Complete genomes of mutualistic bacterial co-symbionts "Candidatus Sulcia muelleri" and "Candidatus Nasuia deltocephalinicola" of the rice green leafhopper Nephotettix cincticeps.

The genomes of obligate bacterial co-symbionts of the green rice leafhopper Nephotettix cincticeps, which is notorious as an agricultural pest, were determined. The streamlined genomes of "Candidatus Sulcia muelleri" and "Candidatus Nasuia deltocephalinicola" exhibited complementary metabolic pathways for synthesizing essential nutrients that contribute to host adaptation.

Genetic variations and microbiome of the poultry red mite Dermanyssus gallinae.

The poultry red mite Dermanyssus gallinae poses a significant threat to the health of hens and poultry production. A comprehensive understanding of D. gallinae is necessary to develop sustainable and efficacious control methods. Here we examined 144 D. gallinae collected from 18 poultry farms throughout the Japanese Archipelago for their genetic variations based on mitochondrial cytochrome c oxidase subunit I (COI) sequences, and microbiome variations based on amplicon sequencing of the 16S ribosomal RNA gene. According to COI sequencing, the Japanese samples were categorized into three haplogroups, which did not reflect the geographical distribution. Microbiome analyses found that the major bacteria associated with D. gallinae were Bartonella, Cardinium, Wolbachia, and Tsukamurella, with Bartonella being most predominant. Among 144 individual mites, all possessed one of the two major types of Bartonella (Bartonella sp. A), while 140 mites possessed the other type (Bartonella sp. B). The presence of the two strains of Bartonella was also confirmed by a single copy gene, rpoB. The presence of Bartonella in laid eggs suggested transovarial vertical transmission. Given that obligate blood-feeding arthropods generally require a supply of B vitamins from symbiotic bacteria, Bartonella may play an important role in mite survival. Rickettsiella, a major symbiont in European D. gallinae populations, and suggested to be an important symbiont by genomic data, was rarely found in Japanese populations. Cardinium detected from D. gallinae fell into a major clade found widely in arthropods, whereas Wolbachia detected in Japanese D. gallinae appear to be a new lineage, located at the base of Wolbachia phylogeny. Of the mitochondrial phylogeny, infection patterns of Cardinium and Wolbachia were strongly correlated, possibly suggesting one or both of the symbionts induce reproductive manipulations and increase spread in the host populations.

A new antimicrobial peptide, Pentatomicin, from the stinkbug Plautia stali.

Antimicrobial peptides (AMPs) play crucial roles in the innate immunity of diverse organisms, which exhibit remarkable diversity in size, structural property and antimicrobial spectrum. Here, we describe a new AMP, named Pentatomicin, from the stinkbug Plautia stali (Hemiptera: Pentatomidae). Orthologous nucleotide sequences of Pentatomicin were present in stinkbugs and beetles but not in other insect groups. Notably, orthologous sequences were also detected from a horseshoe crab, cyanobacteria and proteobacteria, suggesting the possibility of inter-domain horizontal gene transfers of Pentatomicin and allied protein genes. The recombinant protein of Pentatomicin was effective against an array of Gram-positive bacteria but not against Gram-negative bacteria. Upon septic shock, the expression of Pentatomicin drastically increased in a manner similar to other AMPs. On the other hand, unlike other AMPs, mock and saline injections increased the expression of Pentatomicin. RNAi-mediated downregulation of Imd pathway genes (Imd and Relish) and Toll pathway genes (MyD88 and Dorsal) revealed that the expression of Pentatomicin is under the control of Toll pathway. Being consistent with in vitro effectiveness of the recombinant protein, adult insects injected with dsRNA of Pentatomicin exhibited higher vulnerability to Gram-positive Staphylococcus aureus than to Gram-negative Escherichia coli. We discovered high levels of Pentatomicin expression in eggs, which is atypical of other AMPs and suggestive of its biological functioning in eggs. Contrary to the expectation, however, RNAi-mediated downregulation of Pentatomicin did not affect normal embryonic development of P. stali. Moreover, the downregulation of Pentatomicin in eggs did not affect vertical symbiont transmission to the offspring even under heavily contaminated conditions, which refuted our expectation that the antimicrobial activity of Pentatomicin may contribute to egg surface-mediated symbiont transmission by suppressing microbial contaminants.

