Defensive fungal symbiosis on insect hindlegs.

Tympanal organs as "insect ears" have evolved repeatedly. Dinidorid stinkbugs were reported to possess a conspicuous tympanal organ on female's hindlegs. Here we report an unexpected discovery that the stinkbug's "tympanal organ" is actually a novel symbiotic organ. The stinkbug's "tympanum" is not membranous but a porous cuticle, where each pore connects to glandular secretory cells. In reproductive females, the hindleg organ is covered with fungal hyphae growing out of the pores. Upon oviposition, the females skillfully transfer the fungi from the organ to the eggs. The eggs are quickly covered with hyphae and physically protected against wasp parasitism. The fungi are mostly benign Cordycipitaceae entomopathogens and show considerable diversity among insect individuals and populations, indicating environmental acquisition of specific fungal associates. These results uncover a novel external fungal symbiosis in which host's elaborate morphological, physiological and behavioral specializations underpin the selective recruitment of benign entomopathogens for a defensive purpose.

Evolutionary Dynamics of Host Organs for Microbial Symbiosis in Tortoise Leaf Beetles (Coleoptera: Chrysomelidae: Cassidinae).

Diverse insects host specific microbial symbionts that play important roles for their growth, survival, and reproduction. They often develop specialized symbiotic organs for harboring the microbial partners. While such intimate associations tend to be stably maintained over evolutionary time, the microbial symbionts may have been lost or replaced occasionally. How symbiont acquisitions, replacements, and losses are linked to the development of the host's symbiotic organs is an important but poorly understood aspect of microbial symbioses. Cassidine leaf beetles are associated with a specific gammaproteobacterial lineage, Stammera, whose reduced genome is streamlined for producing pectin-degrading enzymes to assist the host's digestion of food plants. We investigated the symbiotic system of 24 Japanese cassidine species and found that (i) most species harbored Stammera within paired symbiotic organs located at the foregut-midgut junction, (ii) the host phylogeny was largely congruent with the symbiont phylogeny, indicating stable host-symbiont association over evolutionary time, (iii) meanwhile, the symbiont was not detected in three distinct host lineages, uncovering recurrent losses of the ancient microbial mutualist, (iv) the symbiotic organs were vestigial but present in the symbiont-free lineages, indicating evolutionary persistence of the symbiotic organs even in the absence of the symbiont, and (v) the number of the symbiotic organs was polymorphic among the cassidine species, either two or four, unveiling a dynamic evolution of the host organs for symbiosis. These findings are discussed as to what molecular mechanisms and evolutionary trajectories underpin the recurrent symbiont losses and the morphogenesis of the symbiotic organs in the herbivorous insect group. IMPORTANCE Insects represent the biodiversity of the terrestrial ecosystem, and their prosperity is attributable to their association with symbiotic microorganisms. By sequestering microbial functionality into their bodies, organs, tissues, or cells, diverse insects have successfully exploited otherwise inaccessible ecological niches and resources, including herbivory enabled by utilization of indigestible plant cell wall components. In leaf beetles of the subfamily Cassininae, an ancient symbiont lineage, Stammera, whose genome is extremely reduced and specialized for encoding pectin-degrading enzymes, is hosted in gut-associated symbiotic organs and contributes to the host's food plant digestion. Here, we demonstrate that multiple symbiont losses and recurrent structural switching of the symbiotic organs have occurred in the evolutionary course of cassidine leaf beetles, which sheds light on the evolutionary and developmental dynamics of the insect's symbiotic organs and provides a model system to investigate how microbial symbionts affect the host's development and morphogenesis and vice versa.

Host's guardian protein counters degenerative symbiont evolution.

