Polyandry may mitigate the negative impact of reproductive interference among bumblebees in Japan.

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.

Integration of satellite remote sensing and MaxEnt modeling for improved detection and management of forest pests.

Forest pests pose a major threat to ecosystem services worldwide, requiring effective monitoring and management strategies. Recently, satellite remote sensing has emerged as a valuable tool to detect defoliation caused by these pests. Lymantria dispar, a major forest pest native to Japan, Siberia, and Europe, as well as introduced regions in North America, is of particular concern. In this study, we used Sentinel-2 satellite imagery to estimate the defoliation area and predict the distribution of L. dispar in Toyama Prefecture, central Japan. The primary aim was to understand the spatial distribution of L. dispar. The normalized difference vegetation index (NDVI) difference analysis estimated a defoliation area of 7.89 km2 in Toyama Prefecture for the year 2022. MaxEnt modeling, using defoliation map as occurrence data, identified the deciduous forests between approximately 35° and 50° at elevations of 400 m and 700 m as highly suitable for L. dispar. This predicted suitability was also high for larval locations but low for egg mass locations, likely due to differences in larval habitats and ovipositing sites. This study is the first attempt to utilize NDVI-based estimates as a proxy for MaxEnt. Our results showed higher prediction accuracy than a previous study based on the occurrence records including larvae, adults, and egg masses, indicating better discrimination of the distribution of L. dispar defoliation. Therefore, our approach to integrating satellite data and species distribution models can potentially enhance the assessment of areas affected by pests for effective forest management.

Diverse Molecular Mechanisms Underlying Microbe-Inducing Male Killing in the Moth Homona magnanima.

Male killing (MK) is a type of reproductive manipulation induced by microbes, where sons of infected mothers are killed during development. MK is a strategy that enhances the fitness of the microbes, and the underlying mechanisms and the process of their evolution have attracted substantial attention. Homona magnanima, a moth, harbors two embryonic MK bacteria, namely, Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), and a larval MK virus, Osugoroshi virus (OGV; Partitiviridae). However, whether the three distantly related male killers employ similar or different mechanisms to accomplish MK remains unknown. Here, we clarified the differential effects of the three male killers on the sex-determination cascades and development of H. magnanima males. Reverse transcription-PCR demonstrated that Wolbachia and Spiroplasma, but not OGVs, disrupted the sex-determination cascade of males by inducing female-type splice variants of doublesex (dsx), a downstream regulator of the sex-determining gene cascade. We also found that MK microbes altered host transcriptomes in different manners; Wolbachia impaired the host dosage compensation system, whereas Spiroplasma and OGVs did not. Moreover, Wolbachia and Spiroplasma, but not OGVs, triggered abnormal apoptosis in male embryos. These findings suggest that distantly related microbes employ distinct machineries to kill males of the identical host species, which would be the outcome of the convergent evolution. IMPORTANCE Many microbes induce male killing (MK) in various insect species. However, it is not well understood whether microbes adopt similar or different MK mechanisms. This gap in our knowledge is partly because different insect models have been examined for each MK microbe. Here, we compared three taxonomically distinct male killers (i.e., Wolbachia, Spiroplasma, and a partiti-like virus) that infect the same host. We provided evidence that microbes can cause MK through distinct mechanisms that differ in the expression of genes involved in sex determination, dosage compensation, and apoptosis. These results imply independent evolutionary scenarios for the acquisition of their MK ability.

Distinct effects of three Wolbachia strains on fitness and immune traits in Homona magnanima.

The endosymbiotic bacterium Wolbachia occasionally increases host fitness or manipulates host reproductions to enhance vertical transmission. Multiple Wolbachia strains can coinfect the same host individual, which alters the density as well as phenotypes of the bacteria. However, the effects of Wolbachia coinfection on host fitness remain largely unknown. Here, we examined the effects of three phylogenetically distinct Wolbachia strains, wHm-a, wHm-b, and wHm-c, on host fitness by comparing non-infected, singly infected, and triply infected Homona magnanima lines within a fixed genetic background. By examining the effects of Wolbachia on host longevity, survivorship, and reproduction, we demonstrated that single infection with either wHm-b or wHm-c reduced host reproduction, but the triple infection led to the highest intrinsic growth rate. Susceptibility to the natural pathogens such as viruses and fungi was not different among the lines regardless of Wolbachia infection status. Cellular and humoral immunities were not affected by Wolbachia in females, whereas phenoloxidase activity was suppressed in males of all Wolbachia-infected lines, implying that it was a result of the mother's curse hypothesis or a strategy of Wolbachia to increase their horizontal transmission efficiency. Although how the host's genetic diversity affects the Wolbachia fitness effects is yet unknown, our findings indicated that the effects of Wolbachia are deeply influenced by infection status and that Wolbachia could change symbiotic strategy depending on host sex and transmission route.

