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.

Molecular evidence linking the larval and adult stages of Mexiconema cichlasomae (Dracunculoidea: Daniconematidae) from Mexico, with notes on its phylogenetic position among Dracunculoidea.

We describe the larval developmental stages and life cycle of the dracunculid nematode Mexiconema cichlasomae in both the intermediate, Argulus yucatanus (Crustacea: Branchiura), and definitive hosts, Cichlasoma urophthalmus (Perciformes: Cichlidae), from the Celestun tropical coastal lagoon, Yucatan, Mexico. The morphological analyses showed significant differences between the total length of L1 found in M. cichlasomae gravid female and L2-L3 in A. yucatanus. This result indicates that the M. cichlasomae larval development occurs in the intermediate host. We obtained sequences from the small subunit (SSU) ribosomal marker from larval stages of M. cichlasomae in A. yucatanus and adult nematodes in C. urophthalmus. Our morphological and molecular results support conspecificity between M. cichlasomae larvae in A. yucatanus and the adult stages in C. urophthalmus. We briefly discuss the phylogenetic position of M. cichlasomae among the Daniconematidae, and provide evidence of the monophyly of the daniconematids associated with branchiurid intermediate hosts. Based on the phylogenetic results, we support the transfer of the Mexiconema genus to the family Skrjabillanidae and do not support the lowering of family Daniconematidae to subfamily.

Species of Philometra (Nematoda, Philometridae) from fishes off the Mediterranean coast of Africa, with a description of Philometra rara n. sp. from Hyporthodus haifensis and a molecular analysis of Philometra saltatrix from Pomatomus saltatrix.

Two gonad-infecting species of Philometra Costa, 1845 (Nematoda, Philometridae) were recorded for the first time from marine perciform fishes off Tunisia and Libya: Philometra rara n. sp. from the rare, deep-water Haifa grouper Hyporthodus haifensis (Serranidae) off Libya and Philometra saltatrix Ramachandran, 1973 from the bluefish Pomatomus saltatrix (Pomatomidae) off Tunisia. Identification of both fish species was confirmed by molecular barcoding. Light and scanning electron microscope studies of Ph. rara n. sp. showed that it is characterized by the length of spicules (216-219 µm) and the gubernaculum (90-93 µm), the gubernaculum/spicules length ratio (1:2.32-2.43), and mainly by the shape and structure of the distal end of the gubernaculum (shovel-shaped with a wide median smooth field in dorsal view), appearing as having a dorsal protuberance in lateral view, and by the structure of the male caudal mound (dorsally interrupted); large subgravid females (70-137 mm long) are characterized by the presence of four oval submedian cephalic elevations, each of them bearing a pair of cephalic papillae of the outer circle. The finding of Ph. saltatrix off Tunisia confirms that this species is widespread throughout the Mediterranean region. A molecular analysis of our Ph. saltatrix specimens and other available philometrid cytochrome c oxidase 1 (COI) sequences showed that most species have robust clades. Sequences of Ph. saltatrix from Tunisia diverge from Ph. saltatrix from Brazil and the USA, suggesting that speciation is currently occurring between populations from both sides of the Atlantic Ocean.

Molecular genomics resource for the parasitic nematode Spirocerca lupi: Identification of 149 microsatellite loci using FIASCO and next generation sequencing.

Understanding genetic diversity and movement patterns in parasitic organisms is paramount to establish control and management strategies. In this study we developed a microsatellite resource as well as a diagnostic multiplex for the cosmopolitan parasitic nematode Spirocerca lupi, known to cause spirocercosis in canids. A combination of microsatellite enrichment and 454 sequencing was used to identify 149 unique microsatellite loci in S. lupi. Twenty loci were characterized further in two sampling sites in South Africa, with 10 loci identified as polymorphic (allele ranges from 4 to 17). These loci were designed into a single diagnostic multiplex suitable for species identification and population genetics studies. The markers were also successful in cross-species amplification in Cylicospirura felineus, Philonema oncorhynchi and Gongylonema pulchrum. Our resource provides a large set of candidate loci for a number of nematode studies as well as loci suitable for diversity and population genetics studies of S. lupi within the South African context as well as globally.

