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Summary A Bursaphelenchus species belonging to the abietinus group was isolated from a bark sample of Fagus japonica infested by an unidentified Dryocoetes bark beetle. The species is typologically characterised by its two-lined lateral field, mitten-shaped spicule possessing a well-developed and dorsally truncate condylus, narrow triangular rostrum and a distinct cucullus, the presence of seven (P1, P2, P3 papilliform and P5 gland-opening-like) male genital papillae, female vulva with side flap and the long, smoothly tapering and ventrally recurved female tail with strongly ventrally recurved distal part. The species is typologically close to B. irokophilus and B. rainulfi but can be distinguished from these two species by the shape of spicule rostrum, which is narrower in the new species. Phylogenetically, the new species forms a well-supported clade with B. rainulfi but can be distinguished from them by the differences in the SSU and D2-D3 LSU sequences. The new species is described as Bursaphelenchus ogawanus n. sp.

Summary Contortylenchus genitalicola is a unique species within the genus. It parasitises the ovary or testis of the longhorn beetle, Monochamus alternatus, and has a neotylenchid-like culturable mycetophagous phase within its life cycle, while the other species in the genus parasitise bark beetles and have a typical unculturable allantonematid life cycle. Because the species was described based solely on morphology, it was necessary to confirm the phylogenetic and taxonomic status of C. genitalicola. A molecular phylogenetic analysis of the species based on ribosomal RNA sequences revealed that C. genitalicola forms a well-supported clade with Contortylenchus spp. isolated from bark beetles, an unidentified allantonematid from Iran, and ‘Deladenus cf. laricis’ isolated from the metallic wood-boring beetle, Agrilus macer LeConte (Coleoptera: Buprestidae), in the USA. The generic status of C. genitalicola was supported by phylogenetic analysis, and the analysis also suggests that the diversity of the genus could be higher than currently expected.

Summary A Schistonchus species was isolated from the syconia of the creeping fig, Ficus pumila, collected from Miyazaki, Japan. The nematode was considered an undescribed species based on its typological characters, molecular profiles (near full length of small subunit and D3 expansion segment of large subunit of RNA) and phylogenetic status inferred from these two loci. The new species is characterised by the presence of flagellate sperm, spicule possessing small condylus, triangular rostrum with bluntly pointed tip and clear dorsal and ventral limbs, structure and arrangement of male caudal papillae, i.e., presence of papilliform P3 and P3a and small and gland opening-like P4 (glandpapillae), relatively short post-uterine sac and female tail forming elongate conoid. It is phylogenetically close to S. hirtus. However, the new species is distinguished from other nominal Schistonchus species by its female tail shape forming elongate conoid, the structure and arrangement of male caudal papillae and several other typological characters. In addition, the host fig of the nematode, F. pumila, is a creeper fig species belonging to subgenus Synoecia (section Rhizocladus; subsection Plagiostigma), from which no fig-associated nematode has been reported so far, i.e., the present study is the first report of Schistonchus (and other syconia-inhabiting nematodes) from the Ficus subgenus Synoecia.

Summary Two Ruehmaphelenchus species were isolated from the ambrosia beetles Euwallacea kuroshio and E. interjectus, collected from Okinawa and Kagoshima, Japan, respectively. Both nematodes were successfully cultured on a lawn of the grey mould Botrytis cinerea. Morphological observation and molecular characterisation revealed that both species are undescribed; therefore, the nematodes are described herein as R. kuroshioi n. sp. and R. interjectus n. sp. Phylogenetically, both species are closely related to R. formosanus, R. fujiensis, R. digitulus and R. asiaticus. Ruehmaphelenchus kuroshioi n. sp. is characterised by a conical male and female tail, which has a terminal projection (mucron), small male spicule with a weakly-developed triangular condylus, dorsal and ventral limbs that are connected to each other by three different tissues, and clearly separated distal pairs of genital papillae (P3 and P4). However, R. kuroshioi n. sp. can be distinguished from its close relatives by a combination of male and female tail characters and its distinctive phylogenetic status. Ruehmaphelenchus interjectus n. sp. is almost identical to R. formosanus, sharing characteristic lateral spikes of the spicule; they form a well-supported phylogenetic clade. However, R. interjectus n. sp. is distinguished from R. formosanus by morphometric values and ribosomal RNA, i.e., a 6-bp difference from the ca 1.7 kb molecular sequence of the 18S ribosomal RNA of R. formosanus, which warrants independent species status.

Summary The nematode cuticle is an important structure that provides protection from abiotic environmental stresses and natural enemies. The cuticle ultrastructure of a Myolaimus species (culture code NKZ384) isolated from Kyoto, Japan, was examined in relation to its avoidance of predation by an aphelenchoidid predator, Seinura caverna. The survivability of Myolaimus sp. co-cultured with the predator was examined and compared with those of four Poikilolaimus spp. previously reported by the present authors. Myolaimus and two of the four Poikilolaimus spp. share a ‘baggy’ cuticle and resisted predation effectively. However, the ultrastructure differed between these two genera: i.e., the cuticle of Myolaimus sp. is seven-zoned, while that of P. regenfussi and P. oxycercus is five-zoned. In addition, Myolaimus sp. does not possess the characteristic osmophilic zone reported in Poikilolaimus spp. Therefore, although the ultrastructure differs, the thick and baggy cuticle found in these two phylogenetically distant genera exhibits functional convergence to resist predation.

