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Phytophagous insect larvae feed on plants containing secondary metabolic products with biological activity against other predatory organisms. Phytophagous insects can use their specialised metabolic systems to covert these secondary metabolic products into compounds with therapeutic properties useful to mankind. Some Asians drink tea decoctions made from phytophagous insect frass which is believed to be effective against inflammatory diseases. However, insects that can convert plant-derived secondary metabolic products into useful human therapeutic agents remain poorly studied. Here, we constructed the TUATinsecta database by integrating publicly plant/insect datasets for the purpose of selecting insect species. Using TUAT-insecta we selected the Asian swallowtail butterfly, Papilio xuthus larvae fed on several species of Rutaceous plants and examined whether the plant-derived secondary metabolites, especially those present in frass, were chemically altered or not. We extracted metabolic products from frass using three organic solvents with different polarities, and evaluated solvent fractions for their cytotoxic effects against several human cell lines. We found that chloroform frass extracts from P. xuthus larvae fed on Poncirus trifoliata leaves contained significant cytotoxic activity. Our findings demonstrate that screening of insect species using the ‘TUATinsecta’ database provides an important pipeline for discovering novel therapeutic agents that might be useful for mankind.

Perhaps, oxidative stress progresses pupation in some Lepidopteran insects; however, the reasons for this remain obscure. In our previous study, we clarified Bombyx mori SOD1 (BmSOD1) and B. mori SOD2 (BmSOD2) proteins respond in common to ultraviolet irradiation (UV) oxidative stress and metamorphosis. This result strongly suggested pupation initiates by oxidative stress and might mediate by down-regulation of expression of BmSOD1 and BmSOD2 proteins. Thus, we examined about these relationships in B. mori in this study. In the microarray data reanalysis, we found the Notch signaling pathways as the common pathways in pupation and UV oxidative stress in B. mori. Also, we showed a molting hormone, 20-hydroxyecdysone, leads not only generation of superoxide but also downregulation of the expression of BmSOD proteins during pupation in B. mori. Our findings can contribute to a deeper understanding of how biological defense systems work against environmental oxidative stress.

Objective Insects are the most evolutionarily successful groups of organisms, and this success is largely due to their flight ability. Interestingly, some stick insects have lost their flight ability despite having wings. To elucidate the shift from wingless to flying forms during insect evolution, we compared the nutritional metabolism system among flight-winged, flightless-winged, and flightless-wingless stick insect groups. Results Here, we report RNA sequencing of midgut transcriptome of Entoria okinawaensis, a prominent Japanese flightless-wingless stick insect, and the comparative analysis of its transcriptome in publicly available midgut transcriptomes obtained from seven stick insect species. A gene enrichment analysis for differentially expressed genes, including those obtained from winged vs wingless and flight vs flightless genes comparisons, revealed that carbohydrate metabolic process-related genes were highly expressed in the winged stick insect group. We also found that the expression of the mitochondrial enolase superfamily member 1 transcript was significantly higher in the winged stick insect group than in the wingless stick insect group. Our findings could indicate that carbohydrate metabolic processes are related to the evolutionary process through which stick insects gain the ability of flight.

For the first time, we have fabricated insect-derived cell sheets by using temperature-responsive culture surfaces having a phase-transition temperature below 25°C. We prepared the temperature-resp...

Temperature is one of the most influential factors for animals. The acclimation (rearing) and challenge temperatures are often more important than the given temperature per se. These effects on physiological responses have been known, but not well understood on immune responses. Here, we investigated the integrated effects of rearing and challenge temperatures on haemocyte populations in larvae of a plusiine moth, Chrysodeixis eriosoma. We hypothesize that the haemocyte concentration is decreased (increased) at higher (lower) temperatures from rearing temperatures and that the proportions of haemocyte types exhibit directional changes at higher (lower) temperatures to compensate for immune reactions. We expect that increasing (decreasing) the challenge temperature from the rearing temperature enhances (reduces) phagocytic activity. We found that higher temperatures slightly decreased the haemocyte concentration. We detected small changes in the proportions of haemocyte types among rearing temperatures, but the changes were non‐directional and most of them were statistically insignificant. We also found the integrated effects only with increases in the challenge temperatures, which resulted in increased phagocytosis, whereas no apparent reactions were detected with decreases in the challenge temperatures. Our results show that the haemocyte concentration is significantly affected by the rearing temperature, which implies that haematopoiesis depends on the ambient temperature.We discuss some adaptive and non‐adaptive components for the positive integrated effects of increases in the challenge temperatures. We also discussed the obtained non‐responsiveness in the integrated effects with decreases in the challenge temperatures., Physiological Entomology, Article in Press.

