Copulation Duration and Sperm Precedence with Reference to Larval Diapause Induction in Monochamus alternatus Hope (Coleoptera: Cerambycidae).

Adults of the pine sawyer Monochamus alternatus are the primary vector of Bursaphelenchus xylophilus, the causative agent of pine wilt disease. A sawyer subspecies in Taiwan (abbreviated 'T') has two generations a year (bivoltinism) due to facultative diapause, whereas another subspecies in Japan (abbreviated 'J') has a one- or two-year life cycle due to obligate diapause. T, with two infection periods a year, will cause more severe disease epidemics than J if it is introduced into Japan. Inter-subspecies hybridization may inhibit the expression of bivoltinism because many F1 hybrids induce diapause. To predict the effects of introducing T into Japan, the present study investigated copulation duration and late-male sperm precedence to fertilize eggs. The results indicated that a single copulation for more than 65 s supplied sufficient sperm to fertilize a lifetime production of eggs. The incidence of larval diapause was 0.15 for the offspring of T females that mated with a T male and increased to 0.292-0.333 after remating with a J male, while the incidence of larval diapause was 0.900-1.000 for hybrids from T females mated with a J male. Consequently, the estimated proportion of second-male sperm used by T females was 0.185-0.217. The effects of introducing T populations into Japan on the severity of disease epidemics were also discussed.

Fungal garden making inside bamboos by a non-social fungus-growing beetle.

In fungus-growing mutualism, it is indispensable for host animals to establish gardens of the symbiotic fungus as rapidly as possible. How to establish fungal gardens has been well-documented in social fungus-farming insects, whereas poorly documented in non-social fungus-farming insects. Here we report that the non-social, fungus-growing lizard beetle Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae) transmits the symbiotic yeast Wickerhamomyces anomalus from the ovipositor-associated mycangium into bamboo internode cavities and disperses the yeast in the cavities to make gardens. Microbial isolation and cryo-scanning electron microscopy observation revealed that W. anomalus was constantly located on the posterior ends of eggs, where larvae came out, and on the inner openings of oviposition holes. Direct observation of oviposition behavior inside internodes revealed that the distal parts of ovipositors showed a peristaltic movement when they were in contact with the posterior ends of eggs. Rearing experiments showed that W. anomalus was spread much more rapidly and widely on culture media and internodes in the presence of the larvae than in the absence. These results suggest that the ovipositors play a critical role in vertical transmission of W. anomalus and that the larvae contribute actively to the garden establishment, providing a novel case of fungal garden founding in non-social insect-fungus mutualism.

Fungal farming in a non-social beetle.

Culturing of microbes for food production, called cultivation mutualism, has been well-documented from eusocial and subsocial insects such as ants, termites and ambrosia beetles, but poorly described from solitary, non-social insects. Here we report a fungal farming in a non-social lizard beetle Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae), which entails development of a special female structure for fungal storage/inoculation, so-called mycangium, and also obligate dependence of the insect on the fungal associate. Adult females of D. bucculenta bore a hole on a recently-dead bamboo culm with their specialized mandibles, lay an egg into the internode cavity, and plug the hole with bamboo fibres. We found that the inner wall of the bamboo internode harboring a larva is always covered with a white fungal layer. A specific Saccharomycetes yeast, Wickerhamomyces anomalus ( = Pichia anomala), was consistently isolated from the inner wall of the bamboo internodes and also from the body surface of the larvae. Histological examination of the ovipositor of adult females revealed an exoskeletal pocket on the eighth abdominal segment. The putative mycangium contained yeast cells, and W. anomalus was repeatedly detected from the symbiotic organ. When first instar larvae were placed on culture media inoculated with W. anomalus, they grew and developed normally to adulthood. By contrast, first instar larvae placed on either sterile culture media or autoclaved strips of bamboo inner wall exhibited arrested growth at the second instar, and addition of W. anomalus to the media resumed growth and development of the larvae. These results strongly suggest a mutualistic nature of the D. bucculenta-W. anomalus association with morphological specialization and physiological dependence. Based on these results, we compare the fungal farming of D. bucculenta with those of social and subsocial insects, and discuss ecological factors relevant to the evolution of fungal farming in a non-social insect.

Exaggerated asymmetric head morphology of female Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae) and ovipositional preference for bamboo internodes.

