ km⁻¹. From the total energy consumption the calculated decline in fat content was 30%. When extrapolating to 6000 km this would have been 60%, leaving only 40% of the total energy reserves for reproduction after arriving at the spawning site. Therefore low cost of transport combined with high fat content are crucial for the capacity of the eel to cross the Atlantic Ocean and reproduce.     

Implications of individual growth status on the future sex of the European eel

Sex-related differences in growth status was demonstrated in eels Anguilla anguilla reared indoors at 17, 20 or 26° C, from the elver stage. Growth status was defined as length increase, weight increase and length–weight relationship. Eels attaining at least 10 g body weight (180–220 mm body length) were tagged with Passive Integrated Transponders (PIT). Length and weight were measured at 6-week intervals, until individuals stopped growing or had attained 150 g weight (380–450 mm). Sex-specific data from potentially undifferentiated eels were provided by retrospective classification of sex. Comparisions between sexes were made within groups graded by length or weight data from the beginning of each 6-week period. There was no consistent difference in absolute length increase between small males and females, but below 40–60 g initial body weight, males displayed on average a higher weight increase than females. Males also had lower length at weight than females, even in the smallest weight groups. Early growth status may influence the future sex of undifferentiated eels, but other approaches are needed for distinction between cause and effect.     

Genome‐wide single‐generation signatures of local selection in the panmictic European eel

Next‐generation sequencing and the collection of genome‐wide data allow identifying adaptive variation and footprints of directional selection. Using a large SNP data set from 259 RAD‐sequenced European eel individuals (glass eels) from eight locations between 34 and 64^oN, we examined the patterns of genome‐wide genetic diversity across locations. We tested for local selection by searching for increased population differentiation using F_ST‐based outlier tests and by testing for significant associations between allele frequencies and environmental variables. The overall low genetic differentiation found (F_ST = 0.0007) indicates that most of the genome is homogenized by gene flow, providing further evidence for genomic panmixia in the European eel. The lack of genetic substructuring was consistent at both nuclear and mitochondrial SNPs. Using an extensive number of diagnostic SNPs, results showed a low occurrence of hybrids between European and American eel, mainly limited to Iceland (5.9%), although individuals with signatures of introgression several generations back in time were found in mainland Europe. Despite panmixia, a small set of SNPs showed high genetic differentiation consistent with single‐generation signatures of spatially varying selection acting on glass eels. After screening 50 354 SNPs, a total of 754 potentially locally selected SNPs were identified. Candidate genes for local selection constituted a wide array of functions, including calcium signalling, neuroactive ligand–receptor interaction and circadian rhythm. Remarkably, one of the candidate genes identified is PERIOD, possibly related to differences in local photoperiod associated with the >30° difference in latitude between locations. Genes under selection were spread across the genome, and there were no large regions of increased differentiation as expected when selection occurs within just a single generation due to panmixia. This supports the conclusion that most of the genome is homogenized by gene flow that removes any effects of diversifying selection from each new generation.     

A simplified method to estimate body growth parameters of the European eel Anguilla anguilla

A simple approach is proposed to fit a body growth model for the European eel Anguilla anguilla to data‐poor case studies. The model is a modified von Bertalanffy curve allowing for delayed sex determination and sexual dimorphism. The proposed procedure provides preliminary estimates of model parameters on the basis of average age and body length of silver eels.     

Persistence of herpesvirus of eel Herpesvirus anguillae in farmed European eel Anguilla anguilla.

Herpesvirus of eel Herpesvirus anguillae (HVA) was isolated repeatedly from farmed eel of an outwardly healthy stock, but virus isolation was much greater in an experimental group of fish that were injected with dexamethasone. The results suggest that HVA can establish a latent infection in eel. Previous exposure of these eels to HVA virus was shown by detection of HVA-specific antibodies. These eels did not show clinical signs after a secondary infection with HVA. Tracing of seropositive eel stocks, which had previous contact with HVA, and of HVA carrier fish can be useful to control disease outbreaks due to HVA infection.     

Size selectivity of fyke nets for European eel Anguilla anguilla

Size selectivity of fyke nets for European eels Anguilla anguilla was investigated by reviewing the results of published experimental studies. A general size selectivity model was then derived that can be easily incorporated into demographic models to simulate population dynamics, assess and monitor abundance and length structure of eel stocks and forecast the consequences of different management options.     

Occurrence of drug-resistant bacteria in two European eel farms

The occurrence of strains that are resistant to oxolinic acid, oxytetracycline, sulfamethoxazole-trimethoprim, and nitrofurantoin among heterotrophic bacteria, including human and fish pathogens, in two freshwater eel farms was investigated. High levels of individual- and multiple-drug-resistant bacteria were detected, although sampling events were not correlated with clinical outbreaks and drug therapy.     

