Presence of non-symbiotic yeasts in a symbiont-transferring organ of a stag beetle that lacks yeast symbionts found in other stag beetles.

Dispersal from wood to wood is essential for wood-inhabiting fungi and wood-inhabiting insects play an important role in the dispersal success of such fungi. However, it is poorly understood whether wood-inhabiting insects which change the habitats from wood to non-wood environments can contribute to the fungal dispersal. Larvae of most stag beetles (Coleoptera: Lucanidae) are wood feeders, while adults are sap feeders. Female adults of lulcanids possess specialized organs (mycetangia) for transportation of fungal symbionts and harbor specific yeasts (e.g., Scheffersomyces spp.) within. Here, we report that the lucanid Aegus subnitidus harbors non-specific yeasts facultatively in mycetangia. We conducted yeast isolation from mycetangia and hindguts of female adults, in a larval gallery in wood-associated materials, and in female-visiting fermented tree sap using culture-dependent methods. Less than half of the females carried a total of 20 yeast species with small amounts using mycetangia and a female harbored up to five species, suggesting the absence of an intimate association with specific yeasts that are found in other lucanids. Yeast species compositions markedly differed between the larval gallery and sap. Most yeasts from the larval galley exhibited xylose-assimilation abilities, while few yeasts from sap did. Mycetangial yeasts comprised a combination from both sources. In hindguts, most yeasts were found in sap (> 70%) with no yeasts in the larval gallery. Sap-associated yeasts in each female mycetangium were also obtained from the female-visiting sap patch, while mycetangial, larval gallery-associated yeasts were absent from the patch, suggesting the survival success of larval gallery-associated yeasts in mycetangia through sap patches. Therefore, wood-inhabiting insects that possess mycetangia can potentially act as vectors of non-symbiotic wood-inhabiting yeasts dispersing from wood to wood via other environments.

Yeast associated with flower longicorn beetle Leptura ochraceofasciata (Cerambycidae: Lepturinae), with implication for its function in symbiosis.

Wood is difficult for most animals to digest due to large amounts of indigestible polymers, but some wood-feeding insects are considered to be able to utilize it as food with the aid of microbial symbionts. Most members of flower longicorn beetles (Coleoptera: Cerambycidae: Lepturinae) feed on nectar and pollen of flowers as adults and wood as larvae. In some lepturines, associations with yeasts are known: female adults possess fungus-storing organs (termed mycetangia) at ovipositors, and larvae also possess such organs (termed mycetomes) in their midguts to carry the associated yeasts. Despite the high diversity of Lepturinae in the world, lepturine-yeast associations, such as the consistency of associated yeasts among the beetle's developmental stages and ecological function of yeast symbionts, have been poorly documented. Here, we investigated the yeast symbiont of the Japanese common lepturine Leptura ochraceofasciata. X-ray computed microtomography revealed that a pair of tube-like, S-shaped mycetangia was located at the basal part of the ovipositor and that a muscle bundle joined the apex of the mycetangium to spiculum ventrale of sternum VIII. All female adults harbored only one yeast species, Scheffersomyces insectosa, in the mycetangia. All larvae harbored S. insectosa exclusively in the mycetomes. Scheffersomyces insectosa was also recovered from surfaces of eggs. Scheffersomyces insectosa assimilated wood-associated sugars including xylose, cellobiose, and xylan in culture. These results suggest the intimate association between L. ochraceofasciata and S. insectosa: S. insectosa is transmitted from the mother to offspring during oviposition and may be related to larval growth in wood.

Nutritional resources of the yeast symbiont cultivated by the lizard beetle Doubledaya bucculenta in bamboos.