Summary To reinforce vector-switching potential of Bursaphelenchus doui in its ecological and evolutionary contexts, we tested our previous hypothesis that “B. doui (or its ancestor) was transferred by Acalolepta fraudatrix, Acalolepta sejuncta, and/or Monochamus subfasciatus (or their ancestral species) from broad-leaved trees to conifers, switched vectors from these cerambycid beetles to Monochamus beetles in conifers, and then evolved into the common ancestor of Bursaphelenchus mucronatus and Bursaphelenchus xylophilus”. The affinity of four B. doui isolates and one B. xylophilus isolate for Monochamus alternatus was tested using our simple nematode-loading method to the beetle, and the affinity was assessed based on the nematode loads on the beetles. Phoretic stages of two B. doui isolates obtained from Monochamus saltuarius and Pinus densiflora showed loading levels similar to that of B. xylophilus, which were significantly higher than those of the other two B. doui isolates obtained from A. fraudatrix and M. subfasciatus. This result indicates that the first two isolates of B. doui derived from a conifer-using beetle and a coniferous tree adapt to M. alternatus better than the last two isolates associated with beetles using broad-leaved trees. The data reinforce that vector switching of B. doui could have occurred during the evolutionary history of the B. xylophilus group.

Summary Bursaphelenchus tadamiensis, which was initially isolated from a stag beetle, Dorcus striatipennis, collected from sap flow of Quercus crispula in Fukushima, north-eastern Japan, was re-isolated three times from sap flow of Quercus spp. in western Japan. The re-isolated populations were mostly morphologically consistent with the type population, but the male bursal flap showed high variations in the newly collected populations. Molecularly, the type and new populations showed differences in the internal transcribed spacer region of ribosomal DNA and mitochondrial cytochrome oxidase subunit I gene, suggesting that the species is genetically divided into several local populations. The isolation pattern of B. tadamiensis, i.e., the species was initially isolated from sap flow-associated beetles and subsequently isolated from sap flow, suggests that the species prefers and is adapted to the sap flow environment as its habitat.

A diplogastrid nematode was isolated from a dung beetle, Onthophagus sp., collected from a rotten mushroom in Kyoto, Japan. The species is characterised by its cheilostomatal shape, separated into 12 narrow plates (rugae), deep stegostom, large ellipsoidal amphids, conical female tail and characteristic receptaculum seminis in the female. Based on its phylogenetic status and stomatal composition, the species is typologically similar to two other diplogastrid genera, Neodiplogaster and Mononchoides. The species can be distinguished from these two genera by the size and shape of the amphid (small pore in Neodiplogaster), female tail shape (long and filiform in Mononchoides) and presence of receptaculum seminis (absence in the two nominal genera), and is described as a monotypic member of a new genus, Onthodiplogaster japonica n. gen., n. sp. Observation of feeding behaviour suggested that O. japonica n. gen., n. sp. does not show clear stomatal dimorphism or polymorphism, which is found in its close relatives, but the species can feed on nematodes (predation), fungi and bacteria. This monomorphic omnivory possibly represents its habitat of dung and other rotten materials, where the environment is biologically divergent, and its condition changes rapidly.

Summary Deladenus nitobei, a hexatylinid species, is a parasite of the wood wasp Sirex nitobei and a fungal feeder, i.e., the nematode parasitises the wasp to be carried to a new environment (a dead tree) where it propagates after feeding on Amylostereum areolatum, a fungus mutualistically associated with the wasp. The complex life history involves four adult forms: mycophagous male, mycophagous female, insect-infective female, and insect-parasitic female. To understand the morphological strategies of D. nitobei, the cuticle ultrastructure of the four adult forms was observed using transmission electron microscopy (TEM). The structures were compared with those of other hexatylinid species, particularly Fergusobia spp. TEM revealed that the insect-infective females of D. nitobei have a more developed cuticle compared with mycophagous males and females, whereas insect-infective females of Fergusobia do not. The developed cuticle enables D. nitobei infective females to move actively and easily penetrate the host cuticle using their hypertrophied stylet. In terms of parasitic stage, the cuticle structure was typical of an insect-parasitic stage, as in Fergusobia, although with some differences. A remnant cuticle is not found in Fergusobia yet retained in D. nitobei. These structural differences imply that D. nitobei is in the middle of evolving transepidermal nutrient acquisition from per os acquisition, whereas Fergusobia is near the end of this evolutionary trend.

Transmission electron microscopy (TEM) was used to compare the ultrastructural differences between healthy male florets (anthers) and one floret parasitized by Ficophagus laevigatus in late phase C syconia of Ficus laevigata from southern Florida. Previous light-microscopic examination of paraffin-sectioned material showed that F. laevigatus-infested anthers of F. laevigata manifested as malformed, often with aberrant pollen and hypertrophied epidermal cells closest to regions containing propagating nematodes. Female florets or fig wasp-parasitized female florets were not observed to be parasitized by nematodes. Considering that plant-feeding in the Aphelenchoididae is purportedly much less specialized than in certain groups of the Tylenchomorpha, where specialized hypertrophied feeder cells are produced in response to nematode feeding, we examined the putative induced response in this unusual aphelenchoidid system with the higher resolution afforded by TEM. TEM confirmed the expression of significant epidermal cell hypertrophy of the anther and anther filament in the presence of propagating nematodes, which was expressed as cell enlargement (2-5X), fractionation of large electron-dense stores into smaller aggregates, irregularly shaped nuclei enclosed by an elongated nuclear envelope, nucleolus enlargement, increased organelle production, and apparent metabolism with increased numbers of mitochondria, pro-plastids, and endoplasmic reticulum, as well as increased thickening of the cell walls. Pathological effects were observed in adjacent cells/tissue (e.g., anther and anther filament parenchymal cells, pollen tubes, pollen, and endothecium) with apparent diminishment as the distance from propagating nematodes increased (which was also probably affected by number of nematodes). Some TEM sections captured previously undocumented ultrastructural highlights of propagating individuals of F. laevigatus.

