Geographical distribution and phoretic associations of the viviparous nematode Tokorhabditis atripennis with Onthophagus dung beetles in Japan.

Viviparity is generally considered to be rare in animals. In nematodes, only six species of Rhabditida are viviparous. Five of these species have been identified in association with Onthophagus dung beetles, with Tokorhabditis atripennis being repeatedly isolated from the dung beetle Onthophagus atripennis in Japan. T. atripennis is easy to culture in a laboratory setting, and its host, O. atripennis, is distributed all over Japan. Therefore, T. atripennis is an ideal candidate for ecological and evolutionary studies on viviparity. However, the extent of their distribution and relationship with dung beetles, as well as habitats, remain unclear. In the present study, we conducted field surveys and successfully isolated 27 strains of viviparous nematodes associated with tunneler dung beetles from various regions of Japan, all of which were identified as T. atripennis. T. atripennis exhibited a strong association with Onthophagus dung beetles, especially O. apicetinctus and O. atripennis. And it was predominantly found in specific anatomical locations on the beetle bodies, such as the 'groove between pronotum and elytron' and the 'back of the wings'. Our findings suggest that Onthophagus species are the primary hosts for T. atripennis, and T. atripennis exhibits a close relationship with the living environments of tunneler beetles. This association may play a significant role in the evolution of viviparity in nematodes.

How did Bursaphelenchus nematodes acquire a specific relationship with their beetle vectors, Monochamus?

For insect-borne pathogens, phoretic ability is important not only to spread more widely and efficiently but also to evolve virulence. Bursaphelenchus xylophilus, the causal agent of pine wilt disease, is transmitted by the cerambycid beetle Monochamus alternatus, which is associated with pine tree host. Their specific phoretic ability to appropriate vectors depending on their life cycle is critical for efficient transfer to the correct host and is expected to enhance virulence. We evaluated how B. xylophilus acquired a specific relationship with M. alternatus with a focus on Bursaphelenchus okinawaensis, a close relative of B. xylophilus that has evolved a relationship with a cerambycid beetle vector. Bursaphelenchus okinawaensis has a single dispersal stage (dauer) larva (third-stage dispersal [DIII] larva), whereas B. xylophilus has two distinct dispersal stages (DIII and fourth-stage dispersal [DIV] larva). Also, the dauer formation in B. okinawaensis is not completely dependent on its beetle vector, whereas DIV larvae of B. xylophilus are induced by volatile from the beetle vector. We investigated the induction conditions of dauer larvae in B. okinawaensis and compared to with B. xylophilus. The dauer percentages of B. okinawaensis significantly increased when the nematode population on the plate increased or when we propagated the nematodes with a crude extract of cultured nematodes, which likely contained dauer-inducing pheromones. In addition, dauer formation tended to be enhanced by the crude extract at high temperatures. Furthermore, when we propagated the nematodes with M. alternatus pupae until the beetles eclosed, B. okinawaensis significantly developed into dauer larvae. However, only 1.3% of dauer larvae were successfully transferred to M. alternatus, the rate lower than that of B. xylophilus. DIII and DIV of B. xylophilus were induced by increasing the nematode population and the presence of the beetle vector, respectively. These results suggest that B. okinawaensis has acquired specificity for the cerambycid beetle through dauer formation, which is efficiently induced in the presence of the beetle, and the DIV larval stage, exclusive to the xylophilus group, may be crucial for high transfer ability to the beetle vector.

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.

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.

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.

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.

Possible stochastic sex determination in Bursaphelenchus nematodes.

Sex determination mechanisms evolve surprisingly rapidly, yet little is known in the large nematode phylum other than for Caenorhabditis elegans, which relies on chromosomal XX-XO sex determination and a dosage compensation mechanism. Here we analyze by sex-specific genome sequencing and genetic analysis sex determination in two fungal feeding/plant-parasitic Bursaphelenchus nematodes and find that their sex differentiation is more likely triggered by random, epigenetic regulation than by more well-known mechanisms of chromosomal or environmental sex determination. There is no detectable difference in male and female chromosomes, nor any linkage to sexual phenotype. Moreover, the protein sets of these nematodes lack genes involved in X chromosome dosage counting or compensation. By contrast, our genetic screen for sex differentiation mutants identifies a Bursaphelenchus ortholog of tra-1, the major output of the C. elegans sex determination cascade. Nematode sex determination pathways might have evolved by "bottom-up" accretion from the most downstream regulator, tra-1.

