Expression of Hairpin-Enriched Mitochondrial DNA in Two Hairworm Species (Nematomorpha).

Nematomorpha (hairworms) is a phylum of parasitic ecdysozoans, best known for infecting arthropods and guiding their hosts toward water, where the parasite can complete its life cycle. Over 350 species of nematomorphs have been described, yet molecular data for the group remain scarce. The few available mitochondrial genomes of nematomorphs are enriched with long inverted repeats, which are embedded in the coding sequences of their genes-a remarkably unusual feature exclusive to this phylum. Here, we obtain and annotate the repeats in the mitochondrial genome of another nematomorph species-Parachordodes pustulosus. Using genomic and transcriptomic libraries, we investigate the impact of inverted repeats on the read coverage of the mitochondrial genome. Pronounced drops in the read coverage coincide with regions containing long inverted repeats, denoting the 'blind spots' of short-fragment sequencing libraries. Phylogenetic inference with the novel data reveals multiple disagreements between the traditional system of Nematomorpha and molecular data, rendering several genera paraphyletic, including Parachordodes.

Heth longquani sp. n.(Rhigonematomorpha: Hethidae)obligate commensalistic nematode of camballoid millipede (Spirostreptida: Cambalopsidae) from Viet Nam.

A new nematode species, Heth longquani sp. n. (Hethidae, Ransonematoidea, Rhigonematomorpha, Rhabditida) is described from the camballoid millipede, Chonecambala crassicauda from Thanh Ha province, Viet Nam. The morphological features of this new species were studied with brightfield and scanning electron microscopies. Partial sequences of LSU rDNA and Cox1 mtDNA are provided. The phylogeny of the nematodes of the genus Heth Cobb, 1898 is discussed.

Invasive Alien Slug Arion vulgaris Moquin-Tandon, 1855 (Gastropoda: Pulmonata: Arionidae) in Moscow Parks and Its Co-introduced Parasite Alloionema appendiculatum Schneider, 1859.

PURPOSE: The present study investigates the origin of Arion vulgaris slugs in the parks of Moscow city and their parasites. METHODS: Snails and slugs inhabiting green areas of Moscow city were collected in the summer season of 2020 and examined on the presence of gastropod-associated nematodes and trematodes using morphological and molecular methods. RESULTS: The presence of the alien slug species, Arion vulgaris, was recorded in several locations, and the mitochondrial gene-based analysis has shown that slug populations inhabited Moscow parks originated from West and Central Europe. Out of a total of 15 gastropod species examined, A. vulgaris was the only species infected by the nematode Alloionema appendiculatum Schneider, 1859, a larval parasite of molluscs. It is the first record of this nematode from the territory of the Russian Federation. COX1 mtDNA sequences of A. appendiculatum obtained from 3 populations of infected slugs were identical with those from Western and Central Europe similarly to their gastropod hosts thus indicating that the nematodes travelled with their hosts. No parasites dangerous for humans or animals were found. CONCLUSION: The complex life cycle of A. appendiculatum includes a free-living stage in soil which offers a source of infection for other potentially susceptible gastropod species but the capacity of A. appendiculatum to change hosts in local conditions needs to be further investigated. The particular susceptibility and tolerance of A. vulgaris to nematodes in our study was in concordance with earlier data while in contradiction with the enemy release hypothesis.

Leidynema bestium sp. n. (Oxyuridomorpha: Thelastomatidae) an intestinal parasite of blaberid cockroaches from Yaroslavl Zoo, Russia.

A new species of thelastomatid Leidynema bestium sp. n. is described from the hind gut lumen of the cockroaches Diploptera punctata (Eschscholtz, 1922) and Elliptorhina chopardi (Lefeuvre, 1966). The new species is characterized by a combination of morphological features: presence of lateral alae in both sexes with posterior margin of alae in females not ending in the shape of a spike; longitudinal striation of the anteriormost annuli in males; four male genital papillae, equatorial position of vulva. In the cladogram inferred from analysis of D2-D3 segment of LSU rDNA L. bestium sp. n. demonstrates a position independent of the Leidynema species with known sequences of this taxonomic marker near the basal node for this genus.

Genetic diversity of Syphacia Seurat, 1916 (Nematoda: Oxyuridae) across the hybrid zone of their rodent hosts in Russia.