Summary Two new Neomisticius species, typologically and phylogenetically close to each other, are described and illustrated from dead Quercus trees and an ambrosia beetle, Platypus quercivorus. The two species share some stylet morphological characteristics, namely, they both possess a long conus occupying more than half of the total stylet length, a long crustaformeria composed of more than 160 cells (eight rows of more than 20 cells each), and a short and broad female tail with a digitate tip. They are distinguished from each other by N. variabilis n. sp. having a wide, spindle-shaped male bursa with a blunt terminus and N. platypi n. sp. having an oval bursa with a rounded terminus. In addition, the males and females of both species have three large rectal glands and the posterior end of the male testis (distal end of the vas deferens) bears three cells that seemingly function as a valve between the vas deferens and the cloacal tube. These characteristics have not been reported in other tylenchids. Currently, the genus contains only three species: the two new species and N. rhizomorphoides, which has a normal stylet with a short conus, a short crustaformeria, and lacks rectal glands and valve cells in the vas deferens. Therefore, the two new species are readily distinguished from N. rhizomorphoides and, based on the previous definition, may even represent a new genus. However, considering their phylogenetic closeness and biological similarities (e.g., association with ambrosia beetles), the generic definition of Neomisticius was emended to include these new species.

Background The first metazoan genome sequenced, that of Caenorhabditis elegans, has motivated animal genome evolution studies. To date > 50 species from the genus Caenorhabditis have been sequenced, allowing research on genome variation. Results In the present study, we describe a new gonochoristic species, Caenorhabditis niphades n. sp., previously referred as C. sp. 36, isolated from adult weevils (Niphades variegatus), with whom they appear to be tightly associated during its life cycle. Along with a species description, we sequenced the genome of C. niphades n. sp. and produced a chromosome-level assembly. A genome comparison highlighted that C. niphades n. sp. has the smallest genome (59 Mbp) so far sequenced in the Elegans supergroup, despite being closely related to a species with an exceptionally large genome, C. japonica. Conclusions The compact genome of C. niphades n. sp. can serve as a key resource for comparative evolutionary studies of genome and gene number expansions in Caenorhabditis species.

Summary A new Devibursaphelenchus species isolated from the bark of a dead Quercus aliena, which had been infected and killed by Japanese oak wilt, was collected in Shiga, Japan. The new species is characterised by the relatively large body in males (661-768 μm) and females (893-1071 μm), conspicuous male bursal flap, male spicule with long condylus and wide blade, female post-vulval uterine sac (PUS) 39-54 μm or 1.6-2.3 times the vulval body diam. long, vestigial female anus, and female tail forming a strongly ventrally recurved elongate conoid with bluntly pointed or narrowly rounded terminus. The new species is typologically similar to D. lini, sharing a large body, conspicuous bursal flap, long PUS, and spicule shape, but can be distinguished from it by the absence of variation in the female tail shape, i.e., the tail of the new species is always long and strongly ventrally curved, while the tail shape varies more in D. lini. A previous molecular phylogenetic analysis suggested that the new species is fairly close to D. lini, but can be distinguished from it by the 1.0% (16 bp) difference within 1.6 kb of the 18S and 3.7% (26 bps) difference within 0.7 kb of the D2-D3 LSU ribosomal RNA genes. The newly found nematode is described and illustrated herein as D. alienae n. sp.

Summary We isolated two tylenchid species belonging to the Sychnotylenchinae, Sychnotylenchus hibisci n. sp. and Neoditylenchus xiphydriae n. sp., from the bark beetle Ernoporus corpulentus and wood wasp Xiphydria ogasawarai, respectively. Sychnotylenchus hibisci n. sp. is characterised by its moderately stout body in both male and female, gubernaculum to spicule length ratio of one-third or less, crustaformeria structure composed of four columns with six large, rounded cells, and a broad female tail with a rounded terminus. The new species is closely related to S. ulmi, with which it shares body shape and gubernaculum to spicule length ratio, but is distinguished by the female tail shape and several morphometric values. Neoditylenchus xiphydriae n. sp. is characterised by its oval to leaf-shaped male bursa, male tail tip reaching the distal end of, and protruding beyond the bursa, a long post-vulval uterine sac (PUS), and a conical female tail with a narrowly rounded terminus. The species is typologically similar to N. pinophilus but is distinguished by its male spicule shape, gubernaculum to spicule length ratio, and longer PUS. Phylogenetically, the Sychnotylenchinae belongs under the Anguinidae (superfamily Sphaerularioidea) and is closely related to several Ditylenchus, Neotylenchus, Neomisticius, and Ficotylus species.

Juvenile mermithid nematodes were found to parasitize winged females (sexuparae) of Erisoma auratum and Tetraneura radicicola. The morphological characteristics of mermithid nematodes are briefly described. The 18S rDNA and 28S rDNA extracted from one nematode were sequenced and used to construct a Bayesian phylogenetic tree, on which the host ranges of mermithid nematodes were represented. Our study indicated that mermithid parasitism of sexuparae led to fewer and smaller sexual female embryos. This is the first record of a mermithid in relation to eriosomatine aphids and the fourth record with respect to Aphididae.

Life in extreme environments is typically studied as a physiological problem, although the existence of extremophilic animals suggests that developmental and behavioral traits might also be adaptive in such environments. Here, we describe a new species of nematode, Tokorhabditistufae, n. gen., n. sp., which was discovered from the alkaline, hypersaline, and arsenic-rich locale of Mono Lake, California. The new species, which offers a tractable model for studying animal-specific adaptations to extremophilic life, shows a combination of unusual reproductive and developmental traits. Like the recently described sister group Auanema, the species has a trioecious mating system comprising males, females, and self-fertilizing hermaphrodites. Our description of the new genus thus reveals that the origin of this uncommon reproductive mode is even more ancient than previously assumed, and it presents a new comparator for the study of mating-system transitions. However, unlike Auanema and almost all other known rhabditid nematodes, the new species is obligately live-bearing, with embryos that grow in utero, suggesting maternal provisioning during development. Finally, our isolation of two additional, molecularly distinct strains of the new genus—specifically from non-extreme locales—establishes a comparative system for the study of extremophilic traits in this model.