Novel Functional Analysis for Pathogenic Proteins of Bursaphelenchus xylophilus in Pine Seed Embryos Using a Virus Vector.

Pine wilt disease (PWD), which is caused by the pine wood nematode Bursaphelenchus xylophilus, is among the most serious tree diseases worldwide. PWD is thought to be initiated by sequential excessive hypersensitive responses to B. xylophilus. Previous studies have reported candidate pathogenic molecules inducing hypersensitive responses in pine trees susceptible to B. xylophilus. The functions of some of these molecules have been analyzed in model plants using transient overexpression; however, whether they can induce hypersensitive responses in natural host pines remains unclear due to the lack of a suitable functional analysis method. In this study, we established a novel functional analysis method for susceptible black pine (Pinus thunbergii) seed embryos using transient overexpression by the Apple latent spherical virus vector and investigated five secreted proteins of B. xylophilus causing cell death in tobacco to determine whether they induce hypersensitive responses in pine. We found that three of five molecules induced significantly higher expression in pathogenesis-related genes ( p < 0.05), indicating hypersensitive response in pine seed embryos compared with mock and green fluorescence protein controls. This result suggests that tobacco-based screening may detect false positives. This study is the first to analyze the function of pathogenic candidate molecules of B. xylophilus in natural host pines using exogenous gene expression, which is anticipated to be a powerful tool for investigating the PWD mechanism.

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.

Comparative Secretome and Functional Analyses Reveal Glycoside Hydrolase Family 30 and Cysteine Peptidase as Virulence Determinants in the Pinewood Nematode Bursaphelenchus xylophilus.

Pine wilt disease, caused by the pinewood nematode, Bursaphelenchus xylophilus, is one of the world's most serious tree diseases. Although the B. xylophilus whole-genome sequence and comprehensive secretome profile have been determined over the past decade, it remains unclear what molecules are critical in pine wilt disease and govern B. xylophilus virulence in host pine trees. Here, a comparative secretome analysis among four isolates of B. xylophilus with distinct virulence levels was performed to identify virulence determinants. The four candidate virulence determinants of B. xylophilus highly secreted in virulent isolates included lipase (Bx-lip1), glycoside hydrolase family 30 (Bx-GH30), and two C1A family cysteine peptidases (Bx-CAT1 and Bx-CAT2). To validate the quantitative differences in the four potential virulence determinants among virulence groups at the protein level, we used real-time reverse-transcription polymerase chain reaction analysis to investigate these determinants at the transcript level at three time points: pre-inoculation, 3 days after inoculation (dai), and 7 dai into pine seedlings. The transcript levels of Bx-CAT1, Bx-CAT2, and Bx-GH30 were significantly higher in virulent isolates than in avirulent isolates at pre-inoculation and 3 dai. A subsequent leaf-disk assay based on transient overexpression in Nicotiana benthamiana revealed that the GH30 candidate virulent factor caused cell death in the plant. Furthermore, we demonstrated that Bx-CAT2 was involved in nutrient uptake for fungal feeding via soaking-mediated RNA interference. These findings indicate that the secreted proteins Bx-GH30 and Bx-CAT2 contribute to B. xylophilus virulence in host pine trees and may be involved in pine wilt disease.

Thaumatin-like proteins and a cysteine protease inhibitor secreted by the pine wood nematode Bursaphelenchus xylophilus induce cell death in Nicotiana benthamiana.

Pine wilt disease (PWD) is an infectious disease of pines that typically kills affected trees. The causal pathogen of PWD is the pine wood nematode (PWN), Bursaphelenchus xylophilus. Understanding of the disease has advanced in recent years through the use of a highly sensitive proteomics procedure and whole genome sequence analysis; in combination, these approaches have enabled identification of proteins secreted by PWNs. However, the roles of these proteins during the onset of parasitism have not yet been elucidated. In this study, we used a leaf-disk assay based on transient overexpression in Nicotiana benthamiana to allow functional screening of 10 candidate pathogenic proteins secreted by PWNs. These proteins were selected based on previous secretome and RNA-seq analyses. We found that five molecules induced significant cell death in tobacco plants relative to a GFP-only control. Three of these proteins (Bx-TH1, Bx-TH2, and Bx-CPI) may have a role in molecular mimicry and likely make important contributions to inducing hypersensitive responses in host plants.