The genetic diversity of Syphacia nematodes (intestinal parasites of rodents) was studied in the hybrid zone of two sister species of common voles, Microtus arvalis and Microtus obscurus, in the Oka River valley, east of Moscow. Syphacia nematodes of other rodent species (Microtus rossiaemeridionalis, Alexandromys oeconomus, Sylvaemus uralensis, and Apodemus agrarius) that inhabit the area were also studied. Phylogenetic trees for the studied nematodes were inferred from the analysis of nuclear ITS1+5.8S+ITS2, LSU rDNA, and mitochondrial CO1 gene partial sequences. Syphacia nematodes of the studied area form three well-defined clades in the phylogenetic tree of this genus. Morphological analysis revealed similarities between the obtained sequences with those of known Syphacia species from the GenBank database, which enabled identifying these three clades up to the species level: S. montana, S. agraria, and S. frederici. Russian haplotypes of Syphacia are different from West European and East Asian haplotypes with pronounced genetic distances. A high level of specificity was reported for two of these three species (S. frederici, only in Sylvaemus uralensis; S. agraria, only in Apodemus agrarius). S. montana was found in different species of voles. Remarkably, S. montana specimens from M. arvalis and M. obscurus were genetically uniform, while S. montana, specimens from hybrids between these two species formed a separate clade distant from those originating from non-hybridised hosts.

The re-description of Synoecnemahirsutum Timm, 1959 (Synoecneminae, Ungellidae, Drilonematoidea) from a pheretimoid earthworm in Vietnam with the analysis of its phylogenetic relationships.

Synoecnemahirsutum Timm, 1959 (Ungellidae, Drilonematoidea), found in the body cavity of the pheretimoid earthworm at the border of Laos and Vietnam, was re-described and illustrated. The mitochondrial genome of S.hirsutum obtained with Illumina HiSeq sequencing is the first annotated mitochondrial genome as a representative of the superfamily Drilonematoidea. The phylogeny inferred from the analysis of 12 mitochondrial genes has shown some similarity of S.hirsutum with a cephalobid Acrobeloidesvarius.

Coding palindromes in mitochondrial genes of Nematomorpha.

Inverted repeats are common DNA elements, but they rarely overlap with protein-coding sequences due to the ensuing conflict with the structure and function of the encoded protein. We discovered numerous perfect inverted repeats of considerable length (up to 284 bp) embedded within the protein-coding genes in mitochondrial genomes of four Nematomorpha species. Strikingly, both arms of the inverted repeats encode conserved regions of the amino acid sequence. We confirmed enzymatic activity of the respiratory complex I encoded by inverted repeat-containing genes. The nucleotide composition of inverted repeats suggests strong selection at the amino acid level in these regions. We conclude that the inverted repeat-containing genes are transcribed and translated into functional proteins. The survey of available mitochondrial genomes reveals that several other organisms possess similar albeit shorter embedded repeats. Mitochondrial genomes of Nematomorpha demonstrate an extraordinary evolutionary compromise where protein function and stringent secondary structure elements within the coding regions are preserved simultaneously.

Coinvasion by the ladybird Harmonia axyridis (Coleoptera: Coccinellidae) and its parasites, Hesperomyces virescens (Ascomycota: Laboulbeniales) and Parasitylenchus bifurcatus (Nematoda: Tylenchida, Allantonematidae), in the Caucasus.

The study of parasites in recently established populations of invasive species can shed light on the sources of invasion and possible indirect interactions between the alien species and native ones. We studied parasites of the global invader Harmonia axyridis (Coleoptera: Coccinellidae) in the Caucasus. In 2012, the first established population of Ha. axyridis was recorded in the Caucasus in Sochi (south of European Russia, Black Sea coast). By 2018, the ladybird had spread to a vast area: Armenia, Georgia and south Russia (Adygea, the Krasnodar territory, the Stavropol territory, Dagestan, Kabardino-Balkaria and North Ossetia). The examination of 213 adults collected in Sochi in 2018 showed that 53% were infested with Hesperomyces virescens fungi (Ascomycota: Laboulbeniales) and that 8% were infested with Parasitylenchus bifurcatus nematodes (Nematoda: Tylenchida, Allantonematidae). The examined Ha. axyridis specimens were free of the parasitic mite Coccipolipus hippodamiae. An analysis of the phylogenetic relationships of P. bifurcatus based on 18S rDNA confirmed the morphological identification of this species. Hesperomyces virescens and P. bifurcatus were first recorded in the Caucasus and Russia, although they are rather widespread in Europe. This likely indicates that they appeared as a result of coinvasion with their host because the populations of Ha. axyridis, He. virescens and P. bifurcatus in the Caucasus are isolated from the main parts of the ranges of these species in Europe. The nearest localities of Ha. axyridis is on another shore of the Black Sea, and the nearest localities of He. virescens and P. bifurcatus are more than 1000 km from the Caucasus. It is impossible to determine whether the first founders of the Caucasian population were infested with the parasites or whether the parasites were introduced by specimens of Ha. axyridis that arrived later from Europe. Harmonia axyridis was released in the region for pest control, but laboratory cultures are always free of He. virescens and P. bifurcatus. Therefore, the detection of He. virescens and P. bifurcatus indicates that the population of Ha. axyridis in the Caucasus could not have derived exclusively from released specimens. We did not find He. virescens on 400 specimens of 29 other ladybird species collected from the same localities as Ha. axyridis in the Caucasus. No reliable correlation between infestation by He. virescens and that by P. bifurcatus has been found. In addition to these two parasites, an unidentified species of the order Mermithida was recorded. This is the first documented case of Ha. axyridis infestation by a parasitic nematode of this order in nature.