The lizard beetle Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae) female has evident asymmetric head morphology. The females excavate small holes in host bamboo internodes for the deposition of eggs. To understand the asymmetry and allometry of male and female adults and larvae of D. bucculenta, mandibular length, genal and head width, and elytral length were measured, and the oviposition preference for different-sized internodes of the bamboo Pleioblastus simonii and the relationship between internode size and emerging adult size were examined. Larval mandibles exhibited no clear asymmetry pattern, and genae showed fluctuating asymmetry in length. Adult male mandibles showed left-directional asymmetry, but genae showed fluctuating asymmetry. Adult female mandibles and genae exhibited marked left-directional asymmetry. The degree of asymmetry of mandibles and genae remained constant regardless of body size. Large females tended to choose large-diameter internodes of P. simonii and to lay eggs successfully, whereas small females tended to choose intermediate-diameter internodes, but to fail in oviposition, suggesting that small females pay a high cost on oviposition. There was a positive correlation between internode size and emerging adult size. Marked directional asymmetry of female mandibles and genae are discussed in relation to greater frequency of cutting bamboo fibers compared with adult males, and the traits of bamboo internode.

Discovery of mycangia and the associated xylose-fermenting yeasts in stag beetles (Coleoptera: Lucanidae).

Most wood-feeding insects need an association with microbes to utilize wood as food, and some have special organs to store and convey the microbes. We report here the discovery of the microbe-storage organ (mycangium) in stag beetles (Coleoptera: Lucanidae), which develop in decayed wood. The mycangium, which was discovered in the abdomen, is present in all adult females of 22 lucanid species examined in this study, but absent in adult males. By contrast, adult insects of both sexes of selected Passalidae, Geotrupidae, and Scarabaeidae, which are related to Lucanidae, lacked mycangia similar to those of the lucanid species. Yeast-like microbes were isolated from the mycangium of five lucanid species. DNA sequence analyses indicate that the microbes are closely related to the xylose-fermenting yeasts Pichia stipitis, Pichia segobiensis, or Pichia sp. known from the gut of a passalid species.

Are stag beetles fungivorous?

Stag beetle larvae generally feed on decaying wood; however, it was unknown whether they can use wood-rotting fungi alone as food. Here, to clarify this, newly hatched larvae of Dorcus rectus (Motschulsky) (Coleoptera: Lucanidae) were reared for 14 days on artificial diets containing a fixed amount of freeze-dried mycelia of the following fungi: Bjerkandera adusta, Trametes versicolor, Pleurotus ostreatus, and Fomitopsis pinicola. The mean incremental gain in larval body mass was greatest on diets containing B. adusta, followed by T. versicolor, P. ostreatus, and F. pinicola. The growth rate of body mass correlated positively with mycelial nitrogen content of the different fungi. It also correlated positively with the mycelial content of B. adusta in the diet. Addition of antibiotics to diets with mycelia nearly halved larval growth, indicating that larvae were able to use fungal mycelia as food without the assistance of associated microbes although the microbes positively affected larval growth. Four newly hatched larvae reared on artificial diets containing B. adusta mycelia developed to the second instar in 21-34 days; and one developed to the third (=final) instar. This study provides evidence that fungi may constitute the bulk of the diet of D. rectus larvae.

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.

The nematocidal activity and the structure-activity relationships of stilbenes.

The pinewood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, is the causative agent of the pine wilt disease which has been devastating forests of Pinus densiflora Sieb.et Zucc. and P. thunbergii Parl. in Japan. To prevent the pine wilt disease, the development of nematocidal compound is required. Twenty-one synthesized stilbenes (1)-(20), (23), salicylic acid (21), and phenylsalicilate (22) were examined for their nematocidal activity against an isolate of B. xylophilus (T-4). Among the tested compounds, two fluorinated stilbenes (15) and (13), were found to be most potent compounds against T-4, demonstrating 99% and 98% lethality at 10 ppm concentration. The LD50 values of compounds 15 and 13 were 3 ppm, respectively.

A new method for loading Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) on adult Monochamus alternatus (Coleoptera: Cerambycidae).

