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Declines in bee populations, in part due to pesticides, especially insecticides, are of global concern. Although most studies have investigated insecticide residues in honeybees and bumblebees, few have focused on non‐Apis solitary bees, which are considered essential pollinators in the field. Most non‐Apis bees are solitary and build their nests in the ground or in tree holes, therefore insecticide exposure pathways would differ from those of honeybees and bumblebees. We analyzed the residues of 20 insecticides in Osmia cornifrons bees and their pollen‐provisions and nesting materials, along with adult honeybees, soil, and wildflowers collected in four apple orchards in two regions in Japan. Few insecticides were common among adult bees, pollen‐provisions, and wildflowers. Insecticides applied in orchards were detected at high frequency: chlorantraniliprole, flubendiamide, and diazinon were detected in almost all samples. Insecticides without a known history of application were detected from various samples at frequencies ranging from 0% to 100%. Even in orchards without a known history of insecticide application, clothianidin was detected in many sample types and at high concentration. A purple deadnettle sample had the highest concentration at 17.5 mg/kg. These results highlight the complexity of pathways of insecticide exposure to O. cornifrons in the environment because insecticides may remain in the environment for long periods and wild bees may forage farther than previously known distances. Furthermore, each sample type contained different insecticides, so wild bees may have been exposed to different insecticides at different life stages. Future research should prioritize wide‐scale measurements of insecticide residues in field conditions and toxicity testing with multiple exposures at different life stages of target species. Environ Toxicol Chem 2023;42:1564–1574. © 2023 SETAC [ABSTRACT FROM AUTHOR]

The ecological impacts of insecticides in aquatic areas around agricultural lands have long been ignored in the regulation scheme of pesticides in Japan. Upon the scheme, the predicted concentration of an insecticide in the main stream of a river is the only parameter considered, suggesting that the ecological impacts of insecticides on local biodiversity around agricultural fields are underestimated. To fill this knowledge gap, we measured insecticide concentrations in surface water and sediment in aquatic areas around paddy fields at 35 locations across Japan. Among the 18 insecticides considered, 15 were detected somewhere in Japan and their concentrations were generally higher in the southwestern region in Japan (e.g. Hiroshima, Saga, or Kagoshima prefectures). Most insecticides were accumulated at higher concentrations in sediment than in surface water, consistent with previous studies. We also detected insecticides applied to nursery boxes at high concentrations in surrounding aquatic areas, although such application is generally considered to have low environmental risks. In addition, derivatives of fipronil, which have similar toxicity as that of fipronil, were often detected in sediment at higher concentrations than fipronil itself. Concentrations of dinotefuran in water at two sampling points were higher than the 5% hazardous concentration (HC5), indicating a possibility of a risk of acute toxicity to aquatic organisms. Our findings indicate that ecological risk assessments of insecticides and their derivatives should be expanded to include concentrations in sediment and water around paddy fields as well. Locations of 11 survey sites and the mean amounts (log+1 transformed) of pesticides detected in sediment (s; μg/kg) and water (w; μg/L) samples. Image 1 • Many insecticides were detected in sediment and water around paddy fields. • Sediment accumulated higher concentration of fipronil derivatives than fipronil. • Dinotefuran concentration often exceeded its community toxicity benchmark. • Environmental risk assessments of pesticides should be more inclusive. We revealed many insecticides and derivatives were detected in sediment and water around paddy fields, arising a possibility of an environmental impact. [ABSTRACT FROM AUTHOR]