Coexistence of Two Male-Killers and Their Impact on the Development of Oriental Tea Tortrix Homona magnanima.

Male-killing, the death of male offspring induced by maternally transmitted microbes, is classified as early, or late, male-killing. The primary advantage afforded by early male-killing, which typically occurs during embryogenesis, is the reallocation of resources to females, that would have otherwise been consumed by males. Meanwhile, the key advantage of late male-killing, which typically occurs during late larval development, is the maximized potential for horizontal transmission. To date, no studies have reported on the associated developmental and physiological effects of host coinfection with early and late male-killers, which may have a significant impact on the population dynamics of the male-killers. Here we used a lepidopteran tea pest Homona magnanima as a model, which is a unique system wherein an early male-killer (a Spiroplasma bacterium) and a late male-killer (an RNA virus) can coexist in nature. An artificially established matriline, coinfected with both Spiroplasma and RNA virus, exhibited embryonic death (early male-killing) as seen in the host line singly infected with Spiroplasma. Moreover, the coinfected line also exhibited developmental retardation and low pupal weight similar to the host line singly infected with the RNA virus. A series of field surveys revealed that Spiroplasma-RNA virus coinfection occurs in nature at a low frequency. Hence, although the two male-killers are capable of coexisting within the H. magnanima population independently, high associated fitness cost appears to limit the prevalence of male-killer coinfection in the field host population.

Closely Related Male-Killing and Nonmale-Killing Wolbachia Strains in the Oriental Tea Tortrix Homona magnanima.

Wolbachia are inherited intracellular bacteria that cause male-specific death in some arthropods, called male-killing. To date, three Wolbachia strains have been identified in the oriental tea tortrix Homona magnanima (Tortricidae, Lepidoptera); however, none of these caused male-killing in the Japanese population. Here, we describe a male-killing Wolbachia strain in Taiwanese H. magnanima. From field-collected H. magnanima, two female-biased host lines were established, and antibiotic treatments revealed Wolbachia (wHm-t) as the causative agent of male-killing. The wsp and MLST genes in wHm-t are identical to corresponding genes in the nonmale-killing strain wHm-c from the Japanese population, implying a close relationship of the two strains. Crossing the Japanese and Taiwanese H. magnanima revealed that Wolbachia genotype rather than the host genetic background was responsible for the presence of the male-killing phenotype. Quantitative PCR analyses revealed that the density of wHm-t was higher than that of other Wolbachia strains in H. magnanima, including wHm-c. The densities of wHm-t were also heterogeneous between host lines. Notably, wHm-t in the low-density and high-density lines carried identical wsp and MLST genes but had distinct lethal patterns. Furthermore, over 90% of field-collected lines of H. magnanima in Taiwan were infected with wHm-t, although not all host lines harboring wHm-t showed male-killing. The host lines that showed male-killing harbored a high density of Wolbachia compared to the host lines that did not show male-killing. Thus, the differences in the phenotypes appear to be dependent on biological and genetic characteristics of closely related Wolbachia strains.

Multiple Infection and Reproductive Manipulations of Wolbachia in Homona magnanima (Lepidoptera: Tortricidae).

Endosymbiotic bacterium Wolbachia interacts with host in either a mutualistic or parasitic manner. Wolbachia is frequently identified in various arthropod species, and to date, Wolbachia infections have been detected in different insects. Here, we found a triple Wolbachia infection in Homona magnanima, a serious tea pest, and investigated the effects of three infecting Wolbachia strains (wHm-a, -b, and -c) on the host. Starting with the triple-infected host line (Wabc), which was collected in western Tokyo in 1999 and maintained in laboratory, we established an uninfected line (W-) and three singly infected lines (Wa, Wb, and Wc) using antibiotics. Mating experiments with the host lines revealed that only wHm-b induced cytoplasmic incompatibility (CI) in H. magnanima, with the intensities of CI different between the Wb and Wabc lines. Regarding mutualistic effects, wHm-c shortened larval development time and increased pupal weight in both the Wc and Wabc lines to the same extent, whereas no distinct phenotype was observed in lines singly infected with wHm-a. Based on quantitative PCR analysis, Wolbachia density in the Wa line was higher than in the other host lines (p < 0.01, n = 10). Wolbachia density in the Wb line was also higher than in the Wc and Wabc lines, while no difference was observed between the Wc and Wabc lines. These results indicate that the difference in the CI intensity between a single or multiple infection may be attributed to the difference in wHm-b density. However, no correlation was observed between mutualistic effects and Wolbachia density.

