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The Southern green shield bug, Nezara viridula, is an invasive piercing and sucking pest insect that feeds on crops and poses a threat to global food production. Insects live in close relationships with microorganisms providing their host with unique capabilities, such as resistance to toxic plant metabolites. In this study, we investigated the resistance to and detoxification of the plant metabolite 3-nitropropionic acid (NPA) by core and transient members of the N. viridula microbial community. Microbial community members showed a different tolerance to the toxin and we determined that six out of eight strains detoxified NPA. Additionally, we determined that NPA might interfere with the biosynthesis and transport of l-leucine. Moreover, our study explored the influence of diet on the gut microbial composition of N. viridula, demonstrating that switching to a single-plant diet shifts the abundance of core microbes. In line with this, testing pairwise microbial interactions revealed that core microbiota members support each other and repress the growth of transient microorganisms. With this work, we provide novel insights into the factors shaping the insect gut microbial communities and demonstrate that N. viridula harbours many toxin-degrading bacteria that could support its resistance to plant defences., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Specialized host-microbe symbioses are ecological communities, whose composition is shaped by various processes. Microbial community assembly in these symbioses is determined in part by interactions between taxa that colonize ecological niches available within habitat patches. The outcomes of these interactions, and by extension the trajectory of community assembly, can display priority effects-dependency on the order in which taxa first occupy these niches. The underlying mechanisms of these phenomena vary from system to system and are often not well resolved. Here, we characterize priority effects in colonization of the squash bug ( Anasa tristis ) by bacterial symbionts from the genus Caballeronia , using pairs of strains that are known to strongly compete during host colonization, as well as strains that are isogenic and thus functionally identical. By introducing symbiont strains into individual bugs in a sequential manner, we show that within-host populations established by the first colonist are extremely resistant to invasion, regardless of strain identity and competitive interactions. By knocking down the population of an initial colonist with antibiotics, we further show that colonization success by the second symbiont is still diminished even when space in the symbiotic organ is available and ostensibly accessible for colonization. We speculate that resident symbionts exclude subsequent infections by manipulating the host environment, partially but not exclusively by eliciting tissue remodeling of the symbiont organ., Importance: Host-associated microbial communities underpin critical ecosystem processes and human health, and their ability to do so is determined in turn by the various processes that shape their composition. While selection deterministically acts on competing genotypes and species during community assembly, the manner by which selection determines the trajectory of community assembly can differ depending on the sequence by which taxa are established within that community. We document this phenomenon, known as a priority effect, during experimental colonization of a North American insect pest, the squash bug Anasa tristis , by its betaproteobacterial symbionts in the genus Caballeronia. Our study demonstrates how stark, strain-level variation can emerge in specialized host-microbe symbioses simply through differences in the order by which strains colonize the host. Understanding the mechanistic drivers of community structure in host-associated microbiomes can highlight both pitfalls and opportunities for the engineering of these communities and their constituent taxa for societal benefit., Competing Interests: The authors declare no conflict of interest.

Fall armyworm (FAW), Spodoptera frugiperda is a generalist pest known to feed on more than 300 plant species, including major staple crops such as rice, maize and sorghum. Biological control of FAW using a combination of a major indigenous egg parasitoid Telenomus remus and entomopathogenic fungi was explored in this study. Metarhizium anisopliae strains (ICIPE 7, ICIPE 41, and ICIPE 78) and Beauveria bassiana ICIPE 621 which demonstrated effectiveness to combat the pest, were evaluated through direct and indirect fungal infection to assess their pathogenicity and virulence against T. remus adults, S. frugiperda eggs and their effects on T. remus parasitism rates. Metarhizium anisopliae ICIPE 7 and ICIPE 78 exhibited the highest virulence against T. remus adults with LT 50 values >2 days. ICIPE 7 induced the highest T. remus mortality rate (81.40 ± 4.17%) following direct infection with dry conidia. Direct fungal infection also had a significant impact on parasitoid emergence, with the highest emergence rate recorded in the M. anisopliae ICIPE 7 treatment (42.50 ± 5.55%), compared to the control ± (83.25 ± 5.94%). In the indirect infection, the highest concentration of 1 x 10 9 conidia ml -1 of ICIPE 78 induced the highest mortality (100 ± 0.00%) of T. remus adults, and the highest mortality (51.25%) of FAW eggs, whereas the least FAW egg mortality (15.25%) was recorded in the lowest concentration 1 x 10 5 conidia ml -1 of ICIPE 41. The number of parasitoids that emerged and their sex ratios were not affected by the different fungal strain concentrations except in ICIPE 7 at high dose. This study showed that potential combination of both M. anisopliae and B. bassiana with T. remus parasitoid can effectively suppress FAW populations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chepkemoi, Fening, Ambele, Munywoki and Akutse.)

Diverse insects are intimately associated with specific symbiotic bacteria, where host and symbiont are integrated into an almost inseparable biological entity. These symbiotic bacteria usually exhibit host specificity, uncultivability, reduced genome size, and other peculiar traits relevant to their symbiotic lifestyle. How host-symbiont specificity is established at the very beginning of symbiosis is of interest but poorly understood. To gain insight into the evolutionary issue, we adopted an experimental approach using the recently developed evolutionary model of symbiosis between the stinkbug Plautia stali and Escherichia coli . Based on the laboratory evolution of P. stali-E. coli mutualism, we selected Δ cyaA mutant of E. coli as an artificial symbiont of P. stali that has established mutualism by a single mutation. In addition, we selected a natural cultivable symbiont of P. stali of relatively recent evolutionary origin. These artificial and natural symbiotic bacteria of P. stali were experimentally inoculated to symbiont-deprived newborn nymphs of diverse stinkbug species. Strikingly, the mutualistic E. coli was unable to establish infection and support growth and survival of all the stinkbug species except for P. stali , uncovering that host specificity can be established at a very early stage of symbiotic evolution. Meanwhile, the natural symbiont was able to establish infection and support growth and survival of several stinkbug species in addition to P. stali , unveiling that a broader host range of the symbiont has evolved in nature. Based on these findings, we discuss what factors are relevant to the establishment of host specificity in the evolution of symbiosis.IMPORTANCEHow does host-symbiont specificity emerge at the very beginning of symbiosis? This question is difficult to address because it is generally difficult to directly observe the onset of symbiosis. However, recent development of experimental evolutionary approaches to symbiosis has brought about a breakthrough. Here we tackled this evolutionary issue using a symbiotic Escherichia coli created in laboratory and a natural Pantoea symbiont, which are both mutualistic to the stinkbug Plautia stali . We experimentally replaced essential symbiotic bacteria of diverse stinkbugs with the artificial and natural symbionts of P. stali and evaluated whether the symbiotic bacteria, which evolved for a specific host, can establish infection and support the growth and survival of heterospecific hosts. Strikingly, the artificial symbiont showed strict host specificity to P. stali , whereas the natural symbiont was capable of symbiosis with diverse stinkbugs, which provide insight into how host-symbiont specificity can be established at early evolutionary stages of symbiosis., Competing Interests: The authors declare no conflict of interest.