Telomere-to-Telomere Genome Assembly of Bursaphelenchus okinawaensis Strain SH1.

Bursaphelenchus okinawaensis is a self-fertilizing, hermaphroditic, fungus-feeding nematode used as a laboratory model for the genus Bursaphelenchus, which includes the important pathogen Bursaphelenchus xylophilus Here, we report the nearly complete genome sequence of B. okinawaensis The 70-Mbp assembly contained six scaffolds (>11 Mbp each) with telomere repeats on their ends, indicating complete chromosomes.

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.

Newly Identified Nematodes from Mono Lake Exhibit Extreme Arsenic Resistance.

Extremophiles have much to reveal about the biology of resilience, yet their study is limited by sampling and culturing difficulties [1-3]. The broad success and small size of nematodes make them advantageous for tackling these problems [4-6]. We investigated the arsenic-rich, alkaline, and hypersaline Mono Lake (CA, US) [7-9] for extremophile nematodes. Though Mono Lake has previously been described to contain only two animal species (brine shrimp and alkali flies) in its water and sediments [10], we report the discovery of eight nematode species from the lake, including microbe grazers, parasites, and predators. Thus, nematodes are the dominant animals of Mono Lake in species richness. Phylogenetic analysis suggests that the nematodes originated from multiple colonization events, which is striking, given the young history of extreme conditions at Mono Lake [7, 11]. One species, Auanema sp., is new, culturable, and survives 500 times the human lethal dose of arsenic. Comparisons to two non-extremophile sister species [12] reveal that arsenic resistance is a common feature of the genus and a preadaptive trait that likely allowed Auanema to inhabit Mono Lake. This preadaptation may be partly explained by a variant in the gene dbt-1 shared with some Caenorhabditis elegans natural populations and known to confer arsenic resistance [13]. Our findings expand Mono Lake's ecosystem from two known animal species to ten, and they provide a new system for studying arsenic resistance. The dominance of nematodes in Mono Lake and other extreme environments and our findings of preadaptation to arsenic raise the intriguing possibility that nematodes are widely pre-adapted to be extremophiles.

Cellomics approach for high-throughput functional annotation of Caenorhabditis elegans neural network.

In Caenorhabditis elegans, which has only 302 neurons, relationships between behaviors and neural networks are not easily elucidated. In this study, we proposed a novel cellomics approach enabling high-throughput and comprehensive exploration of the functions of a single neuron or a subset of neurons in a complex neural network on a particular behavior. To realize this, we combined optogenetics and Brainbow technologies. Using these technologies, we established a C. elegans library where opsin is labeled in a randomized pattern. Behavioral analysis on this library under light illumination enabled high-throughput annotation of neurons affecting target behaviors. We applied this approach to the egg-laying behavior of C. elegans and succeeded in high-throughput confirmation that hermaphrodite-specific neurons play an important role in the egg-laying behavior. This cellomics approach will lead to the accumulation of neurophysiological and behavioral data of the C. elegans neural network, which is necessary for constructing neuroanatomically grounded models of behavior.

Morphological, molecular and developmental characterization of the thelastomatid nematode Thelastoma bulhoesi (de Magalhães, 1900) (Oxyuridomorpha: Thelastomatidae) parasite of Periplaneta americana (Linnaeus, 1758) (Blattodea: Blattidae) in Japan.

The American cockroach Periplaneta americana (Linnaeus, 1758) (Blattodea: Blattidae) has been spreading worldwide by commerce and has successfully adjusted to living with humans. There are many reports of thelastomatid parasitic nematode isolated from P. americana in many countries including USA, Canada, India, Argentina, Bulgaria, but not in Japan. We have investigated the parasitic nematodes in P. americana lab strain and field-captured individuals in Japan and found that Thelastoma bulhoesi (de Magalhães, 1900) (Oxyuridomorpha: Thelastomatidae) parasitizes with high infection rates. We described morphological, molecular, and developmental characters of the parasitic nematode because such information was missing despite it has been discovered more than one hundred years ago. We described morphometrics with DIC microscopy and fine structure of male and female adult with SEM observation. We also reveal the embryonic and postembryonic development of this nematode. This is the first report of a thelastomatid nematode isolated from American cockroach in Japan, and the data showed here is also very useful and fundamental for further analysis of the cockroach and parasite relations.