A new gonad-infecting species of Philometra, P. barnesi sp. n. (Nematoda: Philometridae), from the marine fish Pomadasys argenteus (Haemulidae) off the northern coast of Australia.

A new nematode species, Philometra barnesi sp. n. (Philometridae), is described from the ovary of the marine teleost Pomadasys argenteus (Fosskål) (Haemulidae) off the northern coast of Australia (near Darwin). The new species is characterized by short subequal spicules (84 and 87 µm long), a gubernaculum without a dorsal protuberance at its distal tip, the structure of male anterior and posterior body ends, the body lengths of males (1.67 mm) and gravid females (320-597 mm) and the structure of the oesophagus and caudal end of gravid females. Philometra barnesi is the sixth nominal gonad-infecting species of this genus recorded from marine fishes in Australian waters and the third species of philometrids described from fishes of the family Haemulidae.

Redescription and genetic characterization of Philometra lagocephali Moravec et Justine 2008 (Nematoda: Philometridae) from Lagocephalus lunaris (Bloch and Schneider) (Tetraodontiformes: Tetradontidae) in the South China Sea.

Philometra lagocephali Moravec et Justine, 2008 was redescribed using light and scanning electron microscopy based on the new material collected from the lunartail puffer Lagocephalus lunaris (Bloch et Schneider) (Tetraodontiformes: Tetradontidae) in the South China Sea. Our specimens are identical with the original description in the body size of gravid female, the width of cephalic and caudal end, the number and arrangement of cephalic papillae, the relative length of the oesophagus to body length, the size of ventriculus, the morphology of oesophageal bulb and posterior end, and the body size of larvae. In addition, specimens of P. lagocephali were also characterized using molecular methods by sequencing and analysing the small ribosomal DNA (18S rDNA) and mitochondrial cytochrome c coxidase subunit 1 (cox1) target regions. Phylogenetic analyses of genus Philometra base on 18S rDNA sequences was made using Mega 5.2. The results confirmed P. lagocephali is a valid species. It's the first molecular study of this species. These new morphological and molecular data contributed to a more precise and rapid diagnosis of this hitherto poorly known philometrid nematode.

Gonad-infecting philometrid Philometra philippinensis sp. nov. (Nematoda, Philometridae) from the bigeye barracuda Sphyraena forsteri Cuvier (Sphyraenidae) off Mariveles, Bataan Province, Philippine archipelago.

We conducted a parasitological study to identify nematodes infecting the gonads of bigeye barracuda Sphyraena forsteri Cuvier (Sphyraenidae) caught in the West Philippine Sea, off the Mariveles coast, Bataan Province, the Philippine archipelago. Males and very tiny nongravid female philometrid nematodes were collected from the ovaries of the fish. Based on light and scanning electron microscope observations, and supported by molecular analyses of the partial nucleotide sequences of the 18S small subunit ribosomal DNA, the philometrid nematode was determined to be a new species belonging to the genus Philometra Costa, 1845 in the family Philometridae (Nematoda: Dracunculoidea), and was named P. philippinensis sp. nov. This new species was clearly distinguishable morphologically from other congeners that parasitize the gonads of marine fishes. Males have unique sickle-shaped spicules and gubernaculum, and a vulva-like cloacal opening. Following Dentirumai philippinensis Quiazon et Moravec, 2013 and Philometra robusta Moravec, Möller et Heeger, 1992, this is the third philometrid species and the first gonad-infecting philometrid from the Philippine archipelago.

A synthesis of our current knowledge of philometrid nematodes, a group of increasingly important fish parasites.