A number of insects are associated with gut symbiotic microorganisms, wherein symbiotic partners play pivotal metabolic roles for each other such as nutrient supplementation, diet degradation, and pesticide detoxification. Despite the ecological and evolutionary importance of gut microbial communities in insects, their diversity and dynamics remain unclear in many species. The green plant bug Apolygus spinolae, a notorious grapevine pest in Japan, damages grape shoots and severely reduces grape berry yield and quality. The plant bug possesses a simple tubular gut housing ~ 10 4 bacteria. Here, we investigated geographic, seasonal, and growth-related dynamics of gut microbiota by high-throughput sequencing in 82 individuals (11 nymphs and 71 adults) from five locations in Hokkaido, Japan. In plant bugs, gut microbiota changed dynamically depending on region, season, and developmental stage. Among the gut bacteria, Serratia was consistently and abundantly detected and was significantly affected by seasonal changes. In addition, Caballeronia, known as a specific symbiont in some stinkbug species, was abundantly detected, especially in insects collected in late summer despite A. spinolae complete lack of midgut crypts known as symbiotic organ harboring Caballeronia in other stinkbug species. Considering their prevalence among host bug populations, it is possible these gut microorganisms play a pivotal role in the adaptation of the green plant bug to grapevine fields, although further confirmation through rearing experiments is needed., (© 2024. The Author(s).)

Given the growing interest in manipulating microbiota to enhance the fitness of mass-reared insects for biological control, this study investigated the impact of an artificial diet on the microbiota composition and performance of Orius strigicollis. We compared the microbiota of O. strigicollis fed on an artificial diet and moth eggs via culturing and 16S rRNA gene amplicon sequencing. Subsequently, we assessed life history traits and immune gene expression of O. strigicollis fed on the artificial diet supplemented with Pantoea dispersa OS1. Results showed that microbial diversity remained largely unaffected by the artificial diet, with similar microbiota compositions in both diet groups. OS1, a minor member of the microbiota but significantly enriched in bugs fed on the artificial diet, improved nymphal survival rates and shifted adult longevity-reproduction life history in females. Additionally, OS1 supplementation elevated the transcription of antimicrobial peptide diptericin. According to population parameters, the group receiving OS1 only during the nymphal stage showed higher population growth potential compared to the group supplemented across all life stages. These findings reveal the resilience of O. strigicollis microbiota under distinct dietary conditions and highlight the potential of using natural symbionts and specific supplementation regimes to improve Orius rearing for future biocontrol programs., (© 2024. The Author(s).)

This study aimed to investigate the effects of symbiosis on the life history of host insects and address their implications at the host population level. We evaluated the effects of symbiotic bacteria Caballeronia insecticola on its host Riptortus pedestris (Fabricus) (Hemiptera: Alydidae) from cohorts for nymphal development, adult survivorship, and female reproduction. Then, life table parameters were compared between symbiotic and apo-symbiotic groups, and the effects of symbiosis on the abundance of R. pedestris were simulated for varying proportions of symbiotic individuals in host populations. We found that symbiosis significantly accelerated the nymphal development and reproductive maturation of females. However, symbiosis incurred survival cost on adult females, reducing their longevity by 28.6%. Nonetheless, symbiotic females laid significantly greater numbers of eggs than the apo-symbiotic during early adult ages. This early reproductive investment negated the adverse effect of their reduced longevity, resulting in the mean lifetime fecundity to not significantly differ between the 2 groups. Indeed, total cohort fecundity of the symbiotic group was 1.3-fold greater than that of the apo-symbiotic group. Life table analysis demonstrated shorter generation time and greater population growth rate in the symbiotic population. Finally, the simulation model results indicate that an increase in the proportion of symbiotic R. pedestris favored the population growth, increasing the population size by 1.9 times for every 25% increase in the proportion of symbiotic individuals. Our study demonstrates that symbiont-mediated changes in the life history parameters of host individuals favor the host population growth, despite substantial reduction in the female longevity., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Orius laevigatus (Hemiptera, Anthocoridae) is a generalist predator extensively used for the biocontrol of diverse agricultural pests. Previous studies on O. laevigatus have focused on the improvement of insect genetic traits, but little is known about its association with microbes, especially viruses that may influence its production and efficacy. More than 280 RNA viruses have been described in other Hemiptera insects, in line with the continuous discovery of insect-specific viruses (ISVs) boosted by next-generation sequencing. In this study, we characterized the repertoire of RNA viruses associated with O. laevigatus. Its virome comprises 27 RNA viruses, classified within fourteen viral families, of which twenty-three viruses are specific to O. laevigatus and four are likely associated with fungal microbiota. The analysis of viral abundance in five O. laevigatus populations confirmed the presence of simultaneous viral infections and highlighted the ubiquitous presence and high abundance of one solinvivirus and three totiviruses. Moreover, we identified 24 non-retroviral endogenous viral elements (nrEVEs) in the genome of O. laevigatus, suggesting a long-term relationship between the host and its virome. Although no symptoms were described in the insect populations under study, the high diversity of viral species and the high abundance of certain RNA viruses identified indicate that RNA viruses may be significant for the applicability and efficacy of O. laevigatus in biocontrol programs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The use of biocontrol agents, such as predators and entomopathogenic nematodes, is a promising approach for the effective control of the tomato leafminer Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidaean), an oligophagous insect feeding mainly on Solanaceae species and a major pest of field- and greenhouse-grown tomatoes globally. In this context, the effects of two entomopathogenic nematode species Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae), as well as their respective bacterial symbionts, Xenorhabdus nematophila and Photorhabdus luminescens (Enterobacterales: Morganelaceae), which were applied as bacterial cell suspensions and as crude cell-free liquid filtrates on T. absoluta larvae, were investigated. The results showed that of all treatments, the nematodes S. carpocapsae and H. bacteriophora were the most effective, causing up to 98 % mortality of T. absoluta larvae. Regarding bacteria and their filtrates, the bacterium X. nematophila was the most effective (69 % mortality in young larvae), while P. luminescens and both bacterial filtrates showed similar potency (ca. 48-55 % mortality in young larvae). To achieve a holistic approach of controlling this important pest, the impact of these factors on the beneficial predator Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) was also studied. The results demonstrated that although nematodes and especially S. carpocapsae, caused significant mortality on N. tenuis (87 %), the bacterial cell suspensions of X. nematophila and P. luminescens and crude cell-free liquid filtrates had minimum impact on this beneficial predator (∼11-30 % mortality)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Plant-sucking insects have intricate associations with a diverse array of microorganisms to facilitate their adaptation to specific ecological niches. The midgut of phytophagous true bugs is generally structured into four distinct compartments to accommodate their microbiota. Nevertheless, there is limited understanding regarding the origins of these gut microbiomes, the mechanisms behind microbial community assembly, and the interactions between gut microbiomes and their insect hosts. In this study, we conducted a comprehensive survey of microbial communities within the midgut compartments of a bean bug Riptortus pedestris, soybean plant, and bulk soil across 12 distinct geographical fields in China, utilizing high-throughput sequencing of the 16 S rRNA gene. Our findings illuminated that gut microbiota of the plant-sucking insects predominantly originated from the surrounding soil environment, and plants also play a subordinate role in mediating microbial acquisition for the insects. Furthermore, our investigation suggested that the composition of the insect gut microbiome was probably shaped by host selection and/or microbe-microbe interactions at the gut compartment level, with marginal influence from soil and geographical factors. Additionally, we had unveiled a noteworthy dynamic in the acquisition of core bacterial taxa, particularly Burkholderia, which were initially sourced from the environment and subsequently enriched within the insect midgut compartments. This bacterial enrichment played a significant role in enhancing insect host reproduction. These findings contribute to our evolving understanding of microbiomes within the insect-plant-soil ecosystem, shedding additional light on the intricate interactions between insects and their microbiomes that underpin the ecological significance of microbial partnerships in host adaptation., (© 2024. The Author(s).)

Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacteria, designated strains F2 T and PGU16, were isolated from the midgut crypts of the bordered plant bug Physopelta gutta , collected in Okinawa prefecture, Japan. Although these strains were derived from different host individuals collected at different times, their 16S rRNA gene sequences were identical and showed the highest similarity to Paraburkholderia caribensis MWAP64 T (99.3 %). The genome of strain F2 T consisted of two chromosomes and two plasmids, and its size and G+C content were 9.28 Mb and 62.4 mol% respectively; on the other hand, that of strain PGU16 consisted of two chromosomes and three plasmids, and its size and G+C content were 9.47 Mb and 62.4 mol%, respectively. Phylogenetic analyses revealed that these two strains are members of the genus Paraburkholderia . The digital DNA-DNA hybridization value between these two strains was 92.4 %; on the other hand, the values between strain F2 T and P. caribensis MWAP64 T or phylogenetically closely related Paraburkholderia species were 44.3 % or below 49.1 %. The predominant fatty acids of both strains were C 16 : 0 , C 17 : 0 cyclo, summed feature 8 (C 18 : 1  ω 7 c /C 18 : 1  ω 6 c ), and C 19 : 0 cyclo ω 8 c , and their respiratory quinone was ubiquinone 8. Based on the above genotypic and phenotypic characteristics, strains F2 T and PGU16 represent a novel species of the genus Paraburkholderia for which the name Paraburkholderia largidicola sp. nov. is proposed. The type strain is F2 T (=NBRC 115765 T =LMG 32765 T ).

Specialized host-microbe symbioses canonically show greater diversity than expected from simple models, both at the population level and within individual hosts. To understand how this heterogeneity arises, we utilize the squash bug, Anasa tristis, and its bacterial symbionts in the genus Caballeronia. We modulate symbiont bottleneck size and inoculum composition during colonization to demonstrate the significance of ecological drift, the noisy fluctuations in community composition due to demographic stochasticity. Consistent with predictions from the neutral theory of biodiversity, we found that ecological drift alone can account for heterogeneity in symbiont community composition between hosts, even when 2 strains are nearly genetically identical. When acting on competing strains, ecological drift can maintain symbiont genetic diversity among different hosts by stochastically determining the dominant strain within each host. Finally, ecological drift mediates heterogeneity in isogenic symbiont populations even within a single host, along a consistent gradient running the anterior-posterior axis of the symbiotic organ. Our results demonstrate that symbiont population structure across scales does not necessarily require host-mediated selection, as it can emerge as a result of ecological drift acting on both isogenic and unrelated competitors. Our findings illuminate the processes that might affect symbiont transmission, coinfection, and population structure in nature, which can drive the evolution of host-microbe symbioses and microbe-microbe interactions within host-associated microbiomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In Burkholderia-Riptortus symbiosis, the host bean bug Riptortus pedestris harbors Burkholderia symbionts in its symbiotic organ, M4 midgut, for use as a nutrient source. After occupying M4, excess Burkholderia symbionts are moved to the M4B region, wherein they are effectively digested and absorbed. Previous studies have shown that M4B has strong symbiont-specific antibacterial activity, which is not because of the expression of antimicrobial peptides but rather because of the expression of digestive enzymes, mainly cathepsin L protease. However, in this study, inhibition of cathepsin L activity did not reduce the bactericidal activity of M4B, indicating that there is an unknown digestive mechanism that renders specifically potent bactericidal activity against Burkholderia symbionts. Transmission electron microscopy revealed that the lumen of symbiotic M4B was filled with a fibrillar matter in contrast to the empty lumen of aposymbiotic M4B. Using chromatographic and electrophoretic analyses, we found that the bactericidal substances in M4B existed as high-molecular-weight (HMW) complexes that were resistant to protease degradation. The bactericidal HMW complexes were visualized on non-denaturing gels using protein- and polysaccharide-staining reagents, thereby indicating that the HMW complexes are composed of proteins and polysaccharides. Strongly stained M4B lumen with Periodic acid-Schiff (PAS) reagent in M4B paraffin sections confirmed HMW complexes with polysaccharide components. Furthermore, M4B smears stained with Periodic acid-Schiff revealed the presence of polysaccharide fibers. Therefore, we propose a key digestive mechanism of M4B: bacteriolytic fibers, polysaccharide fibers associated with digestive enzymes such as cathepsin L, specialized for Burkholderia symbionts in Riptortus gut symbiosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

The Southern green shield bug, Nezara viridula, is an invasive piercing and sucking pest insect that feeds on crop plants and poses a threat to global food production. Given that insects are known to live in a close relationship with microorganisms, our study provides insights into the community composition and function of the N. viridula-associated microbiota and its effect on host-plant interactions. We discovered that N. viridula hosts both vertically and horizontally transmitted microbiota throughout different developmental stages and their salivary glands harbor a thriving microbial community that is transmitted to the plant while feeding. The N. viridula microbiota was shown to aid its host with the detoxification of a plant metabolite, namely 3-nitropropionic acid, and repression of host plant defenses. Our results demonstrate that the N. viridula-associated microbiota plays an important role in interactions between insects and plants and could therefore be considered a valuable target for the development of sustainable pest control strategies., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Insects possess complex and dynamic gut microbial system, which contributes to host nutrient absorption, reproduction, energy metabolism, and protection against stress. However, there are limited data on interactions of host-gut bacterial microbiota through miRNA (microRNA) regulation in a significant pest, Riptortus pedestris. Here, we performed the 16S rRNA amplicon sequencing and small RNA sequencing from the R. pedestris gut under three environmental conditions and antibiotic treatment, suggesting that we obtained a large amount of reads by assembly, filtration and quality control. The 16S rRNA amplicon sequencing results showed that the abundance and diversity of gut bacterial microbiota were significantly changed between antibiotic treatment and other groups, and they are involved in metabolism and biosynthesis-related function based on functional prediction. Furthermore, we identified different numbers of differentially expressed unigenes (DEGs) and differentially expressed miRNAs (DEMs) based on high-quality mappable reads, which were enriched in various immune-related pathways, including Toll-like receptor, RIG-I-like receptor, NOD-like receptor, JAK/STAT, PI3K/Akt, NF-κB, MAPK signaling pathways, and so forth, using GO and KEGG enrichment analysis. Later on, the identified miRNAs and their target genes in the R. pedestris gut were predicted and randomly selected to construct an interaction network. Finally, our study indicated that alterations in the gut bacterial microbiota are significantly positively or negatively associated with DEMs of the Toll/Imd signaling pathway with Pearson correlation analysis. Taken together, the results of our study lay the foundation for further deeply understanding the interactions between the gut microbiota and immune responses in R. pedestris through miRNA regulation, and provide the new basis for pest management in hemipteran pests., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The true bugs (Heteroptera) occupy nearly all of the known ecological niches of insects. Among them, as a group containing more than 30,000 species, the phytophagous true bugs are making increasing impacts on agricultural and forestry ecosystems. Previous studies proved that symbiotic bacteria play important roles in these insects in fitting various habitats. However, it is still obscure about the evolutionary and ecological patterns of the microorganisms of phytophagous true bugs as a whole with comprehensive taxon sampling., Results: Here, in order to explore the symbiotic patterns between plant-feeding true bugs and their symbiotic microorganisms, 209 species belonging to 32 families of 9 superfamilies had been sampled, which covered all the major phytophagous families of true bugs. The symbiotic microbial communities were surveyed by full-length 16S rRNA gene and ITS amplicons respectively for bacteria and fungi using the PacBio platform. We revealed that hosts mainly affect the dominant bacteria of symbiotic microbial communities, while habitats generally influence the subordinate ones. Thereafter, we carried out the ancestral state reconstruction of the dominant bacteria and found that dramatic replacements of dominant bacteria occurred in the early Cretaceous and formed newly stable symbiotic relationships accompanying the radiation of insect families. In contrast, the symbiotic fungi were revealed to be horizontally transmitted, which makes fungal communities distinctive in different habitats but not significantly related to hosts., Conclusions: Host and habitat determine microbial communities of plant-feeding true bugs in different roles. The symbiotic bacterial communities are both shaped by host and habitat but in different ways. Nevertheless, the symbiotic fungal communities are mainly influenced by habitat but not host. These findings shed light on a general framework for future microbiome research of phytophagous insects. Video Abstract., (© 2023. The Author(s).)