Single mutation makes Escherichia coli an insect mutualist.

Microorganisms often live in symbiosis with their hosts, and some are considered mutualists, where all species involved benefit from the interaction. How free-living microorganisms have evolved to become mutualists is unclear. Here we report an experimental system in which non-symbiotic Escherichia coli evolves into an insect mutualist. The stinkbug Plautia stali is typically associated with its essential gut symbiont, Pantoea sp., which colonizes a specialized symbiotic organ. When sterilized newborn nymphs were infected with E. coli rather than Pantoea sp., only a few insects survived, in which E. coli exhibited specific localization to the symbiotic organ and vertical transmission to the offspring. Through transgenerational maintenance with P. stali, several hypermutating E. coli lines independently evolved to support the host's high adult emergence and improved body colour; these were called 'mutualistic' E. coli. These mutants exhibited slower bacterial growth, smaller size, loss of flagellar motility and lack of an extracellular matrix. Transcriptomic and genomic analyses of 'mutualistic' E. coli lines revealed independent mutations that disrupted the carbon catabolite repression global transcriptional regulator system. Each mutation reproduced the mutualistic phenotypes when introduced into wild-type E. coli, confirming that single carbon catabolite repression mutations can make E. coli an insect mutualist. These findings provide an experimental system for future work on host-microbe symbioses and may explain why microbial mutualisms are omnipresent in nature.

Endosymbiotic bacteria of the boar louse Haematopinus apri (Insecta: Phthiraptera: Anoplura).

Insects exclusively feeding on vertebrate blood are usually dependent on symbiotic bacteria for provisioning of B vitamins. Among them, sucking lice are prominent in that their symbiotic bacteria as well as their symbiotic organs exhibit striking diversity. Here we investigated the bacterial diversity associated with the boar louse Haematopinus apri in comparison with the hog louse Haematopinus suis. Amplicon sequencing analysis identified the primary endosymbiont predominantly detected from all populations of H. apri with some minor secondary bacterial associates. Sequencing and phylogenetic analysis of bacterial 16S rRNA gene confirmed that the endosymbionts of the boar louse H. apri, the hog louse H. suis and the cattle louse Haematopinus eurysternus form a distinct clade in the Gammaproteobacteria. The endosymbiont clade of Haematopinus spp. was phylogenetically distinct from the primary endosymbionts of other louse lineages. Fluorescence in situ hybridization visualized the endosymbiont localization within midgut epithelium, ovarial ampulla and posterior oocyte of H. apri, which were substantially the same as the endosymbiont localization previously described in H. suis and H. eurysternus. Mitochondrial haplotype analysis revealed that, although the domestic pig was derived from the wild boar over the past 8,000 years of human history, the populations of H. apri constituted a distinct sister clade to the populations of H. suis. Based on these results, we discussed possible evolutionary trajectories of the boar louse, the hog louse and their endosymbionts in the context of swine domestication. We proposed 'Candidatus Haematopinicola symbiotica' for the distinct clade of the endosymbionts of Haematopinus spp.

Effectiveness of orally-delivered double-stranded RNA on gene silencing in the stinkbug Plautia stali.

Development of a reliable method for RNA interference (RNAi) by orally-delivered double-stranded RNA (dsRNA) is potentially promising for crop protection. Considering that RNAi efficiency considerably varies among different insect species, it is important to seek for the practical conditions under which dsRNA-mediated RNAi effectively works against each pest insect. Here we investigated RNAi efficiency in the brown-winged green stinkbug Plautia stali, which is notorious for infesting various fruits and crop plants. Microinjection of dsRNA into P. stali revealed high RNAi efficiency-injection of only 30 ng dsRNA into last-instar nymphs was sufficient to knockdown target genes as manifested by their phenotypes, and injection of 300 ng dsRNA suppressed the gene expression levels by 80% to 99.9%. Knockdown experiments by dsRNA injection showed that multicopper oxidase 2 (MCO2), vacuolar ATPase (vATPase), inhibitor of apoptosis (IAP), and vacuolar-sorting protein Snf7 are essential for survival of P. stali, as has been demonstrated in other insects. By contrast, P. stali exhibited very low RNAi efficiency when dsRNA was orally administered. When 1000 ng/µL of dsRNA solution was orally provided to first-instar nymphs, no obvious phenotypes were observed. Consistent with this, RT-qPCR showed that the gene expression levels were not affected. A higher concentration of dsRNA (5000 ng/µL) induced mortality in some cohorts, and the gene expression levels were reduced to nearly 50%. Simultaneous oral administration of dsRNA against potential RNAi blocker genes did not improve the RNAi efficiency of the target genes. In conclusion, P. stali shows high sensitivity to RNAi with injected dsRNA but, unlike the allied pest stinkbugs Halyomorpha halys and Nezara viridula, very low sensitivity to RNAi with orally-delivered dsRNA, which highlights the varied sensitivity to RNAi across different species and limits the applicability of the molecular tool for controlling this specific insect pest.

