Arboreal or terrestrial: Oviposition site of Zhangixalus frogs affects the thermal function of foam nests.

Temperature is essential for the survival and development of eggs. Some anurans have evolved and developed foam nesting traits, with thermal insulation considered to be among their functions. Foam-nesting frogs tend to exhibit reproductive plasticity. For example, they oviposit on both trees and the ground. How such plasticity affects foam nest function is of major relevance and is likely related to the adaptation of foam nesting frogs. However, this has not been well studied. In this study, we examined the interaction between foam nest site, foam nest function, and egg fate using the Japanese green tree frog, Zhangixalus arboreus, and analysed how nest site differences (arboreal or terrestrial) affect the thermal function of foam nests. We compared the thermal functions of foam nests between arboreal and terrestrial oviposition sites of Z. arboreus. We artificially replaced half of the arboreal nests with terrestrial environments and recorded temperature in and outside of the experimental terrestrial nest and original arboreal nests. We also examined egg survival and hatching rates for all the nests. The results indicated superior heat insulation in terrestrial nests, with warmer temperatures inside than outside the nests, especially at night, which led to a high egg survival rate. Therefore, terrestrial ovipositing should be valid under cold weather conditions. This may be related to the evolutionary history of oviposition site plasticity of this genus, which originally had an arboreal oviposition trait but evolved into terrestrial site use owing to global cooling. Our novel insights into the evolution and adaptivity of foam nesting and oviposition site use in Z. arboreus make an important contribution to animal ecology.

Fusarium kuroshium is the primary fungal symbiont of an ambrosia beetle, Euwallacea fornicatus, and can kill mango tree in Japan.

This study identifies fungi associated with Euwallacea fornicatus and determines whether these fungal species play the role of primary symbiont. E. fornicatus adults that emerged from the branches of infested trees in Okinawa main island, Japan, were collected and used to isolate fungi. Fusarium kuroshium and Penicillium citrinum were the most dominant fungal associates of females and males, respectively. F. kuroshium was much more frequently isolated from the head, including mycangia (fungus-carrying organs), of females than any other body parts. We inoculated healthy mango saplings with F. kuroshium or F. decemcellulare, both of which were symbionts of E. fornicatus females infesting mango trees. F. kuroshium decreased leaf stomatal conductance and rate of xylem sap-conduction area and increased length and area of xylem discoloration of the saplings, thereby weakening and killing some. These results suggest that F. kuroshium, a mycangial fungus of E. fornicatus, inhibits water flow in mango trees. This study is the first to report that F. kuroshium causes wilt disease in mango trees and that it is a primary fungal symbiont of E. fornicatus.

How much do field mice prefer dwarf bamboo seeds? Two-choice experiments between seeds of Sasa borealis and several tree species on the forest floor.

Bambusoideae is a taxon of mass-flowering monocarpic perennials with a long life cycle. Forest ecosystems are affected by Bambusoideae seeding and death events in various ways, including an increased abundance of Apodemus spp. The utilization and preference of dwarf bamboo seeds over tree seeds by field mice remain elusive. Therefore, we aimed to determine whether field mice prefer dwarf bamboo to tree seeds. We examined one dwarf bamboo species (Sasa borealis) against four tree species with varying acorn/fruit traits (Castanea crenata, Quercus crispula, Fagus crenata, and Lindera triloba). The seeds were placed in a container in a forest among dead S. borealis culms, with an automatic camera monitoring the setup. The examined seeds were mainly foraged by two field mouse species, Apodemus speciosus and Apodemus argenteus, with preference in the following order: C. crenata, L. triloba, S. borealis, F. crenata, and Q. crispula. Our findings indicated that during S. borealis mast seeding years, predation pressure on F. crenata and Q. crispula seeds could be considerably reduced. This suggests that mast seeding might disrupt the normal pattern of survival, and seed dispersal patterns, potentially altering the forest vegetation composition.

The Role of Mycangial Fungi Associated with Ambrosia Beetles (Euwallacea interjectus) in Fig Wilt Disease: Dual Inoculation of Fusarium kuroshium and Ceratocystis ficicola Can Bring Fig Saplings to Early Symptom Development.