Cl- absorption in European eel intestine and its regulation

The intestinal epithelium of the euryhaline teleost fish, Anguilla anguilla, absorbs Cl(-) transepithelially. This gives rise to a negative transepithelial potential at the basolateral side of the epithelium and to a measured short circuit current. Cl(-) absorption occurs via bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransport, localized on the luminal membrane. The cotransport operates in parallel with a luminal K(+) conductance that recycles the ion into the lumen. Cl(-) leaves the cell across the basolateral membrane by way of Cl(-) conductance and presumably via a KCl cotransport. The driving force for this process is provided by the electrochemical sodium gradient across the plasma membrane, generated and maintained by the basolateral Na(+)-K(+)-ATPase. The resulting NaCl absorption process is active and enables marine fish to take up water, thereby compensating for water that was lost passively from the body. Fresh water acclimatized eel also absorb Cl(-) actively, although in smaller quantities, utilizing the same ion transport mechanisms as marine eels. This mechanism is basically the same as the model proposed for the thick ascending limb (cTAL). Cl(-) absorption is regulated by a number of cellular factors, such as HCO(3) (-), pH, Ca(2+), cyclic nucleotides, and cytoskeletal elements. It is sensitive to osmotic stress, and therefore is a good physiological model to study ion transport mechanisms that are activated when osmotic stress induces cell volume regulation. The activation of these various ion transport pathways is dependent on cellular transduction mechanisms in which phosphorylation events (mainly by PKC and MLCK for the hypertonic response) and cytoskeletal elements, either microfilaments or microtubules, seem to play key roles.     

Maturation patterns in female European eel: age and size at the silver eel stage

The maturation pattern in the female European eel Anguilla anguilla was studied by investigating age and size patterns of silver eels in different aquatic environments in Sweden, covering limnic, brackish and marine waters. The results neither supported the hypothesis that there is a critical size or age when eels enter the silvery stage, nor that size and age at maturity are positively related. Age at maturity, however, was observed to be negatively related to growth rate in all localities, i.e. the female reproductive tactic apparently is to become sexually mature at the earliest possible opportunity. Furthermore, it was recognized that a significant amount of variation was due to habitat differences, since the female eel maturation pattern deviated systematically between sampling sites, as it did also when the effect of growth rate was eliminated. Thus, the ability of the female eel to adjust maturation to an optimal size and age can be questioned, because the panmictic nature of the eel means local adaptations are unlikely Growth rate dependent differences suggest that variations in maturation patterns between eel environments are linked more to the opportunity for nutrient accumulation than to other aspects of growth.     

Changes in sugar levels during slow growth of Dendrocalamus hamiltonii somatic embryos due to liquid paraffin overlay

A liquid paraffin overlay (LPO) was used for storage of rapidly multiplying somatic embryos of Dendrocalamus hamiltonii under slow-growth conditions. Slow growth was associated with changes in sugar metabolism. In rapidly growing embryogenic tissues, a sharp decline in starch and non-reducing sugars indicated rapid utilization of starch. In contrast, under slow-growth conditions in somatic embryos stored under LPO, a gradual decline in starch indicated its slower utilization. As a result, growth of somatic embryos under LPO was suppressed and subculturing was not required. Following retrieval from growth under LPO after 30, 90, 180, 270, and 365 d of storage, the somatic embryos showed high recovery and germination (79.78%, 77.49%, 71.22%, 67.13%, and 59.99%, respectively) and were able to proliferate following transfer to Murashige and Skoog’s (Physiol. Plant. 15:473–497, 1962) medium containing 1 mgl⁻¹ BAP and 2% sucrose. The study provides useful information on in vitro storage of embryogenic tissue of D. hamiltonii.     

Habitat carrying capacity is reached for the European eel in a small coastal catchment: evidence and implications for managing eel stocks