Summary An undescribed Cryptaphelenchus species was isolated from a bark beetle, Cryphalus piceae that had emerged from a dead log of the Veitch’s fir, Abies veitchii, collected from Sugadaira Montane Research Center, University of Tsukuba, Nagano, Japan. The new species is characterised by its female post-vulval uterine sac (PUS) of less than one vulval body diam. in length and female posterior body end (tail) conical with elongate posterior part and variably shaped tip, seven male genital papillae, which are composed/arranged as a precloacal P1, P2 pair slightly anterior to cloacal opening, and two small pairs of glandpapillae near tail tip and very narrow bursal flap-like cuticular extension surrounding the male tail. In addition to these characters, the male apophysis appears wing-like in ventral view. In possessing an elongate posterior part, Cryptaphelenchus abietis n. sp. shares the female tail shape with C. borlossi, C. leptocaudus and C. sutoricus, and shares a short PUS with C. iranicus, C. varicaudatus, C. baujardi and C. paravaricaudatus. However, the new species can be distinguished from these species by other typological and morphometric characters and its molecular phylogenetic status. Phylogenetically, the new species is closest to Bursaphelenchus minutus, but can be clearly distinguished by morphological and molecular sequence characters.

Summary Although some anti-predator defences have been documented in nematodes, the role of the cuticle in defending against predator attacks remains unclear. Microscopical observations have suggested that certain Poikilolaimus species, including P. oxycercus and P. regenfussi, have thick cuticles relative to their congeners, which may represent an anti-predator defence, but the role of the cuticle in reducing predation has not been examined. To address this knowledge gap, we first quantified cuticle structure using transmission electron microscopy among four Poikilolaimus species (P. oxycercus, P. regenfussi, P. floridensis and P. carsiops). We then examined their survival rates following exposure to the predatory aphelenchoidid nematode, Seinura caverna, and assessed correlations between cuticle thickness and survival rate. The cuticles of P. oxycercus and P. regenfussi were significantly thicker than those of P. floridensis and P. carsiops. In addition, P. oxycercus and P. regenfussi had an osmophilic zone in their cuticle structure, which was not observed in P. floridensis and P. carsiops. Both thickness of specific cuticle zones and total cuticle thickness were positively correlated with survival in the presence of a predator, with the strongest correlation observed with the thickness of the osmophilic zone. We suggest that P. oxycercus and P. regenfussi have uniquely evolved cuticles among members of Poikilolaimus in terms of thickness and structure that play an important role in reducing predation.

Summary A Bursaphelenchus sp. belonging to the B. idius-group was isolated from a bark beetle, Ernoporus corpulentus, which had emerged from dead twigs of Hibiscus tiliaceus collected in Okinawa, in the subtropical region of Japan. In addition to its group-specific characters, i.e., four lateral lines in both male and female and less developed capitulum and relatively broad blade of the male spicules, the species is characterised by a relatively slender female tail with various tip shapes. Molecularly, the species is close to B. fagi and B. tiliae based on SSU and D2-D3 LSU sequences, but can be clearly distinguished from these two species. The species is described and illustrated herein as B. hibisci n. sp.

Summary Aphelenchoides roulingae n. sp. is described and illustrated. This new species was isolated from Xylocopa collaris sauteri collected at Fushan, Yilan county, Taiwan. Typologically, the new species is characterised by a three-lined lateral field, conical female tail with various and not pointed termini, male spicule with well-developed and slightly dorsally truncate condylus, blunt triangular-shaped rostrum and unevenly ventrally curved dorsal contour of the spicule blade (calomus-lamina complex). The combination of typological characters of the new species is unique. Aphelenchoides roulingae n. sp. shares the three-lined lateral field and female tail shape with ‘A. helicus’, which was previously transferred to Robustodorus from Aphelenchoides, but can be differentiated from that species by its longer and more slender female tail, narrower female tail tip, and longer female stylet. In addition, the spicule morphology of A. roulingae n. sp. is unique and not similar to that of any other Aphelenchoides species. Phylogenetically, the new species forms a well-supported clade with the other Xylocopa bee-associated species, A. xylocopae, but can be typologically and molecularly distinguished from the species. In addition to the taxonomic description, some typological characters of the genus Aphelenchoides are discussed.

The first metazoan genome sequenced, that of Caenorhabditis elegans, impulse animal genome evolution studies. To date > 50 species from the genus Caenorhabditis have been sequenced, allowing research on genome variation. In the present study, we describe a new gonochoristic species, Caenorhabditis niphades n. sp., previously referred as C. sp. 36, isolated from adult weevils (Niphades variegatus), with whom they appear to be tightly associated during its life cycle. Along with a species description, we sequenced the genome of C. niphades n. sp. and produced a chromosome-level assembly. A genome comparison highlighted that C. niphades n. sp. has the smallest genome (59 Mbp) so far sequenced in the Elegans supergroup, despite of being closely related with the largest genome species, C. japonica. The compact genome of C. niphades n. sp. can serve as a key resource for comparative evolutionary studies of genome and gene number expansions in Caenorhabditis species.

Summary The nematode associations of two nitidulid beetles, Epuraea ocularis and Lasiodactylus pictus, and their habitat (substrate) were examined in an experimental stand in Kyoto, Japan. Beetles were collected in October 2020 using banana traps. Molecular and morphological identification recognised two nematode species: Sheraphelenchus sucus and Chylorhabditis epuraeae. Sheraphelenchus sucus was isolated exclusively from E. ocularis, while C. epuraeae was isolated from both beetle species. Dauer juveniles (DJ) of S. sucus were isolated from the substrate, rotten banana. The typological characters of S. sucus DJ are illustrated and photo-documented, and S. heterophallus n. comb. (= Aphelenchoides heterophallus Steiner, 1934 and Parasitaphelenchus heterophallus (Steiner, 1934) Rühm, 1956), recognised during a literature survey, is proposed.