Many insects possess symbiotic bacteria in their bodies, and microbial symbionts play pivotal metabolic roles for their hosts. Members of the heteropteran superfamilies Coreoidea and Lygaeoidea stinkbugs harbor symbionts of the genus Caballeronia in their intestinal tracts. Compared with symbiotic associations in Coreoidea, those in Lygaeoidea insects are still less understood. Here, we investigated a symbiotic relationship involving the mulberry seed bug Paradieuches dissimilis (Lygaeoidea: Rhyparochromidae) using histological observations, cultivation of the symbiont, 16S rRNA gene amplicon sequencing, and infection testing of cultured symbionts. Histological observations and cultivation revealed that P. dissimilis harbors Caballeronia symbionts in the crypts of its posterior midgut. 16S rRNA gene amplicon sequencing of field-collected P. dissimilis confirmed that the genus Caballeronia is dominant in the midgut of natural populations of P. dissimilis. In addition, PCR diagnostics showed that the eggs were free of symbiotic bacteria, and hatchlings horizontally acquired the symbionts from ambient soil. Infection and rearing experiments revealed that symbiont-free aposymbiotic individuals had abnormal body color, small body size, and, strikingly, a low survival rate, wherein no individuals reached adulthood, indicating an obligate cooperative mutualism between the mulberry seed bug and Caballeronia symbionts., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Microbial mutualists are pivotal for insect adaptation, which often entails the evolution of elaborate organs for symbiosis. Addressing what mechanisms underpin the development of such organs is of evolutionary interest. Here, we investigated the stinkbug Plautia stali, whose posterior midgut is transformed into a specialized symbiotic organ. Despite being a simple tube in newborns, it developed numerous crypts in four rows, whose inner cavity hosts a specific bacterial symbiont, during the 1st to 2nd nymphal instar stages. Visualization of dividing cells revealed that active cell proliferation was coincident with the crypt formation, although spatial patterns of the proliferating cells did not reflect the crypt arrangement. Visualization of visceral muscles in the midgut, consisting of circular muscles and longitudinal muscles, uncovered that, strikingly, circular muscles exhibited a characteristic arrangement running between the crypts specifically in the symbiotic organ. Even in the early 1st instar stage, when no crypts were seen, two rows of epithelial areas delineated by bifurcated circular muscles were identified. In the 2nd instar stage, crossing muscle fibers appeared and connected the adjacent circular muscles, whereby the midgut epithelium was divided into four rows of crypt-to-be areas. The crypt formation proceeded even in aposymbiotic nymphs, revealing the autonomous nature of the crypt development. We propose a mechanistic model of crypt formation wherein the spatial arrangement of muscle fibers and the proliferation of epithelial cells underpin the formation of crypts as midgut evaginations. IMPORTANCE Diverse organisms are associated with microbial mutualists, in which specialized host organs often develop for retaining the microbial partners. In light of the origin of evolutionary novelties, it is important to understand what mechanisms underpin the elaborate morphogenesis of such symbiotic organs, which must have been shaped through interactions with the microbial symbionts. Using the stinkbug Plautia stali as a model, we demonstrated that visceral muscular patterning and proliferation of intestinal epithelial cells during the early nymphal stages are involved in the formation of numerous symbiont-harboring crypts arranged in four rows in the posterior midgut to constitute the symbiotic organ. Strikingly, the crypt formation occurred normally even in symbiont-free nymphs, revealing that the crypt development proceeds autonomously. These findings suggest that the crypt formation is deeply implemented into the normal development of P. stali , which must reflect the considerably ancient evolutionary origin of the midgut symbiotic organ in stinkbugs.

Lactic acid bacteria (LAB) are beneficial bacteria for humans and animals. However, the characteristics and functions of LAB in insects remain unclear. Here, we isolated LAB from the gut of Riptortus pedestris, a pest that is a significant problem in soybean cultivation in Korea, and identified two Lactococcus lactis and one Enterococcus faecalis using matrix-associated laser desorption/ionization-time of flight and 16S rRNA analyses. All three LAB strains survived at pH 8, and L. lactis B103 and E. faecalis B105 survived at pH 9 for 24 h. In addition, these strains survived well in simulated gastric juice of humans containing pepsin and exhibited high resistance to bile salts. Two strains of L. lactis and one of E. faecalis maintained constant density (> 104 colony-forming units [CFU]/mL) at pH 2.5, but viability at pH 2.2 was strain-dependent. The three LAB were reinoculated into second-instar nymphs of R. pedestris and colonized well, reaching a constant density (> 105 CFU/gut) in the adult insect gut. Interestingly, feeding of these LAB increased the survival rate of insects compared to the negative control, with the largest increase seen for L. lactis B103. However, the LAB did not increase the weight or length of adult insects. These results indicate that insect-derived LAB possess the traits required for survival under gastrointestinal conditions and have beneficial effects on insect hosts. The LAB infection frequency of the wild bean bug populations was 89% (n = 18) in Gyeongsangnam-do, South Korea. These LAB can be utilized as a novel probiotic in the cultivation of beneficial insects. This study provides fundamental information about the symbiosis between insects and LAB, and a novel concept for pest control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Choi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Insect pests introduced in eucalyptus plantations in Brazil are mostly of Australian origin, but native microorganisms have potential for their management. High quality biopesticide production based on entomopathogenic fungi depends on adequate technologies. The objective of this study was to evaluate Mycoharvester® equipment to harvest and separating particles to obtain pure Metarhizium anisopliae conidia to manage Thaumastocoris peregrinus Carpintero & Dellapé, 2006 (Hemiptera: Thaumastocoridae). The Mycoharvester® version 5b harvested and separated M. anisopliae spores. The pure conidia were suspended in Tween 80® (0.1%) and calibrated to the concentrations of 1 x 106, 107, 108 and 109 conidia/ml to evaluate the pathogenicity, lethal concentration 50 and 90 (LC50, LC90) and lethal time 50 and 90 (LT50, LT90) of this fungus to T. peregrinus. This equipment harvested 85% of the conidia from rice, with production of 4.8 ± 0.38 x 109 conidia/g dry mass of substrate + fungus. The water content of 6.36% of the single spore powder (pure conidia) separated by the Mycoharvester® was lower than that of the agglomerated product. The product harvested at the concentrations of 108 and 109 conidia/ml caused high mortality to T. peregrinus third instar nymphs and adults. The separation of conidia produced by solid-state fermentation with the Mycoharvester® is an important step toward optimizing the fungal production system of pure conidia, and to formulate biopesticides for insect pest management., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Velozo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Symbiosis requires the adaptation of symbiotic bacteria to the host environment. Symbiotic factors for bacterial adaptation have been studied in various experimental models, including the Burkholderia -bean bug symbiosis model. Previously identified symbiotic factors of Burkholderia symbionts of bean bugs provided insight into the host environment being stressful to the symbionts. Because DegP, which functions as both a protease and a chaperone, supports bacterial growth under various stressful conditions, we hypothesized that DegP might be a novel symbiotic factor of Burkholderia symbionts in the symbiotic association with bean bugs. The expression level of degP was highly elevated in symbiotic Burkholderia cells in comparison with cultured cells. When the degP -deficient strain competed for symbiotic association against the wild-type strain, the Δ degP strain showed no symbiotic competitiveness. In vivo monoinfection with the Δ degP strain revealed a lower symbiont titer in the symbiotic organ than that of the wild-type strain, indicating that the Δ degP strain failed to persist in the host. In in vitro assays, the Δ degP strain showed susceptibility to heat and high-salt stressors and a decreased level of biofilm formation. To further determine the role of the proteolytic activity of DegP in symbiosis, we generated missense mutant DegP S248A exhibiting a defect in protease activity only. The Δ degP strain complemented with degP S248A showed in vitro characteristics similar to those of the Δ degP strain and failed to persist in the symbiotic organ. Together, the results of our study demonstrated that the proteolytic activity of DegP, which is involved in the stress resistance and biofilm formation of the Burkholderia symbiont, plays an essential role in symbiotic persistence in the host bean bug. IMPORTANCE Bacterial DegP has dual functions as a protease and a chaperone and supports bacterial growth under stressful conditions. In symbioses involving bacteria, bacterial symbionts encounter various stressors and may need functional DegP for symbiotic association with the host. Using the Burkholderia -bean bug symbiosis model, which is a useful model for identifying bacterial symbiotic factors, we demonstrated that DegP is indeed a symbiotic factor of Burkholderia persistence in its host bean bug. In vitro experiments to understand the symbiotic mechanisms of degP revealed that degP confers resistance to heat and high-salt stresses. In addition, degP supports biofilm formation, which is a previously identified persistence factor of the Burkholderia symbiont. Furthermore, using a missense mutation in a protease catalytic site of degP , we specifically elucidated that the proteolytic activity of degP plays essential roles in stress resistance, biofilm formation, and, thus, symbiotic persistence in the host bean bug.

