Several chemical compounds, e.g., linoleic and oleic acids, have been reported to be attractive to the pine wood nematode, Bursaphelenchus xylophilus, in previous studies. To examine the effects of fatty acids on the number of nematodes carried by Monochamus alternatus and Acanthocinus orientalis, unsaturated fatty acids (linoleic, oleic and hexadecenoic acids) and saturated fatty acids (palmitic and stearic acids) around the pupal chambers of individual beetles of the two species were analysed using gas chromatography-mass spectrometry. The number of nematodes carried by individual M. alternatus was large, averaging 1628 (range 2 to 27 200), whilst A. orientalis carried no nematodes. There was no difference in the concentration of linoleic acid around the pupal chambers of M. alternatus and A. orientalis, and the concentration of oleic acid around Monochamus chambers was less than that around Acanthocinus chambers. There were no clear significant relationships between the concentration of linoleic or oleic acids around Monochamus pupal chambers and the numbers of nematodes carried by individual Monochamus beetles emerging from the chambers. It was concluded that the concentration of unsaturated fatty acids around pupal chambers in wilt-killed pine trees could not explain the differences in the numbers of nematodes carried by M. alternatus and by A. orientalis, and the differences in the numbers of nematodes carried by individual Monochamus beetles.
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.
We examined the transfer of Bursaphelenchus okinawaensis associated with Monochamus maruokai into M. alternatus and Psacothea hilaris using a simple nematode loading method in order: i) to clarify the effects of cerambycid beetles on the formation of dauer juveniles of B. okinawaensis; and ii) to clarify whether dauer juveniles transfer to cerambycid beetles other than M. maruokai. Dauer juveniles appeared at a high percentage without M. alternatus or P. hilaris and these beetles did not have any positive effects on their formation. Dauer juveniles transferred to the adults of M. alternatus and P. hilaris. We concluded that dauer juveniles of B. okinawaensis form readily without its vector beetles and may be able to transfer to many kinds of cerambycid beetles, although the numbers carried by a beetle are small.
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.
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.
Diplogasteroides nix n. sp. is described based on its typological characteristics, molecular profile and mating experiments. Diplogasteroides nix n. sp. is characterised by its tube-like stoma with three small dorsal teeth and two subventral ridges, spicule clearly ventrally bent at one-third from the anterior end, gubernaculum with a somewhat squared anterior end and sharply pointed distal end in a lateral view and nine pairs of genital papillae with an arrangement of ⟨v1, (v2, v3d)/v4, ad, ph, (v5, v6, v7), pd⟩, with the very small v5 and v6 protruding from socket-like bases, and v6 has a tripartite tip on males and well developed receptaculum seminis in females. In addition to the general morphological characters, the new species has several small secretory pore-like openings connected to secretory cells just beneath the surface cuticle (tentatively termed as ‘lateral glands’) in both males and females, and two pairs of button-like surface structures with clear internal connections in females. The new species is typologically and molecularly very similar to its tentatively assigned closest relative, D. andrassyi. These two species are morphologically almost identical, forming a cryptic species complex with some very minor morphological differences, e.g., the morphology of gubernaculum somewhat roundish squared vs clearly rounded anterior edge. Molecularly, these two species have identical small subunit sequences and have only 5 bp difference in the D2-D3 extension segments of the large subunit. However, the new species can be distinguished from D. andrassyi by mating incompatibility, yielding only F1 progenies in both male × female and female × male combinations. Biologically, these two species and another close relative were isolated from different carrier Monochamus longhorn beetles, i.e., D. nix n. sp., D. andrassyi and D. asiaticus have been found from M. urussovii, M. grandis and M. alternatus, respectively. However, because these beetle species share the distribution range and host tree species, further collection and identification are necessary to understand their host (carrier) interactions and specificity.
Bursaphelenchus clavicauda n. sp. is described and figured. The new species was isolated from Cryphalus sp., a species of bark beetle emerging from a dead log of Castanopsis cuspidata, and reared on Botrytis cinerea. Type specimens were collected from a 2- and 4-week-old culture on Botrytis cinerea. The new species has a body length of ca 665 μm (male) and 770 μm (female), medium (ca 30-50 for males and ca 30-45 for females) a values, b values of ca 9-13 (male) and 10-13 (female), c values of ca 20-28 (male) and 20-27 (female), c′ values of ca 2 (male) and ca 3-5 (female), ca 9-13 μm long stylet, three lines in the lateral field, relatively small (ca 14 μm) and strongly arcuate spicule with recurved and pointed condylus, seven (1 + 2 + 2 + 2) male caudal papillae and club-like female tail with rounded terminus and with or without various shapes of mucro. The new species is close to B. eremus, B. uncispicularis, B. yongensis and several other species possessing a large, recurved and pointed spicular condylus and is distinguished from these species mainly by male spicule, male caudal papillae and female tail morphologies, number of lateral lines and several morphometric values of females and males. Cultured nematodes were used for sequencing the partial (ca 1.6 kbps) small subunit, ITS regions and large subunit of ribosomal DNA for comparisons with those deposited at GenBank. The DNA sequences of the B. clavicauda n. sp. were close to those of B. eggersi, B. eremus, B. tusciae and B. yongensis.
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.