Symbiotic fungi of wood-inhabiting insects are often considered to aid wood digestion of host insects when the associated fungi can assimilate wood-associated indigestible materials. In most cases, however, the components of wood that are utilized by fungal symbionts remain poorly understood. The lizard beetle Doubledaya bucculenta (Coleoptera, Erotylidae, Languriinae) farms the symbiotic yeast Wickerhamomyces anomalus inside the cavity of host bamboo internodes, which serves as food for larvae. To determine the carbon sources of the internodes serving as nutritional substrates for W. anomalus, we used ion exchange chromatography measurements to analyze free and structural sugar compositions in fresh pith (FP), yeast-cultured pith (YP), and larva-reared pith (LP) of internodes. Glucose and fructose were the major free sugars in FP and markedly decreased in YP and LP. For structural sugars, no sugar significantly decreased in YP or LP compared with FP. Carbon assimilation tests showed that W. anomalus assimilated glucose, mannose, fructose, and sucrose strongly, xylose and cellobiose moderately, and xylan weakly. Elemental analysis revealed that the compositions of carbon, hydrogen, and nitrogen were not significantly different among tissue types. These results suggest that W. anomalus does not consume bamboo-associated indigestible sugars but most free sugars, mainly glucose and fructose, in the pith. Our findings suggest that a symbiont's abilities may not always benefit its host in nature.

Time-dependent oviposition deterrence by an oviposition mark in a fungus-farming lizard beetle.

In insects, the selection of a suitable oviposition site is critical for offspring performance. Avoiding hosts already occupied by conspecific immatures in oviposition is one of the mechanisms to reduce intraspecific competition. The avoidance of such hosts by ovipositing females has been well-documented in parasitoid wasps and phytophagous insects but remains unknown in mycophagous insects. Here, we report that oviposition marks (OMs) of the fungus-farming lizard beetle, Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae), deter conspecific females from oviposition. The females excavate a wall of host bamboo internodes to access cavities for oviposition and plug the holes with excised bamboo fibers after oviposition. A larva completes its development within a single internode. To determine what types of OMs function as oviposition deterrents and how the oviposition-deterring pattern associates with offspring survival, we examined the oviposition behaviors, egg-period lengths, and reactions of larvae to eggs. When a single internode was provided, most females laid eggs in internodes with no OMs, incomplete OMs, or plug-removed completed OMs (COMs), but fewer females laid in internodes with fresh COMs and no females laid in those with old COMs. When eggs were reared under constant temperatures, egg-period lengths varied, suggesting that later-laid eggs sometimes hatch earlier than earlier-laid eggs. When a larva encountered an egg, all eggs were killed shortly. These results suggest that the females avoid internodes associated with high offspring mortality and that the COM's plug contains an oviposition-deterring cue. Given that larvae of oviposition-deterring insects including D. bucculenta inhabit spatiotemporally-limited sites and have limited mobility, high resource competition in the larval stage may have driven the evolution of oviposition deterrence.

Two new species of the genus Anisoderomorpha Arrow, 1925 (Coleoptera: Erotylidae: Languriinae).

The genus Anisoderomorpha Arrow, 1925 (Coleoptera: Erotylidae: Languriinae) is redescribed and two new species of Anisoderomorpha are described: A. tamdaoensis sp. nov. from Vietnam and A. malayana sp. nov. from Malaysia. The adults of A. tamdaoensis sp. nov. were found to be sexually dimorphic in external morphology, particularly in the shape of the head and mandibles: females have a large asymmetrical head and mandibles, whereas males have a symmetrical head with slightly asymmetrical mandibles. All three members of Anisoderomorpha and its related taxon, Celolanguria curvipes Arrow, 1925, are illustrated and the host plant of Anisoderomorpha is predicted based on the morphological similarity of the specified mouthparts to those of other genera.

Heads or tails: exaggerated morphologies in relation to the use of large bamboo internodes in two lizard beetles, Doubledaya ruficollis and Oxylanguria acutipennis (Coleoptera: Erotylidae: Languriinae).