BackgroundPolyembryony is defined as the formation of several embryos from a single egg. This phenomenon can occur in humans, armadillo, and some endoparasitoid insects. However, the mechanism underlying polyembryogenesis in animals remains to be elucidated. The polyembryonic parasitoid waspCopidosoma floridanumoviposits its egg into an egg of the host insect; eventually, over 2000 individuals will arise from one egg. Previously, we reported that polyembryogenesis is enhanced when the juvenile hormone (JH) added to the culture medium in the embryo culture. Hence, in the present study, we performed RNA sequencing (RNA-Seq) analysis to investigate the molecular mechanisms controlling polyembryogenesis ofC. floridanum. Functional annotation of genes is not fully available forC.floridanum; however, whole genome assembly has been archived. Hence, we constructed a pipeline for gene functional annotation inC. floridanumand performed molecular network analysis. We analyzed differentially expressed genes between control and JH-treated molura after 48 h of culture, then used the tblastx program to assign wholeC. floridanumtranscripts to human gene.ResultsWe obtained 11,117 transcripts in the JH treatment group and identified 217 differentially expressed genes compared with the control group. As a result, 76% ofC. floridanumtranscripts were assigned to human genes. Gene enrichment analysis revealed genes associated with platelet degranulation, fatty acid biosynthesis, cell morphogenesis in the differentiation and integrin signaling pathways were fluctuated following JH treatment. Furthermore, Cytoscape analysis revealed a molecular interaction that was possibly associated with polyembryogenesis .ConclusionsWe have constructed a pipeline for gene functional annotation ofC. floridanum, and identified transcripts with high similarity to human genes during early embryo developmental. Additionally, this study reveals new molecular interactions associated with polyembryogenesis; these interactions could indicate the molecular mechanisms underlying polyembryony. Our results highlight the potential utility of molecular interaction analysis in human twins.

Human intestinal absorption is estimated using a human colon carcinoma cell line (Caco-2) cells from human colorectal adenocarcinoma, intestinal perfusion, or a mammalian model. These current evaluation systems are limited in their ability to estimate human intestinal absorption. In addition, in vivo evaluation systems using laboratory animals such as mice and rats entail animal ethics problems, and it is difficult to screen compounds on a large scale at the drug discovery stage. Thus, we propose the use of Bombyx mori larvae for evaluation of intestinal absorption of compounds as an alternative system in this study. First, to compare the characteristics among Caco-2 cells, human intestine, and B. mori larval midgut, we analyzed their RNA-seq data, and we found 26 drug transporters common to humans and B. mori. Next, we quantitatively developed an oral administration technique in B. mori and established a method using silkworm B. mori larvae that can easily estimate the intestinal permeability of compounds. Consequently, we could determine the dose and technique for oral administration in B. mori larvae. We also developed a B. mori model to evaluate the intestinal permeability of orally administered. Our constructed evaluation system will be useful for evaluating intestinal permeability in medical drug development.

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Sperm polymorphism is widely known in invertebrates. In insects, Lepidoptera has two types of sperm: nucleated eupyrene (fertile) sperm and anucleated apyrene (unfertile) sperm. These sperm types are produced during post-embryogenesis, and eupyrene spermatogenesis precedes apyrene spermatogenesis. During overwintering, spermatogenesis stops and a portion of undifferentiated-stage spermatocytes degenerate. After overwintering, spermatogenesis restarts with unaffected spermatogonia. However, how new spermatozoa arise in the adult testes after overwintering is not known in Lepidoptera. In this study, we investigated the spermatogenesis events in the nymphalid butterfly Polygonia c-aureum after overwintering under various environmental conditions. Our results indicate that both eupyrene and apyrene spermatogenesis restart at any stopping stage and sperm of these types are regenerated in no particular order after adult insect overwintering. This suggests that the spermatogenesis occurring after overwintering proceeds without embryogenetic restrictions related to the developmental sequence.

