Reaching the steady state: 30 years of Anguillicola crassus infection of European eel, Anguilla anguilla L., in Northern Germany.

A 30 years long data series on the infection dynamics of European eel (Anguilla anguilla L.) with the non-native invasive nematode Anguillicola crassus Kuwahara, Niimi & Hagaki, 1974 is presented. Parasite burden was evaluated for 30 years in inland and coastal waters in Mecklenburg-Western Pomerania from 1991 to 2020. The total prevalence, mean intensity and damage status of the swim bladders were very high during the first decade (1991­2000), and significantly decreased in both marine and freshwater eel populations in the following decades (2001­2010, 2011­2020). The parasite intensity of eels in coastal waters was significantly lower compared with the freshwater systems (61.3% vs 79.5% in the first decade), indicating the vulnerability of the parasites to brackish water conditions and the fact that the life cycle of A. crassus cannot be completed under high saline conditions. Eel caught in the western part of the Baltic Sea (west of Darss sill) had the lowest mean infection (51.8% in first decade) compared to the eastern part with 63.8%. Thus, besides different infection patterns caused by the environmental conditions, a temporal trend towards a reduced parasite intensity and a more balanced parasite­host relationship developed in the 30 years of interaction after the first invasion. Possible reasons and mechanisms for the observed trends in parasite­host interactions are discussed.

Effects of the invasive swim bladder parasite Anguillicola crassus on health and condition indicators in the European eel.

Parasites negatively affect biological processes within their hosts, which may alter for example health, growth, and reproductive ability. Non-native invasive parasites, in particular, may have large effects on the endemic hosts, given that the hosts lack evolved specific defences against such parasites. The swim bladder nematode Anguillicola crassus, an invasive parasite originating from Asia, is found in the European eel (Anguilla anguilla, L. 1758), since the 1980s. We investigated whether A. crassus affected several indicators related to health of the European eel (spleen- and liver size, body fat content and relative condition). Our results indicate that during the continental residency of the eels, infection by A. crassus had no major negative impacts on the investigated health indicators at the generally low infection intensities present in this study (median 2-3 visible parasites). Given that many of the adult eels were found to have swim bladder damage, concerns about their spawning migration through deeper oceanic environments can still be raised. To allow further investigations, we suggest that quantification of swim bladder damage should be implemented in eel-monitoring programs. Compared to other parasite pressure parameters, swim bladder damage provides additional information about past infections and future problems.

A qPCR-based method to detect the eel parasitic nematode Anguillicola crassus in intermediate and final hosts.

Being able to systematically detect parasitic infection, even when no visual signs of infection are present, is crucial to the establishment of accurate conservation policies. The nematode Anguillicola crassus infects the swimbladder of anguillid species and is a potential threat for eel populations. In North America, naïve hosts such as the American eel Anguilla rostrata are affected by this infection. The accidental introduction of A. crassus following restocking programs may contribute to the actual decline of the American eel in Canada. We present a quantitative real time PCR-based method to detect A. crassus infection in final and intermediate hosts. We tested two protocols on samples from different geographical origins in Canada: 1) a general detection of A. crassus DNA in pools of young final hosts (glass eels) or crustacean intermediate hosts 2) a detection at the individual scale by analyzing swim bladders from elvers, or from adult yellow and silver eels. The DNA of A. crassus was detected in one pool of zooplankton (intermediate host) from the Richelieu River (Montérégie-Québec), as well as in individual swim bladders of 13 elvers from Grande and Petite Trinité rivers (Côte-Nord-Québec). We suggest that our qPCR approach could be used in a quantitative way to estimate the parasitic burden in individual swim bladders of elvers. Our method, which goes beyond most of previous developed protocols that restricted the diagnosis of A. crassus to the moment when it was fully established in its final host, should help to detect early A. crassus infection in nature.

Towards an in situ non-lethal rapid test to accurately detect the presence of the nematode parasite, Anguillicoloides crassus, in European eel, Anguilla anguilla.

Anguillicoloides crassus is an invasive nematode parasite of the critically endangered European eel, Anguilla anguilla, and possibly one of the primary drivers of eel population collapse, impacting many features of eel physiology and life history. Early detection of the parasite is vital to limit the spread of A. crassus, to assess its potential impact on spawning biomass. However accurate diagnosis of infection could only be achieved via necropsy. To support eel fisheries management we developed a rapid, non-lethal, minimally invasive and in situ DNA-based method to infer the presence of the parasite in the swim bladder. Screening of 131 wild eels was undertaken between 2017 and 2019 in Ireland and UK to validate the procedure. DNA extractions and PCR were conducted using both a Qiagen Stool kit and in situ using Whatman qualitative filter paper No1 and a miniPCR DNA Discovery-System™. Primers were specifically designed to target the cytochrome oxidase mtDNA gene region and in situ extraction and amplification takes approximately 3 h for up to 16 individuals. Our in-situ diagnostic procedure demonstrated positive predictive values at 96% and negative predictive values at 87% by comparison to necropsy data. Our method could be a valuable tool in the hands of fisheries managers to enable infection control and help protect this iconic but critically endangered species.