The cavities of bamboos (Poaceae) are used by various animals. Most of the animals access these cavities either by existing cracks or by excavating bamboos with soft walls or small, thin-walled bamboos. Only a few animals excavate into the cavities of large and thick- and hard-walled internodes of mature bamboos. We studied two lizard beetle species (Coleoptera: Erotylidae: Languriinae), Doubledaya ruficollis and Oxylanguria acutipennis, that excavate into large internode cavities of recently dead mature bamboos and have morphological modifications. We observed that females of D. ruficollis used their mandibles to bore oviposition holes on Schizostachyum sp. (mean wall thickness = 3.00 mm) and O. acutipennis did so on Dendrocalamus sp. (3.37 mm) bamboos. Previous studies suggested that the markedly asymmetrical mandibles and needle-like ovipositors of females in the genus Doubledaya are adaptive traits for excavating hard-walled bamboos for oviposition. Therefore, we measured their mandibular lengths and ovipositor lengths. D. ruficollis females had greater asymmetry in the mandibles and shorter and less-sclerotized ovipositors than females of congeners using small bamboos. In contrast, O. acutipennis females had slightly asymmetrical mandibles and elongated, well-sclerotized ovipositors. Oviposition holes of D. ruficollis were cone-shaped (evenly tapering), whereas those of O. acutipennis were funnel-shaped (tube-like at the internal apex). This suggests that D. ruficollis females excavate oviposition holes using the mandibles only, and O. acutipennis females use both the mandibles and ovipositors. These differences suggest different oviposition-associated morphological specialization for using large bamboos: the extremely asymmetrical mandibles in D. ruficollis and elongated, needle-like ovipositors in O. acutipennis.

Fungal garden making inside bamboos by a non-social fungus-growing beetle.

In fungus-growing mutualism, it is indispensable for host animals to establish gardens of the symbiotic fungus as rapidly as possible. How to establish fungal gardens has been well-documented in social fungus-farming insects, whereas poorly documented in non-social fungus-farming insects. Here we report that the non-social, fungus-growing lizard beetle Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae) transmits the symbiotic yeast Wickerhamomyces anomalus from the ovipositor-associated mycangium into bamboo internode cavities and disperses the yeast in the cavities to make gardens. Microbial isolation and cryo-scanning electron microscopy observation revealed that W. anomalus was constantly located on the posterior ends of eggs, where larvae came out, and on the inner openings of oviposition holes. Direct observation of oviposition behavior inside internodes revealed that the distal parts of ovipositors showed a peristaltic movement when they were in contact with the posterior ends of eggs. Rearing experiments showed that W. anomalus was spread much more rapidly and widely on culture media and internodes in the presence of the larvae than in the absence. These results suggest that the ovipositors play a critical role in vertical transmission of W. anomalus and that the larvae contribute actively to the garden establishment, providing a novel case of fungal garden founding in non-social insect-fungus mutualism.

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.

Exaggerated asymmetric head morphology of female Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae) and ovipositional preference for bamboo internodes.

The lizard beetle Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae) female has evident asymmetric head morphology. The females excavate small holes in host bamboo internodes for the deposition of eggs. To understand the asymmetry and allometry of male and female adults and larvae of D. bucculenta, mandibular length, genal and head width, and elytral length were measured, and the oviposition preference for different-sized internodes of the bamboo Pleioblastus simonii and the relationship between internode size and emerging adult size were examined. Larval mandibles exhibited no clear asymmetry pattern, and genae showed fluctuating asymmetry in length. Adult male mandibles showed left-directional asymmetry, but genae showed fluctuating asymmetry. Adult female mandibles and genae exhibited marked left-directional asymmetry. The degree of asymmetry of mandibles and genae remained constant regardless of body size. Large females tended to choose large-diameter internodes of P. simonii and to lay eggs successfully, whereas small females tended to choose intermediate-diameter internodes, but to fail in oviposition, suggesting that small females pay a high cost on oviposition. There was a positive correlation between internode size and emerging adult size. Marked directional asymmetry of female mandibles and genae are discussed in relation to greater frequency of cutting bamboo fibers compared with adult males, and the traits of bamboo internode.