Microbial symbioses significantly contribute to diverse organisms, where long-lasting associations tend to result in symbiont genome erosion, uncultivability, extinction, and replacement. How such inherently deteriorating symbiosis can be harnessed to stable partnership is of general evolutionary interest. Here, we report the discovery of a host protein essential for sustaining symbiosis. Plataspid stinkbugs obligatorily host an uncultivable and genome-reduced gut symbiont, Ishikawaella Upon oviposition, females deposit "capsules" for symbiont delivery to offspring. Within the capsules, the fragile symbiotic bacteria survive the harsh conditions outside the host until acquired by newborn nymphs to establish vertical transmission. We identified a single protein dominating the capsule content, which is massively secreted by female-specific intestinal organs, embedding the symbiont cells, and packaged into the capsules. Knockdown of the protein resulted in symbiont degeneration, arrested capsule production, symbiont transmission failure, and retarded nymphal growth, unveiling its essential function for ensuring symbiont survival and vertical transmission. The protein originated from a lineage of odorant-binding protein-like multigene family, shedding light on the origin of evolutionary novelty regarding symbiosis. Experimental suppression of capsule production extended the female's lifespan, uncovering a substantial cost for maintaining symbiosis. In addition to the host's guardian protein, the symbiont's molecular chaperone, GroEL, was overproduced in the capsules, highlighting that the symbiont's eroding functionality is compensated for by stabilizer molecules of host and symbiont origins. Our finding provides insight into how intimate host-symbiont associations can be maintained over evolutionary time despite the symbiont's potential vulnerability to degeneration and malfunctioning.

Diverse Heat Tolerance of the Yeast Symbionts of Platycerus Stag Beetles in Japan.

The genus Platycerus (Coleoptera: Lucanidae) is a small stag beetle group, which is adapted to cool-temperate deciduous broad-leaved forests in East Asia. Ten Platycerus species in Japan form a monophyletic clade endemic to Japan and inhabit species-specific climatic zones. They are reported to have co-evolutionary associations with their yeast symbionts of the genus Sheffersomyces based on host cytochrome oxidase subunit I (COI) and yeast intergenic spacer (IGS) phylogenies. Here we examined the heat tolerances of the yeast colonies isolated from the mycangia of 37 females belonging ten Japanese Platycerus species. The upper limits of growth and survival temperatures of each colony were decided by cultivating it at ten temperature levels between 17.5 and 40°C. Although both temperatures varied during 25.0-31.25°C, the maximum survival temperatures (MSTs) were a little higher than the maximum growth temperatures (MGTs) in 16 colonies. Pearson's correlations between these temperatures and environmental factors (elevation and 19 bioclimatic variables from Worldclim database) of host beetle collection sites were calculated. These temperatures were significantly correlated with elevation negatively, the maximum temperature of the warmest month (Bio5) positively, and some precipitative variables, especially in the warm season (Bio12, 13, 16, 18) negatively. Sympatric Platycerus kawadai and Platycerus albisomni share the same lineage of yeast symbionts that exhibit the same heat tolerance, but the elevational lower range limit of P. kawadai is higher than that of P. albisomni. Based on the field survey in their sympatric site, the maximum temperature of host wood of P. kawadai larvae is higher about 2-3°C than that of P. albisomni larvae in the summer, which may restrict the elevational range of P. kawadai to higher area. In conclusion, it is suggested that the heat tolerance of yeast symbionts restricts the habitat range of their host Platycerus species or/and that the environmental condition that host Platycerus species prefers affect the heat tolerance of its yeast symbionts.

Evolutionary Relationship Between Platycerus Stag Beetles and Their Mycangium-Associated Yeast Symbionts.