The ambrosia beetle, Euwallacea interjectus, is a wood-boring pest and a vector of Ceratocystis ficicola, a pathogenic fungus causing fig (Ficus carica) wilt disease (FWD) in Japan. The ambrosia fungi, Fusarium kuroshium and Neocosmospora metavorans, have been frequently isolated from heads (including mycangia) of wild and reared adult female E. interjectus, respectively. However, the exact mechanisms driving FWD as well as the interactions between F. kuroshium and C. ficicola in fig orchard remain unclear. To verify the role of the mycangial fungi in the FWD progression, fig saplings were subjected to inoculation treatments (T1, F. kuroshium; T2, N. metavorans, reference positive control; T3, C. ficicola; T4, F. kuroshium + C. ficicola, realistic on-site combination). T3 and T4 saplings began wilting approximately 12 days after inoculation, leading to eventual death. Median duration from inoculation to death of the T4 saplings was approximately four days significantly faster than that of the T3 saplings. Xylem sap-conduction test indicated that dysfunction and necrosis area were considerably wider in the T4 saplings than in T3 saplings. These results demonstrate that the synergistic action of F. kuroshium and C. ficicola contributed to accelerated wilting in the saplings. Based on these discoveries, we proposed a model for system changes in the symbiosis between E. interjectus and its associated fungi in FWD in Japan.

Longer mtDNA Fragments Provide a Better Insight into the Genetic Diversity of the Sycamore Lace Bug, Corythucha ciliata (Say, 1832) (Tingidae, Hemiptera), Both in Its Native and Invaded Areas.

The sycamore lace bug (Corythucha ciliata Say, 1832) is of North American origin, but after its introduction to Europe (1964), South America (1985), Asia (1995), Australia (2006), and Africa (2014), it became an abundant and widespread pest on plane (Platanus spp.) trees. We analysed a 1356 bp long fragment of the mtDNA (COI gene) of 327 sycamore lace bug individuals from 38 geographic locations from Europe, Asia, and North America. Seventeen haplotypes (17 HTs) were detected. C. ciliata populations from North America exhibited higher haplotype diversity (12 HTs) than populations from Europe (6 HTs), Asia (4 HTs), or Japan (2 HTs). The haplotypes formed two haplogroups separated by at least seven mutation steps. One of these mutation steps includes HTs from North America and Japan. Another includes HTs from North America, Europe, and Asia. Haplotypes from Asia Minor, the Caucasus, and Central Asia are linked to haplotypes from Europe, while haplotypes found in Japan are linked to haplotypes found in North America only. The incorporation of published data from the GenBank into our dataset (altogether 517 individuals from 57 locations, but only 546 bp long fragment of the mtDNA) did not show any structure according to the geographic origin of the individuals.

Novel Symbiotic Association Between Euwallacea Ambrosia Beetle and Fusarium Fungus on Fig Trees in Japan.

Ficus carica plantations in Japan were first reported to be infested by an ambrosia beetle species, identified as Euwallacea interjectus, in 1996. The purpose of this study was to determine the symbiotic fungi of female adults of E. interjectus emerging from F. carica trees infected with fig wilt disease (FWD). Dispersal adults (51 females) of E. interjectus, which were collected from logs of an infested fig tree in Hiroshima Prefecture, Western Japan, were separated into three respective body parts (head, thorax, and abdomen) and used for fungal isolation. Isolated fungi were identified based on the morphological characteristics and DNA sequence data. Over 13 species of associated fungi were detected, of which a specific fungus, Fusarium kuroshium, was dominant in female head (including oral mycangia). The plant-pathogenic fungus of FWD, Ceratocystis ficicola, was not observed within any body parts of E. interjectus. We further discussed the relationship among E. interjectus and its associated fungi in fig tree.

Additional description and genome analyses of Caenorhabditis auriculariae representing the basal lineage of genus Caenorhabditis.