Anguillicola (Anguillicoides) crassus: Morphometric characteristics and pathogenicity in eels (Anguilla anguilla) in Greece.

Anguillicola (Anguillicoides) crassus is a swimbladder nematode parasite of Anguilla anguilla (Linnaeus, 1758). The present study investigates the morphology and pathogenicity of A. crassus in European eel, as well as, the effects of different aquatic environments on the infection of A. crassus, in Greece. A total of 268 nematodes were collected from four estuarine systems in Greece. In all collected parasites, measurements carried out to define the morphological characteristics of parasites: parasite length and width, oesophagus max length and max width. Τhe mean parasite length was 23.50 mm (95% CI: 22.42-24.58) for females, 12.95 mm (95% CI: 12.25-13.64) for the males and 6.39 mm (95% CI: 5.27-7.50) for the juveniles. The mean parasite width was 1.99 mm (95% CI: 1.88-2.10) for females, 0.93 mm (95% CI: 0.86-1.00) for males and 0.51 mm (95% CI: 0.39-0.64) for juveniles. In total, the mean intensity was found 3.15 (95% CI: 2.53-3.78), while the highest mean intensity per eel was 8.00 (95% CI: 0.00-29.51) in Tholi Lagoon and the lowest was 2.20 (95% CI: 0.36-4.04) in Amvrakikos Gulf. Longitudinal and transverse histological sections of two adults A. crassus and two swimbladders were carried out. Multiple granules were observed, as a tissue reaction of the organism to swath around the 4th stage larvae (L4) that have entered the swimbladder. Molecular analysis was performed on three female adults A. crassus derived from eel specimens coming from the Vistonis estuarine system. The high reproductive capacity of the parasite reveals that A. anguilla has low effective defense mechanisms against the parasite. Also, the morphometric variations of the A. crassus seems to have a plastic feature which is being differently expressed when exposed to various environments.

DIPLOTRIAENA OBTUSA (NEMATODA: DIPLOTRIAENIDAE) FROM BARN SWALLOWS (HIRUNDO RUSTICA) AND CLIFF SWALLOWS (PETROCHELIDON PYRRHONOTA) COLLECTED DURING MORTALITY EVENTS IN THE UPPER MIDWEST, USA.

Several mortality events involving barn swallows (Hirundo rustica) and cliff swallows (Petrochelidon pyrrhonota) were reported in the Upper Midwestern states in 2017 and 2018. Barn swallow mortality followed unseasonal cold snaps, with the primary cause of death being emaciation with concurrent air sac nematodiasis. Lesions in cliff swallows were consistent with blunt force trauma from suspected car impacts. Examination of air sac nematodes from both bird species revealed morphological characters consistent with Diplotriaena obtusa. Sequence analysis of the partial 18S rRNA gene indicated the samples clustered with other species in the genus Diplotriaena. These nematodes provide a link between morphological specimens and DNA sequence data for D. obtusa.

Morphological and genetic description of two new species of philometrid nematodes (Philometridae) parasitic in needlefishes (Belonidae) from estuaries of Florida, USA.

Two new species of philometrid nematodes (Philometridae) from needlefishes (Belonidae) in Florida are described based on morphological and genetic characteristics: Philometra aequispiculata sp. n. (males and females) collected from the ovary of Strongylura marina (Walbaum) (type host) and Strongylura notata (Poey), and Philometra notatae sp. n. (females) from the swimbladder of S. notata. Both species are described and illustrated based on light and scanning electron microscopical examinations. Morphologically, P. aequispiculata sp. n. differs from all congeners mainly in the unique structure of the distal tip of the gubernaculum, whereas P. notatae sp. n. is mainly characterised by the presence of eight markedly large cephalic papillae of the outer circle in gravid and subgravid females, the body length of the gravid female (54 mm) and by the absence of caudal projections. Molecular characterisation of the new species was assessed from phylogenetic analysis of mitochondrial cytochrome c oxidase I (COI) and SSU rRNA small-subunit ribosomal RNA (SSU) sequences among closely related philometrids by way of Bayesian inference. Phylogenetic reconstructions based on COI and SSU sequences show each of the new species comprise discrete ancestor-descendent lineages.