True bugs (Hemiptera, suborder Heteroptera) constitute the largest suborder of nonholometabolous insects and occupy a wide range of habitats various from terrestrial to semiaquatic to aquatic niches. The transition and occupation of these diverse habitats impose various challenges to true bugs, including access to oxygen for the aquatic species and plant defense for the terrestrial phytophagans. Although numerous studies have demonstrated that microorganisms can provide multiple benefits to terrestrial host insects, a systematic study with comprehensive higher taxa sampling that represents aquatic and semiaquatic habitats is still lacking. To explore the role of symbiotic microorganisms in true bug adaptations, 204 samples belonging to all seven infraorders of Heteroptera were investigated, representing approximately 85% of its superfamilies and almost all known habitats. The symbiotic microbial communities of these insects were analyzed based on the full-length amplicons of the bacterial 16S rRNA gene and fungal ITS region. Bacterial communities varied among hosts inhabiting terrestrial, semiaquatic, and aquatic habitats, while fungal communities were more related to the geographical distribution of the hosts. Interestingly, co-occurrence networks showed that species inhabiting similar habitats shared symbiotic microorganism association types. Moreover, functional prediction analyses showed that the symbiotic bacterial community of aquatic species displayed richer amino acid and lipid metabolism pathways, while plant-feeding true bugs benefited more from the symbiont-provided xenobiotics biodegradation pathway. These results deepened the recognition that symbiotic microorganisms were likely to help heteropterans occupy diverse ecological habitats and provided a reference framework for further studies on how microorganisms affect host insects living in various habitats. IMPORTANCE Symbiotic bacteria and fungi generally colonize insects and provide various benefits for hosts. Although numerous studies have investigated symbionts in terrestrial plant-feeding insects, explorations of symbiotic bacterial and fungal communities in aquatic and semiaquatic insects are rare. In this study, the symbiotic microorganisms of 204 aquatic, semiaquatic, and terrestrial true bugs were explored. This comprehensive taxon sampling covers ~85% of the superfamilies of true bugs and most insect habitats. Analyses of the diversity of symbionts demonstrated that the symbiotic microbial diversities of true bugs were mainly affected by host habitats. Co-occurrence networks showed that true bugs inhabiting similar habitats shared symbiotic microbial association types. These correlations between symbionts and hosts together with the functions of bacterial communities indicated that symbiotic microbial communities may help true bugs adapt to (semi)aquatic habitats.

Background: Effective, safe and practical biocontrol options are greatly needed for combating Lygus lineolaris on protected culture strawberry. This study demonstrated how ultaviolet (UV)-selective plastics can improve the efficacy of the fungal biocontrol agent Beauveria bassiana (Mycotrol) compared to the conventional insecticide acetamiprid (Assail) against L. lineolaris on low tunnel strawberry., Results: We found that UVB-blocking treatments improved B. bassiana spore viability in both in vitro and in vivo laboratory experiments. In the field, survival of Mycotrol-treated sentinel L. lineolaris was lowest under UVB-blocking low tunnels, but this did not translate into significant differences among covering treatments in local L. lineolaris density or fruit damage. In contrast, applying the product Assail resulted in the lowest L. lineolaris density and highest quality yield compared to Mycotrol sprays. This was especially pronounced under low tunnels of any UV-limiting plastic., Conclusions: This study indicates that growing under low tunnels is a useful tool to improve the efficacy of conventional products and biopesticides containing microbial biocontrol agents. The efficacy of both products was improved under low tunnels, and specifically under UVB-blocking plastics for Mycotrol containing B. bassiana. However, there was little evidence that UVB plastics resulted in lower L. lineolaris densities and proportion of damaged fruit for either product in the field. Therefore we conclude that growing under any plastic covering is likely to benefit growers, but the economic value of growing under UVB tunnels is unclear. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

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., (© 2022. The Author(s).)

The damage caused by stink bugs that feed on agricultural crops accounts for such significant losses that transgenic plant resistance to stink bugs would be highly desirable. As the level of toxicity of the Bacillus thuringiensis-derived, ETX/Mtx2 pesticidal protein Mpp83Aa1 is insufficient for practical use against the southern green stink bug Nezara viridula, we employed two disparate approaches to isolate peptides NvBP1 and ABP5 that bind to specific proteins (alpha amylase and aminopeptidase N respectively) on the surface of the N. viridula gut. Incorporation of these peptides into Mpp83Aa1 provided artificial anchors resulting in increased gut binding, and enhanced toxicity. These peptide-modified pesticidal proteins with increased toxicity provide a key advance for potential future use against N. viridula when delivered by transgenic plants to mitigate economic loss associated with this important pest., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Diverse insects are obligatorily associated with microbial symbionts, wherein the host often develops special symbiotic organs and vertically transmits the symbiont to the next generation. What molecular factors underpin the host-symbiont relationship is of great interest but poorly understood. Here we report a novel protein preferentially produced in a female-specific symbiotic organ of the stinkbug Plautia stali, whose posterior midgut develops numerous crypts to host a Pantoea-allied bacterial mutualist. In adult females, several posteriormost crypts are conspicuously enlarged, presumably specialized for vertical symbiont transmission. We detected conspicuous protein bands specific to the female's swollen crypts by gel electrophoresis, and identified them as representing a novel mucin-like glycoprotein. Histological inspections confirmed that the mucin protein is localized to the female's swollen crypts, coexisting with a substantial population of the symbiotic bacteria, and excreted from the swollen crypts to the midgut main tract together with the symbiotic bacteria. Using RNA interference, we successfully suppressed production of the mucin protein in adult females of P. stali. However, although the mucin protein was depleted, the symbiont population persisted in the swollen crypts, and vertical symbiont transmission to the next generation occurred. Possible biological roles and evolutionary trajectory of the symbiosis-related mucin protein are discussed., (© 2022. The Author(s).)