Members of the Philometridae represent the most important group of dracunculoid nematodes parasitizing fishes. In his monograph treating the Dracunculoidea, Moravec (2006) reported a total of 11 genera and 105 species of philometrids parasitizing freshwater, brackish-water and marine fishes. However, during the last six years (2007-2012), an additional 42 new species of Philometridae have been described, representing a 40% increase of the number of nominal species. Most of these species (30) belong to Philometra Costa, 1845, mainly represented by parasites of marine fishes, a few others (8) to Philometroides Yamaguti, 1935, and a single one to each of the following genera: Caranginema Moravec, Montoya-Mendoza et Salgado-Maldonado, 2008, Dentiphilometra Moravec et Wang, 2002, Dentirumai Quiazon et Moravec, 2013* and Spirophilometra Parukhin, 1971. Moreover, three new genera, Afrophilometra Moravec, Charo-Karisa et Jirku, 2009, Caranginema and Dentirumai, were erected. Representatives of seven genera, Afrophilometra, Buckleyella Rasheed, 1963, Caranginema, Dentiphilometra, Dentirumai, Paraphilometroides Moravec et Shaharom-Harrison, 1989 and Rumai Travassos, 1960, were studied using scanning electron microscopy (SEM) for the first time. Thirteen known but poorly described philometrid species were redescribed and, in some species of Caranginema and Philometra, previously unknown conspecific males were discovered and described. The male surface ultrastructure studied by SEM provided new taxonomically important features for species distinction. Gene sequencing was used in several recent studies and advanced our understanding of phylogenetic interrelationships among representatives of seven genera (Afrophilometra, Alinema Rasheed, 1963, Caranginema, Nilonema Khalil, 1960, Philometra, Philometroides and Rumai) and of the extent of the biodiversity of philometrids. New data were obtained on the biology and pathogenicity of several species of Nilonema, Philometra, Philometroides and Rumai. The need to carry out surveys in order to find males and to use SEM and gene sequencing to identify philometrids is emphasized. Appropriate quantitative methods to determine the impact of philometrids in ovarian tissue on host fecundity are recommended. Further detailed studies on philometrids would be significant not only from the theoretical viewpoint, but also because of their practical implications. A list of philometrid nematode species by continents is provided.

Evolutionary divergence of the swim bladder nematode Anguillicola crassus after colonization of a novel host, Anguilla anguilla.

BACKGROUND: Anguillicola crassus, a swim bladder nematode naturally parasitizing the Japanese eel, was introduced about 30 years ago from East Asia into Europe where it colonized almost all populations of the European eel. We conducted a common garden experiment under a reciprocal transfer design infecting both European and Japanese eels with populations of A. crassus from Germany, Poland and Taiwan. We tested, whether differences in infectivity, developmental dynamics and reproductive output between the European and Asian parasite populations occur while harboured in the specimens of native and colonized eel host, and if these differences are genetically based or are plastic responses to the new environment. RESULTS: Under common garden conditions an evolutionary change in the both European parasite populations of A. crassus compared with their Taiwanese conspecifics was observed for infectivity and developmental dynamics, but not for reproductive output. When infecting the European eel, current European populations of the parasite were less infective and developed faster than their Taiwanese conspecifics. In the reciprocally infected Japanese eel the genetically induced differences between the parasite strains were less apparent than in the European eel but higher infectivity, faster development and higher larval mortality of the European parasite populations could be inferred. CONCLUSIONS: The differences in infectivity and developmental dynamics between European and Taiwanese populations of A. crassus found in our study suggest rapid genetic divergence of this parasite after a successful host switch in Europe.

The transcriptome of the invasive eel swimbladder nematode parasite Anguillicola crassus.