Summary Steinernema tielingense was found to be widely distributed in Japan. The molecular characteristics of the nematodes isolated from Japan are basically in accordance with those mentioned in the original description of S. tielingense. However, the Japanese isolates are morphologically distinct from the type population by the following characters: length of the infective juvenile, 711 (671-769) μm, excretory pore of adult males and infective juveniles located posterior mid-pharynx, D% < 50%, smaller H value of 35% and shorter spicules in adult males of both generations. Although discrepancies in morphology and morphometric data between the Chinese isolate and Japanese isolate were observed, the molecular data matched and on this basis the Japanese isolate was identified as being conspecific with S. tielingense. The symbiotic bacterium isolated from the Japanese population of S. tielingense was identified as Xenorhabdus bovienii.

Summary An undescribed Basilaphelenchus species was isolated from the dead and decaying wood of an unidentified broad-leaved tree in Kyoto, Japan. The species was cultured on a lawn of Botrytis cinerea after many attempts and kept as a laboratory strain. The nematode is characterised by the position of its secretory-excretory pore, which differs between males (anterior to median bulb) and females (overlapping or posterior to median bulb), the tail morphology of males (conical with very short mucron) and females (smoothly tapering conical with various shapes at terminus) and male spicule morphology, with spicules possessing a smoothly curved blade (calomus-lamina complex) and a shallow capitulum depression. In addition, the new species is differentiated from other members of the genus by the molecular sequences of its ribosomal RNA region. Phylogenetically, the new species is close to two Iranian species, B. brevicaudatus and B. gorganensis, but can be distinguished from them based upon its separated phylogenetic status. The genus contains five species that were described from Chile and Iran. Thus, the isolation of a species from this genus in Kyoto, Japan, represents the first record of the genus from East Asia. The new species is described and illustrated herein as B. pedrami n. sp.

Summary Two Bursaphelenchus spp. belonging to the eggersi group (subgroup 3) were isolated from bark beetles (Cryphalus spp.). One species, isolated from Cryphalus sp. cf. carpini that emerged from a log of an unidentified broad-leaved tree (considered to be Carpinus sp.) collected in Sugadaira, Nagano, Japan, was identified molecularly and typologically as B. carpini. The other species, isolated from C. abbreviatus that emerged from a dead log of Ficus microcarpa collected in Okinawa, Japan, was differentiated molecularly from other eggersi group species. The new species is characterised by its three-lined lateral field, seven genital papillae with the posteriormost pair forming ‘glandpapillae’, short and stout male spicule with a strongly dorsally recurved and pointed condylus, small and rectangular male bursal flap with a variable terminus, female vulva with a ‘side flap’, broadly rounded female tail tip and molecular sequences of the ribosomal RNA region and partial mtCOI gene. The species is described and illustrated herein as B. microcarpae n. sp.

Summary Three undescribed Ruehmaphelenchus species were isolated from dead wood of Quercus crispula collected in three different localities in Japan. One of these three species was successfully cultured using Botrytis cinerea as its food fungus and is described and figured as R. fujiensis n. sp. The new species is characterised by the following features: male tail, conical with or without a projection; female tail, conical with variously shaped terminal mucron or projection; and male spicule possessing a relatively long and triangular condylus. The new species is close to, or almost a cryptic species of R. digitulus, i.e., the typological characters and morphometric values of these two species are mostly overlapping. In a molecular phylogenetic relationship inferred from near-full-length small subunit (18S) and D2-D3 expansion segments of the large subunit (28S) of ribosomal RNA, the new species is close to R. asiaticus, R. digitulus, R. formosanus, and Ruehmaphelenchus sp. NKZ202; however, it was clearly separated from these species.

A survey for the presence of nematodes on the skin of the native Florida manatee, Trichechus manatus latirostris from Crystal River, Florida was conducted during annual manatee health assessments. A putative isolate of Cutidiplogaster manati (Diplogastridae) and two other nematodes belonging to the same family were recovered from mid-dorsal tail skin-scrapings from all sampled winter-collected healthy wild adult manatees during two successive years (2018–2019). Qualitative abundance estimates of these three species of diplogastrid nematodes suggest that an average wild Florida manatee adult might possess between 30,000 and 120,000 nematodes on its tail dorsum and that the entire body dorsum including the tail might possess 160,000–640,000 nematodes in roughly equal ratios. Attempts to culture these nematodes on a variety of different culture media were unsuccessful but examination of the mouth (stomatal) morphology suggests specialized feeding on microbes such as diatoms or predation on other nematodes. No skin lesions were observed during the 2018–2019 samplings suggesting that under normal conditions these nematodes are highly specialized free-living epibionts of the skin that are tightly bound to this niche and horizontally transferred between individual manatees in an analogous fashion to human skin mites (Demodex folliculorum and D. brevis). Molecular phylogenetic inferences using sequences of near full length SSU and D2–D3 expansion segments of LSU rRNA genes revealed a putative new morphospecies in Cutidiplogaster sister to C. manati that was monophyletic with several named Mononchoides species, and another putative new morphospecies that formed a clade with several undescribed species similar in appearance to Mononchoides as well as Tylopharyx, Eudiplogasterium, Paroigolaimella and Sachsia.