One of the major routes of transmission of rickettsial and ehrlichial diseases is via ticks that infest numerous host species, including humans. Besides mammals, reptiles and amphibians also carry ticks that may harbor Rickettsia and Ehrlichia strains that are pathogenic to humans. Furthermore, reptiles and amphibians are exempt from quarantine in Japan, thus facilitating the entry of parasites and pathogens to the country through import. Accordingly, in the current study, we examined the presence of Rickettsia and Ehrlichia spp. genes in ticks associated with reptiles and amphibians originating from outside Japan. Ninety-three ticks representing nine tick species (genera Amblyomma and Hyalomma) were isolated from at least 28 animals spanning 10 species and originating from 12 countries (Ghana, Jordan, Madagascar, Panama, Russia, Sri Lanka, Sudan, Suriname, Tanzania, Togo, Uzbekistan, and Zambia). None of the nine tick species are indigenous in Japan. The genes encoding the common rickettsial 17-kDa antigen, citrate synthase (gltA), and outer membrane protein A (ompA) were positively detected in 45.2% (42/93), 40.9% (38/93), and 23.7% (22/93) of the ticks, respectively, by polymerase chain reaction (PCR). The genes encoding ehrlichial heat shock protein (groEL) and major outer membrane protein (omp-1) were PCR-positive in 7.5% (7/93) and 2.2% (2/93) of the ticks, respectively. The p44 gene, which encodes the Anaplasma outer membrane protein, was not detected. Phylogenetic analysis showed that several of the rickettsial and ehrlichial sequences isolated in this study were highly similar to human pathogen genes, including agents not previously detected in Japan. These data demonstrate the global transportation of pathogenic Rickettsia and Ehrlichia through reptile- and amphibian-associated ticks. These imported animals have potential to transfer pathogens into human life. These results highlight the need to control the international transportation of known and potential pathogens carried by ticks in reptiles, amphibians, and other animals, in order to improve national and international public health. [ABSTRACT FROM AUTHOR]

Japan's economy depends on the importation of natural resources, and as a result, Japan is subjected to a high risk of biological invasion. Although Japan has quarantine systems to protect ecosystems, agriculture, forestry, fisheries, and human health against alien species, economic globalization has resulted in an ever-increasing risk of invasion. Mite invasion is no exception. Alien species that impact natural ecosystems are regulated in Japan by the Invasive Alien Species Act. However, the law focuses only on visibly recognizable species, so that species too small to see, such as viruses, bacteria, fungi, and mites, are beyond the scope of this law. The Plant Protection Law has limited the introduction of alien pests, including mites, that are harmful to agricultural crops. Recently, the liberalization of global trade policies have increased pressure to loosen regulations on various pests, including spider mites. Infectious diseases and their causative species are quarantined under the Rabies Prevention Law, the Domestic Animal Infectious Diseases Control Law, and the Human Infectious Diseases Control Law, but these laws do not cover wildlife diseases. The most serious problem is that wild reptiles, which can be carriers of ticks and tick-borne diseases, can be freely introduced to Japan. These loopholes in Japan's regulatory system have resulted in mite and tick invasions, which affect not only wildlife communities and human society but also endemism and biological diversity of natural mite populations. [ABSTRACT FROM AUTHOR]

Aim The Argentine ant, Linepithema humile, has been spreading via human activities from its native range in South America across much of the globe for more than a century. This invasive ant was first detected in Japan in 1993. Its successful world-wide expansion is attributed to a social structure, namely supercoloniality, whereby individuals from separate nests cooperate. Here, we examined the genetic structure of L. humile populations to understand its invasion history. Location Japan. Methods We analysed mitochondrial DNA of Linepithema humile workers from native and other introduced populations and then integrated previously registered sequences. Results Sequencing revealed six haplotypes distributed across its introduced ranges, of which five were present in Japan. The first haplotype was shared by the dominant Japanese, European, North American, Australian and New Zealand supercolonies; the second by the Kobe C supercolony and a Florida population; and the third by the Kobe B and secondary Californian supercolonies and North Carolina colonies. The remaining three haplotypes were each restricted to the Kobe A, Tokyo and Catalonian supercolonies, respectively. Each of the five mutually antagonistic supercolonies was fixed for one of the five haplotypes, and multiple supercolonies were found within a small area. Main conclusions The large number of haplotypes found in Japan likely reflects the strong propagule pressure of L. humile resulting from the fact that the country is one of the top five importers of trade commodities world-wide. The short invasion history of L. humile in Japan could explain the maintenance of genetic diversity of each independent introduction. In addition, our sampling mostly occurred at major international shipping ports that are likely to be primary sites of introduction. The several recently established L. humile populations within a small area in Japan provide an opportunity to identify the sources of introduction and the local patterns of spread. [ABSTRACT FROM AUTHOR]

