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.

Infection Density Dynamics of the Citrus Greening Bacterium "Candidatus Liberibacter asiaticus" in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants.

Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, "Candidatus Liberibacter asiaticus," and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of "Ca. Liberibacter asiaticus" in field populations of D. citri with experiments using field-collected insects to address how "Ca. Liberibacter asiaticus" infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from "Ca. Liberibacter asiaticus"-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were "Ca. Liberibacter asiaticus" positive. The infections were systemic across head-thorax and abdomen, ranging from 10(3) to 10(7) bacteria per insect. In spring, the infection densities were low in March, at ∼ 10(3) bacteria per insect, increasing up to 10(6) to 10(7) bacteria per insect in April and May, and decreasing to 10(5) to 10(6) bacteria per insect in late May, whereas the infection densities were constantly ∼ 10(6) to 10(7) bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with "Ca. Liberibacter asiaticus" infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected "Ca. Liberibacter asiaticus"-infected insects suggested that (i) "Ca. Liberibacter asiaticus"-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼ 10(6) bacteria per insect) of "Ca. Liberibacter asiaticus" density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits "Ca. Liberibacter asiaticus" to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.

Discrepancy in the degree of population differentiation between color-morph frequencies and neutral genetic loci in the damselfly Ischnura senegalensis in Okinawa Island, Japan.

Evaluation of relative contribution of natural selection and stochastic processes to population differentiation has been of great interest in evolutionary biology. In a damselfly, Ischnura senegalensis, females show color dimorphism (gynochrome vs. androchrome), and color-morph frequencies are known to greatly vary among local populations within Okinawa Island, a small island of Ryukyu Archipelago, Japan. In this study, to examine the effects of natural selection and stochastic processes on the within-island variation in color-morph frequencies, we compared the degree of population differentiation at the color-morph locus with that at a mitochondrial DNA region and ten nuclear microsatellite loci. F ST values at the neutral loci were close to zero, indicating presence of sufficient gene flow (dispersal of adult individuals) between the local populations. In contrast, F ST values at the color-morph locus were significantly different from zero. These results suggest that variation in female color-morph frequencies observed among local populations in Okinawa Island has been caused by divergent selection acting on the phenotype and/or genes tightly linked with the color locus.

Is elytral color polymorphism in sweetpotato weevil (Coleoptera: Brentidae) a visible marker for sterile insect technique? Comparison of male mating behavior.

The sterile insect technique (SIT) is widely used for suppressing or eradicating target pest insect populations. In the eradication programs using the SIT, a large number of sterile insects are marked and released in the field. In Japan, Cylas fonnrmicarius (F.) group (Coleoptera: Brentidae) weevils are marked with a fluorescent powder dye to monitor the progress of such programs. However, this monitoring technique is not fully effective because of the disappearance or contamination of the dye. Therefore, an alternative marking method is required. Currently, a rare color morph such as piceous elytra (PE) is used for visible marking of C. formicarius group weevils. A PE-monomorphic strain has previously been established by artificial selection from a small locally distributed population; this can lead to reduced survival and genetic changes in behavioral traits due to inbreeding depression. In this study, we evaluated the survival rate and mating behavior of PE males of C. formicarius group. The characteristics of the PE males were similar to those of the wild strain (WS) males. Thus, we considered that PE males were suitable for visible marking in the eradication programs using the SIT.

The neogregarine protozoan Farinocystis sp. reduces longevity and fecundity in the West Indian sweet potato weevil, Euscepes postfasciatus (Fairmaire).

The number of West Indian sweet potato weevils, Euscepes postfasciatus, being mass-reared in a facility for use in sterile insect technique (SIT) eradication programs has undergone a drastic reduction. A neogregarine protozoan pathogen Farinocystis sp. (an undescribed species) was detected in vivo in the mass-reared E. postfasciatus. We investigated the effects of this disease on the longevity and fecundity of host weevils and the incubation time of the disease in the host body under mass-rearing conditions. Our results demonstrated that infection by this Farinocystis sp. decreased both longevity and fecundity in E. postfasciatus. In particular, the pathogen severely limited the production of progeny by infected females compared to healthy females. Therefore, we consider this protozoan infection to be the major cause of the decreased E. postfasciatus production in the mass-rearing facility.