Caenorhabditis auriculariae, which was morphologically described in 1999, was re-isolated from a Platydema mushroom-associated beetle. Based on the re-isolated materials, some morphological characteristics were re-examined and ascribed to the species. In addition, to clarify phylogenetic relationships with other Caenorhabditis species and biological features of the nematode, the whole genome was sequenced and assembled into 109.5 Mb with 16,279 predicted protein-coding genes. Molecular phylogenetic analyses based on ribosomal RNA and 269 single-copy genes revealed the species is closely related to C. sonorae and C. monodelphis placing them at the most basal clade of the genus. C. auriculariae has morphological characteristics clearly differed from those two species and harbours a number of species-specific gene families, indicating its usefulness as a new outgroup species for Caenorhabditis evolutionary studies. A comparison of carbohydrate-active enzyme (CAZy) repertoires in genomes, which we found useful to speculate about the lifestyle of Caenorhabditis nematodes, suggested that C. auriculariae likely has a life-cycle with tight-association with insects.

Characterization of FsXEG12A from the cellulose-degrading ectosymbiotic fungus Fusarium spp. strain EI cultured by the ambrosia beetle.

Despite the threat of Fusarium dieback posed due to ambrosia fungi cultured by ambrosia beetles such as Euwallacea spp., the wood-degradation mechanisms utilized by ambrosia fungi are not fully understood. In this study, we analyzed the 16S rRNA and 18S rRNA genes of the microbial community from the Ficus tree tunnel excavated by Euwallacea interjectus and isolated the cellulose-degrading fungus, Fusarium spp. strain EI, by enrichment culture with carboxymethyl cellulose as the sole carbon source. The cellulolytic enzyme secreted by the fungus was identified and expressed in Pichia pastoris, and its enzymatic properties were characterized. The cellulolytic enzyme, termed FsXEG12A, could hydrolyze carboxymethyl cellulose, microcrystalline cellulose, xyloglucan, lichenan, and glucomannan, indicating that the broad substrate specificity of FsXEG12A could be beneficial for degrading complex wood components such as cellulose, xyloglucan, and galactoglucomannan in angiosperms. Inhibition of FsXEG12A function is, thus, an effective target for Fusarium dieback caused by Euwallacea spp.

Pathogenicity and Distribution of Fusarium solani Isolates Associated with Erythrina Decline in Japan.

Erythrina spp. trees have been declining since the 2000s worldwide, and fungi belonging to Fusarium solani species complex (FSSC) have been suggested to be a causal factor of decline and mortality of Erythrina variegata trees in Okinawa Island, Japan. In addition to the FSSC isolate grouped as "it-1" based on ITS sequence data (previously called strain A), we conducted an inoculation experiment with two isolates grouped as "it-2" (previously strain B), which is genetically close to it-1. Two it-2 isolates originating from two islands showed pathogenicity to E. variegata with the same symptoms as those caused by it-1 isolate. We also found the isolates of it-1 and it-2 were widely distributed, including on Ishigaki Island, ∼400 km south of Okinawa Island across the ocean. All isolates of it-1 and it-2 belong to the ambrosia Fusarium clade of the FSSC, a group of symbionts of ambrosia beetles, including the pathogens of Fusarium dieback in avocados and teas. The detection of ambrosia beetles Euwallacea spp. from our specimens provided information on the vectors of the pathogens. Our present results suggest the fungi of the FSSC could be responsible for the Erythrina decline in other areas with damage.

Landscape-scale genetic differentiation of a mycangial fungus associated with the ambrosia beetle, Xylosandrus germanus (Blandford) (Curculionidae:Scolytinae) in Japan.

In this study, we examined the genetic structures of the ambrosia fungus isolated from mycangia of the scolytine beetle, Xylosandrus germanus to understand their co-evolutionary relationships. We analyzed datasets of three ambrosia fungus loci (18S rDNA, 28S rDNA, and the ß-tubulin gene) and a X. germanus locus dataset (cytochrome c oxidase subunit 1 (COI) mitochondrial DNA). The ambrosia fungi were separated into three cultural morphptypes, and their haplotypes were distinguished by phylogenetic analysis on the basis of the three loci. The COI phylogenetic analysis revealed three distinct genetic lineages (clades A, B, and C) within X. germanus, each of which corresponded to specific ambrosia fungus cultural morphptypes. The fungal symbiont phylogeny was not concordant with that of the beetle. Our results suggest that X. germanus may be unable to exchange its mycangial fungi, but extraordinary horizontal transmission of symbiotic fungi between the beetle's lineages occurred at least once during the evolutionary history of this symbiosis.