Aim Our goal was to use the occurrence of the influenza A virus in wild birds in Japan to create a potential risk map for the spread of avian influenza by migratory birds. Our modelling included a consideration of the multicollinearity and spatial autocorrelation of environmental variables and an examination of the reproducibility of the model results. Location Japan. Methods We used the maximum entropy approach to generate potential distribution models from presence-only data. Independent variables in the model included environmental factors such as winter temperature and precipitation, host factors such as duck population size and habitat abundance and artificial factors such as size of urban areas and poultry density. We used eigenvector-based spatial filters to alleviate spatial autocorrelation. To explore the reliability of the model, we compared the risk indices of localities positive in past winters for the influenza A virus in wild birds with those of all localities. Results The model alleviated spatial autocorrelation with a high degree of accuracy. Dabbling duck population, size of urban area, diving duck population and altitude were the variables that were most strongly correlated with the potential distribution of avian influenza. We used the frequency of occurrence of the influenza A virus in five recent years in localities where wild birds were infected to estimate the repeatability of the high-risk indices; the potential risk indices for avian influenza in wild birds were high in localities where wild birds were infected in past. Main conclusions The dabbling duck population in an area appeared to be the best indicator of high risk for the introduction of avian influenza from abroad. Priority monitoring localities for avian influenza carried by wild birds should be designated in western Japan and along the Pacific coast, which we estimated to be high-risk areas. Poultry farms in these areas should increase their biosecurity to prevent vectors from introducing avian influenza. [ABSTRACT FROM AUTHOR]

Recent increases in international trade have increased the cost to control and eradicate exotic species. Although many species are under quarantine control for agriculture, forestry, and public health, most species invisible to the naked eye are ignored because of the lack of both specialized assessors and risk assessments. We developed a species risk assessment particularly adapted to fungi, nematodes, and mites, that might be unintentionally introduced with exotic forest products and become threats to terrestrial ecosystems. We developed our assessment with reference to existing risk assessments for exotic organisms, including their ecological features such as phoresy and parasitism. We then tested our assessment with well-known organisms and assessed the risks of organisms unintentionally introduced into Japan. The assessment demonstrated scientifically acceptable scores for each organism. We suggest quarantine control of risk pathways as a practical approach for controlling unintentionally introduced organisms that are invisible to the naked eye. [ABSTRACT FROM AUTHOR]

For some wildlife commodities, rare species are especially sought after. The tendency for rare commodities to be of higher value can fuel their exploitation and as numbers dwindle, the demand can increase. Consequently, this can precipitate these rare species into an overexploitation vortex where they become increasingly rare, valued and exploited until eventual extinction. We focus here on the hobby of collecting stag beetles, to ascertain if the market value of these items is driven by rarity and if, consequently, these species are vulnerable to this overexploitation vortex. Stag beetle collections fuel a large and lucrative market in Japan, involving more than 700 species from all over the world, with over 15 million specimens imported a year. Some particularly valued species fetch more than US$5,000 a piece. We assessed the importance of species rarity as an acquisition criterion in this market using two methods: an Internet online questionnaire responded to by 509 participants and through examining the quantities imported in Japan and prices paid by collectors. We discovered that species rarity is one of the main choice criteria for acquisition by collectors: rare stag beetles are valued more than the common species and, consequently, stag beetles are vulnerable to the anthropogenic Allee effect in this market. Because of the sheer size of the market and the pervasive nature of this rarity paradox, the attraction to rarity equates to a potential extinction threat for many rare stag beetles species. [ABSTRACT FROM AUTHOR]

