Regulation of Anguillicola crassus (Nematoda) infrapopulations in their definitive host, the European eel, Anguilla anguilla

The parasitic nematode Anguillicola crassus was recently introduced into populations of the European eel, Anguilla anguilla. We investigated, under experimental conditions, the regulation of A. crassus infrapopulations. We tested the effects of (1) the resource-limited habitat of the parasite and (2) the coexistence of several developmental stages in its niche (the swim-bladder) on the composition of the infrapopulations. The results revealed that the respective effects of these factors differed substantially during the course of the infection. Third-stage larvae (L3s) establishment would not be constrained by the size of the swim-bladder. Their moult to fourth-stage larvae (L4s) would be accelerated as the number of L3s increased. The moulting time of L4s to adults would be reduced by males and would be constrained by the size of the swim-bladder. However, the moult of L4s to adults and their further development would be synchronized with those of the opposite sex. At the time of mating, the number of males and the body weight of adults would depend on the size of the swim-bladder. Soon after the laying of eggs, the developmental constraint on the late L3s would decrease. When adults die, constraints would cease and late larval stages would moult to become adults.     

Influence of Anguillicola crassus (Nematoda) and Ichthyophthirius multifiliis (Ciliophora) on swimming activity of European eel Anguilla anguilla

We investigated the swimming activity of 70 European eels Anguilla anguilla in relation to natural infection with 2 parasite species: the eel-specific swimbladder nematode Anguillicola crassus and the non-specific skin and gill protozoan Ichthyophthirius multifiliis. We measured how long individual eels exposed to a water current in a swimming channel with a steady-stream profile could withstand the water current. The parasites affected the swimming behaviour of eels in different ways. The maximum period of time the fish were able to swim against the current was not correlated with infection by A. crassus. In contrast, infection with I. multifiliis reduced the swimming time. The protozoan has a higher pathogenicity than the swimbladder nematode, at least in closed systems, where I. multifiliis is able to spread within a few days. Reduction in swimming capacity after infection with the ciliate averaged 47 % compared to capacity prior to infection. Thus, our results do not support the previously suggested strong negative relation between swimming activity of eels and intensity of A. crassus infection, at least in the short-term. However, there are indications in the literature that the pathological effects of A. crassus on the eel swimmbladder may involve a higher energy demand, possibly manifested in a prolonged spawning migration. As a result, eels heavily infected with this parasite may arrive too late at the spawning site to participate in mating. This could ensure a selection of 'good genes'.     

Histopathological changes in the swimbladder wall of the European eel Anguilla anguilla due to infections with Anguillicola crassus

The histopathological changes in swimbladders of European eels naturally and experimentally infected with Anguillicola crassus were studied using transmission and scanning electron microscopy. During the course of probably several infections swimbladders undergo characteristic changes. In addition to the thickening of the entire swimbladder wall, and to the folded internal surface of this organ, inflammation, migration of white blood cells, fibrosis and changes in the epithelial cells are frequently seen. Epithelial cells tend to proliferate heavily and form hyperplastic tissues; these processes are accompanied by changes in the internal structure of the cells. The normally cubic cells become spherical or columnar and form folds facing the lumen of the swimbladder. As a consequence, most of these cells lose contact with the blood vessels and show no strict polarity. In heavily affected swimbladders the basal labyrinth of the epithelial cells is reduced, i.e. becomes shorter and less densely packed. The lamina propria shows severe fibrosis with infiltration of white blood cells. Larvae of A. crassus, inhabiting the wall of the swimbladder, were found to be surrounded by cell debris, but this local necrosis does not affect the entire swimbladder in its overall structure. These histological findings can partly explain changes in the gas composition in eels infected with A. crassus.     

Biased sex ratio in the European eel (Anguilla anguilla) swim-bladder parasite Anguillicola crassus, experimentally induced by 11-ketotestosterone

Parasites are intimately connected to the host in which they live, and some may be affected by the polluted environment of their host. The present study describes the effect of a steroid hormone (11-ketotestosterone) on the sex ratio of the invasive hematophagous nematode Anguillicola crassus Kuwahara, Niimi & Itagaki, 1974, when experimentally injected to European eels, Anguilla anguilla. Our results showed that this steroid induced a significant male-biased ratio in the nematode A. crassus infrapopulations, suggesting that the presence of endocrine disruptors in the environment may lead to skewed sex ratios among parasites.     

