The developmental and structural uniqueness of the embryo of the extremophile viviparous nematode, Tokorhabditis tufae.

Viviparity, a reproductive form that supplies nutrients to the embryo during gestation, has repeatedly and independently occurred in multiple lineages of animals. During the convergent evolution of viviparity, various modifications of development, structure, and physiology emerged. A new species of nematode, Tokorhabditis tufae, was discovered in the alkaline, hypersaline, and arsenic-rich environment of Mono lake. Its reproductive form is viviparity because it is obligately live-bearing and the embryo increases in size during development. However, the magnitude of the increase in size and nutrient provisioning are unclear. We measured egg and embryo sizes at three developmental stages in T. tufae. Eggs and embryos of T. tufae at the threefold stage were respectively 2.6- and 3.6-fold larger than at the single-cell stage. We then obtained T. tufae embryos at the single-cell, lima bean, and threefold developmental stages and investigated the egg hatching frequency at three different concentrations of egg salt buffer. Removal of embryos from the uterus halted embryonic development at the single-cell and lima bean stages in T. tufae irrespective of the solution used for incubation, indicating the provision of nutrients within the uterus. Ultrastructural and permeability evaluation showed that the permeability barrier did not form during embryonic development, resulting in increased molecular permeability. This high permeability caused by the absence of the permeability barrier likely enables supply of nutrients from the mother. The structural and physiological modifications in T. tufae are like those in other viviparous animals. We conclude that T. tufae is a viviparous rather than an ovoviviparous nematode. T. tufae will facilitate investigation of the evolution of viviparity in animals.

First record of Stigmatomyces (Ascomycota: Laboulbeniales) on Drosophilidae from Japan.

Three Stigmatomyces species were detected on five drosophilid species from Japan. We report Stigmatomyces majewskii on Drosophila rufa and Drosophila suzukii, Stigmatomyces scaptodrosophilae on Scaptodrosophila coracina and Scaptodrosophila subtilis, and Stigmatomyces sacaptomyzae on Scaptomyza graminum. Except for Scaptomyza graminum, each of these species is a newly identified Stigmatomyces host. Our discovery that D. suzukii is a host of S. majewskii may provide new pest management approaches for this global agricultural pest insect.

Highlighting Potential Physical and Chemical Cues Involved in Conspecific Recognition System in a Predator Nematode, Seinura caverna.

Conspecific recognition is the ability to distinguish and respond to individuals of the same species. In nematodes, this behavior can mediate aggregation, feeding behavior, or mating. Here, we investigated whether and how the predatory nematode Seinura caverna recognizes and avoids conspecifics to prey on. In predation assays, S. caverna did not kill conspecifics, but killed nematodes of three heterospecific species. Interestingly, S. caverna did not kill Ektaphelenchoides spondylis nematodes. Seinura caverna did not eject its stylet when encountering conspecifics or E. spondylis. The characterization of the internal cuticle structure of 13 nematode species suggested that the cuticle may play a role in the preying decision, as E. spondylis and S. caverna exhibited similar, type III, cuticle layers. Chemical extracts from S. caverna further repelled conspecifics. We discuss the potential hierarchical use of physical and chemical cues in S. caverna predation behavior and provide insights into the evolutionary adaptations and behavior of this organism.

Ultrastructural study of anther parasitism of Ficus laevigata by Ficophagus laevigatus (Aphelenchoididae).

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.

Onthodiplogaster japonica n. gen., n. sp. (Rhabditida: Diplogastridae) isolated from Onthophagus sp. (Coleoptera: Scarabaeidae) from Japan.

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.

Nematode and Strepsipteran Parasitism in Bait-Trapped and Hand-Collected Hornets (Hymenoptera, Vespidae, Vespa).

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.

The compact genome of Caenorhabditis niphades n. sp., isolated from a wood-boring weevil, Niphades variegatus.

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.

Tokorhabditis Tauri n. Sp. and T. Atripennis n. Sp. (Rhabditida: Rhabditidae), Isolated from Onthophagus Dung Beetles (Coleoptera: Scarabaeidae) from the Eastern USA and Japan.

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.

Association of Fig Pollinating Wasps and Fig Nematodes inside Male and Female Figs of a Dioecious Fig Tree in Sumatra, Indonesia.