A serious disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis was first found in Japan in December 2006 in imported pet frogs. This was the first report of chytridiomycosis in Asia. To assess the risk of pandemic chytridiomycosis to Japanese frogs, we surveyed the distribution of the fungus among captive and wild frog populations. We established a nested PCR assay that uses two pairs of PCR primers to amplify the internal transcribed spacer (ITS) region of a ribosomal RNA cassette to detect mild fungal infections from as little as 0.001 pg (1 fg) of B. dendrobatidis DNA. We collected swab samples from 265 amphibians sold at pet shops, 294 bred at institutes and 2103 collected at field sites from northern to southwestern Japan. We detected infections in native and exotic species, both in captivity and in the field. Sequencing of PCR products revealed 26 haplotypes of the B. dendrobatidis ITS region. Phylogenetic analysis showed that three of these haplotypes were specific to the Japanese giant salamander ( Andrias japonicus) and appeared to have established a commensal relationship with this native amphibian. Many other haplotypes were carried by alien amphibians. The highest genetic diversity of B. dendrobatidis was found in the American bullfrog ( Rana catesbeiana). Some strains of B. dendrobatidis appeared to be endemic to Japanese native amphibians, but many alien strains are being introduced into Japan via imported amphibians. To improve chytridiomycosis risk management, we must consider the risk of B. dendrobatidis changing hosts as a result of anthropogenic disturbance of the host-specific distribution of the fungus. [ABSTRACT FROM AUTHOR]

Kin selection theory predicts a high coefficient of genetic relatedness among nestmates, explaining the frequent evolution of eusociality in a social hymenopteran colony. The bumble bee is a primitively eusocial hymenoptera whose colonies are founded by a single queen. In such a monogynous colony, mating frequency of the queen is the sole factor that affects genetic relatedness among nestmates. Although the queens of most bumble bee species are known to be monandrous, there are some species that are known to be polyandrous. Here, we estimated the effective paternity in the native colonies of Japanese bumble bees, Bombus ardens, B. diversus, and B. honshuensis, using genetic polymorphic data of microsatellites. We found no evidence for polyandry in any investigated colonies, which suggests that within-colony genetic relatedness is very high for all of these colonies. [ABSTRACT FROM AUTHOR]

A commercial colony ofBombus terrestris(L.) was introduced to Japan in 1992 for crop pollination in greenhouses. Since then wild colonies have developed and spread in some regions. In the present study, we measured the spatial distribution and temporal change in abundance ofB. terrestrisin the Chitose River Basin, Hokkaido, Japan to elucidate the relation of greenhouses to the bee’s distribution and to evaluate its potential effects on native bumblebees. Bumblebees were collected with window traps in windbreak forests roughly 1, 2, 4, and 6 km NNW and SSE of a large greenhouse. The peak catch ofB. terrestrisqueens occurred in early June, suggesting that they had successfully hibernated in the field. The distributions ofB. terrestrisand the nativeB. ardenswere mutually exclusive, while the nativeB. hypocritaappeared at all sites. Catches ofB. terrestriswere restricted to within 4 km of the nearest greenhouse, suggesting that the invasion was still in the initial phase in this area. The reduction in abundance of the native bumblebees in the sites of highB. terrestrisabundance suggests the presence of interspecies competition betweenB. terrestrisand the native bumblebees during the early part of the colony activity, although such reduction inB. ardenscan be explained by habitat suitability. [ABSTRACT FROM AUTHOR]