Microbial symbionts are critical for the development and survival of many eukaryotes. Recent research suggests that the genes enabling these relationships can be localized in horizontally transferred regions of microbial genomes termed "symbiotic islands." Recently, a putative symbiotic island was found that may facilitate symbioses between true bugs and numerous Burkholderia species, based on analysis of five Burkholderia symbionts. We expanded on this work by exploring the putative island's prevalence, origin, and association with colonization across the bacterial family Burkholderiaceae . We performed a broad comparative analysis of 229 Burkholderiaceae genomes, including 8 new genomes of insect- or soil-associated Burkholderia sequenced for this study. We detected the region in 23% of the genomes; these were located solely within two Burkholderia clades. Our analyses suggested that the contiguous region arose at the common ancestor of plant- and insect-associated Burkholderia clades, but the genes themselves are ancestral. Although the region was initially discovered on plasmids and we did detect two likely instances of horizontal transfer within Burkholderia , we found that the region is almost always localized to a chromosome and does not possess any of the mobility elements that typify genomic islands. Finally, to attempt to deduce the region's function, we combined our data with information on several strains' abilities to colonize the insect's symbiotic organ. Although the region was associated with improved colonization of the host, this relationship was confounded with, and likely driven by, Burkholderia clade membership. These findings advance our understanding of the genomic underpinnings of a widespread insect-microbe symbiosis. IMPORTANCE Many plants and animals form intricate associations with bacteria. These pairings can be mediated by genomic islands, contiguous regions containing numerous genes with cohesive functionality. Pathogen-associated islands are well described, but recent evidence suggests that mutualistic islands, which benefit both host and symbiont, may also be common. Recently, a putative symbiosis island was found in Burkholderia symbionts of insects. We determined that this genomic region is located in only two clades of Burkholderia (the plant- and insect-associated species) and that although it has undergone horizontal transfer, it is most likely a symbiosis-associated region rather than a true island. This region is associated with improved host colonization, although this is may be due to specific Burkholderia clades' abilities to colonize rather than presence of the region. By studying the genomic basis of the insect- Burkholderia symbiosis, we can better understand how mutualisms evolve in animals.

Many beneficial symbioses between bacteria and their terrestrial arthropod hosts are vertically transmitted from mother to offspring, ensuring that the progeny acquire necessary partners. Unusually, in several families of coreoid and lygaeoid bugs (Hemiptera), nymphs must instead ingest the beneficial symbiont, Burkholderia ( sensu lato ), from the environment early in development. We studied the effects of Burkholderia on development of two species of leaf-footed bug (Coreidae) in the genus Leptoglossus , Leptoglossus zonatus and Leptoglossus phyllopus. We found no evidence for vertical transmission of the symbiont but found stark differences in performance between symbiotic and aposymbiotic individuals. Symbiotic nymphs grew more rapidly, were approximately four times more likely to survive to adulthood than aposymbiotic bugs, and were two times larger. These findings suggest that Burkholderia is an obligate symbiont for the Leptoglossus species. We also tested for variation in fitness effects conferred by four symbiont isolates representing different species within the Burkholderia insect-associated stinkbug beneficial and environmental (SBE) clade. While three isolates conferred similar benefits to hosts, nymphs associated with the fourth isolate grew more slowly and weighed significantly less as adults. The effects of the four isolates were similar for both Leptoglossus species. This work indicates that both Burkholderia acquisition and isolate identity play critical roles in the growth and development of Leptoglossus . IMPORTANCE Leptoglossus zonatus and L. phyllopus are important polyphagous pests, and both species have been well-studied but generally without regard to their dependance on a bacterial symbiont. Our results indicate that the central role of Burkholderia in the biology of these insects, as well as in other leaf-footed bugs, should be considered in future studies of coreid life history, ecology, and pest management. Our work suggests that acquisition of Burkholderia is critical for the growth and development of Leptoglossus species. Further, we found that there was variation in performance outcomes according to symbiont identity, even among members of the stinkbug beneficial and environmental clade. This suggests that although environmental acquisition of a symbiont can provide extraordinary flexibility in partner associations, it also carries a risk if the partner is suboptimal.

Fusarium oxysporum f. sp. vasinfectum VCG 0114 (race 4; i.e., FOV4) is an emerging pathogen that causes severe root rot and wilt of cotton. FOV4 is seed-borne, but the mode of seed invasion is uncertain. In an initial study, seeds in bolls that were puncture inoculated with FOV4 conidia when they were 25- or 30-days old became infected but remained viable. Because stink bugs can ingest and introduce bacterial and yeast pathogens into cotton bolls, we hypothesized that stink bugs may ingest and transmit FOV4. Southern green stink bugs and brown stink bugs were exposed to potato dextrose agar cultures of FOV4 and subsequently caged with cotton bolls to assess transmission potential. Both species fed on the cultures and acquired FOV4, and brown stink bugs transmitted FOV4 to cotton bolls. Thus, management of FOV4 may require management of stink bugs to mitigate the spread of the disease in cotton.

Two novel kojic acid derivatives, kojicones A and B (1 and 2), along with the precursors kojic acid (3) and (2R,4R)-4-hydroxy-5-methoxy-2,4-dimethyl-2- [(2R)-2-methylbutyryloxy]-5-cyclohexen-1,3-dione (4), were isolated from a fungal strain Aspergillus versicolor. Their structures and absolute configurations were accurately confirmed by HRESIMS data, NMR analysis, and electronic circular dichroism (ECD) calculations. Kojicones A and B were the first examples of kojic acid adducts with cyclohexen-1,3-dione possessing unprecedented tricycle skeletons. Compounds 1-3 were found to have inhibition on the NO production of murine RAW 264.7 cells. They can also reduce the mRNA expression of four cytokines (IL-6, IL-1β, TNF-α, and iNOS) and promote the expression of IL-4 at 20 μM. Moreover, kojic acid (3) could treat the DSS (dextran sulfate sodium)-induced colitis on mice with the effectiveness similar to that of the positive control. The results suggested that kojic acid and its derivatives could be a promising anti-inflammatory source for the medicinal and cosmetic industry., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nutritional symbioses between bacteria and insects are prevalent and diverse, allowing insects to expand their feeding strategies and niches. A common consequence of long-term associations is a considerable reduction in symbiont genome size likely influenced by the radical shift in selective pressures as a result of the less variable environment within the host. While several of these cases can be found across distinct insect species, most examples provide a limited view of a single or few stages of the process of genome reduction. Stink bugs (Pentatomidae) contain inherited gamma-proteobacterial symbionts in a modified organ in their midgut and are an example of a long-term nutritional symbiosis, but multiple cases of new symbiont acquisition throughout the history of the family have been described. We sequenced the genomes of 11 symbionts of stink bugs with sizes that ranged from equal to those of their free-living relatives to less than 20%. Comparative genomics of these and previously sequenced symbionts revealed initial stages of genome reduction including an initial pseudogenization before genome reduction, followed by multiple stages of progressive degeneration of existing metabolic pathways likely to impact host interactions such as cell wall component biosynthesis. Amino acid biosynthesis pathways were retained in a similar manner as in other nutritional symbionts. Stink bug symbionts display convergent genome reduction events showing progressive changes from a free-living bacterium to a host-dependent symbiont. This system can therefore be used to study convergent genome evolution of symbiosis at a scale not previously available.

