Hisashi Kajimura

Dynamics of the fungal symbionts in the gallery system and the mycangia of the ambrosia beetle, Xylosandrus mutilatus (Blandford) (Coleoptera: Scolytidae) in relation to its life history

The dynamics of the fungal symbionts in the gallery system and the mycangia of the ambrosia beetle,Xylosandrus mutilatus, were studied in relation to its life history using both isolation experiments and scanning electron microscopy (SEM). In the galleries,Ambrosiella sp. was predominant during the larval stages but its relative dominance gradually decreased during the development of the larvae. In contrast, yeasts (mainlyCandida sp.) andPaecilomyces sp. dominated continuously in the galleries after eclosion.Ambrosiella sp. was consistently stored in the mycangia in all adult stages, except in the teneral and overwintering adults when the other fungi were dominant. No fungal spores occurred in the mycangia of the adult beetles reared under aseptic conditions from the pupal stage, while onlyAmbrosiella sp. was stored in those reared from the teneral-adult stage. These results suggest that: (i) Xmutilatus is associated with at least three fungal species, among whichAmbrosiella sp. is the most essential food resource for development of the broods; (ii) immediately after eclosion, new female adults may take at least four associated fungal species, with no or incomplete selection, into their mycangia from the walls of the cradles; and (iii) conditions may well be produced in the mycangia of both matured and dispersing beetles whereby only the spores ofAmbrosiella sp. can proliferate.

Guild structures of seed insects in relation to acorn development in two oak species

We investigated patterns of acorn growth in Quercus variabilis Blume and Quercus serrata Thunb., seasonal trends in emergence of seed insects found in the acorns, oviposition periods of the insects and falling periods of insect-infested acorns. In Q. variabilis, two insect guilds were associated with acorn development: (i) the immature acorn feeding (IAF) guild [Curculionidae sp., Poecilips cardamomi (Schaufuss), and Characoma ruficirra (Hampson)]; and (ii) the mature acorn feeding (MAF) guild [Curculio robustus (Roelofs), Curculio sikkimensis (Heller), and Cydia glandicolana (Danilevsky)]. In Q. serrata, there were three guilds: (i) the pistillate flower feeding (PFF) guild (cynipid wasp); (ii) the IAF guild [sap absorption by Mechoris ursulus (Roelofs)]; and (iii) the MAF guild (M. ursulus, C. sikkimensis, Cydia danilevskyi (Kuznetzov), C. glandicolana and Autostichidae sp.). The succession of guilds during acorn development may be a consequence of the use by different species of the limited food resource. The lack of a PFF guild in Q. variabilis that was found in our field site would have a positive effect on IAF guilds in utilizing the acorns.

Effects of Insect Predation on Hypocotyl Survival and Germination Success of Mature Quercus variabilis Acorns

The rates of hypocotyl and radicle survival and of germination success were investigated in mature acorns ofQuercus variabilis Blume in relation to endosperm loss due to seed insects. The acorns were damaged by curculio weevils (Coleoptera: Curculionidae) and moths, including tortricid moths (Lepidoptera: Tortricidae); the former were more abundant than the latter. Acorns damaged by curculio weevils showed a significantly lower germination rate when there was a large endosperm loss than the rate for sound acorns. The survival rate of the hypocotyl and radicle also decreased as endosperm loss increased. These results suggest that acorn germination is directly inhibited by damage to the hypocotyl and radicle, which depends on the amount of endosperm eaten by the weevils. However, the germination rate of acorns with hypocotyl and radicle was consistently high, irrespective of the degree of endosperm loss, which suggests that weevil-damaged acorns probably germinate, provided the hypocotyl and radicle survive until the cessation of damage.

Superinfection of five Wolbachia in the alnus ambrosia beetle, Xylosandrus germanus (Blandford) (Coleoptera: Curuculionidae)