As a signature of reproductive interference (RI), we reviewed hybrid production in eusocial bumblebees in Japan, by comparing introduced Bombus terrestris with native B. ignitus in Honshu (main island of Japan) and with native B. hypocrita sapporoensis in Hokkaido (northern island of Japan). In this review, we present additional new data showing hybrid production between introduced B. terrestris and native B. ignitus in Honshu. Interspecific mating with introduced B. terrestris disrupts the reproduction of native B. h. sapporoensis and B. ignitus, which belong to the same subgenus of Bombus, through inviable egg production. This interference appears to facilitate species replacement on Hokkaido. Simultaneously, the mating frequencies for queens of B. terrestris have increased, suggesting that polyandry might evolve in response to the extent of RI between B. terrestris and B. h. sapporoensis. To suppress the population size of B. terrestris in Hokkaido, two methods have been proposed: the mass release of B. h. sapporoensis males to induce RI between the two species and the spraying of insecticides against foraging workers so that the workers will carry the insecticides back to their colonies, killing the immature bees within the colonies. A candidate insecticide type is insect growth regulator, which may disrupt larval development without any apparent effect on foraging workers. [ABSTRACT FROM AUTHOR]

Abstract: The infrequency of tsunamis results in a lack of knowledge regarding the impact they have on ecosystems. Comparison of the floristic composition before and after tsunamis using permanent plots is an effective approach to estimate the ecological impacts of tsunamis in coastal environments. Here, we report the floristic changes in beach vegetation in northern Tohoku, Japan caused by the 2011 Tohoku-oki tsunami. Among the beaches studied, we observed differences in the vegetative characteristics of the beach flora, whereas similarities were found in vegetation response patterns, particularly for herbaceous vegetation. No significant interaction was found between the overall number of vascular plant species or the number of non-beach species and the tsunami disturbance; conversely, on beaches that experienced relatively small impacts, the number of typical/characteristic beach species, Pielou evenness index values, and Shannon-Wiener diversity index values were significantly lower after the tsunami. Following the tsunami, the herbaceous vegetation species composition on all of the beaches was characterized by a few non-beach species that are known to tolerate strong anthropogenic disturbances; in contrast, the damage to shrub vegetation was lower than that to herbaceous vegetation. Our findings are similar to what was observed following the 2004 Indian Ocean tsunami in Thailand, suggesting that the initial response patterns of beach vegetation to tsunamis are similar, regardless of the affected climatic zone and species composition. Long-term field monitoring is needed to elucidate post-tsunami recovery, vegetation succession, and the ecological impact of the increase of non-beach species. [Copyright &y& Elsevier]

Pesticides may have serious negative impacts on bee populations. The pesticide exposure of bees could depend on the surrounding landscapes in which they forage. In this study, we assess pesticide exposure across various land-use categories, while targeting the Japanese honey bee, Apis cerana japonica, a native subspecies of the eastern honey bee. In a project involving public participation, we measured the concentrations of major pesticides in honey and beeswax collected from 175 Japanese honey bee colonies across Japan and quantitatively analyzed the relationships between pesticide presence/absence or pesticide concentration and land-use categories around the colonies. Our findings revealed that the surrounding environment in which bees live strongly influences pesticide residues in beehive materials, whether the pesticides are systemic or not, with a clear trend for each land-use category. Agricultural lands, particularly paddy fields and orchards, and urban areas resulted in higher pesticide exposure, whereas forests presented a lower risk of exposure. To effectively control pesticide exposure levels in bees, it is essential to understand pesticide usage patterns and to develop appropriate regulatory systems in non-agricultural lands, similar to those in agricultural lands., (© 2024. The Author(s).)

We devised a method to detect the classical swine fever virus (CSFV) in tail-wiped swabs from wild boars. The CSFV gene in swabs was detected with high sensitivity using nested real-time polymerase chain reaction (PCR), which is a combination of reverse transcription-PCR (RT-PCR) and real-time PCR. We compared CSFV gene detection from boar tissue using the conventional and our tail-wiped swab method. The tail-wiped swab method showed sensitivity and specificity of 100% (26/26) and 98.8% (172/174), respectively compared to the conventional method. Thus, the swab-based CSFV detection method was considered to have detection sensitivity comparable to that of conventional methods. Additionally, we conducted surveillance for CSFV in wild boars on Awaji Island. CSFV was detected in 10.7% (45/420) of samples.