Diversity and function of multicopper oxidase genes in the stinkbug Plautia stali.

Multicopper oxidase (MCO) genes comprise multigene families in bacteria, fungi, plants and animals. Two families of MCO genes, MCO1 (laccase1) and MCO2 (laccase2), are conserved among diverse insects and relatively well-characterized, whereas additional MCO genes, whose biological functions have been poorly understood, are also found in some insects. Previous studies reported that MCO1 participates in gut immunity and MCO2 plays important roles in cuticle sclerotization and pigmentation of insects. In mosquitoes, MCO2 was reported to be involved in eggshell sclerotization and pigmentation, on the ground that knockdown of MCO2 caused deformity and fragility of the eggshell. Here we identified a total of 7 MCO genes, including PsMCO1 and PsMCO2, and investigated their expression and function in the brown-winged green stinkbug Plautia stali. RNA interference (RNAi) knockdown of MCO genes by injecting double-stranded RNA (dsRNA) into nymphs revealed that MCO2, but not the other 6 MCOs, is required for cuticle sclerotization and pigmentation, and also for survival of P. stali. Trans-generational knockdown of MCO2 by injecting dsRNA into adult females (maternal RNAi) resulted in the production of unhatched eggs despite the absence of deformity or fragility of the eggshell. These results suggested that MCO2 plays an important role in sclerotization and pigmentation of the cuticle but not in eggshell integrity in P. stali. Maternal RNAi of any of the other 6 MCO genes and 3 tyrosinase genes affected neither survival nor eggshell integrity of P. stali. Contrary to the observations in the red flour beetle and the brown rice planthopper, RNAi knockdown of MCO6 (MCORP; Multicopper oxidase related protein) exhibited no lethal effects on P. stali. Taken together, our findings provide insight into the functional diversity and commonality of MCOs across hemipteran and other insect groups.

Functional crosstalk across IMD and Toll pathways: insight into the evolution of incomplete immune cascades.

In insects, antimicrobial humoral immunity is governed by two distinct gene cascades, IMD pathway mainly targeting Gram-negative bacteria and Toll pathway preferentially targeting Gram-positive bacteria, which are widely conserved among diverse metazoans. However, recent genomic studies uncovered that IMD pathway is exceptionally absent in some hemipteran lineages like aphids and assassin bugs. How the apparently incomplete immune pathways have evolved with functionality is of interest. Here we report the discovery that, in the hemipteran stinkbug Plautia stali, both IMD and Toll pathways are present but their functional differentiation is blurred. Injection of Gram-negative bacteria and Gram-positive bacteria upregulated effector genes of both pathways. Notably, RNAi experiments unveiled significant functional permeation and crosstalk between IMD and Toll pathways: RNAi of IMD pathway genes suppressed upregulation of effector molecules of both pathways, where the suppression was more remarkable for IMD effectors; and RNAi of Toll pathway genes reduced upregulation of effector molecules of both pathways, where the suppression was more conspicuous for Toll effectors. These results suggest the possibility that, in hemipterans and other arthropods, IMD and Toll pathways are intertwined to target wider and overlapping arrays of microbes, which might have predisposed and facilitated the evolution of incomplete immune pathways.

Re-examination of the maternal control of progeny size and body color in the desert locust Schistocerca gregaria: Differences from previous conclusions.