Evaluation of an ELISA and immunoblotting for studying the humoral immune response in Anguillicola crassus infected European eel Anguilla anguilla

The applicability of an enzyme-linked immunosorbent assay (ELISA) for the detection of anguillicolosis in feral eels was examined using a crude antigen preparation from the body wall of adult Anguillicola crassus. The screening consisted of samples from 100 feral European eels Anguilla anguilla. As a reference the actual status of infection was determined by dissection of the eels' swim-bladders. The ELISA results were compared with a background value calculated from the results obtained from 43 non-infected farm eels. The screened samples had a high prevalence of A. crassus (83 %); however, the specificity and the negative predictive value of the ELISA were low compared to the high positive predictive value. Nonetheless, the reproducibility (precision) of the test was satisfactory, and for the non-infected reference group specificity was 97.7 %. Although the ELISA, as used in the present study, is not applicable for diagnostic purposes, it represents a useful tool for the investigation of the specific humoral immune response of eels against A. crassus under controlled experimental conditions. Immunoblots using crude antigen preparations from different parts of adult A. crassus as well as a crude somatic third-stage (L3) antigen preparation illustrated that only antigens associated with the body wall of adult A. crassus are potentially suitable for diagnostic purposes. Despite the fact that antibodies against Raphidascaris acus cross-reacted with 3 body wall antigens of A. crassus, the most encouraging results were obtained with the antigen preparation from the outer cuticle of adult A. crassus which yielded a conspicuous, broad band at about 100 kDa.     

Vaccination of eels (Anguilla japonica and Anguilla anguilla) against Anguillicola crassus with irradiated L3

The original host of the swimbladder nematode Anguillicola crassus, the Japanese eel (Anguilla japonica) and the recently colonized European eel (Anguilla anguilla) were immunized with 40 irradiated (500 Gy) 3rd-stage larvae (L3) of this parasite and challenged with an infection of 40 normal L3. The immunization induced a significant reduction of the number of adult worms developing from the challenge infection in A. japonica, but not in A. anguilla. The induced resistance (calculated using the relation of the number of adult worms in immunized eels and in non-immunized control eels) in A. japonica was 87.3%+/-30.4%. Following a single infection, the percentage of adult worms found in A. japonica was lower as compared to A. anguilla, and the few adult worms were much smaller, revealing a lower susceptibility of A. japonica to A. crassus in comparison to A. anguilla. Both eel species developed an antibody response against A. crassus, but the level of antibody responses was not positively correlated with the protection against infection, suggesting that the antibody response is not a key element in resistance of eels against A. crassus. This study suggests that the original host of A. crassus is able to mount efficient protective immune responses against its parasite, whereas the newly acquired host seems to lack this ability.     

The swimbladder nematode Anguillicola crassus in the European eel Anguilla anguilla and the Japanese eel Anguilla japonica: differences in susceptibility and immunity between a recently colonized host and the original host

The swimbladder nematode Anguillicola crassus originates from Asia where it is a parasite of the Japanese eel Anguilla japonica. After its introduction to Europe about 25 years ago, the parasite spread rapidly within the indigenous populations of the European eel Anguilla anguilla and subsequently the prevalence and mean intensity appeared to stabilize. Under experimental and aquaculture conditions the na?ve new host appears to be more susceptible to A. crassus compared to the original host. Both eel species develop a immune response against A. crassus. The antibody response is well characterized for the European eel, but poorly characterized for the Japanese eel. It remains unclear if antibodies have any protective function against A. crassus. Encapsulation of larvae of A. crassus can be observed in naturally infected European eels. However, encapsulation of larvae following experimental infection has not been detected in European eels, but only in Japanese eels. Reinfection experiments and intraperitoneal injection of A. crassus homogenates failed to demonstrate the development of acquired immunity in European eels. Immunization with irradiated third stage larvae provided preliminary evidence for acquired immunity against A. crassus in the Japanese eel, but not in the European eel.     

Physiological responses to acute temperature increase in European eels Anguilla anguilla infected with Anguillicola crassus

The swimbladder parasite, Anguillicola crassus has infected, and spread rapidly, through European eel Anguilla anguilla (L.) populations over the past 20 to 25 yr. Our aim in the present studies was to elucidate whether the presence of A. crassus in these eels alters their rapid physiological responses to an acute temperature increase, compared to the response of uninfected fish. Both infected and uninfected fish showed significant increases in plasma cortisol after 2 h at a raised environmental temperature with increased plasma glucose after 6 h. However, infected eels exhibited a slight lag in glucose mobilisation, which may be due to the metabolic cost of harbouring a sanguiverous parasite. Both infected and uninfected fish showed a significant increase in haematocrit after 6 h of temperature elevation, but only uninfected fish exhibited a significant increase in haemoglobin at this point. However, there were no significant changes in mean erythrocyte haemoglobin concentration in either group. Our results suggest that acute temperature increase alone is unlikely to cause significant mortality of A. crassus-infected European eels; however, the effects of chronic increases in temperature in combination with other factors such as toxicants and hypoxia requires examination.     