The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes. Electronic supplementary material The online version of this article (doi:10.1007/s11626-017-0140-7) contains supplementary material, which is available to authorized users.

Self-sacrifice is very rare among organisms. Here, we report a new and astonishing case of adaptive self-sacrifice in a polyembryonic parasitic wasp, Copidosoma floridanum. This wasp is unique in terms of its larval cloning and soldier larvae. Male clone larvae have been found to be killed by female soldier larvae, which suggests intersexual conflict between male and female larvae. However, we show here that mass killing is adaptive to all the killed males as well as the female soldiers that have conducted the killing because the killing increases their indirect fitness by promoting the reproduction of their clone sibs. We construct a simple model that shows that the optimal number of surviving males for both male and female larvae is very small but not zero. We then compare this prediction with the field data. These data agree quite well with the model predictions, showing an optimal killing rate of approximately 94–98% of the males in a mixed brood. The underlying mechanism of this mass kill is almost identical to the local competition for mates that occurs in other wasp species. The maternal control of the sex ratio during oviposition, which is well known in other hymenopterans, is impossible in this polyembryonic wasp. Thus, this mass kill is necessary to maximize the fitness of the female killers and male victims, which can be seen as an analogy of programmed cell death in multicellular organisms.

Some polyembryonic parasitoid species produce two morphologically different types of larvae from a single egg. The specialized larval morph that forms in addition to the reproductive larvae has been variously described as asexual (Silvestri 1906), teratoid (Parker and Thompson 1928), and precocious (Doutt 1947, 1952) larvae and is characterized by its slender body and developed mandibles. In Copidosoma floridanum, many reproductive larvae appear at the final-instar stage of the host and eventually develop into adults, whereas a small number of precocious larvae first appear at the early-instar stage of the host and then die without undergoing metamorphosis (Fig. 4.1). Thus, the precocious larvae represent a sterile caste. There are two hypotheses for why precocious larvae are produced: (1) to act as soldier larvae that defend sibling embryos against competitors invading the same host and (2) to adjust the sex ratio in mixed-sex broods. Therefore, the function and evolution of precocious larvae have mostly been examined from the viewpoint of sociobiology.

The phenomenon of polyembryony in insects was first recognized by Marchal (1898) in the chalcidoid family Encyrtidae. This large family of small, solitary egg–larval endoparasitoids includes 460 genera, two of which exhibit polyembryony: Ageniaspis and Copidosoma (Ivanova-Kasas 1972).

Entry into the host body is a prerequisite for successful completion of the endoparasitoid life cycle. Most endoparasitoids achieve this by laying eggs directly inside the body cavity of the host. In most braconid and ichneumonid parasitoids, the ovipositor is inserted within the host hemocoel to lay eggs, and the hatched larvae grow and develop rapidly inside the host hemolymph as host development advances and finally consume the host tissues, leading to death of the host, which is why parasitic wasps are usually referred to as “parasitoids.” By contrast, egg–larval endoparasitoids, such as those in the genus Ascogaster (family Braconidae), lay their eggs inside the host embryo or alternatively in the yolk of the host egg, following which the newly hatched larvae enter the host embryo. However, the polyembryonic egg–larval endoparasitoid Copidosoma floridanum cannot employ this strategy due to its prolonged morula stage, so this species has evolved a novel approach for entering the host body that involves tissue-compatible invasion by the motile morula.

Polyembryony is a unique form of asexual reproduction whereby multiple offspring are produced from a single egg or zygote. To use Craig et al.’s (1997) words, polyembryony is a paradoxical combination of two reproductive modes—sexual and asexual reproduction. Nevertheless, it has evolved and been maintained in a wide range of taxa, including rust fungi (Alexopoulos 1952; Craig et al. 1997), algae (Searles 1980), and animals (Craig et al. 1997; Skold et al. 2009), including the well-known phenomenon of identical twins in humans.