1. Despite carrying capacity being one of the most important parameters in population management and modelling, we lack substantial evidence for habitat limitations on freshwater species. Here we tested the ideal free distribution (IFD) hypothesis using an indirect behaviour‐based method for small closed populations assuming that animals can effectively estimate habitat suitability and distribute themselves accordingly in time and space. 2. We analysed spatiotemporal variations in the density of the European eel Anguilla, a catadromous species with good colonisation abilities in a small coastal catchment in France. The general linear model used enabled us to test simultaneously the effect of temporal, macro‐ and meso‐scale habitat factors on the presence and abundance of eels at 30 sites over an 8‐year period. 3. Almost every site sampled had eels, whatever its location on the catchment and its habitat characteristics. Density estimates (overall mean ± SD of 0.40 ± 0.48 m^?2) were at the upper range of other values for European catchments. Moreover, eel densities were mainly influenced by the availability of suitable habitats (rocky substratum and instream cover), which suggests that their distribution reflects an IFD. 4. Despite marked variability in recruitment, the density of the oldest size‐class remained stable over the study, suggesting that density‐dependent mortality occurred, probably due to intraspecific competition for space and food and to predation. 5. These findings suggest that eel habitats are saturated in the Frémur. Therefore, we suggest that the mean abundance of eels observed could serve as a threshold value for other male‐dominated river stocks (provided they have a similar overall percentage of suitable habitats) that are common in small, low gradient streams on the north‐Atlantic coast of Europe.     

Effects of diet supplementation with carp ovary on gonad differentiation and growth of the European eel

Treating elvers of European eel Anguilla anguilla with mature carp ovary for 3–6 months during early growth induced female differentiation in 51·6–66·7% of treated animals compared with c . 5% in controls. The treatment also induced differentiation of ovaries in eels <13 cm L _T and a higher number of Syrski organs with ambisexual characters, and was most effective when administered at an early growth stage. The results could be attributed to the natural steroid content of the carp ovary. The total weight of treated animals at the end of the farm experiment was 84·7% higher than controls. The specific growth rate for weight was significantly higher in female yellow eels than in males, for both control and treated groups. The enhanced growth was related to induced feminization. A diet supplementation with mature carp ovary could be a good approach to control of sex differentiation and growth in eels.     

A global viability assessment of the European eel

The global European eel (Anguilla anguilla) stock is critically endangered according to the IUCN, and the European Commission has urged the development of conservation plans aimed to ensure its viability. However, the complex life cycle of this panmictic species, which reproduces in the open ocean but spends most of its prereproductive life in continental waters (thus embracing a huge geographic range and a variety of habitat types), makes it difficult to assess the long‐term effectiveness of conservation measures. The interplay between local and global stressors raises intriguing cross‐scale conservation challenges that require a comprehensive modelling approach to be addressed. We developed a full life cycle model of the global European eel stock, encompassing both the oceanic and the continental phases of eel's life, and explicitly allowing for spatial heterogeneity in vital rates, availability of suitable habitat and settlement potential via a metapopulation approach. We calibrated the model against a long‐term time series of global European eel catches and used it to hindcast the dynamics of the stock in the past and project it over the 21st century under different management scenarios. Although our analysis relies on a number of inevitable simplifying assumptions and on data that may not embrace the whole range of variation in population dynamics at the small spatiotemporal scale, our hindcast is consistent with the general pattern of decline of the stock over recent decades. The results of our projections suggest that (i) habitat loss played a major role in the European eel decline; (ii) the viability of the global stock is at risk if appropriate protection measures are not implemented; (iii) the recovery of spawner escapement requires that fishing mortality is significantly reduced; and (iv) the recovery of recruitment might not be feasible if reproductive output is not enhanced.     

A comparison of techniques for studying oogenesis in the European eel Anguilla anguilla

A multi‐technique approach was used to study the changes occurring in European eel Anguilla anguilla ovaries during hormonally‐induced vitellogenesis. Aside from classic techniques used to monitor the vitellogenic process, such as ovary histology, fat content analysis, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and vitellogenin enzyme linked immunosorbent assay (ELISA), a new technique, Fourier‐transform infrared (FT‐IR) microspectroscopy, was used to analyse A. anguilla ovaries. The results from the different techniques provided different ways of approaching the same process. Although it is considered a time consuming approach, of all the employed techniques, histology provided the most direct evidences about vitellogenesis. SDS–PAGE and ELISA were also useful for studying vitellogenesis, whereas fat analysis cannot be used for this purpose. The FT‐IR analysis provided a representative IR spectrum for each ovarian stage (previtellogenic stage, early vitellogenic stage, mid‐vitellogenic stage and late vitellogenic stage), demonstrating that it is a valid method able to illustrate the distribution of the oocytes within the ovary slices. The chemical maps obtained confirmed changes in lipid concentrations and revealed their distribution within the oocytes at different maturational stages. When the results and the accuracy of the FT‐IR analysis were compared with those of the traditional techniques commonly used to establish the vitellogenic stage, it became evident that FT‐IR is a useful and reliable tool, with many advantages, including the fact that it requires little biological material, the costs involved are low, analysis times are short and last but not least, the fact that it offers the possibility of simultaneously analysing various biocomponents of the same oocyte.     