Development of the swimbladder in the European eel (Anguilla anguilla)

The swimbladder of the adult eel, Anguilla anguilla, with its bipolar countercurrent system, the rete mirabile, is a widely used model for swimbladder function, but very little is known about the development of this swimbladder. Our histological studies on the developing swimbladder revealed that during metamorphosis the swimbladder becomes present as a dorsal outgrowth of the esophagus. It is filled with surfactant, and gas was not detected in the swimbladder. In the young glass-eel, the epithelial (gas gland) cells of the swimbladder are columnar, but do not yet have the typical basolateral labyrinth established in adult animals. Few blood vessels are found in the swimbladder tissue, and the submucosa is present as a thick layer of connective tissue, giving a large diffusion distance between blood vessel and swimbladder lumen. Within the next 2 or 3 months of development, gas gland cells develop their typical basolateral labyrinth, and the thickness of the submucosa is significantly reduced, resulting in a short diffusion distance between blood vessels and the swimbladder lumen. The first filling of the swimbladder with gas is observed while the gas gland cells are still in a poorly differentiated status and it appears unlikely that these cells can accomplish their typical role in gas deposition. The presence of small gas bubbles in the swimbladder as well as in the ductus pneumaticus at the time of initial swimbladder inflation suggests that the swimbladder is filled by air gulping or possibly by taking up gas bubbles from the water.     

Infection status of the swimbladder worm, Anguillicola crassus in silver stage European eel, Anguilla anguilla, from three different habitats in Danish waters

The infection status of silver stage eels, Anguilla anguilla , infected with Anguillicola crassus from three locations, ranging from a lake to seawater, were investigated. Data show a significant difference in mean intensity of Anguillicola crassus in silver stage eels according to the salinity of the habitats.     

Colonization, larval survival and epidemiology of the nematode Anguillicola crassus, parasitic in the eel, Anguilla anguilla, in Britain

In late 1987, immature Anguillicola crassus were reported for the first time in Britain from eels from two river systems. By late 1988, gravid adults were present in a number of rivers in the east of England in two discrete centres of distribution: one in East Anglia correlated with the route taken by lorries exporting eels to the continent, and one in the R. Thames correlated with the import of eels to London. The parasite was firmly established in the R. Trent, where prevalence levels reached 100% in some places. Laboratory investigations showed that adult parasites and their eggs remained viable even after infected eels had been maintained for 4 weeks in 100% sea water. Hatching of eggs declined with increasing salinity, but was not totally inhibited by sea water. Survival and infectivity of freeliving second stage larvae were maximal in natural fresh water (95% survival for 4 months, and 50% still infective to copepod intermediate hosts after 70 days), but declined in alkaline water and with increased salinity. Nevertheless, in 100% sea water, 50% of larvae were still infective after 8 days. Specificity to the intermediate host was low, and eels of all sizes could be infected. These characteristics, plus a high reproductive potential, give the parasite exceptional colonization potential and ability, enabling it to survive natural movements of eels from catchment to catchment and to increase rapidly within a new locality. The ability of free-living larvae to adhere to the substratum and survive in sea water enables them to survive in eel-transport lorries from which they will not readily be removed by flushing, the normal cleansing procedure. It is concluded that there were two separate introductions of the parasite to Britain; via the eel import trade through London, and, totally unexpectedly, via the eel export trade in lorries traversing East Anglia. The parasite is now firmly established in Britain and will continue to spread by natural movements of eels but especially by human-assisted movements of infected eels for stocking and market. This latter practice is recognized as a major factor in introducing and disseminating fish parasites.     

Replication banding in the chromosomes of the European eel (Anguilla anguilla)

The chromosomes of the European eel Anguilla anguilla have been analyzed with a replication banding technique from lymphocyte cultures treated with 5-BrdU. This technique allows us to identify with high resolution the individual chromosome pairs and to differentiate classes of chromatin by the order of replication. The replication banding obtained on the chromosomes of European eel can be related with the structural bands described in this species.     