Sexual reproduction is the most common mode of reproduction in many multicellular organisms, including insects. The evolutionary success of sexual reproduction has been attributed to the generation of variation among offspring, which is important for the survival and future reproduction of the population. Consequently, populations that reproduce sexually can leave more offspring than those that reproduce asexually. This variation is created by the development of heritable mutations in the germ-cell lines in sexually reproducing organisms. These mutations are continuously reshuffled by the mixing and recombination of genes from two parents and are transferred to the next generation. More importantly, sexual reproduction will also eliminate accumulated, often harmful, alterations in the DNA that occur during meiosis and recombination—that is, individuals with harmful genes are unable to pass their genes to the next generation as a result of natural selection. On the other hand, the harmless portion of genes that are created during the process of meiosis from the harmful mutated genes may increase the chance of survival. By contrast, asexual reproduction results in the transfer of the full maternal genotype, which must be optimal in the present environment, and so asexually reproducing individual can successfully colonize new habitats and develop immediately (Williams 1975).

Synergy is known to be vital for the group collaboration among non-kin individuals. In order to evaluate the condition of synergy that initiates group living, we build a model of food intake based on three types of functional response. We show that type III functional response is prerequisite for synergy to allow group living.The optimal number of gathering individuals can be also evaluated from Type III functional response curve. Type III functional response consists of terms depending linearly and bilinearly on the number of individuals and the bilinear term represents synergy. For a fixed value of the linear coefficient, there are upper and lower boundaries of the bilinear coefficient for synergistic collaboration. The dilution effect can be incorporated into the model through the functional response of the predator. Thus, the functional response of the predator as well as that of the prey contribute to the group living of the prey. Our model shows that group livings can be categorized into three types, namely those due to (1) synergy effect of the own group, (2) dilution effect against predators, and (3) both effects contributing together. The predator's functional response plays a decisive role in the last two types, where the predator response should be of anti-Type III (i.e., Type II)., Ecological Complexity, 44, art.no.100874; 2020

Insects are well adapted to changing environmental conditions. They have unique systems for eliminating reactive oxygen species (ROS). Superoxide dismutase (SOD) is a key enzyme that plays a primary role in removing ROS. Bombyx mori is a lepidopteran insect, whose body size is larger than the model insect Drosophila melanogaster, which enabled us to more easily examine gene expression at the tissue level. We searched B. mori SOD (BmSOD) genes using genome database, and we analyzed their function under different type of oxidative stress. Consequently, we identified four new types of BmSODs in addition to the three types already known. Two of the seven types had a unique domain architecture that has not been discovered previously in the SOD family, and they were expressed in different tissues and developmental stages. Furthermore, these BmSODs responded differently to several kinds of stressors. Our results showed that the seven types of BmSODs are likely to play different roles in B. mori; therefore, B. mori could be used to distinguish the functions of each SOD for resistance to oxidative stress that changes with the environmental conditions.

Human intestinal absorption is estimated using a human colon carcinoma cell line (Caco-2) cells from human colorectal adenocarcinoma, intestinal perfusion, or a mammalian model. These current evaluation systems are limited in their ability to estimate human intestinal absorption. In addition, in vivo evaluation systems using laboratory animals such as mice and rats entail animal ethics problems, and it is difficult to screen compounds on a large scale at the drug discovery stage. Thus, we propose the use of Bombyx mori larvae for evaluation of intestinal absorption of compounds as an alternative system in this study. First, to compare the characteristics among Caco-2 cells, human intestine, and B. mori larval midgut, we analyzed their RNA-seq data, and we found 26 drug transporters common to humans and B. mori. Next, we quantitatively developed an oral administration technique in B. mori and established a method using silkworm B. mori larvae that can easily estimate the intestinal permeability of compounds. Consequently, we could determine the dose and technique for oral administration in B. mori larvae. We also developed a B. mori model to evaluate the intestinal permeability of orally administered. Our constructed evaluation system will be useful for evaluating intestinal permeability in medical drug development.

