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.

Cytoplasmic incompatibility in the semivoltine longicorn beetle Acalolepta fraudatrix (Coleoptera: Cerambycidae) double infected with Wolbachia.

Wolbachia are obligatory endosymbiotic α-proteobacteria found in many arthropods. They are maternally inherited, and can induce reproductive alterations in the hosts. Despite considerable recent progress in studies on the associations between Wolbachia and various taxonomic groups of insects, none of the researches have revealed the effects of Wolbachia on longicorn beetles as the host insect. Acalolepta fraudatrix is a forest longicorn beetle that is distributed in East Asia. In this study, the relationship between Wolbachia and A. fraudatrix was investigated. Out of two populations of A. fraudatrix screened for Wolbachia using the genes ftsZ, wsp, and 16S rRNA, only one of the populations showed detection of all three genes indicating the presence of Wolbachia. Electron microscopy and fluorescent in situ hybridization also confirmed that the A. fraudatrix population was infected with Wolbachia. Sequencing the wsp genes derived from single insects revealed that two strains of Wolbachia coexisted in the insects based on the detection of two different sequences of the wsp gene. We designated these strains as wFra1 and wFra2. The bacterial titers of wFra1 were nearly 2-fold and 3-fold higher than wFra2 in the testes and ovaries, respectively. The two strains of Wolbachia in the insects were completely eliminated by rearing the insects on artificial diets containing 1% concentration of tetracycline for 1 generation. Reciprocal crosses between Wolbachia-infected and Wolbachia-uninfected A. fraudatrix demonstrated that only eggs produced by the crosses between Wolbachia-infected males and Wolbachia-uninfected females did not hatch, indicating that Wolbachia infecting A. fraudatrix causes cytoplasmic incompatibility in the host insect. This is the first report showing the effect of Wolbachia on reproductive function in a longicorn beetle, A. fraudatrix.

Potential vector switching in the evolution of Bursaphelenchus xylophilus group nematodes (Nematoda: Aphelenchoididae).

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.

Diplogasteroides asiaticus n. sp. is Associated with Monochamus alternatus in Japan.

Diplogasteroides asiaticus n. sp. is described and illustrated, and its molecular profile and phylogenetic status within the family Diplogastridae are inferred. Morphologically, the new species is characterized by its stomatal structure, a tube-like stoma with three small, rod-like dorsal teeth and two subventral ridges; a spicule clearly ventrally bent at 1/3 from the anterior end; a gubernaculum with a rounded anterior end and sharply pointed distal end in lateral view; nine pairs of genital papillae with an arrangement of ; a short tail spike in males; and a well-developed receptaculum seminis, i.e., the antiparallel blind sacs of the uteri beyond the vulva region and elongated conical tail in females. This new species is morphologically similar to D. haslacheri, but it can be distinguished by the morphology of the somewhat shorter tail in females. D. asiaticus n. sp. shares high sequence conservation with D. andrassyi as there is only one base pair difference in the nearly full-length 18S rDNA and seven base pair differences in the D2-D3 expansion segments of the 28S rDNA. Despite this sequence conservation, the species status of D. asiaticus n. sp. was confirmed using the biological species concept, as D. asiaticus n. sp. and D. andrassyi failed to generate viable F2 progeny in hybridization tests.

An entomoparasitic adult form in Bursaphelenchus doui (Nematoda: Tylenchomorpha) associated with Acalolepta fraudatrix.

The nematode family Aphelenchoididae (Rhabditida: Tylenchomorpha) includes species with various feeding habitats. Bursaphelenchus, a member of the family, has for a long time been considered as a home for plant parasitic or mycophagous species (or both). However, recent intensive biological studies on the family revealed that the genus contains several insect parasitic species. Dauer juveniles of Bursaphelenchus doui were isolated from Acalolepta fraudatrix during a field study of longhorn beetle-Bursaphelenchus nematode associations. Two different insect-associated forms, an "entomoparasitic adult form" and a regular dauer juvenile, were isolated from a single individual beetle in a subsequent laboratory investigation of the B. doui-A. fraudatrix relationship. Thus these 2 distinct, insect-associated forms were confirmed to occur simultaneously. The entomoparasitic form is morphologically similar to that of Bursaphelenchus luxuriosae, with a dome-shaped head and vacuole-like spots assumed to be an internal structure of sensory organ, a stylet, a metacorpus (median bulb), and a moderately-developed and seemingly fully functional reproductive system. It is distinguishable from B. luxuriosae based on male spicule morphology and female tail morphology. A degenerate ingestive-digestive system distinguishes the entomoparasitic form from the propagative form and, unlike dauer juveniles, it has a moderately-developed reproductive system. The presence of this characteristic parasitic adult form is known only in these 2 Bursaphelenchus species. However, these 2 species did not form a clear monophyletic clade within the Bursaphelenchus xylophilus group and, thus, this characteristic parasitic form may occur independently in each species.