BACKGROUND: Anguillicola crassus is an economically and ecologically important parasitic nematode of eels. The native range of A. crassus is in East Asia, where it infects Anguilla japonica, the Japanese eel. A. crassus was introduced into European eels, Anguilla anguilla, 30 years ago. The parasite is more pathogenic in its new host than in its native one, and is thought to threaten the endangered An. anguilla across its range. The molecular bases for the increased pathogenicity of the nematodes in their new hosts is not known. RESULTS: A reference transcriptome was assembled for A. crassus from Roche 454 pyrosequencing data. Raw reads (756,363 total) from nematodes from An. japonica and An. anguilla hosts were filtered for likely host contaminants and ribosomal RNAs. The remaining 353,055 reads were assembled into 11,372 contigs of a high confidence assembly (spanning 6.6 Mb) and an additional 21,153 singletons and contigs of a lower confidence assembly (spanning an additional 6.2 Mb). Roughly 55% of the high confidence assembly contigs were annotated with domain- or protein sequence similarity derived functional information. Sequences conserved only in nematodes, or unique to A. crassus were more likely to have secretory signal peptides. Thousands of high quality single nucleotide polymorphisms were identified, and coding polymorphism was correlated with differential expression between individual nematodes. Transcripts identified as being under positive selection were enriched in peptidases. Enzymes involved in energy metabolism were enriched in the set of genes differentially expressed between European and Asian A. crassus. CONCLUSIONS: The reference transcriptome of A. crassus is of high quality, and will serve as a basis for future work on the invasion biology of this important parasite. The polymorphisms identified will provide a key tool set for analysis of population structure and identification of genes likely to be involved in increased pathogenicity in European eel hosts. The identification of peptidases under positive selection is a first step in this programme.

Merging species? Evidence for hybridization between the eel parasites Anguillicola crassus and A. novaezelandiae (Nematoda, Anguillicolidea).

BACKGROUND: The eel parasitic nematodes Anguillicola crassus (originating from Asia) and Anguillicola novaezelandiae (originating from New Zealand) were both introduced to Europe, but occurred in sympatry only in Lake Bracciano in Italy, where they both infected the European eel (Anguilla anguilla). A. novaezelandiae was introduced to the lake in 1975 and disappeared soon after A. crassus was also found there in 1993. We tested the hypothesis if hybridization of the two species might be an explanation for the findings at Lake Bracciano. FINDINGS: After laboratory infection of one European eel with 10 third stage larvae of each parasite, two living female and 4 male adults of each species were found to co-occur in the swim bladder after 222 days post exposure. In 9 out of 17 eggs, isolated in total from uteri of the two A. novaezelandiae females, alleles were detected by microsatellite analysis that are characteristic for A. crassus, suggesting the hybrid origin of these eggs. In contrast, none of the eggs isolated from A. crassus females possessed alleles different from those found in A. crassus adults, but it was revealed that one female can be inseminated by several males. CONCLUSION: Our results show that A. crassus and A. novaezelandiae can co-infect a single eel and can mature together in the same swim bladder. We also provide evidence for the possibility of hybridization of A. crassus males with A. novaezelandiae females. Therefore, hybridization might be an explanation for the disappearance of A. novaezelandiae from Lake Bracciano.

Philometrids of the southern flounder Paralichthys lethostigma: a multidimensional approach to determine their diversity.

Two species of philometrid nematode, Philometra overstreeti and Philometroides paralichthydis, infect the southern flounder, Paralichthys lethostigma. Individuals of P. overstreeti are located between the teeth and inside the bony part of the branchial arches of the fish. Individuals of P. paralichthydis are associated with the bones of the buccal cavity and among muscles that control the dorsal and anal fins. Sequencing of part of the cytochrome oxidase I gene revealed 4 distinct genetic clades, each corresponding exactly to the 4 respective locations of the parasites in the host, suggesting the need for taxonomic revision. We hypothesized that each clade represented a separate species and, because the worms are morphologically indistinguishable, compared population level parameters of the clades comprising each currently recognized species. For each currently recognized species, the presence of worms from 1 clade was negatively correlated with the presence of worms from the other. Results also indicated significant differences between the clades in prevalences relative to both biotic and abiotic factors. Results clearly indicated major differences in the ecology of the philometrids constituting each clade. Taken as a whole, molecular and ecological data support the contention that the 4 genetic clades are likely 4 distinct species.

Systematics of Mexiconema cichlasomae (Nematoda: Daniconematidae) based on sequences of SSU rDNA.