To show the importance of vector switching of nematodes in the evolution of the Bursaphelenchus xylophilus group, we tested a hypothesis that “Bursaphelenchus doui (or its ancestor) was transferred by Acalolepta fraudatrix, Acalolepta sejuncta, and/or Monochamus subfasciatus (or their ancestral species) from broad‐leaved trees to conifers, switched vectors from these cerambycid beetles to Monochamus beetles in conifers, and then evolved into the common ancestor of Bursaphelenchus mucronatus and B. xylophilus.” We used a simple nematode‐loading method to beetles and produced 20 binary combinations of five B. xylophilus group species and four cerambycid beetle species in the tribe Lamiini. The affinity of the nematodes for the beetles was examined based on phoretic stage formation of the nematodes. Phoretic stages of B. doui appeared in all beetle species examined, namely Acalolepta luxuriosa, Psacothea hilaris, A. fraudatrix, and Monochamus alternatus, although the affinity of the nematode for M. alternatus was weak. This finding indicates that B. doui could switch vectors to conifer‐using Monochamus beetles after transfer by A. fraudatrix from broad‐leaved trees to conifers. We conclude that vector switching of nematodes could have potentially happened during the evolutionary history of the B. xylophilus group., To clarify the role of vector switching of nematodes in the evolution of the Bursaphelenchus xylophilus group, we used a simple nematode‐loading method to beetles and produced 20 binary combinations of five B. xylophilus group species and four cerambycid beetle species in the tribe Lamiini. The affinity of the nematodes for the beetles was examined based on phoretic stage formation of the nematodes. The results showed that vector switching of nematodes could have potentially happened during the evolutionary history of the B. xylophilus group.

Summary A new aphelenchoidid entomoparasitic nematode was isolated from the body cavity of overwintering individuals of a tenebrionid beetle, Uloma marseuli, collected at Shiga, Japan. The nematode is characterised by its six equal-sized lips forming a dome shape continuous with body contour. It has a moderately thick stylet, with wide lumen, a long two-part long (conus + conophore) and elongate oval or pear-shaped metacorpus with glandular anterior part. The male spicules are separate with a well-developed condylus, triangular rostrum and smoothly and strongly curved calomus-lamina complex. A gubernaculum or apophysis is absent. There are two pairs of papilliform male genital papillae. Females lack a post-vulval uterine sac, have a seemingly vestigial rectum and anus, and conical tail. The combination of the typological characters of the species does not fit any currently accepted aphelenchoidid genus and is somewhat intermediate between the Ektaphelenchinae and Entaphelenchinae. The molecular phylogenetic analysis also suggested that the nematode is close to both Ektaphelenchoides (Ektaphelenchinae) and Peraphelenchus (Entaphelenchinae). Thus, the nematode is described and illustrated as Lenisaphelenchus ulomae n. gen., n. sp. and tentatively placed in the Ektaphelenchinae.

Summary A new rhabditid entomophilic nematode was isolated from adult Epuraea (Haptoncus) ocularis collected from brown to light brown sap on the trunk of Ulmus parvifolia in Kyoto, Japan. The typological characters of the species, anteriorly opened peloderan bursa with nine bursal rays with the first, fifth, and eighth pairs directed dorsally, very long and two-part spicule with broad tip, and elongate conical female tail, do not fit the current nominal rhabditid genera. While phylogenetic analyses based on the near-full-length small subunit (18S) ribosomal RNA sequence tentatively suggest that the species is the sister of Buetschlinema, the typological and biological characters of the new species clearly differ from that genus, and so a new species is described herein as Chylorhabditis epuraeae n. gen., n. sp.

Summary Stylet (feeding) dimorphism of Bursaphelenchus sinensis was experimentally confirmed during a recent study and its predatory form was considered similar in morphology to species of Berntsenus. Therefore, available type and voucher materials for members of Berntsenus were examined for their morphology and compared with Bursaphelenchus sinensis and B. aberrans, another related species showing feeding dimorphism. The typological characters of their co-habiting aphelenchoidid species were also examined. The type materials of Berntsenus spp. have characteristic squared lips, a thick stylet, large median bulb and short and stout pharyngeal glands; males, when present, have a P1 papilla and a tape-like bursa, suggesting that they are the predatory form of improperly classified Bursaphelenchus species. In addition, Aphelenchoides pityokteini and Ektaphelenchus tenuidens (= Aphelenchoides tenuidens), co-occurring with their respective host bark beetle isolates of Berntsenus, were typologically confirmed to belong to the aberrans-group of Bursaphelenchus and considered to be conspecific to each co-habiting Berntsenus species. Herein, Berntsenus is synonymised with Bursaphelenchus, and Bursaphelenchus brachycephalus n. comb. (= Berntsenus brachycephalus and E. tenuidens) and Bursaphelenchus pityokteini n. comb. (= A. pityokteini and Berntsenus labiosus) are proposed.

Summary To investigate the phoretic association between nematodes in the genus Bursaphelenchus and cerambycid beetles in north-eastern Japan, Pinus densiflora logs were placed in two pine forests in the towns of Yamada and Hiranai as beetle oviposition sites. At 1-2 years after oviposition, adults of Monochamus saltuarius emerged from the logs placed in Yamada and adults of Acalolepta sejuncta and A. fraudatrix emerged from the logs placed in Hiranai, and Bursaphelenchus nematodes were recovered from two of these species (M. saltuarius and A. sejuncta). Morphometrics and a phylogenetic analysis of the internal transcribed spacer regions 1 and 2 and the 5.8S ribosomal RNA gene indicated that both of these nematode populations were B. doui, which has previously only been known to use M. subfasciatus and A. fraudatrix as vectors. Therefore, this study demonstrated that this nematode utilises at least four species of beetles belonging to two genera as vectors in the field.

Phenotypic plasticity is one of the most important strategies used by organisms with low mobility to survive in fluctuating environments. Phenotypic plasticity plays a vital role in nematodes because they have small bodies and lack wings or legs and thus, cannot move far by themselves. Bursaphelenchus xylophilus, the pathogenic nematode species that causes pine wilt disease, experiences fluctuating conditions throughout their life history; i.e., in both the phytophagous and mycetophagous phases. However, whether the functional morphology changes between the life phases of B. xylophilus remains unknown. Our study revealed differences in the ultrastructure of B. xylophilus between the two phases. Well-developed lateral alae and atrophied intestinal microvilli were observed in the phytophagous phase compared with the mycetophagous phase. The ultrastructure in the phytophagous phase was morphologically similar to that at the dauer stage, which enables the larvae to survive in harsh environments. It suggests that the living tree represents a harsh environment for B. xylophilus, and ultrastructural phenotypic plasticity is a key strategy for B. xylophilus to survive in a living tree. In addition, ultrastructural observations of obligate plant-parasitic species closely related to B. xylophilus revealed that B. xylophilus may be in the process of adapting to feed on plant cells.