Adult females of stag beetles (Coleoptera: Lucanidae) possess an ovipositor-associated mycangium for conveying symbiotic microorganisms. In most lucanid species, their mycangium contains yeast symbionts of the genus Scheffersomyces Kurtzman and Suzuki that are known for their xylose-fermenting capability. The lucanid genus Platycerus Geoffroy, 1762 is a group of small blue stag beetles, in which ten Japanese species constitute a monophyletic clade. Here we examined the evolutionary relationships of these Japanese Platycerus species and their yeast symbionts, together with a Korean Platycerus species and other lucanid species as outgroup taxa. Based on the internal transcribed spacer (ITS) and the intergenic spacer (IGS) sequences, the yeast symbionts of all Platycerus species were closely related to each other and formed a monophyletic clade. There is no variation in ITS sequences of the yeast symbionts of the Japanese Platycerus species. Based on IGS sequences, the yeast symbionts formed clusters that largely reflected the geographic distribution of the host insects, being shared by sympatric Platycerus species except for P. delicatulus Lewis, 1883 and P. viridicuprus Kubota & Otobe, The symbiont phylogeny was globally not congruent with the host COI-based phylogeny, although some local congruences were observed. Statistically significant correlations were detected between the genetic distances of COI sequences of the host insects and those of IGS sequences of the yeast symbionts in Japan. These results suggest that, at least to some extent, the host insects and the yeast symbionts may have experienced co-evolutionary associations. While the Japanese Platycerus species formed a monophyletic clade in the COI phylogeny, the yeast symbionts of Japanese P. viridicuprus were very closely related to those of Korean P. hongwonpyoi Imura & Choe, 1989, suggesting the possibility that a recent secondary contact of the two beetle species during a marine withdrawal, e.g., in the last glacial period, might have resulted in an inter-specific horizontal transmission of the yeast symbiont.

Recurrent symbiont recruitment from fungal parasites in cicadas.

Diverse insects are associated with ancient bacterial symbionts, whose genomes have often suffered drastic reduction and degeneration. In extreme cases, such symbiont genomes seem almost unable to sustain the basic cellular functioning, which comprises an open question in the evolution of symbiosis. Here, we report an insect group wherein an ancient symbiont lineage suffering massive genome erosion has experienced recurrent extinction and replacement by host-associated pathogenic microbes. Cicadas are associated with the ancient bacterial co-obligate symbionts Sulcia and Hodgkinia, whose streamlined genomes are specialized for synthesizing essential amino acids, thereby enabling the host to live on plant sap. However, our inspection of 24 Japanese cicada species revealed that while all species possessed Sulcia, only nine species retained Hodgkinia, and their genomes exhibited substantial structural instability. The remaining 15 species lacked Hodgkinia and instead harbored yeast-like fungal symbionts. Detailed phylogenetic analyses uncovered repeated Hodgkinia-fungus and fungus-fungus replacements in cicadas. The fungal symbionts were phylogenetically intermingled with cicada-parasitizing Ophiocordyceps fungi, identifying entomopathogenic origins of the fungal symbionts. Most fungal symbionts of cicadas were uncultivable, but the fungal symbiont of Meimuna opalifera was cultivable, possibly because it is at an early stage of fungal symbiont replacement. Genome sequencing of the fungal symbiont revealed its metabolic versatility, presumably capable of synthesizing almost all amino acids, vitamins, and other metabolites, which is more than sufficient to compensate for the Hodgkinia loss. These findings highlight a straightforward ecological and evolutionary connection between parasitism and symbiosis, which may provide an evolutionary trajectory to renovate deteriorated ancient symbiosis via pathogen domestication.

Elementary budget of stag beetle larvae associated with selective utilization of nitrogen in decaying wood.

Wood degradation by insects plays important roles for the forest matter cycling. Since wood is deficient in nitrogen compared to the insect body, wood-feeding insects need to assimilate the nitrogen selectively and discard an excess carbon. Such a stoichiometric imbalance between food and body will cause high metabolic cost; therefore, wood-feeding insects may somehow alleviate the stoichiometric imbalance. Here, we investigated the carbon and nitrogen budgets of the larvae of stag beetle, Dorcus rectus, which feed on decaying wood. Assimilation efficiency of ingested wood was 22%, and those values based on the carbon and nitrogen were 27 and 45%, respectively, suggesting the selective digestion of nitrogen in wood. Element-based gross growth efficiency was much higher for nitrogen (45%) than for carbon (3%). As a result, the larvae released 24% of the ingested carbon as volatile, whereas almost no gaseous exchange was observed for nitrogen. Moreover, solubility-based elementary analysis revealed that the larvae mainly utilized alkaline-soluble-water-insoluble fraction of wood, which is rich in nitrogen. Actually, the midgut of the larvae was highly alkaline (pH 10.3). Stag beetle larvae are known to exhibit coprophagy, and here we also confirmed that alkaline-soluble-water-insoluble nitrogen increased again from fresh feces to old feces in the field. Stable isotope analysis suggested the utilization of aerial nitrogen by larvae; however, its actual contribution is still disputable. Those results suggest that D. rectus larvae selectively utilize alkaline-soluble nitrogenous substrates by using their highly alkaline midgut, and perhaps associate with microbes that enhance the nitrogen recycling in feces.