The first report of a putative "entomoparasitic adult form" in Bursaphelenchus.

A putative "entomoparasitic adult form" of Bursaphelenchus luxuriosae was found in the tracheal system and body cavity of its cerambycid vector beetle, Acalolepta luxuriosa. Morphologically, this form is intermediate between the usual mycophagous adult and the phoretic dauer juvenile, i.e., it shares the primary and secondary reproductive features with the mycophagous phase but shares a degenerate digestive tract with JIV dauer juveniles. In addition, the "parasitic form" has specialized characters relative to the mycophagous phase analogous to dicyclic Hexatylina, e.g., vacuole-like dots (assumed to be sensory organs) at the anterior end, a very long vulval flap, conical female tail, and elongated male spicules. The presence of insect-parasitic juveniles has been reported in several Bursaphelenchus species, but this is the first confirmed report of a putative "entomoparasitic adult form" in the genus. Thus, the "parasitic adult form" of B. luxuriosae is hypothesized to be an autapomorphic character of this species in the genus. The physiological impact of the parasitism on the host beetle is assumed to be weak because no clear symptoms were observed in the infested beetles. Also, no nematode eggs or propagative juveniles were observed in the beetle host, suggesting that more research is needed to confirm the nature of the association (parasitic vs. endophoretic).

First report of parthenogenesis in the genus Bursaphelenchus Fuchs, 1937: a description of Bursaphelenchus okinawaensis sp. nov. isolated from Monochamus maruokai (Coleoptera: Cerambycidae).

An evolutionarily interesting nematode, Bursaphelenchus okinawaensis sp. nov., is described and illustrated. The new species has several characteristic morphological traits, i.e., four lateral lines in both sexes, lack of a vulval flap in females, and a triangular spicule shape and stout P4 caudal papillae in males, and characteristic biological traits, including phoretic association with Monochamus maruokai, a species of longhorn beetle, parthenogenetic reproduction, and a high frequency of dauer production. Bursaphelenchus okinawaensis sp. nov. shares several important traits with various phylogenetic groups within the genus. The new species shares its spicule shape with B. hellenicus and B. hofmanni. It shares four lateral lines, P4 caudal papillar structure (size and position), and female vulval shape with the 'hunti' group, although it was molecularly inferred to be phylogenetically closer to the 'xylophilus' group and B. africanus. The autapomorphic traits of B. okinawaensis sp. nov. are parthenogenetic reproduction and high frequency of dauer production. All other nominal Bursaphelenchus nematodes have bisexual reproduction and tightly synchronized dauer production. The unique morphological and biological traits of B. okinawaensis sp. nov. suggest genetic flexibility within the genus. The importance of the morphology and arrangement of the caudal papillae is discussed relative to the phylogeny of the genus.

Maturation feeding and transmission of Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) by Monochamus alternatus (Coleoptera: Cerambycidae) inoculated with Beauveria bassiana (Deuteromycotina: Hyphomycetes).

We examined the amount of maturation feeding and transmission of pinewood nematodes, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Nematoda: Parasitaphelenchidae), to healthy pine (Pinus spp.) trees by pine sawyer Monochamus alternatus Hope (Coleoptera: Cerambycidae) adults infected with Beauveria bassiana (Balsamo) Vuill. (Deuteromycotina: Hyphomycetes). Inoculated beetles fed less than noninoculated beetles, probably because feeding by inoculated beetles began to decrease at about 4 d postinoculation and inoculated beetles ceased to feed for several days before their death. In inoculated beetles carrying >1,000 nematodes, some beetles died before nematode departure. The remaining heavily nematode-infested beetles lived until the beginning of nematode departure, but they had stopped feeding, preventing the nematodes from entering pine twigs. We suggest that microbial control of pine sawyer adults by B. bassiana may be effective in preventing transmission of pine wilt disease to healthy pine trees.