Nesidiocoris tenuis (Reuter) is an efficient predatory biological control agent used throughout the Mediterranean Basin in tomato crops but regarded as a pest in northern European countries. From the family Miridae, it is an economically important insect yet very little is known in terms of genetic information and no genomic or transcriptomic studies have been published. Here, we use a linked-read sequencing strategy on a single female N. tenuis. From this, we assembled the 355 Mbp genome and delivered an ab initio, homology-based and evidence-based annotation. Along the way, the bacterial "contamination" was removed from the assembly. In addition, bacterial lateral gene transfer (LGT) candidates were detected in the N. tenuis genome. The complete gene set is composed of 24 688 genes; the associated proteins were compared to other hemipterans (Cimex lectularis, Halyomorpha halys and Acyrthosiphon pisum). We visualized the genome using various cytogenetic techniques, such as karyotyping, CGH and GISH, indicating a karyotype of 2n = 32. Additional analyses include the localization of 18S rDNA and unique satellite probes as well as pooled sequencing to assess nucleotide diversity and neutrality of the commercial population. This is one of the first mirid genomes to be released and the first of a mirid biological control agent., (© 2020 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug Riptortus pedestris , Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont, Burkholderia insecticola Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects., Competing Interests: The authors declare no competing interest.

Phytophagous stink bugs typically harbor nutritional symbiotic bacteria in their midgut, to integrate their unbalanced diet. In the Pentatomidae, most symbionts are affiliated to the genus Pantoea, and are polyphyletic. This suggests a scenario of an ancestral establishment of symbiosis, followed by multiple symbiont replacement events by akin environmental bacteria in different host lineages. In this study, a novel Pantoeaspecies ('CandidatusPantoea persica') was characterized from the gut of the pentatomid Acrosternum arabicum, and shown to be highly abundant in a specific portion of the gut and necessary for the host development. The genome of the symbiont (2.9 Mb), while presenting putative host-supportive metabolic pathways, including those for amino acids and vitamin synthesis, showed a high level of pseudogenization, indicating ongoing genome reduction. Comparative analyses with other free-living and symbiotic Pantoea highlighted a convergent pattern of genome reduction in symbionts of pentatomids, putatively following the typical phases modelized in obligate nutritional symbionts of insects. Additionally, this system has distinctive traits, as hosts are closely related, and symbionts originated multiple independent times from closely related free-living bacteria, displaying convergent and independent conspicuous genome reduction. Due to such peculiarities, this may become an ideal model to study genome evolutionary processes in insect symbionts., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Southern green stink bug [Nezara viridula (L.)] adults and other pentatomid pests can transmit pathogens (e.g., the bacterium Pantoea agglomerans) that cause disease in cotton (Gossypium hirsutum L.) and other high-value cash crops worldwide. First instars of N. viridula were recently shown to ingest P. agglomerans strain Sc1R with rifampicin-resistance, and to retain the pathogen to the 2nd instar. The objective of this study was to determine the acquisition of P. agglomerans Sc1R by early instars of N. viridula and determine persistence of P. agglomerans Sc1R across subsequent stadia. In three trials, early instars (1st and 2nd) were exposed to P. agglomerans Sc1R and subsequently maintained to adulthood; cohorts were sampled at 3rd and 5th instars, as well as adults. In every trial, P. agglomerans Sc1R was detected in all stadia, including adults, but significantly higher frequencies of infection than expected were observed at the initial stage of infection (either 1st or 2nd instar). Higher densities of P. agglomerans Sc1R were detected in 1st and 2nd instars, and lower densities were observed in subsequent stadia. Densities of innate microbiota were generally lower when the initial stage of exposure was at 1st instar than when the initial stage of exposure was at the 2nd instar. Overall, half of the adults possessed P. agglomerans Sc1R. These findings demonstrated that N. viridula nymphs can acquire P. agglomerans Sc1R and retain the pathogen to adulthood. Potential avenues of research to further elucidate the implications of nymphs harboring pathogens to adulthood are discussed., Competing Interests: The authors have declared that no competing interests exist.

Over the last decades, a few fungal species have been mentioned in the literature associated with the rubber tree lace bug, Leptopharsa heveae Drake & Poor (Hemiptera: Tingidae). The rubber plantation area treated with these biocontrol agents has been steady since the 1990s, estimated in 10,000-15,000 ha per year. A few large-scale rubber operations, one biocontrol company, and three government-owned laboratories were, and currently one still is, involved in their commercialization. One species, currently referred to as Sporothrix insectorum, has been historically deployed in biocontrol applications in Brazilian rubber farms. However, L. heveae-infecting isolates have only been identified through morphological examinations; therefore, proper molecular assessments are needed for accurate identifications. Hence, DNA of six L. heveae-infecting isolates (five of which have been deployed in field applications) were extracted and sequenced. Multigene phylogeny found that both Simplicillium lanosoniveum and Cordyceps (formerly Isaria) sp. have been sprayed on rubber plantations to manage L. heveae populations, although the former is the only one currently applied. Simplicillium lanosoniveum and Cordyceps sp. have no relation whatsoever to true Sporothrix species associated with human and animal diseases. Therefore, our molecular data may encourage biocontrol companies to register mycoinsecticides targeting L. heveae. We also added unpublished historical accounts after contacting key contributors to the launching of this not so well-known biocontrol program in the 1980s.

The brown marmorated stink bug Halyomorpha halys (Stål) is a globally invasive species that harbours the primary bacterial symbiont 'Candidatus Pantoea carbekii'. In this work, P. carbekii was used as another genetic marker to investigate the biodiversity and biogeographical patterns of this important pest, in native and newly invaded areas, especially in Italy. The correlation between the genetic structure of the symbiont and that of its host was studied through the analyses of one bacterial and one host marker, the putative pseudogene ΔybgF and the mitochondrial gene COI, respectively. As a result, five new P. carbekii haplotypes were identified, and an association pattern between host-symbiont haplotypes was observed. Host species showed higher haplotype diversity than symbiont, which can be expected in a long term host-symbiont association. Populations from the north-eastern Italy showed the highest values of genetic diversity for both markers, highlighting that this particular Italian area could be the result of multiple ongoing introductions. Moreover, some of the symbiont-host haplotypes observed were shared only by populations from north-eastern Italy and native areas, especially Japan, suggesting further introductions from this native country to Italy. Overall, our findings improve the understanding of the potential origin of multiple accidental introductions of H. halys in Italy.

Plants of the genus Eucalyptus, cultivated in many countries, have great importance for the world economy. In Brazil, this culture occupies a total of 5.7 million hectares, but native and exotic insect pests can reduce its productivity. Thaumastocoris peregrinus Carpintero & Dellapé (Hemiptera: Thaumastocoridae), an exotic Australian pest, damages Eucalyptus plants. Biological control using the egg parasitoid Cleruchoides noackae Lin & Huber (Hymenoptera: Mymaridae), Heteroptera predators and entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae, have potential for managing T. peregrinus. Chemical insecticides, including bifenthrin and acetamiprid + bifenthrin, also control this insect. The compatibility of chemical and biological control methods favors integrated pest management. The objective of this study was to evaluate the selectivity of commercial products based on B. bassiana, M. anisopliae and the chemical bifenthrin on the parasitoid C. noackae and its parasitism on T. peregrinus eggs. The selectivity test followed the standards recommended by the International Organization for Biological Control (IOBC). Beauveria bassiana has selectivity to parasitism as well as viability, but was slightly harmful to C. noackae adults; M. anisopliae was innocuous to adults and to the viability of the offspring of this parasitoid, but it reduced the parasitism rate; and bifenthrin did not show selectivity to this parasitoid.

