Noritoshi Maehara

Bursaphelenchus firmae n. sp. (Nematoda: Aphelenchoididae), isolated from Monochamus grandis Waterhouse that emerged from dead firs, Abies firma Sieb. et Zucc.

Bursaphelenchus firmae n. sp. is described. This new species was isolated during a field survey of longhorn beetle-associated nematodes. The fourth-stage dispersal (dauer) juveniles of the new species were recovered from dissected bodies (tracheal system) of Monochamus grandis, which emerged from dead logs of Japanese fir, Abies firma, collected from Hachioji, Tokyo, Japan. The new species is mid-sized for the genus, with females 603-828 μm and males 530-698 μm long. Four lateral lines occur on the body surface and seven genital papillae are found in males (P1 ventral single papilla and P2-P4 pairs). A long and arcuate spicule forms a trapezium in lateral view and a rather large, sub-squared, bursal flap and a vulva with conspicuous vulval flap are present. The female tail is smoothly tapered and possesses a conspicuous and blunt mucro. Based upon its diagnostic morphological characters, the new species belongs to the B. xylophilus group of the genus, and is closely related to B. fraudulentus, B. mucronatus, B. doui, B. macromucronatus and B. populi. It is distinguished from these five species by the morphology of the male bursal flap and the female mucro and several morphometric values. Molecular phylogenetic analyses inferred from D2/D3 LSU suggest that the new species is close to B. mucronatus and B. xylophilus, i.e., these three species form a well supported monophyletic clade within the genus. Although the new species has a weak pathogenicity to pine trees, it does not seem to be a severe risk to native pine forests.

Description of Bursaphelenchus kiyoharai n. sp. (Tylenchina: Aphelenchoididae) with remarks on the taxonomic framework of the Parasitaphelenchinae Rühm, 1956 and Aphelenchoidinae Fuchs, 1937

A Bursaphelenchus species was isolated from an ambrosia beetle, Xyleborus seriatus, during a biodiversity survey of entomophilic nematodes in a conserved forest in the Shirakami mountain range, Aomori, Japan. The nematode has several interesting morphological characters that do not fit the morphological definition of Bursaphelenchus, e.g., males have a tail spike instead of a bursal flap and lack the P1 ventral single papilla, and females have a very long and tapering tail (filiform). The molecular phylogenetic status of the new species inferred by the near-full-length small subunit (SSU: 18S) and the D2/D3 expansion segments of the large subunit (LSU: 28S) suggests, however, that the nematode is included in the B. fungivorus group, and is most closely related to B. thailandae and B. willibaldi. Despite these unique morphological characters, its spicule morphology, which is almost identical to that of B. gonzalezi, B. thailandae and B. willibaldi, and vulval structure, i.e., lacking a vulval flap, are in accordance with the molecular phylogenetic status of the nematode. The new species, which shows evidence of secondary loss of the bursal flap and P1 papilla, is described herein as B. kiyoharai n. sp. The typological descriptions of the genus and Parasitaphelenchinae were reconsidered and the taxonomic framework of the Aphelenchoidinae and Parasitaphelenchinae revised. Bursaphelenchus asiaticus n. comb. is proposed for Ruehmaphelenchus asiaticus.

Effect of fungus inoculation on the number of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) carried by Monochamus alternatus (Coleoptera: Cerambycidae)

Blue-stain fungi were mainly isolated from the wood of pine wilt-killed Pinus densiflora. Intense blue-stain on the pupal chamber walls of the Japanese pine sawyer (Monochamus alternatus) increased the number of pinewood nematodes (Bursaphelenchus xylophilus) aggregating around such chambers and the number carried by the beetles that emerged from the chambers. There were differences in the numbers of nematodes carried by beetles among individual trees from which the beetles emerged. The beetles emerging from dry chambers carried relatively few nematodes.

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

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

Bursaphelenchus doui Braasch, Gu, Burgermeister & Zhang, 2005 (Aphelenchida: Parasitaphelenchidae), an associate of Monochamus subfasciatus Bates (Coleoptera: Cerambycidae) and Pinus densiflora Sieb. & Zucc.

Summary – Bursaphelenchus doui was isolated from a dead Japanese red pine, Pinus densiflora, in Shizuoka, and from the tracheal system of a species of longhorn beetle, Monochamus subfasciatus, collected at Tama Forest Science Garden of Forestry and Forest Products Research Institute, Tokyo, Japan. The Japanese populations of B. doui were compared with the original description of material obtained from coniferous packaging materials imported from Taiwan and Korea to continental China. Additional characters from the Japanese population include a constricted female mucron with a step-like appearance and several morphometric values. The molecular profiles of the Japanese B. doui populations were determined by DNA sequencing and ITS-RFLP profiles and were compared with those of the Taiwanese and Chinese populations of B. doui and other species in the genus. The phylogenetic analysis of the small subunit and large subunit ribosomal DNA indicated that B. doui is clearly included in the xylophilus-group of the genus Bursaphelenchus and may be close to B. conicaudatus and B. luxuriosae. The potential risk of B. doui for pine species is considered to be relatively low because B. doui did not display any pathogenicity to Japanese black pine in an inoculation test.

First Report of Parthenogenesis in the Genus Bursaphelenchus Fuchs, 1937: A Description of Bursaphelenchus okinawaensis sp. nov. Isolated from Monochamus maruokai (Coleoptera: Cerambycidae)

Abstract 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.

AN ENTOMOPARASITIC ADULT FORM IN BURSAPHELENCHUS DOUI (NEMATODA: TYLENCHOMORPHA) ASSOCIATED WITH ACALOLEPTA FRAUDATRIX

Abstract:  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.

Presence of the cerambycid beetles Psacothea hilaris and Monochamus alternatus affecting the life cycle strategy of Bursaphelenchus xylophilus

Although fourth-stage dispersal juveniles (JIV) of Bursaphelenchus xylophilus, the pinewood nematode (PWN), developed in the presence of both Monochamus alternatus and Psacothea hilaris, the numbers and the percentage of JIV were far higher in the presence of the former than of the latter. JIV first appeared 7 days after pupation of M. alternatus and the number increased from the day of beetle eclosion to the third day thereafter, then remained stable. We conclude that the presence of specific vectors affects both the life history of the PWN and the numbers of nematodes carried by vectors emerging from killed pine trees.

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

Abstract 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).

Factors affecting the number of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) carried by several species of beetles

Numerous pinewood nematodes (Bursaphelenchus xylophilus) aggregated around pupal chambers of Monochamus alternatus in pine wilt-killed trees and the beetle carried many nematodes. However, few nematodes aggregated around pupal chambers of the other beetles, i.e., Acanthocinus griseus, Pissodes obscurus and Trogossita japonica, and these beetles carried few or no nematodes. The higher percentage of M. alternatus pupal chambers with intense blue-stain of wood would cause more nematodes to aggregate around such chambers, as blue-stain fungi are suitable for B. xylophilus propagation.