Summary Bursaphelenchus acaloleptae n. sp. is described and figured based upon its morphological characters and molecular profiles. The new species belongs to the xylophilus-group of the genus and is typologically characterised by its secretory-excretory pore being located slightly posterior to the median bulb, weakly tapering, relatively broad and straight female tail with variously shaped terminus, and rounded male bursal flap with squared terminus. The new species is phylogenetically and biologically closest to B. luxuriosae. These cryptic species share the host (habitat) tree species, Aralia elata (Araliaceae), the same carrier insect species, Acalolepta luxuriosa (Cerambycidae), and a characteristic phoretic adult form. However, the new species is typologically distinguished from all other xylophilus-group species by the combination of the above-mentioned characters and molecular profiles (SSU and LSU molecular barcodes). Further, B. acaloleptae n. sp. can be distinguished from its closest relative, B. luxuriosae, by mating experiments, i.e., these two species did not produce a viable F1 generation in reciprocal crossings, thereby confirming separate species status using the biological species concept.

A diplogastrid nematode species was isolated from a termite-associated tenebrionid beetle, Ziaelas formosanus, collected from several localities in Taiwan. The new species shares a characteristic feeding structure, i.e., an extremely long and thin stoma with two genera, Rhabditolaimus and Mehdinema. However, the new species has highly characteristic male and female reproductive system, i.e., arrangement of male genital papillae, spicule and gubernaculum morphologies, and presence of female receptaculum seminis, that are similar to those of Acrostichus. A molecular phylogenetic analysis based on near-full-length small subunit and D2-D3 expansion segments of large subunit of ribosomal RNA gene sequences suggested that the species belongs to Acrostichus. The highly derived feeding characters of the new species accentuates the plasticity of the feeding structures of diplogastrid nematodes. In addition to the species description, the generic characters of Acrostichus are emended, and a list of the species in the genus is provided.

Summary Two populations of an aphelenchoidid nematode were recovered from natural forests in Golestan province, Iran. Both populations resembled each other morphologically. The females of the first population, from rotten wood of Fagus orientalis in Golestan National Park, measured 294-338 μm long, and females of the second population, from forests of Ramian county, measured 365-464 μm long. A reverse taxonomic approach, based upon SSU and LSU phylogenies using a maximal number of Aphelenchoididae genera, revealed the two isolates to be conspecific and representatives of a new species of Basilaphelenchus. Basilaphelenchus magnabulbus n. sp. is described and illustrated herein and is mainly characterised by the unique form of the metacorpus, this being elongate and filling the corresponding body region similar to the common form in other aphelenchoidids (vs smaller with well posteriorly located small valve in previously described species of the genus). The new species has an elevated lip region, fine stylet with three elongate posteriorly directed knobs, female tail elongate conoid and ventrally bent, and male tail short conical with three pairs of caudal papillae. It was morphologically compared with four previously known species of the genus, viz., B. persicus, B. brevicaudatus, B. gorganensis and B. grosmannae, and three species of Aphelenchoides having a broadly similar morphology.

The parasitism of two groups of host-manipulating parasites of hornets was examined in Kyoto, Japan. Vespa mandarinia (661 individuals), V. simillima (303), V. analis (457), V. ducalis (158), V. crabro (57), and V. dybowskii (4) were collected either by bait trap or hand collection with an insect net, and examined for their parasites. An endoparasitic nematode, Sphaerularia vespae was isolated from three overwintered gynes of V. mandarinia and a gyne of V. ducalis. While endoparasitic insects, Xenos spp., were recovered from 13 V. mandarinia, 77 V. analis, two V. ducalis, and three V. crabro, and those recovered from V. analis and others were molecularly identified as X. oxyodontes and X. moutoni, respectively. Comparing Xenos parasitism level and capturing methods, the parasitism level was significantly higher in trapped hosts than in hand-collected ones, suggesting that stylopized hosts are more strongly attracted to the food source (bait trap) compared with unparasitized hosts. The genotypes of S. vespae were identical to each other, and near identical to its type population. While each of the two Xenos spp. showed four mitochondrial DNA haplotypes. A phylogenetic comparison suggested that Xenos haplotypes found in the present study are close to those previously reported from Japan and other Asian countries.

Nematodes of the genus Auanema are interesting models for studying sex determination mechanisms because their populations consist of three sexual morphs (males, females, and hermaphrodites) and produce skewed sex ratios. Here, we introduce a new undescribed species of this genus, Auanema melissensis n. sp., together with its draft nuclear genome. This species is also trioecious and does not cross with the other described species A. rhodensis or A. freiburgensis. Similar to A. freiburgensis, A. melissensis’ maternal environment influences the hermaphrodite versus female sex determination of the offspring. The genome of A. melissensis is ~60 Mb, containing 11,040 protein-coding genes and 8.07% of repeat sequences. Using the estimated ancestral chromosomal gene content (Nigon elements), it was possible to identify putative X chromosome scaffolds.

Two new species of Tokorhabditis, T. tauri n. sp. and T. atripennis n. sp., which were isolated from multiple Onthophagus species in North America and from O. atripennis in Japan, respectively, are described. The new species are each diagnosed by characters of the male tail and genitalia, in addition to molecular barcode differences that were previously reported. The description of T. tauri n. sp. expands the suite of known nematode associates of O. taurus, promoting ecological studies using a beetle that is an experimental model for insect–nematode–microbiota interactions in a semi-natural setting. Furthermore, our description of a third Tokorhabditis species, T. atripennis n. sp., sets up a comparative model for such ecological interactions, as well as other phenomena as previously described for T. tufae, including maternal care through obligate vivipary, the evolution of reproductive mode, and extremophilic living.