Armigeres subalbatus (Coquillett) is a medically important mosquito and a model species for immunology research. We successfully established two cell lines from the neonate larvae of A. subalbatus using two different media. To our knowledge, this is the first report of an established Armigeres mosquito cell line. The cell lines, designated as Ar-3 and Ar-13, consist of adherent and diploid cells with compact colonies. Both these cell lines grow slowly after passage at a split ratio of 1:5 and a population doubling time of 2.7 and 3.0 d, respectively. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to confirm that these lines correspond to the species of origin and are clearly distinct from seven other insect cell lines. Furthermore, reverse-transcription PCR was used to demonstrate that the Ar-3 cell line is susceptible to the Japanese encephalitis virus and two insect flaviviruses associated with Culex and Aedes mosquitoes but relatively insensitive to dengue virus. These data indicate that the newly established cell lines are cellular models of A. subalbatus as well as beneficial tools for the propagation of viruses associated with the Armigeres mosquito.

The male sex pheromone of the longicorn beetle, Xylotrechus pyrrhoderus pyrrhoderus Bates (Cerambycidae: Tribe Clytini) plays an important role in attracting females. This pheromone is produced by the pheromone gland located in the prothorax. However, the detailed structure and underlying developmental process of this gland are still unknown. We investigated the gland structure by using histological analysis and confirmed that the gland consists of the following parts: gland cell mass, a unique spherical space in the cuticle layer, and ductules connecting the gland cells with the spherical space and conducting canals to the outer opening. The gland structure first appeared male-specific in the late pupal stage, during which the epidermal cells began depositing the exocuticle; the development of the gland was completed after adult emergence. Furthermore, we verified the structural equivalents of the X. p. pyrrhoderus male pheromone gland in 11 species of 2 tribes, Clytini and Anaglyptini. The glands of these insects could be classified into four types on the basis of the absence or presence of the spherical space and the division of the gland cell mass layer. Most noteworthy, all the species with the spherical space and division-type gland were restricted to the Xylotrechus clade, as inferred from the molecular phylogenetic analysis. These results suggest that Clytini and Anaglyptini species share a fundamental process of male pheromone gland development, and that the Japanese Xylotrechus species might have established their current status by developing distinct structural features in the male pheromone gland.

Males of the cerambycid beetle Xylotrechus pyrrhoderus release a mixture of (S)-2-hydroxy-3-octanone [(S)-1] and (2S,3S)-2,3-octanediol [(2S,3S)-2] as a sex pheromone that attracts conspecific females. The chemical structures of these pheromone components include a common motif and are assumed to be biosynthetically related. Here, we show that deuterated (S)-1, applied on the cuticle of a pronotal pheromone gland, was converted into (2S,3S)-2, that included deuterium atoms, but a reverse conversion did not take place. These results reveal a carbonyl reductase to be active in the pheromone gland, and that the ketol is a biosynthetic precursor of the diol. Males did not produce (R)-1; however, deuterated (R)-1 was converted into (2R,3R)-2, indicating an attack of the enzyme from the opposite side of the hydroxyl group at the 2-position. Furthermore, to understand the substrate specificity of the enzyme, racemates of 2-hydroxy-3-hexanone and 2-hydroxy-3-decanone were synthesized and applied to the gland. Their conversion into the corresponding diols suggests that the enzyme reduces the carbonyl group at the 3-position, regardless of the chain length.

Background Various insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases. Results Enolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis. Conclusions We identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3455-y) contains supplementary material, which is available to authorized users.

The queenless ant, Pristomyrmex punctatus (F. Smith) reproduces parthenogenetically. The workers lay unfertilized eggs, which develop into female workers. This mode of reproduction generates hereditary clones. A previous research shows that when genetically monomorphic colonies were split, the workers tended to reassemble after being split into two groups, but when genetically polymorphic colonies split, they remained as two separate colonies. However, it remains unclear whether the workers can recognize individual genotype. Here, it was investigated whether individuals from geographically divergent, genetically monomorphic colonies would assemble with individuals of the same genotype. Two artificially fused colonies were prepared, A and B, comprising 200 individuals and 100 individuals, respectively. Each half of the artificially fused colony was composed of workers from two different genetically monomorphic source colonies. The workers assembled as a single colony when the genotype of the source colonies was identical. However, when the genotypes of source colonies were different, the workers did not assemble into one colony, but split into two groups according to genotype. These results suggest that P. punctatus can potentially recognize individual genotype, and select colony members based on an individual’s genotype.