The molecular characterization of the daniconematid dracunculoid Mexiconema cichlasomae Moravec, Vidal, and Salgado-Maldonado, 1992 through the sequencing of SSU rDNA from adult individuals is presented herein. Additionally, preliminary genetic relationships of this nematode are inferred from alignment of sequences generated previously for other dracunculoids. Maximum parsimony and maximum likelihood analyses recovered identical trees. As anticipated by previous taxonomic work, M. cichlasomae is putatively closely related to skrjabillanid dracunculoids represented by Molnaria intestinalis (Dogiel and Bychovsky, 1934) and Skrjabillanus scardinii Molnár, 1966 SSU rDNA sequences, but the relationships of this newly discovered clade to other dracunculoid clades remain unresolved.

Genetic signatures in an invasive parasite of Anguilla anguilla correlate with differential stock management.

In this article, it is shown that available genetic tools for the omnipresent parasite Anguillicoloides crassus in European eels Anguilla anguilla are sensitive to different immigration rates into local A. anguilla stocks for two separated river systems. Relying on four highly polymorphic microsatellite markers, it was inferred that under natural recruitment, nematode samples meet Hardy-Weinberg expectations for a single panmictic population, while genetic signals show signs for a strong Wahlund effect most likely due to very recent population mixing under frequent restocking of young A. anguilla. This was indicated by a low but significant F(ST) value among within-host populations (infrapopulations) along with high inbreeding indices F(IS) consistent over all loci. The latter signal is shown to stem from high levels of admixture and the presence of first-generation migrants, and alternative explanations such as marker- and sex-specific biases in the nematode populations could be dismissed. Moreover, the slightly increased degree of relatedness within infrapopulations in the stocked river system cannot explain the excessive inbreeding values found and are most likely a direct consequence of recent influx of already infected fish harbouring parasites with different genetic signatures. Applying a simulation approach using known variables from the nematode's invasion history, only the artificial introduction of a Wahlund effect leads to a close match between simulated and real data, which is a strong argument for using the parasite as a biological tag for detecting and characterizing fish translocation.

Population structure of the parasitic nematode Anguillicola crassus, an invader of declining North Atlantic eel stocks.

Probably half of all animal species exhibit a parasitic lifestyle and numerous parasites have recently expanded their distribution and host ranges due to anthropogenic activities. Here, we report on the population genetic structure of the invasive nematode Anguillicola crassus, a parasite in freshwater eels, which recently spread from Asia to Europe and North America. Samples were collected from the newly colonized naive host species Anguilla anguilla (Europe) and Anguilla rostrata (North America), and from indigenous Anguilla japonica in Taiwan and Japan. Using seven microsatellite loci and one mitochondrial marker, we show that the parasite's population structure in Europe mirrors the zoogeographic Boreal-Lusitanian break along the English Channel. Both the north-to-south decline of nuclear allelic diversity and the loss of private alleles in the same direction are consistent with a significant isolation-by-distance pattern based on rho(ST) values. In combination with the specific topology of the distance tree among nematode populations, our data suggest that Europe was invaded only once from Taiwan, and that subsequently, genetic diversity was lost due to random drift. On the contrary, the North American sample shares distinct nuclear and mitochondrial signatures with Japanese specimens. We propose that the genetic structure in Europe was shaped by long-range anthropogenic eel host transfers in the north and a single dispersal event into the southwest. The genetically distinct Brittany sample at the edge of the Boreal-Lusitanian boundary is indicative of natural dispersal of fish hosts since recruitment occurs naturally there and invertebrate host dissemination is interrupted due to oceanic currents.

Evolutionary relationships of Spirurina (Nematoda: Chromadorea: Rhabditida) with special emphasis on dracunculoid nematodes inferred from SSU rRNA gene sequences.

The analysis of 26 new small subunit rRNA sequences obtained from helminths that primarily parasitize fishes sampled from five continents provided well-supported trees, allowing us to study the phylogenetic relationships among spirurid nematodes. The analyses have shown that Dracunculoidea is a paraphyletic taxon and Anguillicolidae and Gnathostomatidae constitute the basal branch of the suborder Spirurina. The genera Philometra and Philometroides appear to be paraphyletic, while on the higher taxonomic level, good correlation between the morphology-based system and molecular data was observed. Neither co-evolution of the studied helminths with their hosts, nor phylogeographic pattern, are apparent in our dataset.