A new method was developed for loading the pinewood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, on the beetle Monochamus alternatus Hope. Postdiapause beetle larvae were sterilized with 70 and 99.9% aqueous ethanol and placed singly in flasks where B. xylophilus reproduced on the fungus Ophiostoma minus (Hedgcock) H. et P. Sydow that had been grown on autoclaved barley grain and Pinus densifiora Sieb. et Zucc. wood chips. The fungus produced a large nematode population that developed to a high proportion of third-stage dispersal juveniles that molted to the fourth-stage dispersal juveniles. The survival rate was 80%, and the mean nematode load was 10,096. It took a mean of 5 wk to obtain the nematode-infested beetles after the initiation of nematode rearing.

Inhibitory Effect of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on B. xylophilus Boarding Adult Monochamus alternatus (Coleoptera: Cerambycidae).

Inhibitory effects of Bursaphelenchus mucronatus on the number of B. xylophilus carried by an adult Monochamus alternatus were investigated using artificial pupal chambers. When pupal chambers were infested with either B. xylophilus or B. mucronatus, the load of B. xylophilus onto the beetle was greater (P < 0.001) than that of B. mucronatus. However, within the pupal chamber there was no difference in the abundance of the third-stage dispersal juveniles, which would molt to the fourth-stage dispersal juveniles to board beetles. The nematode load on beetles that emerged from pupal chambers infested with both Bursaphelenchus species was smaller (P = 0.015) than that of beetles with B. xylophilus alone but greater (P < 0.001) than that of beetles with B. mucronatus alone, suggesting an inhibitory effect of B. mucronatus. As a result of this study, the rate of inhibition of B. mucronatus on molting of third-stage dispersal juveniles of B. xylophilus to fourth-stage dispersal juveniles was 0.65, which resulted in great inhibition on boarding beetles at a rate of 0.7.

Horizontal Transmission of Bursaphelenchus xylophilus between Sexes of Monochamus alternatus.

Four experiments were conducted using nematode-infested and nematode-free adults of the cerambycid beetle, Monochamus alternatus, to determine horizontal transmission pathways of Bursaphelenchus xylophilus. When nematode-infested beetles of one sex and nematode-free beetles of the opposite sex were paired in containers for 48 or 72 hours, the number of nematodes carried by nematode-free beetles tended to increase with increased number of nematodes carried by nematode-infested beetles. The nematodes acquired by "nematode-free" beetles could be transmitted to pine. A female beetle that received 13 nematodes from a male transmitted one nematode to a Pinus densiflora bolt via an oviposition wound. When the nematode-infested and nematode-free beetles were observed continuously, it was observed that the number of nematodes carried by nematode-free beetles at the end of the first sexual mounting increased as the number of nematodes carried by nematode-infested beetles just before mounting increased. The number of nematodes transferred to nematode-free beetles was positively related to duration time of mounting. There was no difference in transmission efficacy between male-to-female transmission and female-to-male transmission. The horizontal transmission pathways are discussed relative to the persistence of B. xylophilus in resistant pine forests and the control of pine wilt disease.

Newly Discovered Transmission Pathway of Bursaphelenchus xylophilus from Males of the Beetle Monochamus alternatus to Pinus densiflora Trees via Oviposition Wounds.

The transmission of Bursaphelenchus xylophilus from Monochamus alternatus males to Pinus densiflora trees via oviposition wounds has been determined. Nematode-infested males, with mandibles fixed experimentally to prevent feeding, were placed for 48 hours with pine bolts containing oviposition wounds that had been made by nematode-free females. After removal of the nematode-infested males, the pine bolts were held for 1 month and then examined for the presence of nematodes. Reproducing nematode populations were recovered from pine bolts that were exposed to male beetles carrying a high number of nematodes. No reproducing nematode population could be recovered from pine bolts exposed to beetles with a small number of nematodes. Nematode reproduction in the pine bolts was not related to the number of oviposition wounds per bolt. Fourth-stage dispersal B. xylophilus juveniles, collected from beetle body surfaces, were inoculated on pine bolt bark 0, 5, 10, and 15 cm away from a single artificial, small hole. These dauer juveniles successfully entered some bolts. The probability of successful nematode reproduction decreased with increased distance between inoculation point and artificial hole. The results indicated that B. xylophilus can move a significant distance to oviposition wounds along the bark surface and enter a tree via the wounds. The new transmission pathway is considered important for the nematode to persist in pine forests such as in North America where pine wilt disease does not occur.