Specificity and genetic diversity of xylose-fermenting Scheffersomyces yeasts associated with small blue stag beetles of the genus Platycerus in East Asia.

Among insect-fungus relationships, xylose-fermenting Scheffersomyces yeasts are well known for their potential in utilizing wood hemicelluloses and their association with various wood-feeding insects. However, their specificity to host insects or strain-level diversity within host species has not been clearly elucidated. In the insect family Lucanidae, larvae usually feed on decaying wood, and adult females consistently possess a fungus-storage organ, called the mycangium, near the abdominal tip. Here the authors investigated host-symbiont relationships between Scheffersomyces yeast symbionts and small blue stag beetles of the genus Platycerus (Coleoptera: Lucanidae) in East Asia by using intergenic spacer (IGS) region as a genetic marker. All yeast strains isolated from the female mycangium of three Platycerus species, P. hongwonpyoi from Korea and P. acuticollis and P. delicatulus from Japan, were allied to Scheffersomyces segobiensis based on the sequences of the nrDNA 26S and internal transcribed spacer (ITS), in which no sequence difference was observed among those strains. However, IGS regions showed clear genetic differentiation within the yeast symbionts of P. hongwonpyoi, as well as between those of Korean and Japanese Platycerus species. In the IGS sequences, nucleotide substitutions were mainly distributed in the whole stretch of IGS1 and the anterior half of IGS2, whereas nucleotide gaps were localized at IGS1 and the middle of IGS2. Despite the conserved association between the Platycerus beetles and the specific strains of S. segobiensis in East Asia, geophylogenetic divergence patterns of the yeast symbionts were not concordant with those of the insect hosts.

Laboratory Rearing System for Ischnura senegalensis (Insecta: Odonata) Enables Detailed Description of Larval Development and Morphogenesis in Dragonfly.

In an attempt to establish an experimental dragonfly model, we developed a laboratory rearing system for the blue-tailed damselfly, Ischnura senegalensis. Adoption of multi-well plastic plates as rearing containers enabled mass-rearing of isolated larvae without cannibalism and convenient microscopic monitoring of individual larvae. Feeding Artemia brine shrimps to younger larvae and Tubifex worms for older larvae resulted in low mortality, synchronized ecdysis, and normal development of the larvae. We continuously monitored the development of 118 larvae every day, of which 49 individuals (41.5%) reached adulthood. The adult insects were fed with Drosophila flies in wet plastic cages, attained reproductive maturity in a week, copulated, laid fertilized eggs, and produced progeny. The final larval instar varied from 9th to 12th, with the 11th instar (56.5%) and the 12th instar (24.2%) constituting the majority. From the 1st instar to the penultimate instar, the duration of each instar was relatively short, mainly ranging from three to 11 days. Afterwards, the duration of each instar was prolonged, reaching 7-25 days for the penultimate instar and 14-28 days for the final instar. Some larvae of final, penultimate and younger instars were subjected to continuous and close morphological examinations, which enabled developmental staging of larvae based on size, shape, and angle of compound eyes and other morphological traits. This laboratory rearing system may facilitate the understanding of physiological, biochemical, and molecular mechanisms underlying metamorphosis, hormonal control, morphogenesis, body color polymorphism, and other biological features of dragonflies.

Small genome symbiont underlies cuticle hardness in beetles.