The desert locust shows conspicuous phase polyphenism of various traits in response to crowding conditions. Gregarious females lay larger eggs that produce black hatchlings, whereas solitarious females lay smaller eggs that produce green hatchlings. Previous studies have shown that changes in egg size and hatchling body color occurred easily in the laboratory upon exposure of the female parent to crowding or isolation for as few as 2 days. Based on these observations, these studies concluded that female adults perceive crowding stimuli with their antennae and require light for perception of the stimuli, with contact chemicals present on the integument of sexually mature males that are responsible for the crowding effect. We undertook this study to identify the chemical structures of the reported contact substances, which remain unknown to date. However, we could not reproduce the main results reported in the aforementioned studies and found that egg size and hatchling body color did not alter easily, even after crowding or isolation of the female parent for 2 days or longer. We were not able to observe a change in the progeny crowding characteristics by stimulating the antennae of female adults through either physical contact with male adults or using hexane extracts of their body surfaces. Similarly, the importance of light for gregarization or solitarization was also ruled out. These results indicate that some of the conclusions of previous studies should be reconsidered.

Novel bacteriocyte-associated pleomorphic symbiont of the grain pest beetle Rhyzopertha dominica (Coleoptera: Bostrichidae).

BACKGROUND: The lesser grain borer Rhyzopertha dominica (Coleoptera: Bostrichidae) is a stored-product pest beetle. Early histological studies dating back to 1930s have reported that R. dominica and other bostrichid species possess a pair of oval symbiotic organs, called the bacteriomes, in which the cytoplasm is densely populated by pleomorphic symbiotic bacteria of peculiar rosette-like shape. However, the microbiological nature of the symbiont has remained elusive. RESULTS: Here we investigated the bacterial symbiont of R. dominica using modern molecular, histological, and microscopic techniques. Whole-mount fluorescence in situ hybridization specifically targeting symbiotic bacteria consistently detected paired bacteriomes, in which the cytoplasm was full of pleomorphic bacterial cells, in the abdomen of adults, pupae and larvae, confirming previous histological descriptions. Molecular phylogenetic analysis identified the symbiont as a member of the Bacteroidetes, in which the symbiont constituted a distinct bacterial lineage allied to a variety of insect-associated endosymbiont clades, including Uzinura of diaspidid scales, Walczuchella of giant scales, Brownia of root mealybugs, Sulcia of diverse hemipterans, and Blattabacterium of roaches. The symbiont gene exhibited markedly AT-biased nucleotide composition and significantly accelerated molecular evolution, suggesting degenerative evolution of the symbiont genome. The symbiotic bacteria were detected in oocytes and embryos, confirming continuous host-symbiont association and vertical symbiont transmission in the host life cycle. CONCLUSIONS: We demonstrate that the symbiont of R. dominica constitutes a novel bacterial lineage in the Bacteroidetes. We propose that reductive evolution of the symbiont genome may be relevant to the amorphous morphology of the bacterial cells via disruption of genes involved in cell wall synthesis and cell division. Genomic and functional aspects of the host-symbiont relationship deserve future studies.

The mechanism controlling phenotypic plasticity of body color in the desert locust: some recent progress.

Schistocerca gregaria exhibits density-dependent body color polyphenism. Nymphs occurring at low population densities show green-brown polyphenism. They show phase polyphenism and develop black patterns at high population densities. Recent studies suggest a third type of polyphensim, that is, homochromy, a response to background color. Laboratory experiments that considered homochromy suggest that humidity is not directly involved in green-brown polyphenism and that odor from other individuals does not induce black patterns. Black patterns can be induced in isolated nymphs by video images of locusts and tadpoles. Juvenile hormone and [His7]-corazonin control body color in locusts. The gene encoding the latter has been identified for S. gregaria and Locusta migratoria, and its key role in controlling black patterning has been demonstrated.

Egg hatching of two locusts, Schistocerca gregaria and Locusta migratoria, in response to light and temperature cycles.

The present study showed that the eggs of the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria, responded to photoperiod by hatching when placed on sand in the laboratory. S. gregaria mainly hatched during the dark phase and L. migratoria during the light phase. The importance of light as a hatching cue depended on the magnitude of the temperature change during the thermoperiod; photoperiod played a more important role in the control of hatching time in both species when the magnitude of the temperature change was small. In addition, the eggs of the two species that were covered with sand did not respond to photoperiod and hatched during both the light and dark phases, indicating that light did not penetrate through the sand. Because locust eggs are normally laid as egg pods and a foam plug is deposited between the egg mass and the ground surface, we tested a possibility that naturally deposited eggs perceived light through the foam plug. The eggs that were deposited and left undisturbed in the sand hatched during the light and dark phases at similar frequencies. These results suggest that the eggs of both locust species responded to light and controlled their hatching timing accordingly but would not use light as a hatching cue in the field. The evolutionary significance of the ability of eggs to respond to light in these locusts was discussed.