In social hymenopterans, monandry of the queen is an ancestral trait, and polyandry is a derived trait. Polyandry of the queen is the norm in a limited number of lineages, such as honeybees, leaf-cutting ants, Pogonomyrmex ants, and Vespula wasps, which presumably provide fitness advantages for the whole colony. The queen of the introduced bumblebee, Bombus terrestris, is polyandrous in Japan, whereas it is monandrous in native regions. We hypothesize that polyandry can evolve in a process that avoids the negative impacts of reproductive interference caused by interspecific mating and conducted genetic studies of the invasive species B. terrestris and two native subspecies, Bombus hypocrita sapporoensis and Bombus hypocrita hypocrita, in Japan. Our results revealed that although the native queens of B. hypocrita hypocrita allopatric with B. terrestris were strictly monandrous, the native queens of B. hypocrita sapporoensis sympatric with B. terrestris were polyandrous. These results suggested that the queens of native B. hypocrita sapporoensis do not experience negative impacts on interspecific mating from the invasive B. terrestris. We discuss the possibility that reproductive interference is a driving force in selection for multiple mating through an arms race between sympatric species., (© 2024. The Author(s).)

There is limited information about virus epidemiology of shorebirds (family Charadriidae and Scolopacidae) in the East Asia-Australasia flyway. We investigated the prevalence of avian influenza viruses (AIVs) in shorebirds in Hokkaido, Japan, the stopover site of the flyway, to understand the ecology of AIV translocation in the flyway from 2006 to 2010. In total, 1,698 shorebirds belonging to 26 species were captured and released into two different sites using mist nets. Tracheal and cloacal swabs were collected from each bird using cotton swabs. The RNA of influenza A viruses was detected using reverse transcription loop-mediated isothermal amplification. One AIV-positive sample was obtained from a Lesser Sand Plover ( Charadrius mongolus ) captured in September 2010 at Lake Komuke. Full lengths of hemagglutinin (HA), neuraminidase (NA), polymerase acidic protein, nucleoprotein, matrix protein 1, and nuclear export protein genes were successfully amplified from the AIV-positive sample. All sequences showed the highest identity with sequences obtained from virus strains from Anseriformes species. Shorebirds migrated to Japan 1 mo earlier than did Anseriformes species. Therefore, the Lesser Sand Plover could have been infected by the virus from Anseriformes species on the breeding grounds. The HA sequence showed the highest identity with the H10 sequence whereas the NA sequence exhibited the highest identity with the N7 sequence. Phylogenic analysis showed that the detected subtype H10N7 belongs to the Eurasia lineage and the related strain might have widely spread in Asia in 2009.