Beetles, representing the majority of the insect species diversity, are characterized by thick and hard cuticle, which plays important roles for their environmental adaptation and underpins their inordinate diversity and prosperity. Here, we report a bacterial endosymbiont extremely specialized for sustaining beetle's cuticle formation. Many weevils are associated with a γ-proteobacterial endosymbiont lineage Nardonella, whose evolutionary origin is estimated as older than 100 million years, but its functional aspect has been elusive. Sequencing of Nardonella genomes from diverse weevils unveiled drastic size reduction to 0.2 Mb, in which minimal complete gene sets for bacterial replication, transcription, and translation were present but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the Nardonella genomes was the tyrosine synthesis pathway, identifying tyrosine provisioning as Nardonella's sole biological role. Weevils are armored with hard cuticle, tyrosine is the principal precursor for cuticle formation, and experimental suppression of Nardonella resulted in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine production for host's cuticle formation and hardening. Notably, Nardonella's tyrosine synthesis pathway was incomplete, lacking the final step transaminase gene. RNA sequencing identified host's aminotransferase genes up-regulated in the bacteriome. RNA interference targeting the aminotransferase genes induced reddish and soft weevils with low tyrosine titer, verifying host's final step regulation of the tyrosine synthesis pathway. Our finding highlights an impressively intimate and focused aspect of the host-symbiont metabolic integrity via streamlined evolution for a single biological function of ecological relevance.

A Novel Symbiotic Ciliate (Ciliophora: Peritrichia) in the Hindgut of a Stag Beetle (Coleoptera: Lucanidae).

Bell-shaped ciliates of the subclass Peritrichia, such as Vorticella, Carchesium and Epistylis, are commonly found in freshwater and other aquatic environments, either solitary or colonial. Peritrichs attach to a substratum via a contractile or non-motile stalk, and collect food particles by water current using ciliary rows around the edge of the bell, called the peristome. Some peritrichs are epibiotic and ectocommensalistic associates of aquatic insects and other animals, settling on the surface of their specific hosts. Only a few peritrichs are known to establish a more internal association with their hosts, locating within the preoral cavity or esophagus of water beetles and presumably subsisting on food materials chewed and ingested by the insects. To date, no endoparasitic or endocommensalistic peritrichs have been reported from insects. Host insects reported to date have all been aquatic, and given the aquatic lifestyle of peritrichs, terrestrial hosts have been considered unlikely. In the present study, we report a dense population of bizarre microbes within the gut of a terrestrial insect, and histological, ultrastructural and molecular phylogenetic analyses identified it as a peritrich ciliate. The highly-developed hindgut of the stag beetle Aegus currani contained oval colonial peritrichs connected by branched stalks resembling grape clusters. Each zooid exhibited a reduced peristome without disc, a vestibulum with active ciliary movement inside, and an elongated macronucleus. These features are morphologically reminiscent of but distinct in some respects from those in Operculariella parasitica, known from the esophagus of dysticid diving beetles. Taxonomic, ecological and functional aspects of this gut-dwelling peritrich warrant future study.

The presence of a mycangium in European Sinodendron cylindricum (Coleoptera: Lucanidae) and the associated yeast symbionts.

Part of the exoskeleton of some wood-inhabiting insects is modified to form a mycangium, which is a specialized organ used to convey fungal spores or yeasts to their offspring. Although most stag beetles (Coleoptera: Lucanidae) are known to have female-specific mycangia and associated yeast symbionts, the evolutionary origin of the mycangium in this group remains unresolved. Here, we report the presence of a mycangium and associated yeast symbionts in the European horned stag beetle Sinodendron cylindricum (L.), which belongs to an ancestral clade of the Lucanidae. The mycangium of S. cylindricum is shown to be female-specific and have the same developmental origin as that of other stag beetles. A total of five yeast strains were isolated from adult mycangia and larval gut of S. cylindricum Of these, we suggest that SICYAM1 is an undescribed yeast with taxonomic novelty, and have identified SICYLG3 as the xylose-fermenting yeast Scheffersomyces insectosa using nuclear ribosomal RNA and ITS sequences. The remaining three yeast strains, SICYAM2, SICYLG1, and SICYLG2, were assigned to the genus Sugiyamaella Yeast density in the adult mycangium was lower than that of the more evolutionarily advanced stag beetles, the European Lucanus cervus (L.) and Dorcus parallelipipedus (L.), which were also examined in this study. No living yeasts were isolated from the adult guts. However, a third instar larva of S. cylindricum harbored 10(4)-10(6) living yeasts in each gut region, which suggests that gut yeasts play an important role in these wood-feeding larvae.