Wolbachia bacteria are among the most common endosymbionts in insects. In Wolbachia research, the Wolbachia surface protein (wsp) gene has been used as a phylogenetic tool, but relationships inferred by single-locus analysis can be unreliable because of the extensive genome recombination among Wolbachia strains. Therefore, a multilocus sequence typing (MLST) method for Wolbachia, which relies upon a set of five conserved genes, is recommended. In this study, we examined whether the alnus ambrosia beetle, Xylosandrus germanus (Blandford), is infected with Wolbachia using wsp and MLST genes. Wolbachia was detected from all tested specimens of X. germanus (n=120) by wsp amplification. Five distinct sequences (i.e. five alleles) for wsp were found, and labeled as wXge1-5. MLST analysis and molecular phylogeny of concatenated sequences of MLST genes identified wXge3 and wXge5 as closely-related strains. The detection rate of wXge4 and wXge1 was 100% and 63.3%, respectively; wXge2, wXge3 and wXge5 were detected from less than 15% of specimens. We performed mitochondrial haplotype analyses that identified three genetic types of X. germanus, i.e. Clades A, B and C. Wsp alleles wXge1, wXge2 and wXge4 were detected in all clade A beetles; wXge2 allele was absent from Clades B and C. We concluded that (i) five wsp alleles were found from X. germanus, (ii) use of MLST genes, rather than the wsp gene, are more suited to construct Wolbachia phylogenies and (iii) wsp alleles wXge2 and wXge3/wXge5 would infect clade A and clade B/C of X. germanus, respectively.

Discovery of Mycangia and Mucus in Adult Female Xiphydriid Woodwasps (Hymenoptera: Xiphydriidae) in Japan

Abstract Dissection of emerging adult xiphydriid woodwasps, Xiphydria ogasawarai Matsumura, occurring in eastern, central, and western Japan during a 2- to 4-yr period, revealed that adult females possessed mycangia filled with fungal spores, whereas the mycangia were absent in males. Two masses of the spore were located on the slit-like 1st and 2nd valvulae of the ovipositor sting directly below the subgenital plate, and were covered with the ventral membranes. This is a new type of mycangia in woodwasps. The spores were globular or ovoid in shape and ≈5–30 μm in diameter, and had no clamp connections. All isolates from the mycangia of the xiphydriid populations, originating from different sites and years, developed the same colonies on potato-dextrose agar. This suggests that female adults of X. ogasawarai carry a single fungal species in their mycangia. It was also found that X. ogasawarai produced a slime-like secretion from glandular organs in the body cavity (mucus reservoir), which is homologous to that present in the Siricinae.

Symbiotic Fungal Flora in Leaf Galls Induced by Illiciomyia yukawai (Diptera: Cecidomyiidae) and in Its Mycangia

We investigated the association between a gall midge, Illiciomyia yukawai, and its symbiotic fungi on Japanese star anise, Illicium anisatum. The number of fungal species isolated from the galls increased with development of the galls, whereas those from the leaves showed a different trend. Botryosphaeria dothidea was dominant in the galls from June to October, and after that Phomopsis sp. 1, Colletotrichum sp., and Pestalotiopsis sp. became dominant. Although B. dothidea was not isolated from the leaves, it was detected from mycangia (abdominal sternite VII) of egg-laying adults at a high isolation frequency (>90%). However, B. dothidea was not isolated from mycangia of adults emerging from galls that were enclosed by plastic bags. This indicates that I. yukawai is closely associated with B. dothidea and that its newly emerged adults do not take the fungus into mycangia directly from the galls where they had developed. Also, the fungus from the fungal layers of ambrosia galls has less ability to propagate on artificial media despite the presence of its mycelial mass in mature galls.

History of the Exotic Ambrosia Beetles Euwallacea interjectus and Euwallacea validus (Coleoptera: Curculionidae: Xyleborini) in the United States

ABSTRACT Exotic insects are constantly intercepted at U.S. ports-of-entry. Of these, wood-boring beetles, particularly xyleborine ambrosia beetles, are sometimes missed during port inspections and become established in the United States. Euwallacea validus (Eichhoff) and Euwallacea interjectus (Blandford) are morphologically similar Asian ambrosia beetle species that vary by their fungal associates and their potential to cause economic damage. Euwallacea validus and E. interjectus were first discovered in New York (1975) and Hawaii (1976), respectively. Euwallacea validus was collected multiple times from widely separated localities and is assumed to have spread throughout the eastern United States. The discovery of E. interjectus in Florida (2011) and Texas (2011) prompted our review of the E. validus specimens because of the potential misidentification of the species. In addition, using mitochondrial cytochrome oxidase I (COI) DNA data and phylogenetic analysis, we tested the hypothesis that multiple introductions account for the U.S. populations of E. interjectus and E. validus. Our review of 7,184 specimens revealed an earlier introduction to the mainland for E. interjectus, which was first collected from Louisiana in 1984. This species is distributed in the South while E. validus occurs in the North with a known area of syntopy in northeastern Georgia. The extent of the syntopy within the United States is unknown and further investigation is required. Phylogenetic analysis of 24 E. interjectus and 20 E. validus individuals resolved clades that associated with each species and gross geographic provenance. Four well-supported clades represented E. interjectus which included the following localities: 1) Hawaii and Thailand; 2) Vietnam, Taiwan, and Texas; 3) Okinawa (Japan); and 4) Japan and several southern U.S. states. One clade comprised all E. validus specimens from Japan and the mainland United States. Four and two haplotypes were found for the E. interjectus and E. validus specimens, respectively, in mainland United States. Except for the Texas specimen, the haplotypes differed by one nucleotide. The relationship of the haplotypes and their sequence similarity suggested that the provenance of E. validus and the majority of E. interjectus haplotypes was Japan while the Texas haplotype originated later and from a location near Taiwan. Given the high nucleotide sequence difference between the Hawaiian and Thai haplotypes, the exact origin of the Hawaiian E. interjectus is unknown but likely Southeast Asia. A broader investigation including more SE Asian individuals will help to further explain the introduction of E. interjectus into Hawaii and Texas.