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.

The impact of Anguillicoloides crassus (Nematoda) on European eel swimbladder: histopathology and relationship between neuroendocrine and immune cells.

The swimbladder functions as a hydrostatic organ in most bony fishes, including the European eel, Anguilla anguilla. Infection by the nematode Anguillicoloides crassus impairs swimbladder function, significantly compromising the success of the eel spawning migration. Swimbladders from 32 yellow eels taken from Lake Trasimeno (Central Italy) were analysed by histopathology- and electron microscopy-based techniques. Sixteen eels (50%) harboured A. crassus in their swimbladders and intensity of infection ranged from 2 to 17 adult nematodes per organ (6.9 ± 1.6, mean ± s.e.). Gross observations of heavily infected swimbladders showed opacity and histological analysis found a papillose aspect to the mucosa and hyperplasia of the lamina propria, muscularis mucosae and submucosa. Inflammation, haemorrhages, dilation of blood vessels and epithelial erosion were common in infected swimbladders. In the epithelium of parasitized swimbladders, many empty spaces and lack of apical junctional complexes were frequent among the gas gland cells. In heavily infected swimbladders, we observed hyperplasia, cellular swelling and abundant vacuolization in the apical portion of the gas gland cells. Numerous mast cells and several macrophage aggregates were noticed in the mucosal layer of infected swimbladders. We found more nervous and endocrine elements immunoreactive to a panel of six rabbit polyclonal antibodies in infected swimbladders compared to uninfected.

Diplotriaena-An Air Sac Nematode Found in a New Holland Honeyeater (Phylidonyris novaehollandiae).

A New Holland honeyeater (Phylidonyris novaehollandiae), a common species found mostly in the southern parts of Australia, was examined after a presumed traumatic incident but died several hours after presentation. At necropsy, a Diplotriaena nematode was found in a cranial air sac. Although no obvious gross pathologic changes were attributed to the nematode, histologic changes of bronchial epithelial hyperplasia and goblet cell metaplasia, consistent with chronic airway irritation were found in the lungs associated with Diplotriaena eggs. Diplotriaena species are parasitic nematodes found in the air sacs of birds. These parasites have an indirect life cycle; larval stages develop in insects, and when eaten by the host, these larvae migrate to the air sac and become adults. Although Diplotriaena species have been found in birds worldwide, none, to our knowledge, have previously been described in New Holland honeyeaters.

Air Sac Nematode ( Serratospiculum tendo) Infection in an Austral Peregrine Falcon ( Falco peregrinus cassini) in Argentina.

We report a case of air sac nematode ( Serratospiculum tendo) infection in an adult male Austral Peregrine Falcon ( Falco peregrinus cassini) admitted to a rehabilitation center in Mendoza Province, Argentina, in September 2017. This case of air sac nematodes reported in an Argentine raptor is only the second report of S. tendo in South America. We recommend examination of all raptors, especially those falcon species that include insects in their diet and inhabit open lands and those in rehabilitation centers or kept for falconry, education, or captive breeding. Fecal analysis and microscopic examination of oral swabs for evidence of parasites are simple noninvasive diagnostic procedures that allow easy detection of these parasites under field and captive circumstances.

Molecular evidence of infection with air sac nematodes in the great tit (Parus major) and the captive-bred gyrfalcon (Falco rusticolus).

Serratospiculiasis is a parasitic disease caused by filariid nematodes of the genus Serratospiculum that parasitise the air sacs of various species of falcons, bald eagles and Cooper's hawks around the world. An infection with Serratospiculum was recently confirmed in a nonspecific host, the great tit, in Slovakia. Parasitic material from this host was fixed for molecular analysis. Nematode found in the air sacs from a captive-bred gyrfalcon was also stored. Analysis of small subunit (18S) ribosomal DNA (18S rDNA) gene indicated that sequences from Serratospiculum sp. and Serratospiculoides amaculata were closely related to a reference sequence from Serratospiculum tendo, in agreement with morphology. This study is the first to generate molecular data and infer the phylogenetic position of S. amaculata as the first representative of the genus Serratospiculoides.

Opisthotonos and unilateral internal hydrocephalus associated with aberrant migration of Serratospiculum sp. or Serratospiculoides sp. in a prairie falcon.