Suppression of Bedbug's Reproduction by RNA Interference of Vitellogenin.

Recent resurgence of the bedbug Cimex lectularius is a global problem on the public health. On account of the worldwide rise of insecticide-resistant bedbug populations, exploration of new approaches to the bedbug control and management is anticipated. In this context, gene silencing by RNA interference (RNAi) has been considered for its potential application to pest control and management, because RNAi enables specific suppression of target genes and thus flexible selection of target traits to be disrupted. In this study, in an attempt to develop a control strategy targeting reproduction of the bedbug, we investigated RNAi-mediated gene silencing of vitellogenin (Vg), a major yolk protein precursor essential for oogenesis. From the bedbug transcriptomes, we identified a typical Vg gene and a truncated Vg gene, which were designated as ClVg and ClVg-like, respectively. ClVg gene was highly expressed mainly in the fat body of adult females, which was more than 100 times higher than the expression level of ClVg-like gene, indicating that ClVg gene is the primary functional Vg gene in the bedbug. RNAi-mediated suppression of ClVg gene expression in adult females resulted in drastically reduced egg production, atrophied ovaries, and inflated abdomen due to hypertrophied fat bodies. These phenotypic consequences are expected not only to suppress the bedbug reproduction directly but also to deteriorate its feeding and survival indirectly via behavioral modifications. These results suggest the potential of ClVg gene as a promising target for RNAi-based population management of the bedbug.

Symbiont-supplemented maternal investment underpinning host's ecological adaptation.

Maternal investment for offspring's growth and survival is widespread among diverse organisms. Vertical symbiont transmission via maternal passage is also pivotal for offspring's growth and survival in many organisms. Hence, it is expected that vertical symbiont transmission may coevolve with various organismal traits concerning maternal investment in offspring. Here we report a novel phenotypic syndrome entailing morphological, histological, behavioral, and ecological specializations for maternal investment and vertical symbiont transmission in stinkbugs of the family Urostylididae. Adult females develop huge ovaries exaggerated for polysaccharide excretion, possess novel ovipositor-associated organs for vertical transmission of a bacterial symbiont ("Candidatus Tachikawaea gelatinosa"), and lay eggs covered with voluminous symbiont-supplemented jelly. Newborns hatch in midwinter, feed solely on the jelly, acquire the symbiont, and grow during winter. In spring, the insects start feeding on plant sap, wherein the symbiont localizes to a specialized midgut region and supplies essential amino acids deficient in the host's diet. The reduced symbiont genome and host-symbiont cospeciation indicate their obligate association over evolutionary time. Experimental deprivation of the jelly results in nymphal mortality, whereas restoration of the jelly leads to recovered nymphal growth, confirming that the jelly supports nymphal growth in winter. Chemical analyses demonstrate that the galactan-based jelly contains a sufficient quantity of amino acids to sustain nymphal growth to the third instar. The versatile biological roles of the symbiont-containing egg-covering jelly highlight intricate evolutionary interactions between maternal resource investment and vertical symbiont transmission, which are commonly important for offspring's growth, survival, and ecological adaptation.

Gene expression in gut symbiotic organ of stinkbug affected by extracellular bacterial symbiont.