Iron-rich particles in European eel (Anguilla anguilla L.)

Magnetic material in the European eel (Anguilla anguilla L.) was investigated by a combination of magnetic susceptibility measurements, energy dispersive X-ray fluorescence analysis and transmission electron microscopy. It was shown that the magnetic material is associated with iron. The main part of the iron is present in the form of iron-rich particles with irregular shapes about 100-3000 A large. The structures of magnetite (Fe3O4), hematite (alpha-Fe2O3) and alpha-iron (bcc structure) were identified. The particles are composed of more than one of these phases with magnetite being a minority phase when present. The iron-rich particles found in the eel are different from the materials reported for bacteria or bees.     

Growth of European eel Anguilla anguilla along the southern Baltic coast of Germany and implication for the eel management

To detect growth differences of European eel Anguilla anguilla along the southern German Baltic coast 728 yellow eels, with total lengths ranging from 256 to 944 mm and ages ranging from 3 to 15 years were collected from six coastal areas from 2005 to 2009. The estimation of the growth performance was based on the otolith increments. The mean growth rate of the female yellow eels varied from 56 to 62 mm?year^–1. No significant differences in the mean growth rate were detected between eels from inner and open coastal areas. The overall mean annual increment of eels was estimated at 59 mm?year^?1. Specific growth rates (SGR) of female yellow eels decreased with increasing age from 0.68 %?day^?1 in the first year to 0.05 %?day^?1 in the tenth year. Results indicate that no separation is needed in the development of population models or management initiatives based on the growth performance of eel in inner and open coastal waters of the southern German Baltic coast.     

Agonistic behaviour and growth in the European eel,Anguilla anguilla L., in relation to warm-water aquaculture*

Agonistic behaviours were studied at high densities and in tournament experiments with pairs of small eels with and without prior isolation. Although innate differences probably exist, eels appear to learn their average positions in diffuse social and size hierarchies from the outcome of repeated agonistic encounters. Dominance or avoidance were most marked when the larger fingerling eels were about 1.5 times the body weight of the smaller. Larger eels tended to attack more readily, be dominant in threat encounters, be less active and spend less time swimming high in the water, consume more food, and show higher growth rates and lower mortality. Implications for warm-water aquaculture are discussed, and practical management techniques to minimize mortality and maximize productivity are suggested.     

Evidence for isolation by time in the European eel (Anguilla anguilla L.)

Life history traits of highly vagile marine species, such as adult reproductive success and larval dispersal, are strongly determined by oceanographic and climatic forces. Nevertheless, marine organisms may show restricted dispersal in time and space. Patterns of isolation by distance (IBD) have been repeatedly observed in marine species. If spawning time is a function of geographical location, temporal and spatial isolation, can easily be confounded or misinterpreted. In this study, we aimed at discriminating between various forces shaping the genetic composition of recruiting juveniles of the European eel (Anguilla anguilla L.). By controlling for geographical variation, we assessed temporal variation and tested for possible isolation by time (IBT) between recruitment waves within and between years. Using 12 polymorphic allozyme and six variable microsatellite loci, we show that genetic differentiation was low (F(ST) = 0.01-0.002) and significant between temporal samples. Regression analysis between genetic and temporal distance, was consistent with a subtle interannual pattern of IBT. Our data suggest that the population dynamics of the European eel may be governed by a double pattern of temporal variance in genetic composition: (i) a broad-scale IBT of spawning cohorts, possibly as a consequence of the large migration loop in anguillids and strong variance in annual adult reproductive contribution; and (ii) a smaller-scale variance in reproductive success (genetic patchiness) within cohorts among seasonally separated spawning groups, most likely originating from fluctuating oceanic and climatic forces. The consistency of both mechanisms remains to be verified with fine-scale analyses of both spawning/migrating aged adults and their offspring to confirm the stochastic/deterministic nature of the IBT pattern in eel.     

Effects of temperature on the growth and survival of the European eel, Anguilla anguilla L.

Experiments to determine the growth rate of eels ( Anguilla anguilla L.) at different temperatures are described and show the optimum temperature for growth to be 22–23° C. The ultimate upper lethal temperature was found to be 38° C and the critical thermal maximum varied from 33 to 39° C for fish acclimated at 14 to 29° C. An attempt was also made to determine lower lethal temperatures. Eels enter a state of torpor at temperatures varying from 3° C for fish acclimated at 29° C to less than 1° C for fish acclimated at 23° C or below. The results have been used to estimate the growth rates expected from eels cultured in power station cooling water using different types of temperature control.