A juvenile, wild-caught prairie falcon ( Falco mexicanus) kept for falconry was presented to a veterinary hospital for intermittent opisthotonos and torticollis. Clinical examination, complete blood count, serum biochemistry panel, and fecal analysis were unremarkable. Clinical signs did not resolve, and the bird was euthanized 6 mo after the appearance of clinical signs. Autopsy revealed a mild, unilateral hydrocephalus and nematodes within the thoracic air sac. Histopathology demonstrated mild, unilateral hydrocephalus; scattered glial nodules; meningeal nematode sections; and meningeal and intraventricular embryonated eggs. Morphology and molecular characterization were consistent with the air sac nematode Serratospiculum or Serratospiculoides spp. Air sac nematode infection can be associated with air sacculitis or pneumonia in falcons. Aberrant migration of air sac filariid nematodes Serratospiculum or Serratospiculoides spp. into the nervous system resulting in clinical disease is rare, but should be included in the differential diagnosis of neurologic diseases in falcons.

Transcript levels of members of the SLC2 and SLC5 families of glucose transport proteins in eel swimbladder tissue: the influence of silvering and the influence of a nematode infection.

The rate of glucose metabolism has been shown to be correlated to glucose uptake in swimbladder gas gland cells. Therefore, it is assumed that in the European eel silvering, i.e., the preparation of the eel for the spawning migration to the Sargasso Sea, coincides with an enhanced capacity for glucose uptake. To test this hypothesis expression of all known glucose transport proteins has been assessed at the transcript level in yellow and in silver eels, and we also included Anguillicola crassus infected swimbladders. Glucose uptake by rete mirabile endothelial cells could be crucial for the countercurrent exchange capacity of the rete. Therefore, this tissue was also included in our analysis. The results revealed expression of ten different members of the slc2 family of glucose transporters, of four slc5 family members, and of kiaa1919 in gas gland tissue. Glucose transporters of the slc2 family were expressed at very high level, and slc2a1b made up about 80% of all slc2 family members, irrespective of the developmental state or the infection status of the eel. Overall, the slc5 family contributed to only about 8% of all detected glucose transport transcripts in gas gland tissue, and the slc2 family to more than 85%. In rete capillaries, the contribution of sodium-dependent glucose transporters was significantly higher, leaving only 66% for the slc2 family of glucose transporters. Neither silvering nor the infection status had a significant effect on the expression of glucose transporters in swimbladder gas gland tissue, suggesting that glucose metabolism of eel gas gland cells may not be related to transcriptional changes of glucose transport proteins.

Hiding in the swamp: new capillariid nematode parasitizing New Zealand brown mudfish.

The extent of New Zealand's freshwater fish-parasite diversity has yet to be fully revealed, with host-parasite relationships still to be described from nearly half the known fish community. While advances in the number of fish species examined and parasite taxa described are being made, some parasite groups, such as nematodes, remain poorly understood. In the present study we combined morphological and molecular analyses to characterize a capillariid nematode found infecting the swim bladder of the brown mudfish Neochanna apoda, an endemic New Zealand fish from peat-swamp-forests. Morphologically, the studied nematodes are distinct from other Capillariinae taxa by the features of the male posterior end, namely the shape of the bursa lobes, and shape of spicule distal end. Male specimens were classified into three different types according to differences in the shape of the bursa lobes at the posterior end, but only one was successfully characterized molecularly. Molecular analysis indicated that the studied capillariid is distinct from other genera. However, inferences about the phylogenetic position of the capillariid reported here will remain uncertain, due to the limited number of Capillariinae taxa characterized molecularly. The discovery of this new capillariid, which atypically infects the swim bladder of its host, which itself inhabits a very unique ecosystem, underlines the very interesting evolutionary history of this parasite, which for now will remain unresolved.

The effect of foraging and ontogeny on the prevalence and intensity of the invasive parasite Anguillicola crassus in the European eel Anguilla anguilla.

Infection patterns of the invasive Anguillicola crassus nematode were investigated in a population of the European eel Anguilla anguilla where parasite invasion is very recent, Loch Lomond, Scotland. Intensity levels of the parasite were associated with differences in fish ontogeny and trophic ecology. Although eels foraged on both fish and invertebrates, individuals which were smaller and fed on invertebrates (>70% contribution to diet) were found to contain a greater number of swim bladder parasites compared to larger eel with a predominance of fish (>60% contribution) in their diet. Within affected fish, a significant negative relationship was found between fish length and parasite intensity, with smaller individuals having higher parasite intensity than larger individuals. This study indicates that food intake and infection risk are linked in this recently infected host-parasite system. From a management perspective increasing our understanding of how infection intensity and repeated exposure is linked to resource use in an ecosystem is important for the future management of this endangered species in Europe.