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.

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

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.

Equilibrium frequency of endosymbionts in multiple infections based on the balance between vertical transmission and cytoplasmic incompatibility.

Cytoplasmic incompatibility (CI)-inducing endosymbiotic bacteria, such as Wolbachia and Cardinium, have been well studied through field data and validations on the basis of numerical simulations. However, the analytically derived equilibrium frequency of multiple infections has not yet been determined, although the equilibrium for cases of single infection has been reported. In this study, we considered the difference equation for endosymbionts using three parameters: the probability of the failure of vertical transmission ([Formula: see text]), CI strength ([Formula: see text]), and the level of host inbreeding ([Formula: see text]). To analyze this model, we particularly focused on [Formula: see text], i.e., the frequency of host individuals completely infected with all [Formula: see text]-bacterial strains in the population. [Formula: see text], [Formula: see text] at the equilibrium state, was analytically calculated in the cases where [Formula: see text] and [Formula: see text] is any arbitrary value. We found that [Formula: see text] can be described using two parameters: [Formula: see text] and [Formula: see text], which is identical to [Formula: see text]. [Formula: see text] has a larger value in a system with a smaller [Formula: see text]. In addition, [Formula: see text] determines the maximum number of strains that infect a single host. Our results revealed the following: i) three parameters can be reduced to a single parameter, i.e., [Formula: see text] and ii) the threshold of the maximum number of infections is defined by [Formula: see text], which prevents additional invasions by endosymbionts.

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.

Fungi associated with Scolytogenes birosimensis (Coleoptera: Curculionidae) infesting Pittosporum tobira.

The bark beetle Scolytogenes birosimensis Niijima is suspected to be involved in the decline of Pittosporum tobira (Thunb. ex Murray) Aiton in the coastal areas of Japan. We isolated fungi from adult S. birosimensis in nine different localities in Japan to assess their potential association and predict their contribution to the success of the beetle. Results from morphological identification of associated fungi showed that the beetle was associated with Fusarium solani and Candida spp. Furthermore, molecular analysis showed that F. solani was most closely related to the plant pathogenic fungus F. solani f. sp. mori. Fungal isolation from surface-sterilized, dissected beetles and scanning electron miscroscope (SEM) observation of the body surface suggested that the associated fungi were carried in the pits on the beetles' elytra. These findings contribute to the understanding of the relationships between S. birosimensis and its associated fungi.

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.

Genetic structure of Japanese populations of Xylosandrus brevis (Curculionidae: Scolytinae).

We examined the genetic structure of populations of an ambrosia beetle, Xylosandrus brevis (Eichhoff) (Curculionidae: Scolytinae), to understand its colonization dynamics. We collected specimens from 20 sites in Japan and studied the genetic structure of these populations using portions of the mitochondrial cytochrome oxidase I gene (COI). A phylogenetic analysis showed three distinct lineages (clades A, B, and C) within X. brevis. Clade A had 65 haplotypes from all the populations, except for populations from Nagano (Chiisagata-gun) and the Ryukyu Islands (Ishigaki), whereas clade B had 14 haplotypes from nine populations (Yamagata, Saitama, Shimo-Minochi-gun, Chiisagata-gun, Toyota, Watarai-gun, Wakayama, Tottori, and Kochi), and clade C had 6 haplotypes from the Ryukyu Islands (Ishigaki) population (and nowhere else). Nested clade phylogeographic analysis showed 65 clade A haplotypes and 14 clade B haplotypes geographically structured in two clades, respectively. A contact zone was identified in the Chubu and Tokai areas. Our results suggest that gene flow and individual movement of X. brevis between the Ryukyu Islands and the other three main islands of Japan has been rare, and X. brevis may be divided into two populations, eastern-central and northern-western, with little gene flow between the four areas of western Japan (Kinki, Chugoku, Shikoku, and Kyushu).