Nematodes can grow within the inflorescences of many fig trees (Ficus spp., Moraceae); however, the feeding behaviour of most nematodes is not known. Fig pollinating wasps (Hymenoptera: Agaonidae) transfer nematodes into young figs upon the wasps' entry into the figs to deposit their eggs. Most Asian fig trees, however, are functionally dioecious, and the pollinating wasps that enter female figs are unable to reproduce. They fail to produce the offspring required to carry the new generations of nematodes. We examined whether female figs of F. hispida can nonetheless support the development of phoretic nematode populations. Nematodes were extracted from male and female figs sampled in Sumatra, Indonesia, to compare the growth of their populations within the figs. We found three species of nematodes that grew within figs of male and female trees of F. hispida: Ficophagus cf. centerae (Aphelenchoididae), Martininema baculum (Aphelenchoididae) and Caenorhabditis sp (Rhabditidae). The latter species (Caenorhabditis sp.) has never been reported to be associated with F. hispida before. Nematode populations peaked at around 120-140 individuals in both sexes of figs, at the time when a succeeding generation of adult fig wasps appeared within male figs. The female figs could support the growth and reproduction of the three nematodes species; however, the absence of vectors meant that female figs remained as traps from which there could be no escape.

Bursaphelenchus suri n. sp.: A second Bursaphelenchus syconial parasite of figs supports adaptive radiation among section Sycomorus figs.

The nematode genus Bursaphelenchus is 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 new Bursaphelenchus species, B. suri n. sp. was isolated from fresh syconia (figs) of Ficus sur and 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 to B. sycophilus, i.e., these two species share the characteristic feeding structures and form a well-supported clade within the B. fungivorus group in the genus. Biologically, these two species are both isolated from fresh figs of the section Sycomorus. However, the new species differs from B. sycophilus by 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 the B. suri n. sp. and B. sycophilus clade and section Sycomorus figs is hypothesized as an example of adaptive radiation with more species to be discovered.

De Novo Genome Assembly of Auanema Melissensis, a Trioecious Free-Living Nematode.

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.

Tokorhabditis n. gen. (Rhabditida, Rhabditidae), a comparative nematode model for extremophilic living.

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, Tokorhabditis tufae, 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.

Molecular diversity and relationships of fig associated nematodes from South Africa.

Nematodes of figs and fig wasps have received limited attention in Africa since their discovery in 1973. Sixteen of the 25 species of native South African figs were sampled for nematode associates using molecular barcoding with three loci (SSU, LSU D2-D3 and mtCOI) and fourteen (93%) were positive for at least one nematode species. Thirty-three putative species of nematodes were identified and classified according to the loci that were amplified and successfully sequenced. Fourteen putative nematode species were classified as Aphelenchoididae, of which nine were identified as Ficophagus from four species of Ficus from the section Galoglychia (i.e., five ex F. burkei including one shared with F. natalensis, one ex F. glumosa, one ex F. lutea, and one ex F. stuhlmannii) and one species ex F. sur from the section Sycomorus. In addition, there were four nematode species classified as Schistonchus s.s. from section Galoglychia figs (i.e., one ex F. burkei, two ex F. trichopoda, and one ex F. glumosa). There was also one species of Bursaphelenchus nematode recovered from F. sur from the section Sycomorus. Sixteen putative nematode species were classified as Diplogastridae, of which eight occurred in two clades of what is currently called Parasitodiplogaster with one (P. salicifoliae) being recovered from two Ficus species in the section Urostigma (F. salicifolia and F. ingens) and seven diplogastrids being associated with six species of Ficus from the section Galoglychia (i.e., two ex F. burkei including P. sycophilon, one ex F. stuhlmannii, one ex F. burtt-davyi, one ex F. trichopoda, one ex F. abutilifolia and one ex F. sansibarica). Three Acrostichus spp., a Teratodiplogaster and a Pristionchus species were recovered from F. sur and two Teratodiplogaster spp. and Pristionchus sycomori were recovered from F. sycomorus with both Ficus species belonging to the subgenus and section Sycomorus. The identities of the previously described T. martini and Parasitodiplogaster doliostoma (= Pristionchus sp. 35) are discussed. Lastly, there was a panagrolaimid identified from F. petersii.

Pristionchus trametes n. sp. (Diplogastridae) isolated from the mushroom Trametes orientalis in Kyoto, Japan.