We developed and characterized nine microsatellite loci for the polyembryonic wasp Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae). Eight of the nine loci were polymorphic and did not deviate from Hardy–Weinberg equilibrium. Our results will contribute to studies on the population genetic structure of C. floridanum.

Defense against enemies is a type of natural selection considered fundamentally equivalent between the sexes. In reality, however, whether males and females differ in defense strategy is unknown. Multiparasitism necessarily leads to the problem of defense for a parasite (parasitoid). The polyembryonic parasitic wasp Copidosoma floridanum is famous for its larval soldiers’ ability to kill other parasites. This wasp also exhibits sexual differences not only with regard to the competitive ability of the soldier caste but also with regard to host immune enhancement. Female soldiers are more aggressive than male soldiers and their numbers increase upon invasion of the host by other parasites. In this report, in vivo and in vitro competition assays were used to test whether females have a toxic humoral factor; if so, then its strength was compared with that of males. We found that females have a toxic factor that is much weaker than that of males. Our results imply sexual complementarity between host humoral toxicity and larval soldiers. We discuss how this sexual complementarity guarantees adaptive advantages for both males and females despite the one-sided killing of male reproductives by larval female soldiers in a mixed-sex brood.

Insect gustatory receptors (Grs) are members of a large family of proteins with seven transmembrane domains that provide insects with the ability to detect chemical signals critical for feeding, mating, and oviposition. To date, 69 Bombyx mori Grs (BmGrs) genes have been identified via genome studies. BmGr9 has been shown to respond specifically to fructose and to function as a ligand-gated ion channel selectively activated by fructose. However, the sites where this Gr are expressed remain unclear. We demonstrated using reverse transcription (RT)-PCR that BmGr9 is widely expressed in the central nervous system (CNS), as well as oral sensory organs. Additionally, immunohistochemistry was performed using anti-BmGr9 antiserum to show that BmGr9 is expressed in cells of the oral sensory organs, including the maxillary galea, maxillary palps, labrum, and labium, as well as in putative neurosecretory cells of the CNS. Furthermore, double immunohistochemical analysis showed that most BmGr9-expressing cells co-localized with putative neuropeptide F1-expressing cells in the brain, suggesting that BmGr9 is involved in the promotion of feeding behaviors. In addition, a portion of BmGr9-expressing cells in the brain co-localized with cells expressing BmGr6, a molecule of the sugar receptor clade, suggesting that sugars other than fructose are involved in the regulation of feeding behaviors in B. mori larvae.

A large number of cells die via programmed cell death during the normal development of the Drosophila optic lobe. In this study, we report the precise spatial and temporal pattern of cell death in this organ. Cell death in the developing optic lobe occurs in two distinct phases. The first phase extends from the start of metamorphosis to the mid-pupal stage. During this phase, a large number of cells die in the optic lobe as a whole, with a peak of cell death at an early pupal stage in the lamina and medulla cortices and the region of the T2/T3/C neurons, and a smaller number of dead cells observed in the lobula plate cortex. The second phase extends from the mid-pupal stage to eclosion. Throughout this period, a small number of dying cells can be observed, with a small peak at a late pupal stage. Most of the dying cells are neurons. During the first phase, dying cells are distributed in specific patterns in cortices. The lamina cortex contains two distinct clusters of dying cells; the medulla cortex, four clusters; the lobula plate cortex, one cluster; and the region of the T2/T3/C neurons, one cluster. Many of the clusters maintain their distinct positions in the optic lobe but others extend the region they cover during development. The presence of distinct clusters of dying cells at different phases suggests that distinct mechanisms control cell death during different stages of optic lobe development in Drosophila.