Adaptive difference in daily timing of hatch in two locust species, Schistocerca gregaria and Locusta migratoria: the effects of thermocycles and phase polyphenism.

This study examined the effects of temperature and phase polyphenism on egg hatching time in the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria. The two species exhibited differences and similarities in hatching behavior when exposed to different temperature conditions. In 12-h thermocycles of various temperatures, the S. gregaria eggs hatched during the cryoperiod (low temperature period), whereas L. migratoria eggs hatched during the thermoperiod (high temperature period). The eggs of both species hatched during the species-specific period of the thermoperiod in response to a temperature difference as small as 1 °C. Furthermore, the locusts adjusted hatching time to a new thermal environment that occurred shortly before the expected hatching time. In both species, the hatching of the eggs was synchronized to a specific time of the day, and two hatching peaks separated by approximately 1 day were observed at a constant temperature after the eggs were transferred from thermocycles 3 days before hatching. Eggs laid by gregarious females hatched earlier than those laid by solitarious females in S. gregaria but this difference was not observed in L. migratoria.

Behavioral phase shift in nymphs of the desert locust, Schistocerca gregaria: special attention to attraction/avoidance behaviors and the role of serotonin.

Schistocerca gregaria exhibits a phase-specific behavior in response to crowding. Nymphs occurring at low population densities (solitarious phase) tend to avoid one another, whereas those occurring at high population densities (gregarious phase) are attracted to one another. This study examined how this attraction/avoidance behavior changed after isolation or crowding. The behavior of the test nymphs was assessed by determining their positioning with respect to a stimulus cup, which contained 12 gregarious nymphs, placed at one end of an elliptical arena and an empty cup placed at the opposite end. Gregarious (crowd-reared) nymphs were most frequently observed close to the stimulus cup, whereas solitarious (isolated-reared) nymphs tended to avoid it. This tendency was easily changed by exposing the nymphs to isolation or crowding. However, contrary to a previously reported conclusion that behavioral gregarization occurs in 4-8 h of crowding, the nymphs required at least 3 days to achieve a significant change in the attraction/avoidance behavior in either direction, from solitarious to gregarious or the converse. The discrepancies between the present study and previous studies appear to result from the different behaviors observed. The present study focused on the attraction/avoidance behavior that is most likely the most important element in the gregarization and solitarization processes, whereas other studies used a mixture of various activity-related behaviors. No evidence was obtained for a trans-generational accumulation in the attraction/avoidance behaviors. Serotonin, a biogenic amine that is suggested by others to be involved in behavioral phase changes in S. gregaria, had no influence on this behavior or body color when injected into the solitarious nymphs.

Polymorphisms of the Dopamine D4 Receptor Gene in Stabled Horses are Related to Differences in Behavioral Response to Frustration.

In stabled horses, behavioral responses to frustration are often observed, especially around feeding time. These behavioral responses are a useful indicator of their welfare. In this study, we investigated the association between this behavioral indicator and DRD4 gene polymorphisms in stabled horses. Twenty one horses housed in two stables were used. The horses were observed for approximately 4 h around feeding over three or more days using focal-sampling and instantaneous-sampling. Horses were genotyped for the A-G substitution in the DRD4 gene. The effects of the A-G substitution (with or without the A allele in the DRD4 gene), the stables, and their interaction on the frequency of behavioral responses to frustration were analyzed using general linear models. The total time budget of behavioral responses to frustration was higher in horses without the A allele than in those with the A allele (P = 0.007). These results indicate that the A-G substitution of the DRD4 gene is related to frustration-related behavioral responses in stabled horses. Appropriate consideration should be made for the DRD4 gene polymorphism when the welfare of stabled horses is assessed, based on this behavioral indicator.

Environmental and genetic controls of soldier caste in a parasitic social wasp.