A new species of Pristionchus was isolated from fruiting bodies of the wood-decaying fungus Trametes orientalis collected from Kyoto, Japan. Attempts to culture it using bacteria, yeast, and freeze-killed wax moth larvae as food or substrate failed. The eurystomatous form of the species was not found in the collected material, and the species is typologically characterized by: its 'small' stoma with thin, membrane-like cheilostomatal plates, a small triangular right subventral tooth, thorn-like dorsal tooth, and small left subventral denticles; a short, blunt male tail spike; and a short, conical female tail. Although the posterior probability support was not high (66%), phylogenetic analysis of both small and large ribosomal RNA gene subunits suggests that the species is closely related to P. elegans and P. bucculentus. The new species can be distinguished from those two by its diagnostic characters comprising the stomatal morphology and male and female tail characters.

Nine new Pristionchus (Nematoda: Diplogastridae) species from China.

The model organism Pristionchus pacificus and the genus Pristionchus, Kreis, 1932 have been intensively studied in the last decade with contemporary work focusing on the development, evolution, ecology, behavior, neurobiology, and genomics of this group of organisms. In particular, mechanistic studies on the development and evolution of mouth-form plasticity, predation and associated self-recognition processes enabled unique insight into life history strategies and the evolution of novelty. These studies include a comparative research agenda making use of the 39 available species of Pristionchus, all of which can be studied in living cultures. Sampling efforts revealed that Asia represents a biodiversity hotspot for Pristionchus worms. However, previous samplings have a bias towards northern and island areas, largely for logistic reasons. Here, we report on two extensive sampling trips to the Yunnan and Shaanxi provinces in Mainland China. We report the isolation of nine new Pristionchus species by morphology, morphometrics, mating experiments and genome-wide sequence analysis.

Additional description and genome analyses of Caenorhabditis auriculariae representing the basal lineage of genus Caenorhabditis.

Caenorhabditis auriculariae, which was morphologically described in 1999, was re-isolated from a Platydema mushroom-associated beetle. Based on the re-isolated materials, some morphological characteristics were re-examined and ascribed to the species. In addition, to clarify phylogenetic relationships with other Caenorhabditis species and biological features of the nematode, the whole genome was sequenced and assembled into 109.5 Mb with 16,279 predicted protein-coding genes. Molecular phylogenetic analyses based on ribosomal RNA and 269 single-copy genes revealed the species is closely related to C. sonorae and C. monodelphis placing them at the most basal clade of the genus. C. auriculariae has morphological characteristics clearly differed from those two species and harbours a number of species-specific gene families, indicating its usefulness as a new outgroup species for Caenorhabditis evolutionary studies. A comparison of carbohydrate-active enzyme (CAZy) repertoires in genomes, which we found useful to speculate about the lifestyle of Caenorhabditis nematodes, suggested that C. auriculariae likely has a life-cycle with tight-association with insects.

Three Seinura species from Japan with a description of S. shigaensis n. sp. (Tylenchomorpha: Aphelenchoididae).

A preliminary survey of Seinura spp. was conducted in the Kyoto area, Western Japan. The survey yielded four new strains of Seinura spp., including two strains of S. caverna, a strain of S. italiensis, and a strain of an undescribed species. Molecularly, the two strains of S. caverna were nearly identical to the type strain but showed some minor variations, particularly in the mitochondrial cytochrome oxidase subunit I gene. The small subunit and D2-D3 large subunit sequences of the Japanese strain of S. italiensis were nearly identical and identical to its original description, respectively, and the difference in the small subunit was due to mis-reading of the sequences. The new species, S. shigaensis n. sp., was phylogenetically close to S. caverna and S. persica, although these three species were clearly different phylogenetically. The new species was typologically similar or nearly identical to several other Seinura spp., including S. chertkovi, S. christiei, S. italiensis, S. steineri, and S. tenuicaudata, but it can be distinguished from those species by the morphometric values. Because the new species is phylogenetically very close to S. caverna, it could be a good comparative system for S. caverna as a potential satellite model for the predatory nematode.

Nematode epibionts on skin of the Florida manatee, Trichechus manatus latirostris.

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.

Ultrastructural plasticity in the plant-parasitic nematode, Bursaphelenchus xylophilus.

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.