Trematodes from the Northern Lapwing, Vanellus vanellus (Charadriidae), from Central Germany.

Seventy-five previously frozen Northern Lapwings, Vanellus vanellus (Linnaeus, 1758) were dissected and examined for trematodes. The available birds were euthanized after suffering severe injuries from a hail storm. Trematode specimens were found in the air sacs, body cavities and kidneys. They were cleared, stained in Alaun-Carmine and examined via light microscopy. Trematodes were assigned to two different families and three different species: Uvitellina vanelli and Selfcoelum sp. from the family Cyclocoelidae and Tanaisia valida of the family Eucotylidae. A prevalence of 12% (for both, Cyclocoelid and Eucotylid infections) was detected. While trematodes of the respiratory system have been discovered in V. vanellus before, the present study describes renal trematodiasis in this bird species for the first time. Furthermore, the first recovering of a trematode of the species Selfcoelum sp. in the Northern Lapwing is described. Full measurements and figures of trematodes are given. Criteria and critical points in specifying and recovering the parasites are discussed.

The life cycle of Huffmanela huffmani Moravec, 1987(Nematoda: Trichosomoididae), an endemic marine-relict parasite of Centrarchidae from a Central Texas spring.

The life cycle of the swim bladder nematode Huffmanela huffmani Moravec, 1987 (Trichinelloidea: Trichosomoididae), an endemic parasite of centrarchid fishes in the upper spring run of the San Marcos River in Hays County, Texas, USA, was experimentally completed. The amphipods Hyalella cf. azteca (Saussure), Hyalella sp. and Gammarus sp. were successfully infected with larvated eggs of Huffmanela huffmani. After ingestion of eggs of H. huffmani by experimental amphipods, the first-stage larvae hatch from their eggshells and penetrate through the digestive tract to the hemocoel of the amphipod. Within about 5 days in the hemocoel of the experimental amphipods at 22 °C, the larvae presumably attained the second larval stage and were infective for the experimental centrarchid definitive hosts, Lepomis spp. The minimum incubation period before adult nematodes began laying eggs in the swim bladders of the definitive hosts was found to be about 7.5 months at 22 °C. This is the first experimentally completed life cycle within the Huffmanelinae.

The Hsp70 response of Anguillicola species to host-specific stressors.

The present study is based on infection experiments of two different swim bladder parasite species, Anguillicola crassus Kuwahara et al., 1974 and Anguillicola novaezelandiae Moravec and Taraschewski, 1988, which were experimentally transferred to the two eel species Anguilla anguilla Linnaeus, 1758 and Anguilla japonica Temmink and Schlegel, 1846, respectively. The host-parasite groups were selected due to their different grades of mutual adaptation. The main aim of this study was to analyze the stress responses within the parasites, which were confronted with different hosts, i.e. with different stressors related to the respective host. For this purpose, mean intensities, recovery rates, larvae output, and levels of synthesized heat shock proteins (Hsp70) were determined in nematodes of each infection group. Increased stress responses were detected in the endemic system of A. crassus parasitizing A. japonica and A. crassus in its recently acquired host A. anguilla, which seems to be associated with the immune response of the particular host species and the expenditure of energy on producing larvae. A. novaezelandiae showed overall weak activities in its unknown host species A. japonica, with the lowest recovery rate of all examined groups neither featuring elevated Hsp responses, nor a high mean intensity, nor any reproductive output. On the contrary, in A. anguilla, the parasite reached higher recovery rates, mean intensities, and reproductive output, but no increased Hsp70 levels could be detected. The four considered factors proved partially interdependent, whereas few results did not follow a clear pattern.

Cyclocoelid ( Morishitium sp.) Trematodes from an Air Sac of a Purple Sandpiper, Calidris maritima (Brünnich).

Like many shorebirds, purple sandpipers ( Calidris maritima [Brünnich] Charadriiformes: Scolopacidae) have experienced population declines in recent years, but causes of these declines have not been established. As part of a larger study to identify causes of these declines, we collected and examined 25 purple sandpipers in coastal Nova Scotia, Canada during March 2013. In the course of dissections to collect tissue samples, we detected a cluster of trematodes in the air sac of 1 bird that were subsequently identified as cyclocoelids belonging to the genus Morishitium Witenberg, 1928, which we believe is the first report of this genus of parasites in this host. Cyclocoelids have been reported from other scolopacids and other shorebird families, but we are unaware of reports of serious pathology arising from these trematodes. Given this and the low prevalence (4%) in our sample, our data cannot ascribe declines in purple sandpiper populations to these trematodes, but our sample is limited both spatially and temporally.