An ascovirus isolated from Spodoptera litura (Noctuidae: Lepidoptera) transmitted by the generalist endoparasitoid Meteorus pulchricornis (Braconidae: Hymenoptera).

The family Ascoviridae is a recently described virus family whose members are transmitted by parasitoids and cause chronic and lethal infections in lepidopteran insects. Little is known about the biology and ecology of ascoviruses, and few isolates have been found outside the United States. We report here the isolation of a new ascovirus variant from Spodoptera litura in Japan. Full genome sequence and phylogenetic analyses showed that this virus was closely related to variants in Heliothis virescens ascovirus-3a, and it was named HvAV-3j. HvAV-3j has a DNA genome of 191 718 bp, with 189 putative ORFs and a GC content of 45.6 %, and is highly similar to HvAV-3h, which was isolated in China. In a field survey, the endoparasitoid Meteorus pulchricornis caused a high percentage of parasitization in populations of S. litura larvae, and under laboratory conditions M. pulchricornis was able to transmit HvAV-3j from infected to uninfected larvae by oviposition. Meteorus pulchricornis is thus likely to be a major vector for HvAV-3j transmission in Japan. This species is recognized here for the first time as a vector of ascoviruses that parasitizes a range of host species that extends across families.

Differential expression of a fructose receptor gene in honey bee workers according to age and behavioral role.

Honey bee (Apis mellifera) workers contribute to the maintenance of colonies in various ways. The primary functions of workers are divided into two types depending on age: young workers (nurses) primarily engage in such behaviors as cleaning and food handling within the hive, whereas older workers (foragers) acquire floral nutrients beyond the colony. Concomitant with this age-dependent change in activity, physiological changes occur in the tissues and organs of workers. Nurses supply younger larvae with honey containing high levels of glucose and supply older larvae with honey containing high levels of fructose. Given that nurses must determine both the concentration and type of sugar used in honey, gustatory receptors (Gr) expressed in the chemosensory organs likely play a role in distinguishing between sugars. Glucose is recognized by Gr1 in honey bees (AmGr1); however, it remains unclear which Gr are responsible for fructose recognition. This study aimed to identify fructose receptors in honey bees and reported that AmGr3, when transiently expressed in Xenopus oocytes, responded only to fructose, and to no other sugars. We analyzed expression levels of AmGr3 to identify which tissues and organs of workers are involved in fructose recognition and determined that expression of AmGr3 was particularly high in the antennae and legs of nurses. Our results suggest that nurses use their antennae and legs to recognize fructose, and that AmGr3 functions as an accurate nutrient sensor used to maintain food quality in honey bee hives.

Morphological properties of the occlusion body of Adoxophyes orana granulovirus.

A granulovirus (GV) that produces occlusion bodies (OBs) having an unusual morphology was found in an Adoxophyes sp. (Lepidoptera: Tortricidae) larva in a tea field in Miyazaki Prefecture, Japan. This isolate is considered to be a mutant of Adoxophyes orana granulovirus, designated AdorGV-M, because the nucleotide sequence of its genome is 99.7% identical to that of an English isolate of AdorGV, AdorGV-E. AdorGV-E produces typical ovocylindrical OBs that contain one occlusion-derived virus (ODV) per OB. On the other hand, AdorGV-M produces large cuboidal OBs, but the number of ODVs per OB was unknown. In this study, we quantified viral DNA in OBs of both AdorGV-E and -M, and determined the number of ODVs occluded in an OB of AdorGV-M. The two isolates had the same quantity of viral DNA in each OB, and we thus confirmed that one OB of AdorGV-M contains one ODV. To investigate the process of OB formation, fat body tissue of A. honmai larvae inoculated with each isolate was observed in a time course by transmission electron microscopy, and OB sizes were measured from micrographs. The main difference in OB formation was that AdorGV-M required more time to mature than AdorGV-E. In AdorGV-E, ODVs began to be covered from one end with an ovocylindrical OB at 96 h post-inoculation (hpi), and most of them were completely occluded at 120 hpi. Occlusion of AdorGV-M ODVs also began at 96 hpi, but the OB shape was cuboidal. Moreover, the OB size of AdorGV-M was similar to that of AdorGV-E at 120 hpi, but continued to grow until 192 hpi. AdorGV-M thus took more time to complete OB formation. Consequently, AdorGV-E has mature OBs with a diameter 0.22 µm and length 0.39 µm, but those of AdorGV-M are 1.34 × 1.23 µm.