The bean bug Riptortus pedestris possesses a specialized symbiotic organ in a posterior region of the midgut, where numerous crypts harbor extracellular betaproteobacterial symbionts of the genus Burkholderia. Second instar nymphs orally acquire the symbiont from the environment, and the symbiont infection benefits the host by facilitating growth and by occasionally conferring insecticide resistance. Here we performed comparative transcriptomic analyses of insect genes expressed in symbiotic and non-symbiotic regions of the midgut dissected from Burkholderia-infected and uninfected R. pedestris. Expression sequence tag analysis of cDNA libraries and quantitative reverse transcription PCR identified a number of insect genes expressed in symbiosis- or aposymbiosis-associated patterns. For example, genes up-regulated in symbiotic relative to aposymbiotic individuals, including many cysteine-rich secreted protein genes and many cathepsin protease genes, are likely to play a role in regulating the symbiosis. Conversely, genes up-regulated in aposymbiotic relative to symbiotic individuals, including a chicken-type lysozyme gene and a defensin-like protein gene, are possibly involved in regulation of non-symbiotic bacterial infections. Our study presents the first transcriptomic data on gut symbiotic organ of a stinkbug, which provides initial clues to understanding of molecular mechanisms underlying the insect-bacterium gut symbiosis and sheds light on several intriguing commonalities between endocellular and extracellular symbiotic associations.

Fungal farming in a non-social beetle.

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

Reductive genome evolution, host-symbiont co-speciation and uterine transmission of endosymbiotic bacteria in bat flies.

Bat flies of the family Nycteribiidae are known for their extreme morphological and physiological traits specialized for ectoparasitic blood-feeding lifestyle on bats, including lack of wings, reduced head and eyes, adenotrophic viviparity with a highly developed uterus and milk glands, as well as association with endosymbiotic bacteria. We investigated Japanese nycteribiid bat flies representing 4 genera, 8 species and 27 populations for their bacterial endosymbionts. From all the nycteribiid species examined, a distinct clade of gammaproteobacteria was consistently detected, which was allied to endosymbionts of other insects such as Riesia spp. of primate lice and Arsenophonus spp. of diverse insects. In adult insects, the endosymbiont was localized in specific bacteriocytes in the abdomen, suggesting an intimate host-symbiont association. In adult females, the endosymbiont was also found in the cavity of milk gland tubules, which suggests uterine vertical transmission of the endosymbiont to larvae through milk gland secretion. In adult females of Penicillidia jenynsii, we discovered a previously unknown type of symbiotic organ in the Nycteribiidae: a pair of large bacteriomes located inside the swellings on the fifth abdominal ventral plate. The endosymbiont genes consistently exhibited adenine/thymine biased nucleotide compositions and accelerated rates of molecular evolution. The endosymbiont genome was estimated to be highly reduced, ~0.76 Mb in size. The endosymbiont phylogeny perfectly mirrored the host insect phylogeny, indicating strict vertical transmission and host-symbiont co-speciation in the evolutionary course of the Nycteribiidae. The designation 'Candidatus Aschnera chinzeii' is proposed for the endosymbiont clade.

Laccase2 is required for cuticular pigmentation in stinkbugs.

During the maturation of insect cuticle, protein-protein and protein-chitin crosslinkages are formed by the action of diphenoloxidases. Two types of diphenoloxidases, laccases and tyrosinases, are present in the insect cuticle. In coleopteran and hymenopteran insects, laccase2 gene has been identified as encoding an enzyme principally responsible for cuticular pigmentation and hardening, whereas biological roles of laccase genes in hemimetabolous insects remain to be established. Here we identified laccase2 genes from three hemipteran stinkbugs, Riptortus pedestris (Alydidae), Nysius plebeius (Lygaeidae) and Megacopta punctatissima (Plataspidae). In R. pedestris, laccase2 gene was highly expressed in epidermal tissues prior to molting. When the gene expression was suppressed by an RNA interference technique, cuticular pigmentation after molting were blocked depending on the dose of injected double-stranded RNA targeting the laccase2 gene. Similar results were obtained for N. plebeius and M. punctatissima. In all the stinkbug species, injecting 20 ng of double-stranded RNA was sufficient to prevent the cuticular maturation. These results indicate that laccase2 gene is generally required for cuticular pigmentation in different stinkbug families, highlighting its conserved biological function across diverse insect taxa.

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

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

Are stag beetles fungivorous?

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