Trichinella nativa haplotypes in Russia show diversity in cytochrome oxidase mtDNA gene.

Partial nucleotide sequences of the two mitochondrial genes (cytB and COI) were obtained for 13 Trichinella isolates from different regions of the Russian Federation. All the cytB mtDNA sequences were identical with the sequences of T. nativa from Canada, showing no nucleotide differences between isolates. Analysis of partial COI mtDNA sequence confirmed the species identification and revealed differences between the studied isolates. Two T. nativa haplotypes, represented in the studied material by four samples, demonstrate limited geographical distribution.

Synoecnema watinagii (Drilonematoidea: Ungellidae: Synoecneminae), a new nematode species parasitic in earthworms from the Philippines with the first molecular and SEM data for the genus.

A new species of the genus Synoecnema-S. watinagii sp. n.-is described and illustrated. The species is characterized by its small size, lack of sexual dimorphism apart from sexual characters, males with pericloacal disk, females with anterior vulva position and lacking an anus, and similar caudal organs in both sexes in the shape of long, deep slits on the surface of the posterior half of body. Sequences of D2-D3 LSU and SSU rDNA and SEM images were obtained for the first time for the genus Synoecnema. Phylogenetic analysis of these sequences supported the validity of the genus and elucidated its relationships within Synoecneminae and Drilonematoidea. The earthworm host was characterized by its CoxI mt DNA sequence.

Heth impalutiensis n. sp. (Nematoda: Ransomnematoidea: Hethidae) a millipede parasite from Central Mindanao, Philippines.

The nematode Heth impalutiensis n. sp. is described from an unidentified spirostreptid millipede (Harpagophoridae) from the Bukidnon Province of Mindanao, the Philippines. Based on morphological characters, H. impalutiensis n. sp. is closest to Asian-Pacific representatives of the genus. Females of H. impalutiensis n. sp. are close to H. dimorphum and H. vietnamensis in body size and form of the lateral lappets, but can be distinguished by the significantly longer tail. Males of H. impalutiensis n. sp. strongly resemble that of H. xaniophora by the presence of such a rare character combinations as mammiform papillae and a bursa-like cuticular fold, but can be easily differentiated by the numbers of genital papillae (7 vs 6 pairs, respectively). Heth impalutiensis n. sp. can be distinguished from all nominal species by hypertrophy of the anterior anal lip in females which overlaps the anal aperture. Phylogenetic analysis based on the newly obtained set of sequences did not provide an evidence of infraorder Rhigonematomorpha monophyly as two superfamilies Ransomnematoidea and Rhigonematoidea formed independent clades in the frames of ascaridid-spirurid-oxyurid super clade (Clade III of Nadler et al., 2007).

A new genus and two new nematode species (Drilonematoidea: Ungellidae: Synoecneminae) parasitic in two morphs of Drawida ghilarovi Gates, endemic earthworm from the Russian Far East.

Drasico n. g. is erected to accommodate two new species of nematode, Drasico nemoralis n. sp. and D. paludigenus n. sp., recovered from coelomic cavities of Drawida ghilarovi Gates, endemic earthworms of the Russian Far East. The new genus is characterised by the following unique for the Synoecneminae characters: apical portion of the head attenuated, cephalic hooks displaced to the base of attenuated portion, amphids displaced posterior to cephalic hooks, excretory duct short and weak, males possessing several genital papilliform sensilla. The new species are differentiated by the size, number and disposition of the male genital sensilla (larger and more numerous in D. nemoralis n. sp.); the body shape of females (with thinner neck and wider mid-body in D. paludigenus n. sp.) and the ovarian tube arranged in transversal folds in D. paludigenus (vs longitudinal folds in D. nemoralis n. sp.). Nucleotide sequences of D2-D3 expansion segment of 28S rDNA for the two new species differed at 13 positions. Phylogenetic analysis revealed close relationships of Drasico n. g. with species of Siconema Timm, 1966. The host species was represented by two morphs (blue-grey forest and tar-black meadow-swamp morph) with intraspecific divergence of 16-17% for cytochrome c oxidase subunit 1 (COI) gene, and each host morph was found infected by a different nematode species. A co-infection with the plectid nematode Creagrocercus drawidae Ivanova & Spiridonov, 2011 was recorded together with D. nemoralis n. sp. in the blue-grey forest morph.