Midgut-based resistance to oral infection by a nucleopolyhedrovirus in the laboratory-selected strain of the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae).

A strain of Adoxophyes honmai resistant to Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) was established from a field-collected colony by repeated selection. Fifth-instar larvae of this resistant strain (R-strain) had over 66 666-fold greater resistance in terms of 50 % lethal concentration values to oral infection of AdhoNPV than non-selected strain larvae (susceptible for AdhoNPV; S2-strain). In this study, the mechanism of resistance to AdhoNPV was determined in R-strain larvae. An assessment of viral genome replication in AdhoNPV-infected S2- and R-strain larvae by quantitative PCR showed no viral genome replication occurring in R-strain larvae. Transcription of AdhoNPV ie-1, vp39 and polyhedrin genes was also not detected in R-strain midgut cells. Besides, a fluorescent brightener had no effect on AdhoNPV infection in either S2- or R-strain. However, binding and fusion of occlusion-derived virus with R-strain were significantly lower than those of S2-strain. These findings suggest that R-strain Adoxophyeshonmai larvae possess a midgut-based resistance to oral infection by AdhoNPV in which midgut epithelial cells are infected less efficiently.

Identification of an Early Male-Killing Agent in the Oriental Tea Tortrix, Homona magnanima.

Arthropods are frequently infected with inherited symbionts, which sometimes confer fitness benefits on female hosts or manipulate host reproduction. Early male killing, in which infected males die during embryogenesis, is induced by some bacteria, such as Wolbachia and Spiroplasma. A female-biased sex ratio has been found in Homona magnanima, collected from a tea plantation in Japan. Here, we examined the male-killing trait in H. magnanima and identified the agent that induces early male killing. The sex ratio distortion (SR) strain produced only females and no males, and its egg hatch rate was significantly lower than that of the normal (N) strain. The N strain was infected with only Wolbachia, whereas the SR strain was infected with both Wolbachia and Spiroplasma. Antibiotic treatment with 0.10% tetracycline restored the 1:1 sex ratio in the SR strain. Females treated with 0.05% tetracycline were positive for Spiroplasma but not for Wolbachia and showed a female-biased sex ratio, whereas Wolbachia-positive females did not revert to male killing. When inoculated with a homogenate of the SR strain female, females infected with only Spiroplasma produced female-biased offspring. Sequence analysis of the 16S rRNA gene revealed that Spiroplasma sp. of H. magnanima belonged to the ixodetis clade. These results indicate that Spiroplasma was responsible for male killing in H. magnanima. Late male killing is induced in H. magnanima by an RNA-like virus, and therefore this is the first case in which different male-killing agents expressed at different times in the life cycle have been found within one host species.

Different acute toxicity of fipronil baits on invasive Linepithema humile supercolonies and some non-target ground arthropods.

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.

Estimating bee distributions and their functional range to map important areas for protecting bee species and their functions.

The decline of wild bee populations causes the decline of bee-pollinated plant populations through the deterioration of pollination services. Since high bee species richness generally involves high functional group diversity, protecting areas of high bee species richness will help to maintain pollination services for plants. However, those areas do not always include the habitats of bee species with specialized functions that expand the range of plants being pollinated. To map important areas for protecting native bee species and their functions, we estimated the distributions and functional range of 13 bumble bee species and 1 honey bee species in Japan. The distributions were estimated from an ensemble of six species distribution models using bee occurrence data and environmental data. The functional range of bee species was estimated by combining the estimated distributions and proboscis length, which frequently corresponds to the floral shape of the plant species they pollinate. The estimated species richness was high in western Hokkaido and the estimated functional range was wide in central Honshu. Our method is useful to see whether areas important for high species richness of pollinators differ from those for rare species or their functions.

Conserved infections and reproductive phenotypes of Wolbachia symbionts in Asian tortrix moths.

Wolbachia is a ubiquitous endosymbiotic bacterium that manipulates insect reproduction. A notable feature of Wolbachia is male killing (MK), whereby sons of infected females are killed during development; however, the evolutionary processes by which Wolbachia acquired the MK ability remain unclear. The tea tortrix moth Homona magnanima (Tortricidae) harbours three non-MK Wolbachia strains (wHm-a, wHm-b and wHm-c) and an MK strain wHm-t. Although wHm-t and wHm-c are closely related, only wHm-t has an MK-associated prophage region. To understand the evolutionary processes underlying the emergence of MK wHm-t, we examined Wolbachia infections and phenotypes in 62 tortricid species collected from 39 localities across Japan, Taiwan, Vietnam and Indonesia. PCR assays detected wHm-c relatives in 51 species and triple infection of wHm-a, wHm-b and wHm-c in 31 species. Apart from Taiwanese H. magnanima, no species exhibited the MK phenotype and were positive for the wHm-t-specific prophage. While wHm-t infection was dominant in Taiwanese H. magnanima, wHm-a, wHm-b and wHm-c were dominant in Japanese H. magnanima populations. These results suggest that wHm-a, wHm-b and wHm-c strains descended from a common ancestor with repeated infection loss and that wHm-t evolved from the wHm-c acquiring MK ability in allopatric populations of H. magnanima.