Mate Choice Behavior of Female Field Crickets Is Not Affected by Exposure to Heterospecific Calling Songs.

Many animals produce acoustic signals to mark territories and attract mates. When different species produce acoustic signals simultaneously, the signals create a noisy environment, with potential acoustic interference between species. Theoretical studies suggest that such reproductive interference may have strong effects on species interaction. For example, the inferior resource competitor can survive if its disadvantage is counterbalanced by superiority in reproductive interference. Two field cricket species, Teleogryllus occipitalis (Audinet-Serville) (Orthoptera: Gryllidae) and Loxoblemmus equestris Saussure (Orthoptera: Gryllidae), cooccur in the same habitat. A previous study has shown that L. equestris is an inferior species to T. occipitalis in terms of resource competition. Therefore, we predicted that mate location and choice behavior of female T. occipitalis would be negatively affected by the acoustic signals of L. equestris and tested this with a series of playback experiments. The mate choice behavior of female T. occipitalis was not significantly affected by the calling song of L. equestris. Our results suggest that the acoustic interference does not explain the cooccurrence of the two species in the same habitat.

Evaluation of toxicity of biorational insecticides against larvae of the alfalfa weevil.

The alfalfa weevil, Hypera postica (Coleoptera: Curculionidae), is a major pest of alfalfa Medicago sativa L. (Fabaceae). While H. postica usually causes the most damage before the first cutting, in summer of 2015 damaging levels of the pest persisted in Montana well after the first harvest of alfalfa. Although conventional insecticides can control H. postica, these chemicals have adverse effects on non-target organisms including pollinators and natural enemy insects. In this context, use of biorational insecticides would be the best alternative options, as they are known to pose less risk to non-target organisms. We therefore examined the six commercially available biorational insecticides against H. postica under laboratory condition: Mycotrol® ESO (Beauveria bassiana GHA), Aza-Direct® (Azadirachtin), Met52® EC (Metarhizium brunneum F52), Xpectro OD® (B. bassiana GHA + pyrethrins), Xpulse OD® (B. bassiana GHA + Azadirachtin) and Entrust WP® (spinosad 80%). Concentrations of 0.1, 0.5, 1.0, and 2.0 times the lowest labelled rates were tested for all products. However, in the case of Entrust WP, additional concentrations of 0.001 and 0.01 times the lowest label rate were also assessed. Mortality rates were determined at 1-9 days post treatment. Based on lethal concentrations and relative potencies, this study clearly showed that Entrust was the most effective, causing 100% mortality within 3 days after treatment among all the tested materials. With regard to other biorational, Xpectro was the second most effective insecticide followed by Xpulse, Aza-Direct, Met52, and Mycotrol. Our results strongly suggested that these biorational insecticides could potentially be applied for H. postica control.

Biological role of Nardonella endosymbiont in its weevil host.

Weevils constitute the most species-rich animal group with over 60,000 described species, many of which possess specialized symbiotic organs and harbor bacterial endosymbionts. Among the diverse microbial associates of weevils, Nardonella spp. represent the most ancient and widespread endosymbiont lineage, having co-speciated with the host weevils for over 125 million years. Thus far, however, no empirical work on the role of Nardonella for weevil biology has been reported. Here we investigated the biological role of the Nardonella endosymbiont for the West Indian sweet potato weevil, Euscepes postfasciatus. This insect is an experimentally tractable pest insect that can easily be reared on a natural diet of sweet potato root as well as on an agar-based artificial diet. By larval feeding on an antibiotic-containing artificial diet, Nardonella infection was effectively eliminated from the treated insects. The antibiotic-treated insects exhibited significantly lighter body weight and lower growth rate than the control insects. Then, the antibiotic-treated insects and the control insects were respectively allowed to mate and oviposit on fresh sweet potatoes without the antibiotic. The offspring of the antibiotic-treated insects, which were all Nardonella-negative, exhibited significantly lighter body weight, smaller body size, lower growth rate and paler body color in comparison with the offspring of the control insects, which were all Nardonella-positive. In conclusion, the Nardonella endosymbiont is involved in normal growth and development of the host weevil. The biological role of the endosymbiont probably underlies the long-lasting host-symbiont co-speciation in the evolutionary course of weevils.