Morphological and molecular characterisation of Steinernema costaricense Uribe-Lorío, Mora & Stock, 2007 (Panagrolaimorpha: Steinernematidae) isolate from Bush Augusta State Park, Missouri, USA.

A new isolate of Steinernema costaricense Uribe-Lorío, Mora & Stock, 2007 (Panagrolaimorpha: Steinernematidae) was discovered from the Bush Augusta State Park, Missouri, USA, and its morphological and molecular examination was carried out. Morphologically, adults of the Missouri isolate are very close to S. costaricense from Costa Rica. Infective juveniles (IJs) from Missouri are characterised by mean body length of 843 µm, mean body width of 33 µm, mean tail length of 80 µm, mean pharynx length of 143 µm, excretory pore at 62 µm from anterior extremity, high cephalic papillae, and lateral field formula 2-6-2 with two central ridges less prominent. They resemble IJs of S. costaricense from Costa Rica in having high cephalic papillae, similar lateral field pattern and pharynx length, but are twice shorter. Based on the nearly complete identity between the sequence of D2-D3 domains of LSU rDNA of the Missouri isolate and those of S. costaricense retrieved from GenBank and the high similarity of their bacterial symbionts, the new isolate was identified as S. costaricense. The phylogenetic affinities among S. costaricense and the species of the "bicornutum" group proposed by the authors of its original description based on LSU rDNA analysis, is debated. In the present study, all three methods of analysis for the ITS region showed that four species of Steinernema from the Americas (S. rarum de Doucet, 1986, S. scarabaei Stock & Koppenhöfer, 2003, S. unicornum Edgington, Buddie, Tymo, France, Merino & Hunt, 2009 and S. costaricense Missouri isolate) formed a weakly supported clade although bootstrap support for the sister-group relationship between the new isolate and S. scarabaei was always high. In phylogenies inferred from D2-D3 LSU rDNA S. costaricense never formed a clade with S. rarum and S. unicornum.

Molecular phylogeny of slug-parasitic nematodes inferred from 18S rRNA gene sequences.

Terrestrial molluscs are diverse and are infected by many nematodes. We propose a phylogeny of slug-parasitic nematodes using 18S rRNA gene sequences from nematodes isolated from slugs collected from six countries. Eight species, representing six families of nematodes were identified and trees inferred placed them within four (I, III, IV and V) out of the five clades of Nematoda, indicating multiple origins of slug parasitism. Five species representing three families formed a monophyletic group in clade V. Although these species are closely related, their morphology has changed greatly, suggesting adaptive radiation to fill different niches within the host.

Habitat associations of two entomopathogenic nematodes: a quantitative study using real-time quantitative polymerase chain reactions.

1. Despite nematodes being the most abundant animals on earth, very few animal ecologists study them, probably because of the difficulties of identifying them to species by morphological methods. 2. A group of nematodes that are important both ecologically and economically is the entomopathogenic nematodes, which play a key role in regulating soil food webs and are sold throughout the world as biological insecticides, yet for which very little is known of their population ecology. 3. A novel detection and quantification method was developed for soil nematodes using real-time polymerase chain reaction (PCR), and the technique was used to estimate numbers of two closely related species of entomopathogenic nematodes, Steinernema kraussei and S. affine in 50 soil samples from 10 sites in Scotland representing two distinct habitats (woodland and grassland). 4. There was a high degree of correlation between our molecular and traditional morphological estimates of population size and our data clearly showed that Steinernema affine occurred only in grassland areas, whereas S. kraussei was found in grassland and woodland samples to a similar degree. 5. Real-time PCR offers a rapid and accurate method of detecting individual nematode species from soil samples without the need for a specialist taxonomist, and has much potential for use in studies of nematode population ecology.

Aulacnema monodelphis n. g., n. sp. and Angiostoma coloaense n. sp. (Nematoda: Rhabditida: Angiostomatidae) from terrestrial molluscs of Vietnam.

Two species of nematodes, Angiostoma coloaense n. sp. and Aulacnema monodelphis n. g., n. sp. (Angiostomatidae: Rhabditida) are described from terrestrial molluscs of Vietnam. Both species are characterised by a long bowl-shaped buccal cavity and typically angiostomatid male bursal alae, spicules and gubernaculum. The new genus, Aulacnema n. g., is erected for the first reported monodelphic angiostomatid. Angiostoma coloaense n. sp. can be distinguished from other species of Angiostoma Dujardin, 1845 by having a buccal cavity length greater than its width and the absence of tail spikes on both males and females.