Infection Prevalence of Microsporidia Vairimorpha (Nosema) spp. in Japanese Bumblebees.

Microsporidia are spore-forming intracellular parasites of various invertebrates and vertebrates. Vairimorpha bombi negatively affects the fitness of bumblebees and its prevalence correlates with declining bumblebee populations. The invasive alien species Bombus terrestris colonized Japan and possibly introduced new parasites. To assess the infection prevalence of V. bombi in Japanese bumblebees and B. terrestris, we investigated V. bombi infections using PCR and microscopy. The prevalence of sporulating V. bombi infections in three Bombus s. str. species/subspecies was low, whereas that of non/low-sporulating Vairimorpha sp. infections in three Diversobombus species/subspecies was high. Invasive B. terrestris showed low prevalence of non/low-sporulating V. bombi infections and shared the same V. bombi haplotype with B. hypocrita found in Hokkaido, where B. terrestris is present, and in Honshu, where B. terrestris is absent. Although V. bombi may have been introduced with B. terrestris colonies imported from Europe, it seems to be originally distributed in Japan. Furthermore, a new Vairimorpha sp. was found in Japanese bumblebee species. V. bombi and Vairimorpha sp. showed different organ and host specificities in bumblebees. There are no reports on the specific effects of other Vairimorpha spp. on bumblebees; further studies are needed to clarify the individual characteristics of Vairimorpha spp.

Combined actions of bacteriophage-encoded genes in Wolbachia-induced male lethality.

Some Wolbachia endosymbionts induce male killing, whereby male offspring of infected females are killed during development; however, the origin and diversity of the underlying mechanisms remain unclear. In this study, we identified a 76 kbp prophage region specific to male-killing Wolbachia hosted by the moth Homona magnanima. The prophage encoded a homolog of the male-killing gene oscar in Ostrinia moths and the wmk gene that induces various toxicities in Drosophila melanogaster. Upon overexpressing these genes in D. melanogaster, wmk-1 and wmk-3 killed all males and most females, whereas Hm-oscar, wmk-2, and wmk-4 had no impact on insect survival. Strikingly, co-expression of tandemly arrayed wmk-3 and wmk-4 killed 90% of males and restored 70% of females, suggesting their conjugated functions for male-specific lethality. While the male-killing gene in the native host remains unknown, our findings highlight the role of bacteriophages in male-killing evolution and differences in male-killing mechanisms among insects.

Potential increase in mating frequency of queens in feral colonies of Bombus terrestris introduced into Japan.

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.

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects.

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

Male-killing mechanisms vary between Spiroplasma species.

Male-killing, a male-specific death of arthropod hosts during development, is induced by Spiroplasma (Mollicutes) endosymbionts of the Citri-Poulsonii and the Ixodetis groups, which are phylogenetically distant groups. Spiroplasma poulsonii induces male-killing in Drosophila melanogaster (Diptera) using the Spaid toxin that harbors ankyrin repeats, whereas little is known about the origin and mechanisms of male-killing induced by Spiroplasma ixodetis. Here, we analyzed the genome and the biological characteristics of a male-killing S. ixodetis strain sHm in the moth Homona magnanima (Tortricidae, Lepidoptera). Strain sHm harbored a 2.1 Mb chromosome and two potential plasmids encoding Type IV effectors, putatively involved in virulence and host-symbiont interactions. Moreover, sHm did not harbor the spaid gene but harbored 10 ankyrin genes that were homologous to those in other S. ixodetis strains. In contrast to the predominant existence of S. poulsonii in hemolymph, our quantitative PCR assays revealed a systemic distribution of strain sHm in H. magnanima, with particularly high titers in Malpighian tubules but low titers in hemolymph. Furthermore, transinfection assays confirmed that strain sHm can infect cultured cells derived from distantly related insects, namely Aedes albopictus (Diptera) and Bombyx mori (Lepidoptera). These results suggest different origins and characteristics of S. ixodetis- and S. poulsonii-induced male-killing.