To conserve local biodiversity and ensure the provision of pollination services, it is essential to understand the impact of pesticides on wild honey bees. Most studies that have investigated the effects of pesticides on honey bees have focused on the European honey bee (Apis mellifera (Hymenoptera: Apidae)), which is commonly domesticated worldwide. However, the Asian honey bee (Apis cerana) is widely distributed throughout Asia, and toxicity data are lacking for this species. This study aimed to fill this important knowledge gap. In this study, we determined the acute contact toxicity in A. cerana to various pesticides, including neonicotinoids, fipronil, organophosphorus, synthetic pyrethroids, carbamate, and anthranilic diamide. Based on the test duration of 48 h of contact LD50 tests, A. cerana was most sensitive to dinotefuran (0.0014 μg/bee), followed by thiamethoxam (0.0024 μg/bee) and fipronil (0.0025 μg/bee). Dinotefuran is used extensively in Asia, thereby potentially creating a substantial hazard. More generally, A. cerana was approximately one order of magnitude more sensitive than was A. mellifera to most of the pesticides evaluated. The results of our study suggest that neonicotinoid pesticides should not be considered as a single group that acts uniformly on all honey bees, and that more careful management strategies are required to conserve A. cerana populations than A. mellifera., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Fipronil is one of the most effective insecticides to control the invasive ant Linepithema humile, but its effectiveness has been assessed without considering the genetic differences among L. humile supercolonies. We hypothesized that the susceptibility of the ant to fipronil might differ among supercolonies. If so, dosage and concentration of fipronil may need to be adjusted for effective eradication of each supercolony. The relative sensitivities of four L. humile supercolonies established in Hyogo (Japan) to fipronil baits were examined based on their acute toxicity (48-h LC(50)). Toxicities of fipronil to seven ground arthropods, including four native ant species, one native isopoda, and two cockroaches were also determined and compared to that of L. humile supercolonies using species sensitivity distributions. Marked differences in susceptibility of fipronil were apparent among the supercolonies (P < 0.008), with the 'Japanese main supercolony' (271 μg L(-1)) being five to ten times more sensitive to fipronil than other colonies (1183-2782 μg L(-1)). Toxicities to non-target species (330-2327 μg L(-1)) were in the same range as that of L. humile, and SSDs between the two species groups were not significantly different (t = -1.389, P = 0.180), suggesting that fipronil's insecticidal activity is practically the same for L. humile as for non-target arthropods. Therefore, if the invasive ant is to be controlled using fipronil, this would also affect the local arthropod biodiversity. Only the 'Japanese main supercolony' can be controlled with appropriate bait dosages of fipronil that would have little impact on the other species.

The international trade in amphibians is believed to have increased the spread of Batrachochytrium dendrobatidis (Bd), the fungal pathogen responsible for chytridiomycosis, which has caused a rapid decline in amphibian populations worldwide. We surveyed amphibians imported into Japan and those held in captivity for a long period or bred in Japan to clarify the Bd infection status. Samples were taken from 820 individuals of 109 amphibian species between 2008 and 2011 and were analyzed by a nested-PCR assay. Bd prevalence in imported amphibians was 10.3% (58/561), while it was 6.9% (18/259) in those in private collections and commercially bred amphibians in Japan. We identified the genotypes of this fungus using partial DNA sequences of the internal transcribed spacer (ITS) region. Sequencing of PCR products of all 76 Bd-positive samples revealed 11 haplotypes of the Bd ITS region. Haplotype A (DNA Data Bank of Japan accession number AB435211) was found in 90% (52/58) of imported amphibians. The results show that Bd is currently entering Japan via the international trade in exotic amphibians as pets, suggesting that the trade has indeed played a major role in the spread of Bd.

With the exception of several species, bumblebees are monandrous. We examined mating frequency in feral colonies of the introduced bumblebee Bombus terrestris in Japan. Using microsatellite markers, genotyping of sperm DNA stored in the spermatheca of nine queens detected multiple insemination paternities in one queen; the others were singly mated. The average effective paternity frequency estimated from the genotypes of queens and workers was 1.23; that estimated from the workers' genotype alone was 2.12. These values were greater than those of laboratory-reared colonies in the native ranges of B. terrestris. The genotypes of one or two workers did not match those of their queens or showed paternities different from those of their nestmates; this may have arisen from either queen takeover or drifting of workers. These alien workers were responsible for the heterogeneous genotype distribution within each B. terrestris colony, resulting in higher estimates of paternity frequency than of insemination frequency. The high mating frequency of introduced B. terrestris may have occurred by artificial selection through mass breeding for commercialization. Moreover, polyandrous queens may be selectively advantageous, because reproduction by such queens is less likely to be disturbed by interspecific mating than that by monandrous queens.