A larval army caste is found in some parasitic wasps with polyembryonic or clonal proliferation, where many clone larvae emerge from a single egg. In contrast to non-parasitic eusocial Hymenoptera, sterile soldier larvae that protect their clonal reproductives are found in both females and males. Recently, the proportion of soldier larvae has been found to vary radically, depending on the internal conditions of the host, such as multiparasitism by other larval parasites. However, the proportion of male soldier larvae is constant, irrespective of the host internal environment. It is unknown if these traits are heritable. Here we show that a high heritability is found in both sexes, while, in the 6th instar hosts, substantially lower heritability is found in females. These results imply that the structure of the larval caste is determined genetically by both female and male embryonic cells, but more likely modified environmentally in females.

Do desert locust hoppers develop gregarious characteristics by watching a video?

Various sensory stimuli have been suggested to induce gregarious body coloration in locusts, but most previous studies ignored the importance of substrate color. This study tested the effects of visual, olfactory and tactile stimuli from other locusts on the induction of gregarious body coloration in single (isolated-reared) Schistocerca gregaria nymphs housed in yellow-green cups. Odor from gregarious (crowd-reared) locusts, which is believed to induce black patterns in single locusts, had little effect when applied to visually isolated nymphs at the 2nd stadium onward, and all test nymphs remained green without black patterns at the last stadium, as in controls reared without odor and visual stimuli. Visual stimuli alone induced black patterns when a single solitarious nymph was allowed to see other locusts in another cup. The degree of black patterns increased as the number of locusts shown increased, and some test nymphs developed body coloration typically observed in gregarious forms. A classical morphometric ratio (hind femur length/head width) shifted toward the value typical of gregarious forms when the single nymphs were allowed to see 5 or 10 locusts. Single nymphs also developed black patterns when presented green conspecific nymphs and adults of two hemipteran species kept in another cup. No synergetic effects of visual and odor stimuli were detected. Movies of locusts, crickets and tadpoles were found effective in inducing black patterns in single locusts. Ontogenetic variation in the sensitivity to crowding and experimental methodology might be responsible for some discrepancies in the conclusions among different researchers.

Re-examination of the roles of environmental factors in the control of body-color polyphenism in solitarious nymphs of the desert locust Schistocerca gregaria with special reference to substrate color and humidity.

This study re-examines the effects of environmental factors including substrate color, humidity, food quality, light intensity and temperature on the green-brown polyphenism, black patterning and background body color of solitarious (isolated-reared) nymphs of Schistocerca gregaria. All individuals reared in yellow-green or yellow containers became green morphs, whereas those reared in white, ivory-colored, blue, grey, brown, zinc-colored and black containers produced brown morphs in similar proportions. The intensity of black patterns was negatively correlated with the brightness of the substrate color of the containers. Humidity, which previous studies claimed controls green-brown polyphenism in this species, exerted no significant influence on either the green-brown polyphenism or the black patterning. Food quality also had little effect on body color. High temperature tended to inhibit darkening. The background body color on the thorax was greatly influenced by the substrate color of rearing containers and a close correlation was found between these two variables, indicating that, in contrast to what has been suggested by others, this species exhibits homochromy to match the body color to the substrate color of its habitat. Similar responses were observed in another strain, although some quantitative differences occurred between the two strains examined. Based on these results, a new model explaining the control of body-color polyphenism in this locust is proposed and the ecological significance of black patterns in solitarious nymphs is discussed.

Clonal structure affects the assembling behavior in the Japanese queenless ant Pristomyrmex punctatus.

The queenless ant Pristomyrmex punctatus (Hymenoptera: Myrmicinae) has a unique society that differs from those of other typical ants. This species does not have a queen, and the workers lay eggs and produce their clones parthenogenetically. However, a colony of these ants does not always comprise members derived from a single clonal line. In this study, we examined whether P. punctatus changes its "assembling behavior" based on colony genetic structure. We prepared two subcolonies--a larger one comprising 200 individuals and a smaller one comprising 100 individuals; these subcolonies were established from a single stock colony. We investigated whether these subcolonies assemble into a single nest. The genetically monomorphic subcolonies (single clonal line) always fused into a single nest; however, the genetically polymorphic subcolonies (multiple clonal lines) did not tend to form a single colony. The present study is the first to demonstrate that the colony genetic structure significantly affects social viscosity in social insects.