Ryusei Tanaka

Diversity of Stag Beetle-Associated Nematodes in Japan

ABSTRACT A survey of stag beetle (Lucanidae)-associated nematodes was conducted in Japan to construct a preliminary species inventory and to obtain their molecular profiles as a first step for elucidating their host fidelity and ecological roles. Eight species of stag beetles (94 individuals in total), Dorcus rubrofemoratus, D. montivagus, D. striatipennis, D. rectus, D. titanus, Prismognathus angularis, Prosopocoilus inclinatus, and Lucanus maculifemoratus were collected from 12 localities in Japan, dissected and examined for nematode associates. Isolated nematodes were used to attempt the establishment of voucher cultures. Successfully cultured nematodes were observed under a light microscope for morphological identification at genus or species level, and were sequenced for their near full length SSU and D2/D3 LSU. All investigated lucanid individuals harbored at least one species of nematode, and eight species (Bursaphelenchus sp., Koerneria luziae, K. lucani, Pristionchus cf. pacificus, Pristionchus sp., Pseudodiplogasteroides composites, Pseudodiplogasteroides sp., and Rhabditidoides sp.: 40 populations of nematodes in total), were successfully cultured from the insects. Within these eight species, four species were considered to predominate and had low carrier specificity and wide distribution, i.e., K. luziae, P. composites, Pristionchus sp. and Rhabditidoides sp. were isolated from various species of beetles and from several different localities, and K. luziae, K. lucani, and P. composites have been previously described from native lucanids in Germany. Considering the recent world-wide trade of living insects, especially lucanids as pets, we suggest the use of caution to prevent the invasion of their associated nematodes and other associated organisms.

New plant-parasitic nematode from the mostly mycophagous genus Bursaphelenchus discovered inside figs in Japan.

A new nematode species, Bursaphelenchus sycophilus n. sp. is described. The species was found in syconia of a fig species, Ficus variegata during a field survey of fig-associated nematodes in Japan. Because it has a well-developed stylet and pharyngeal glands, the species is considered an obligate plant parasite, and is easily distinguished from all other fungal-feeding species in the genus based upon these characters. Although B. sycophilus n. sp. shares an important typological character, male spicule possessing a strongly recurved condylus, with the “B. eremus group” and the “B. leoni group” of the genus, it was inferred to be monophyletic with the “B. fungivorus group”. The uniquely shaped stylet and well-developed pharyngeal glands is reminiscent of the fig-floret parasitic but paraphyletic assemblage of “Schistonchus”. Thus, these morphological characters appear to be an extreme example of convergent evolution in the nematode family, Aphelenchoididae, inside figs. Other characters shared by the new species and its close relatives, i.e., lack of ventral P1 male genital papilla, female vulval flap, and papilla-shaped P4 genital papillae in males, corroborate the molecular phylogenetic inference. The unique biological character of obligate plant parasitism and highly derived appearance of the ingestive organs of Bursaphelenchus sycophilus n. sp. expands our knowledge of the potential morphological, physiological and developmental plasticity of the genus Bursaphelenchus.

Bursaphelenchus niphades n. sp. (Tylenchina: Aphelenchoididae) amensally associated with Niphades variegatus (Roelofs) (Coleoptera: Curculionidae)

A Bursaphelenchus species was isolated from a Japanese native wood-boring weevil, Niphades variegatus, and dead Pinaceae trees. The nematode is associated with the weevils as dauer (dispersal third stage) juveniles and the dauers enter the weevil tracheal system forming an abnormal expansion on the weevil trachea (atrium). Thus, the nematode is hypothesised to be an amensal/phoretic associate of the weevil because the abnormal expansion appeared to inhibit weevil respiration. The propagative stages of the nematode are associated with dead trees (wood and bark materials) and are thought to feed there on naturally propagated fungi. Morphologically, the new species is considered an undescribed species close to B. antoniae, B. chengi and B. hylobianum. Within these four species, the new species, which is described herein as B. niphades n. sp., is closest to B. chengi, i.e., the typological character of these two species are almost identical to each other and is distinguished by some minor characters (structure of the male P4 genital papillae and spicule length). The molecular phylogenetic analysis supported the morphological observations. Bursaphelenchus niphades n. sp. formed a well supported subclade with the four species and is intermediate between B. hylobianum and B. chengi; however, it is distinguished by the molecular sequences of some ribosomal RNA genes. Because three of these four species are associated with weevil species, the subclade is considered a ‘weevil-associated’ species group.