The purpose of this study was to establish a method for eradicating a chytrid fungus (Batrachochytrium dendrobatidis; Bd) from the Japanese giant salamander Andrias japonicus. The emerging agent (Bd) has a high rate of detection in this endangered amphibian species, which is designated as a special natural monument in Japan. Four Japanese giant salamanders with Bd confirmed by PCR assay were bathed in 0.01% itraconazole for 5 min d-1 over 10 successive days. PCR assays were conducted prior to treatment, on Days 5 and 10 of treatment, and on Days 7 and 14 post-treatment. By treatment Day 5, all individuals tested negative for Bd and remained negative until the end of the experiment. No side effects associated with itraconazole were observed. The present method appears to be a safe and effective approach for Bd eradication and may contribute to reducing the threat and spread of Bd among endangered amphibians. Notably, this study represents the first reported Bd eradication experiment involving Japanese giant salamanders.

The European bumblebee, Bombus terrestris, is an invasive eusocial species whose distribution is expanding greatly beyond its native range because numerous colonies are imported to or locally produced in non-native countries for pollination of agricultural crops. Closely related species exist in Japan where the unrestricted import and use of B. terrestris has resulted in the establishment of wild colonies. Laboratory studies previously showed that B. terrestris and Japanese native species can copulate and produce fertilized eggs. Although these eggs do not hatch, the interspecific mating can cause a serious reproductive disturbance to native bumblebees. In this study, we determined the frequencies of interspecies mating between B. terrestris males and native bumblebee queens in the wild on the islands of Hokkaido and Honshu by analyzing the DNA sequences of spermatozoa stored in spermathecae of native queens. We found that 20.2% of B. hypocrita hypocrita queens and 30.2% of B. hypocrita sapporoensis queens had spermatozoa of B. terrestris males in their spermathecae. Given that a Bombus queen generally mates only once in her life, such high frequencies of interspecific mating with B. terrestris pose serious threats to the populations of native bumblebees in Japan.

The genetic structure of populations is not necessarily reflected in the geographical proximity of individuals, because environmental gradients such as those of vegetation or climate can function as cryptic barriers to gene flow. We examined polymorphisms at nine microsatellite loci to determine and discuss whether a distinctive genetic structure was detectable in a spatially continuous population of the sika deer (Cervus nippon) on the Boso Peninsula of central Japan. Spatially explicit Bayesian analysis revealed that two genetically distinctive clusters exist in the Boso population. The spatial boundary of the two clusters approximately conformed to the border defined previously from a mitochondrial DNA dataset. By combining information on the geomorphic features surrounding the boundary and that on the lineage of 1970s population, we propose a schematic scenario for characterizing the population genetic structure to the present. The current population consists of genetically different lineages, and spatially discontinuous clusters have come into contact in the vicinity of a local road running along a steep-walled ravine that could act as principal barrier to gene flow. Biological factors such as distribution of vegetation and philopatric behavior might also have helped strengthen the cryptic genetic structure of the Boso population.

The bumblebee Bombus terrestris is not only an effective pollinator, but also a potential invasive alien species outside its native range. Recently, nearly 30% of queens of the Japanese native species Bombus hypocrita sapporoensis and B. hypocrita hypocrita were estimated to copulate with B. terrestris males in the field, suggesting that indigenous bumblebees could be genetically deteriorated through hybrid production with the introduced species. In this study, we evaluated hybrid production between the introduced B. terrestris and the indigenous B. hypocrita sapporoensis under laboratory conditions. The hatching rate of eggs derived from interspecific matings was 0% and 8.6% depending on the direction of the cross, which was significantly lower than that from intraspecific matings of B. terrestris (76.9%) and B. hypocrita sapporoensis (78.9%). Genetic studies using microsatellite markers revealed that both haploid and diploid individuals were present in the egg stage, whereas all hatched larvae were haploid. In addition, histological studies revealed that eggs derived from interspecific matings terminated development 2 days after oviposition. These results strongly suggested that eggs derived from interspecific matings are inviable due to post-mating isolation mechanisms. Mass release of exotic pollinators could cause serious population declines of native bumblebee species.