Phoretic nematodes associated with two mass-occurring populations of the millipede Parafontaria laminata were examined, focusing on Pristionchus spp. The nematodes that propagated on dissected millipedes were genotyped using the D2-D3 expansion segments of the 28S ribosomal RNA gene. Four Pristionchus spp. were detected: P. degawai, P. laevicollis, P. fukushimae, and P. entomophagus. Of the four, P. degawai dominated and it was isolated from more than 90% of the millipedes examined. The haplotypes of partial sequences of mitochondrial cytochrome oxidase subunit I examined for Pristionchus spp. and P. degawai showed high haplotype diversity.

Phenotypic plasticity has been widely reported in animals and can drive investment in new biological characters that engender ecological adaptability. The nematode family Diplogastridae, especially Pristionchus pacificus with its dramatic stomatal (feeding) dimorphism, has become an important model system to analyze the evolutionary and developmental aspects of polyphenism. However, this plasticity has not been confirmed in other nematode groups. In the present study, we experimentally examined the feeding dimorphism of a fungal feeding free-living nematode, Bursaphelenchus sinensis. In a laboratory culturing experiment, the nematode expressed dimorphism, i.e., a small proportion of the population manifested as a predatory form. This form only occurred in females and was not clearly influenced by the presence of potential prey species. In addition, the ratio of the predatory form to the mycophagous form varied among different fungal food species grown in monoculture on different culture media. The predatory form of B. sinensis was typologically similar to the monomorphic (specialized) predators belonging to the same family. However, some essential morphological characters were slightly different from the specialized predators, and their behaviours were clearly disparate, suggesting that predation in B. sinensis is derived from a different phylogenetic origin than that of the specialized predators.

Summary We morphologically and molecularly characterized segmented filamentous bacteria (SFB) associated with Rhigonema sp. nematodes in millipede hindguts. Seventy-three Riukiaria sp. millipedes were collected from a broad-leaf forest in Japan, and nematodes were excised from the millipede’s hindguts. The occurrence rate of SFB associated with nematodes was 24 % (10/41) for males, 47 % (14/30) for females, and 100 % (2/2) for juveniles. Genomic DNA was extracted from four SFB-rich nematode heads, and we obtained 40 bacterial clones via analysis of nearly full-length 16S rDNA gene sequences. At the phylum level, Firmicutes, Proteobacteria, and Verrucomicrobia accounted for 55 %, 40 %, and 5 % of SFB, respectively. In Firmicutes, Clostridiaceae (28 %) and Lachnospiraceae (15 %) were the dominant groups. Our sequences were divided into seven and three subclades between Firmicutes and Proteobacteria in the phylogenetic tree. In the Firmicutes clade, eight sequences were classified as Lachnospiraceae with a bootstrap value >83 %. A phylogenetic tree involving known uncultured Lachnospiraceae sequences characterized the phylogenetic position of SFB associated with nematodes. Our results suggest that the association of SFB with nematode bodies was probably incidental and that SFB are not always present in millipede hindguts. Our bacterial groups corresponded to those of arthropod hindgut, and SFB associated with nematodes were inferred to belong to Lachnospiraceae. Because the Lachnospiraceae sequences obtained in this study showed specific lineages that differed from all the known deposited sequence data, these groups may be unique to Riukiaria sp.

Sexual dimorphism in motility-related traits is widespread among animals, including several species of Nematoda. However, no study has examined motility-related structural components and compared them between sexes. We examined the motility-related components in four species: Bursaphelenchus conicaudatus Kanzaki, Tsuda and Futai, 2000; Bursaphelenchus rainulfi Braasch and Burgermeister, 2002; Bursaphelenchus doui Braasch, Gu, Burgermeister and Zhang, 2005; Parasitaphelenchus costati Kanzaki, Ekino, Ide, Masuya and Degawa, 2018. We measured the structure and amount of cuticle and body-wall muscles and estimated their relationship to body diameter or total cross-sectional area. Although no structural differences were observed in muscle, the relevant muscle area of B. doui and P. costati was significantly smaller in females than in males. This difference was greatest in P. costati. In all but B. doui, the relative cuticle thickness was significantly smaller in females than in males. Furthermore, only P. costati females had no striated basal zones in their cuticles; these are thought to be cross-linked proteins that provide strength to nematode cuticle during body movement. These results indicate that sexual dimorphism in motility-related structural components is present in P. costati and that females invest less energy in the components than do males.

The nematode genusBursaphelenchusis a highly divergent group. This genus mainly consists of mycophagous entomophilic species, but some species have specialized as obligate or facultative plant parasites, facultative insect parasites, or exhibit feeding dimorphism (phenotypic plasticity) leading to mycophagous and predatory forms. In the present study, a newBursaphelenchusspecies,B.surin. sp. was isolated from fresh syconia (figs) ofFicus surand is described and illustrated based on its typological characters and molecular phylogenetic status. The new species is characterized by its highly derived feeding structures found in obligate plant parasites, lip possessing a labial disc and a long and thick stylet with a long conus and extremely well-developed basal swellings. In addition, slender body of both sexes is characteristic of the species. The new species is phylogenetically and typologically closely related toB.sycophilus, i.e., these two species share the characteristic feeding structures and form a well-supported clade within theB.fungivorusgroup in the genus. Biologically, these two species are both isolated from fresh figs of the sectionSycomorus. However, the new species differs fromB.sycophilusby the length of the female post-uterine sac and the shape of the male spicule, i.e., the new species has a long post-uterine sac and spicule condylus without dorsal recurvature. Thus, the new species is the second obligate fig parasite of the genus, and the evolutionary relationship between theB.surin. sp. andB.sycophilusclade and sectionSycomorusfigs is hypothesized as an example of adaptive radiation with more species to be discovered.