Phylogenetic Status of Insect Parasitism in the Subfamily Entaphelenchinae Nickle with Description of Peraphelenchus orientalis n. sp. (Tylenchomorpha: Aphelenchoididae)

Abstract:  The nematode family Aphelenchoididae is a highly divergent group that contains plant parasites, predators, fungal feeders, and insect parasites. It is taxonomically separated into 7 subfamilies. Although molecular phylogenetic relationships among 6 of the subfamilies have been clarified, the phylogenetic position of the subfamily Entaphelenchinae, which is composed of endoparasites of insects, remains unclear. Here, a new entaphelenchid species, Peraphelenchus orientalis n. sp., was isolated from the body cavities of burying beetles, Nicrophorus quadripunctatus, with a 14% prevalence (5 out of 36). The phylogenetic position of the subfamily within the family Aphelenchoididae was determined using morphological and molecular data for the new species. The rRNA sequences suggested that the new species belongs to Clade 3 of Aphelenchoididae, which mostly consists of predators and insect parasites. Although molecular sequences from other entaphelenchid species were not available, the subfamily appears to be monophyletic. The new species is characterized by the absence of a functional rectum and anus and by the presence of 3 pairs of male genital papillae, a loosely coiled male body, and a W-shaped male spicule. Compared with the original description of Peraphelenchus necrophori, P. orientalis n. sp. has significant typological differences, possibly because of misinterpretations during the original description of P. necrophori. Excluding these questionable characters, i.e., presence–absence of functional rectum and anus and number of male genital papillae, the new species is distinguished from P. necrophori by minor morphological characters and morphometric values.

Assessment of Helminth Biodiversity in Wild Rats Using 18S rDNA Based Metagenomics

Parasite diversity has important implications in several research fields including ecology, evolutionary biology and epidemiology. Wide-ranging analysis has been restricted because of the difficult, highly specialised and time-consuming processes involved in parasite identification. In this study, we assessed parasite diversity in wild rats using 18S rDNA-based metagenomics. 18S rDNA PCR products were sequenced using an Illumina MiSeq sequencer and the analysis of the sequences using the QIIME software successfully classified them into several parasite groups. The comparison of the results with those obtained using standard methods including microscopic observation of helminth parasites in the rat intestines and PCR amplification/sequencing of 18S rDNA from isolated single worms suggests that this new technique is reliable and useful to investigate parasite diversity.

Species-specific recognition of the carrier insect by dauer larvae of the nematode Caenorhabditis japonica

SUMMARY Host recognition is crucial during the phoretic stage of nematodes because it facilitates their association with hosts. However, limited information is available on the direct cues used for host recognition and host specificity in nematodes. Caenorhabditis japonica forms an intimate association with the burrower bug Parastrachia japonensis. Caenorhabditis japonica dauer larvae (DL), the phoretic stage of the nematode, are mainly found on adult P. japonensis females but no other species. To understand the mechanisms of species-specific and female carrier-biased ectophoresy in C. japonica, we investigated whether C. japonica DL could recognize their hosts using nematode loading and chemoattraction experiments. During the loading experiments, up to 300 C. japonica DL embarked on male and female P. japonensis, whereas none or very few utilized the other shield bugs Erthesina fullo and Macroscytus japonensis or the terrestrial isopod Armadillidium vulgare. In the chemoattraction experiments, hexane extracts containing the body surface components of nymphs and both adult P. japonensis sexes attracted C. japonica DL, whereas those of other shield bugs did not. Parastrachia japonensis extracts also arrested the dispersal of C. japonica DL released at a site where hexane extracts were spotted on an agar plate; i.e. >50% of DL remained at the site even 60 min after nematode inoculation whereas M. japonensis extracts or hexane alone did not have the same effect. These results suggest that C. japonica DL recognize their host species using direct chemical attractants from their specific host to maintain their association.