Genetic structure of Japanese populations of an ambrosia beetle, Xylosandrus germanus (Curculionidae: Scolytinae)

We examined the genetic structures of 13 Japanese populations of an ambrosia beetle, Xylosandrus germanus (Curculionidae: Scolytinae), to understand the effects of geographical barriers on the colonization dynamics of this species. The genetic structure was studied using portions of the mitochondrial cytochrome oxidase I (COI) gene. A phylogenetic analysis revealed three distinct lineages (clades A, B and C) within X. germanus. Clade A contained 21 haplotypes from all 13 populations; whereas clade B contained eight haplotypes from Hokkaido (Sapporo and Furano), Iwate and Nagano populations; and clade C contained only a single a haplotype from the Hokkaido (Furano) population. In the analysis of molecular variance (amova), the greatest amount of genetic variation was detected between populations in Hokkaido and those in Honshu and other southern islands. Between these two groups of populations, all the values of the coefficient of gene differentiation were significantly larger than zero, except for the Hokkaido (Sapporo) versus Nagano comparison. Our results confirm that for X. germanus, gene flow has been interrupted between Hokkaido and Honshu since the last glacial maximum.

Two novel ascomycetous yeast species, Wickerhamomyces scolytoplatypi sp. nov. and Cyberlindnera xylebori sp. nov., isolated from ambrosia beetle galleries

Thirteen strains of yeasts were isolated from ambrosia beetle galleries at several sites in Japan. Based on the morphological and biochemical characteristics and phylogenetic analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene of the yeasts, 10 strains were shown to represent a novel species of the genus Wickerhamomyces, described as Wickerhamomyces scolytoplatypi sp. nov. (type strain NBRC 11029(T) = CBS 12186(T)), and were closely related to Wickerhamomyces hampshirensis. The three other strains represented a novel species of the genus Cyberlindnera, described as Cyberlindnera xylebori sp. nov. (type strain NBRC 11048(T) = CBS 12187(T)), and were closely related to Cyberlindnera euphorbiiphila. It is suggested that these species are associated with ambrosia beetles and we consider ambrosia beetle galleries as good sources of novel yeasts.

Wolbachia endosymbionts in haplodiploid and diploid scolytine beetles (Coleoptera: Curculionidae: Scolytinae).

Haplodiploidy is a sex determination system in which fertilized diploid eggs develop into females and unfertilized haploid eggs develop into males. The evolutionary explanations for this phenomenon include the possibility that haplodiploidy can be reinforced by infection with endosymbiotic bacteria, such as Wolbachia. The subfamily Scolytinae contains species with haplodiploid and diploid sex determination systems. Thus, we studied the association with Wolbachia in 12 diploid and 11 haplodiploid scolytine beetles by analyzing wsp and multilocus sequence typing (MLST) of five loci in this endosymbiont. Wolbachia genotypes were compared with mitochondrial (COI) and nuclear (EF) genotypes in the scolytines. Eight of the 23 scolytine species were infected with Wolbachia, with haplodiploids at significantly higher rates than diploid species. Cloning and sequencing detected multiple infections with up to six Wolbachia strains in individual species. Phylogenetic analyses of wsp and five MLST genes revealed different Wolbachia strains in scolytines. Comparisons between the beetle and Wolbachia phylogenies revealed that closely related beetles were infected with genetically different Wolbachia strains. These results suggest the horizontal transmission of multiple Wolbachia strains between scolytines. We discuss these results in terms of the evolution of different sex determination systems in scolytine beetles.