The queenless ant, Pristomyrmex punctatus (F. Smith) reproduces parthenogenetically. Workers lay unfertilized eggs, which develop into female workers. This mode of reproduction generates hereditary clones, though a colony is not necessarily constructed from single clonal line. In a previous study, where a colony was separated into two subcolonies, it was found that genetically monomorphic colonies tended to reassemble but genetically polymorphic colonies did not. Here, we used multiple clonal colonies to investigate whether P. punctatus could recognize “individual” relatedness and assemble with subcolony members of the same clonal type. Results show individuals did not assemble with individuals of the same clonal type in each subcolony. This suggests they cannot recognize individual relatedness.

The biology of the immature stage of Neochrysocharis formosa, a larval idiobiont-endoparasitoid of agromyzid leaf miners, was studied to gain a better understanding of N. formosa and in vitro rearing. Eggs have a smooth surface with a thin, transparent chorion. The length and width are about 190 μm and 60 μm, respectively. Eggs hatched in an average of 32.6 h. Three larval stages are decided by observation of the pre-ecdysis phase. Larvae are hymenopteriform with small mandibles and have 13 body segments. Larval duration at each instar is as follows: 1st instar 35.1 h, 2nd instar 35.7 h and 3rd instar 46.5 h. Mature 3rd-instar larvae escaped from the host and pupated within the mine of the host larvae. Pupal duration was 8 days. The host larvae stopped moving a few minutes after parasitoid oviposition. To examine the oviposition site, longitudinal sections of parasitized host larvae showed all eggs to be within the host’s hemocoel. Fewer eggs were oviposited in the anterior part than the middle and posterior part of the host larvae. No encapsulation or melanization were observed around the parasitoid eggs.

Adult males of the grape borer, Xylotrechus pyrrhoderus, secrete (S)-2-hydroxy-3-octanone [(S)-1] and (2S,3S)-2,3-octanediol [(2S,3S)-2] from their nota of prothoraces as sex pheromone components. Their structural similarity suggests that one of them is the biosynthetic precursor of the other component. In order to confirm the biochemical conversion, deuterated derivatives of both components were synthesized by starting from a Wittig reaction between hexanal and an ylide derived from D(5)-iodoethane and ending with enantiomeric resolution by chiral HPLC. The molecular ions of 1 and 2 could scarcely be detected by using a GC-MS analysis, and the labeled compounds showed similar mass spectra to the unlabeled pheromone components. However, several fragment ions, including four deuterium atoms, were observed in the mass spectra of their acetate derivatives, indicating that the conversion could be confirmed by examining a compound with the diagnostic ions after acetylation of the volatiles collected from insects treated with the labeled precursors.

We present a bioactuator powered by insect dorsal vessel tissue which can work for a long time at room temperature without maintenance. Previously reported bioactuators which exploit contracting ability of mammalian heart muscle cell have required precise environmental control to keep the cell alive and contracting. To overcome this problem, we propose a bioactuator using dorsal vessel tissue. The insect tissue which can grow at room temperature is generally robust over a range of culture conditions compared to mammalian tissues and cells. First, we confirm that a dorsal vessel tissue of lepidoptera larva Ctenoplusia agnata contracts spontaneously for at least 30 days without medium replacement at 25 degrees C. Using the dorsal vessel tissue cultured under the same conditions, we succeed in driving micropillars 100 microm in diameter and 1000 microm in height for more than 90 days. The strongest displacement of the micropillar top occurs on the 42(nd) day and is 23 microm. Based on these results, the contracting force is roughly estimated as 4.7 microN which is larger than that by a few mammalian cardiomyocytes (3.4 microN). Definite displacements of more than 10 microm are observed for 58 days from the 15(th) to the 72(nd) days. The number of life cycles can be roughly calculated as 7.5 x 10(5) times for the average frequency of about 0.15 Hz, which is no less than that of conventional mechanical actuators. These results suggest that the insect dorsal vessel tissue is a more promising material for bioactuators used at room temperature than other biological cell-based materials.