Specialist versus generalist life histories and nucleotide diversity in Caenorhabditis nematodes

Species with broad ecological amplitudes with respect to a key focal resource, niche generalists, should maintain larger and more connected populations than niche specialists, leading to the prediction that nucleotide diversity will be lower and more subdivided in specialists relative to their generalist relatives. This logic describes the specialist-generalist variation hypothesis (SGVH). Some outbreeding species of Caenorhabditis nematodes use a variety of invertebrate dispersal vectors and have high molecular diversity. By contrast, Caenorhabditis japonica lives in a strict association and synchronized life cycle with its dispersal host, the shield bug Parastrachia japonensis, itself a diet specialist. Here, we characterize sequence variation for 20 nuclear loci to investigate how C. japonicas life history shapes nucleotide diversity. We find that C. japonica has more than threefold lower polymorphism than other outbreeding Caenorhabditis species, but that local populations are not genetically disconnected. Coupled with its restricted range, we propose that its specialist host association contributes to a smaller effective population size and lower genetic variation than host generalist Caenorhabditis species with outbreeding reproductive modes. A literature survey of diverse organisms provides broader support for the SGVH. These findings encourage further testing of ecological and evolutionary hypotheses with comparative population genetics in Caenorhabditis and other taxa.

Parapristionchus giblindavisi n. gen., n. sp. (Rhabditida: Diplogastridae) isolated from stag beetles (Coleoptera: Lucanidae) in Japan

A new species of diplogastrid nematode, isolated in a previous survey of nematodes associated with stag beetles in Japan, is described as Parapristionchus giblindavisi n. gen., n. sp. Parapristionchus n. gen. differs from other diplogastrid genera chiefly by its stomatal morphology. Distinguishing the genus are the presence of a claw-like dorsal tooth in both the eurystomatous and stenostomatous forms and the division of the cheilostom into 12 plates lacking apical flaps. According to phylogenetic analysis of nine ribosomal protein gene sequences, Parapristionchus n. gen. shows deep divergence from other known genera. Molecular evidence strongly supports P. giblindavisi n. gen., n. sp. + Pristionchus spp. as monophyletic with respect to all other diplogastrids examined. Congruent with a clade of P. giblindavisi n. gen., n. sp. + Pristionchus spp. is the shared presence of a bifurcate P7 genital papilla. Discovery and description of a close sister group to Pristionchus, a model biological system, enables character polarisation in macroevolutionary studies of Pristionchus nematodes.

Sheraphelenchus sucus n. sp. (Tylenchina: Aphelenchoididae) isolated from sap flow of Quercus serrata in Japan.

A Sheraphelenchus species was isolated from a sample of sap exuding from a scar on the bark of Quercus serrata. Besides its generic characteristics, i.e., posteriorly located vulva in the female, male spicule with conspicuous dorsal limb and male tail with spike-like projection, the new species is characterised by a short stylet with a small basal swelling. The near-full-length of 18S and D2/D3 expansion segments of ribosomal RNA genes (near-full SSU and D2/D3LSU) were determined as its molecular barcode sequences and the phylogenetic status of the species (= genus Sheraphelenchus) was estimated using the near-full SSU. The molecular phylogenetic analysis revealed that the new species is included in the genus Bursaphelenchus, and is close to B. anatolius and B. kevini. Although Sheraphelenchus is phylogenetically inferred to occur within Bursaphelenchus, the new species is described herein as S. sucus n. sp., thereby retaining the genus Sheraphelenchus until additional data about morphological similarities between these two taxa are obtained.

Comparative genomics of Taphrina fungi causing varying degrees of tumorous deformity in plants.

Taphrina fungi are biotrophic plant pathogens that cause plant deformity diseases. We sequenced the genomes of four Taphrina species—Taphrina wiesneri, T. deformans, T. flavorubra, and T. populina—which parasitize Prunus, Cerasus, and Populus hosts with varying severity of disease symptoms. High levels of gene synteny within Taphrina species were observed, and our comparative analysis further revealed that these fungi may utilize multiple strategies in coping with the host environment that are also found in some specialized dimorphic species. These include species-specific aneuploidy and clusters of highly diverged secreted proteins located at subtelomeres. We also identified species differences in plant hormone biosynthesis pathways, which may contribute to varying degree of disease symptoms. The genomes provide a rich resource for investigation into Taphrina biology and evolutionary studies across the basal ascomycetes clade.