Sex Attraction and Mating in Bursaphelenchus okinawaensis and B. xylophilus.

The fungal feeding, hermaphroditic Bursaphelenchus okinawaensis is a laboratory model to understand the biology of Bursaphelenchus. The extent to which B. okinawaensis can be used to model Bursaphelenchus xylophilus mating was investigated. A chemotaxis assay was conducted to examine whether B. xylophilus and B. okinawaensis produce and respond to volatile sex attractants. Unmated B. xylophilus females were found to attract B. xylophilus males. Similarly, old (sperm depleted) but not young (sperm repleted) B. okinawaensis hermaphrodites attract B. okinawaensis males. Thus, in both species, sperm status corresponds to its ability to attract males. B. xylophilus males also produce a volatile pheromone that attracts both mated and unmated females. A second assay, in which the behavior of males on petri plates in the presence of different females or hermaphrodites of Bursaphelenchus was observed, revealed that B. xylophilus unmated females attract B. okinawaensis males, and B. okinawaensis old hermaphrodites attract B. xylophilus males. These observations suggested that the pheromones of Bursaphelenchus work to some extent across species. Mating behavior through spicule insertion occurs across species, suggesting that postcopulatory mechanisms prevent production of interspecific progeny. The hermaphroditic B. okinawaensis will be a useful model to conduct genetic studies for the understanding of the molecular mechanisms underlying mating behavior in Bursaphelenchus nematodes.

Catalases Induction in High Virulence Pinewood Nematode Bursaphelenchus xylophilus under Hydrogen Peroxide-Induced Stress.

Considered an EPPO A2 quarantine pest, Bursaphelenchus xylophilus is the causal agent of the pine wilt disease and the most devastating plant parasitic nematode attacking coniferous trees in the world. In the early stages of invasion, this nematode has to manage host defence mechanisms, such as strong oxidative stress. Only successful, virulent nematodes are able to tolerate the basal plant defences, and furthermore migrate and proliferate inside of the host tree. In this work, our main objective was to understand to what extent B. xylophilus catalases are involved in their tolerance to oxidative stress and virulence, using as oxidant agent the reactive oxygen species hydrogen peroxide (H2O2). After 24 hours of exposure, high virulence isolates of B. xylophilus could withstand higher H2O2 concentrations in comparison with low virulence B. xylophilus and B. mucronatus, corroborating our observation of Bxy-ctl-1 and Bxy-ctl-2 catalase up-regulation under the same experimental conditions. Both catalases are expressed throughout the nematode intestine. In addition, transgenic strains of Caenorhabditis elegans overexpressing B. xylophilus catalases were constructed and evaluated for survival under similar conditions as previously. Our results suggest that catalases of high virulence B. xylophilus were crucial for nematode survival under prolonged exposure to in vitro oxidative stress, highlighting their adaptive response, which could contribute to their success in host conditions.

Evidence of hermaphroditism and sex ratio distortion in the fungal feeding nematode Bursaphelenchus okinawaensis.

Nematodes have many different reproductive strategies along with their divergent life histories; the ability of hermaphrodite to self- and cross-fertilize is useful for genetic manipulation. Here, we demonstrate the hermaphroditism of the fungal feeding nematode Bursaphelenchus okinawaensis, which was formerly described as a parthenogenetic nematode, and we show its other unique sexual characteristics. To determine that it is hermaphroditic, we performed the following experiments: observation of the pronuclear and chromosome behavior during oogenesis and early embryogenesis; observation of spermatogenesis during the fourth larval stage; investigation of sperm utilization; and investigation of phenotypic segregation after cross-mating using a chemically induced visible mutant. We then investigated the mating preferences and spermatid size difference between males and hermaphrodites. B. okinawaensis males successfully mated only with sperm-depleted old hermaphrodites, and the spermatid sizes of males were almost the same as those of hermaphrodites. Moreover, the sex ratio of cross-fertilized progeny was highly skewed toward hermaphrodites. B. okinawaensis is phylogenetically distant from established model nematodes such as C. elegans and is more closely related to some economically relevant parasitic nematodes. This newly discovered hermaphroditic nematode has great potential for evolutionary and parasitological research.