Here we propose a bioactuator using an insect dorsal vessel (heart muscle) tissue which can drive autonomously at room temperature for a long term. Living muscle cells have been attracted much interest as an actuator because a muscle tissue is totally superior to artificial muscles. Previously reported bioactuators using mammalian heart muscle cells require precise environmental control to keep cells alive and contracting. On the other hand, an insect tissue is robust compared to mammalian cells; they can grow at room temperature for a long term without medium replacement. In this paper, we succeeded in driving micropillars by the dorsal vessel tissue for more than 90 days. The strongest displacement was 23 [μm] . Based on the results of displacement, the driving force was estimated 4.7 [μN] . The definite displacements more than 10 [μm] were observed for 58 days from the 15th day to the 72nd day. Therefore, the life cycle can be calculated at 7.5×105 times as the average frequency was about 0.15 [Hz] . These results suggest that the insect heart tissue is more promising material for a bioactuator at room temperature compared with the other biological cell based actuators.

Crude extracts from larvae, pupae and adults of cabbage white butterflies, Pieris rapae and Pieris brassicae, and green-veined butterfly, Pieris napi, have an ability to induce apoptosis in the human cancer cell lines. As apoptosis inducing protein, pierisin-1 and -2 have been isolated from pupae of P. rapae and P. brassicae, respectively, and shown to exhibit DNA ADP-ribosylating activity. Although the highest activity was detected in the late phase of larvae and early phase of pupae, certain activity was found in adult butterflies. In order to investigate distribution of substances having pierisin-like activities in butterflies, many species need to be analyzed. However, fresh samples of larvae and pupae are hard to obtain, especially if samples are of scarce species or from overseas. The usage of adult butterflies is practical to examine the distribution of pierisin-like activity in many species. In this study, we examined the cytotoxicity of crude extracts from adults of P. rapae against HeLa cells and DNA ADP-ribosylation ability during storage for 1, 2 and 8 weeks at room temperature after killing adult butterflies after eclosion. Body weights decreased to 18% for 8 weeks through dehydration. Cytotoxicity of samples from butterfly kept for 1, 2 and 8 weeks decreased to 47, 39 and 22%, respectively, of the control value. DNA ADP-ribosylating activity of the samples also decreased to 30, 27 and 23%. Similar reduction was observed on western blot analysis with anti-pierisin-1 antibody. Fortunately, these results suggest that cytotoxic and DNA ADP-ribosylating activity persists to some extent in the body after killing, at least for 8 weeks. Thus, butterfly adult samples kept for two months at room temperature can still be useful for examination of the presence of substance having pierisin-like activity.

Cells must coordinate adjustments in genome expression to accommodate changes in their environment. We hypothesized that the amount of transcriptome change is proportional to the amount of environmental change. To capture the effects of environmental changes on the transcriptome, we compared transcriptome diversities (defined as the Shannon entropy of frequency distribution) of silkworm fat-body tissues cultured with several concentrations of phenobarbital. Although there was no proportional relationship, we did identify a drug concentration "tipping point" between 0.25 and 1.0 mM. Cells cultured in media containing lower drug concentrations than the tipping point showed uniformly high transcriptome diversities, while those cultured at higher drug concentrations than the tipping point showed uniformly low transcriptome diversities. The plasticity of transcriptome diversity was corroborated by cultivations of fat bodies in MGM-450 insect medium without phenobarbital and in 0.25 mM phenobarbital-supplemented MGM-450 insect medium after previous cultivation (cultivation for 80 hours in MGM-450 insect medium without phenobarbital, followed by cultivation for 10 hours in 1.0 mM phenobarbital-supplemented MGM-450 insect medium). Interestingly, the transcriptome diversities of cells cultured in media containing 0.25 mM phenobarbital after previous cultivation (cultivation for 80 hours in MGM-450 insect medium without phenobarbital, followed by cultivation for 10 hours in 1.0 mM phenobarbital-supplemented MGM-450 insect medium) were different from cells cultured in media containing 0.25 mM phenobarbital after previous cultivation (cultivation for 80 hours in MGM-450 insect medium without phenobarbital). This hysteretic phenomenon of transcriptome diversities indicates multi-stability of the genome expression system. Cellular memories were recorded in genome expression networks as in DNA/histone modifications.