Cloning and Expression of Guanylin from the European eel (Anguilla anguilla)

Extracts of intestinal epithelia from the European eel (Anguilla anguilla) stimulated cGMP production in the T84 human colon carcinoma cell line which suggested the presence of a guanylin-like peptide in this teleost fish. Degenerate oligonucleotide primers were subsequently used in RT-PCR resulting in the amplification, cloning, and sequencing of two cDNAs which represent possible 5' spliceoforms of an eel homologue of the mammalian peptide, guanylin. Northern blotting indicated that the main site of expression of the eel peptide is in the intestine with much lower signals also detected in the kidney. Intestinal expression of guanylin mRNA is up-regulated in both nonmigratory "yellow" and the more sexually mature, migratory "silver" eels following acclimation to the seawater environment. These results suggest that this peptide signalling system may play a role in osmoregulation in euryhaline teleost fish during migration between the marine and freshwater environments.     

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.     

Dynamics and predicted decline of Anguillicola crassus infection in European eels, Anguilla anguilla, in Neusiedler See, Austria

The eel population in Neusiedler See has been maintained by regular massive stocking since 1958. After the establishment of the National Park Neusiedler See-Seewinkel in 1993, eel stocking was prohibited and the population, together with the specific parasites of eels, was predicted to decline to extinction within 10 years. This investigation was undertaken to document the decline and extinction of the Anguillicola crassus population in eels. From 1994 to 2001, 720 eels were collected from two sites in the lake. Prevalence and abundance of A. crassus were lower in spring than in summer and autumn and larger eels harboured more parasites than smaller ones. Neither year of study nor sampling site were correlated with parasite infection levels. No significant trend in the population parameters of A. crassus was detected over the 8 years of the survey. This suggested that there had been no significant decline in the eel population. This suggestion was confirmed by investigations of the fishery, which also found evidence of regular illegal stocking. The stability of the A. crassus population over the past decade seems to reflect the lack of change in eel population density. No mass mortalities of eels occurred over the period despite the many similarities between Neusiedler See and Lake Balaton in Hungary. Differences in eel size, eel diet and the lack of large-scale insecticide use are discussed as possible explanations for the absence of eel mass mortalities in Neusiedler See.     

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.     

Swim bladder nematodes (Anguillicoloides crassus) disturb silvering in European eels (Anguilla anguilla)

The introduced parasite Anguillicoloides crassus is thought to play an important role in the decline of freshwater eel (Anguilla spp.) populations. These nematodes are known to negatively affect many fitness-related traits in eels. We used experimental infections to study the effect of A. crassus on the relative size or mass of organs, and the expression of functionally relevant genes (total of 12 parameters) that are involved in the silvering process of Anguilla anguilla. Our results showed that the liver mass, the hemoglobin α-chain, and androgen receptors α expression levels were significantly higher in infected eels, whereas the freshwater rod opsin expression level and the gut mass were significantly lower in infected eels. Our results suggested that infected eels were at a more advanced stage in the silvering process than uninfected counterparts of similar size. These results may be explained by 2 hypotheses. First, A. crassus could trigger physiological mechanisms involved in the silvering process as a side-effect of infection. Second, eels may adjust their life history traits in response to infection. The implications for eel migration and reproductive success may be either negative or positive, depending on whether the response to A. crassus infection results in an additional cost of the parasite or is due to the phenotypic plasticity of the host.     

An enhanced humoral immune response against the swimbladder nematode, Anguillicola crassus, in the Japanese eel, Anguilla japonica, compared with the European eel, A. anguilla.

The humoral immune response in the two eel species, Anguilla japonica and Anguilla anguilla against two fractions of antigens in Anguillicola crassus were studied. Within species, both eel species showed significantly elevated titres compared with controls when immunized with antigens from Anguillicola crassus. In interspecific comparison, Anguilla japonica showed significantly elevated titres in comparison with Anguilla anguilla. Immunization of Anguilla anguilla caused a significantly decrease in the plasma levels of protein in comparison with control fish and all groups of Anguilla japonica. In contrast, Anguilla japonica showed significantly lower plasma levels of Ig in all groups compared with Anguilla anguilla. The different susceptibilities to Anguillicola crassus between the natural host, Anguilla japonica, and the naive, Anguilla anguilla, is partly due to differences in the ability of the two eel species to mount a humoral immune response.