Tibraca limbativentris (Hemiptera: Pentatomidae) is a major pest in Neotropical rice agroecosystems. In autumn-winter, this pest takes refuge in rice stubble and in native vegetation, which allows it to re-colonize the crop during the rice growing season. However, it is still unknown whether this vegetation is a pest sink or is actually a natural trap that contributes to pest population biocontrol. Here we present information on the insect's alternative host plants, a preliminary outline of the relationship between plant phylogeny and insect abundance, and the impact that winter natural enemies have in pest population. Also, we include a proposed methodology for pest density analysis in winter hosts. Our results show significant differences in the abundance/density that T. limbativentris reaches in the 12 host plant species present in our study areas, with a plant-use pattern significantly related to the phylogenetic clade of Poales. Stink bug winter populations mainly comprised diapause adults, and 40% of insect digestive tracts had content. Survival of T. limbativentris was 56.92% in winter hosts. About mortality, 10% was due to undetermined causes and 33.08% due to entomopathogenic fungi, showing a natural regulation of the pest population. Our results suggest that native vegetation impacts winter survival of T. limbativentris. Although these plants offer shelter, they offer a greater contribution to Integrated Pest Management: the natural regulation of winter pest populations through entomopathogenic attack. Further studies on T. limbativentris population dynamics and the preservation of native areas near rice fields will be required for the development of best control practices.

Plant-sucking stinkbugs are especially associated with mutualistic gut bacterial symbionts. Here, we explored the symbiotic relationship of a pistachio stinkbug, Acrosternum heegeri Fieber by histological, fluorescence in situ hybridization (FISH), real-time PCR and molecular phylogenetic techniques. Furthermore, the effects of the symbiont on the resting/wandering behaviors of the newborn nymphs, pre-adult survival rates, and stage compositions were investigated. Transmission electron microscopy and real-time PCR analyses showed that a rod-shaped gammaproteobacterium was persistently located within the posterior midgut crypts. Molecular phylogenetic and FISH techniques strongly suggested that this symbiont should be placed in the genus Pantoea of the Enterobacteriales. Scanning electron microscopy confirmed the presence of the bacterial cells on the egg surface which the surface sterilization of the eggs resulted in the successful removal of the symbiont from the eggs. Symbiotic and aposymbiotic A. heegeri showed no significant differences in the wandering behaviors of the first nymphal stages, while the symbiont-free insects suffered retarded growth and lower survivability. Together, the results highlight the habitat and acquisition features of Pantoea symbiont and its contribution in A. heegeri biology that might help us for better pest management in the future.

Bacterial cell shapes may be altered by the cell cycle, nutrient availability, environmental stress, and interactions with other organisms. The bean bug Riptortus pedestris possesses a symbiotic bacterium, Burkholderia insecticola, in its midgut crypts. This symbiont is a typical rod-shaped bacterium under in vitro culture conditions, but changes to a spherical shape inside the gut symbiotic organ of the host insect, suggesting the induction of morphological alterations in B. insecticola by host factors. The present study revealed that a deletion mutant of a peptidoglycan amidase gene (amiC), showing a filamentous chain form in vitro, adapted a swollen L-form-like cell shape in midgut crypts. Spatiotemporal observations of the ΔamiC mutant in midgut crypts revealed the induction of swollen cells, particularly prior to the molting of insects. To elucidate the mechanisms underlying in vivo-specific morphological alterations, the symbiont was cultured under 13 different conditions and its cell shape was examined. Swollen cells, similar to symbiont cells in midgut crypts, were induced when the mutant was treated with fosfomycin, an inhibitor of peptidoglycan precursor biosynthesis. Collectively, these results strongly suggest that the Burkholderia symbiont in midgut crypts is under the control of the host insect via a cell wall-attacking agent.

The adaptation of herbivorous insects to new host plants is key to their evolutionary success in diverse environments. Many insects are associated with mutualistic gut bacteria that contribute to the host's nutrition and can thereby facilitate dietary switching in polyphagous insects. However, how gut microbial communities differ between populations of the same species that feed on different host plants remains poorly understood. Most species of Pyrrhocoridae (Hemiptera: Heteroptera) are specialist seed-feeders on plants in the family Malvaceae, although populations of one species, Probergrothius angolensis, have switched to the very distantly related Welwitschia mirabilis plant in the Namib Desert. We first compared the development and survival of laboratory populations of Pr. angolensis with two other pyrrhocorids on seeds of Welwitschia and found only Pr. angolensis was capable of successfully completing its development. We then collected Pr. angolensis in Namibia from Malvaceae and Welwitschia host plants, respectively, to assess their bacterial and fungal community profiles using high-throughput amplicon sequencing. Comparison with long-term laboratory-reared insects indicated stable associations of Pr. angolensis with core bacteria (Commensalibacter, Enterococcus, Bartonella and Klebsiella), but not with fungi or yeasts. Phylogenetic analyses of core bacteria revealed relationships to other insect-associated bacteria, but also found new taxa indicating potential host-specialized nutritional roles. Importantly, the microbial community profiles of bugs feeding on Welwitschia versus Malvaceae revealed stark and consistent differences in the relative abundance of core bacterial taxa that correlate with the host-plant switch; we were able to reproduce this result through feeding experiments. Thus, a dynamic gut microbiota may provide a means for insect adaptation to new host plants in new environments when food plants are extremely divergent., (© 2019 John Wiley & Sons Ltd.)

Despite the omnipresence of specific host-symbiont associations with acquisition of the microbial symbiont from the environment, little is known about how the specificity of the interaction evolved and is maintained. The bean bug Riptortus pedestris acquires a specific bacterial symbiont of the genus Burkholderia from environmental soil and harbors it in midgut crypts. The genus Burkholderia consists of over 100 species, showing ecologically diverse lifestyles, and including serious human pathogens, plant pathogens, and nodule-forming plant mutualists, as well as insect mutualists. Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that nonsymbiotic Burkholderia and even its outgroup Pandoraea could stably colonize the gut symbiotic organ and provide beneficial effects to the bean bug when inoculated on aposymbiotic hosts. However, coinoculation revealed that the native symbiont always outcompeted the nonnative bacteria inside the gut symbiotic organ, explaining the predominance of the native Burkholderia symbiont in natural bean bug populations. Hence, the abilities for colonization and cooperation, usually thought of as specific traits of mutualists, are not unique to the native Burkholderia symbiont but, to the contrary, competitiveness inside the gut is a derived trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont., Competing Interests: The authors declare no competing interest.

Host-microbe symbioses often evolved highly complex developmental processes and colonization mechanisms for establishment of stable associations. It has long been recognized that many insects harbour beneficial bacteria inside specific symbiotic cells (bacteriocytes) or organs (bacteriomes). However, the evolutionary origin and mechanisms underlying bacterial colonization in bacteriocyte/bacteriome formation have been poorly understood. In order to uncover the origin of such evolutionary novelties, we studied the development of symbiotic organs in five stinkbug species representing the superfamily Lygaeoidea in which diverse bacteriocyte/bacteriome systems have evolved. We tracked the symbiont movement within the eggs during the embryonic development and determined crucial stages at which symbiont infection and bacteriocyte formation occur, using whole-mount fluorescence in situ hybridization. In summary, three distinct developmental patterns were observed: two different modes of symbiont transfer from initial symbiont cluster (symbiont ball) to presumptive bacteriocytes in the embryonic abdomen, and direct incorporation of the symbiont ball without translocation of bacterial cells. Across the host taxa, only closely related species seemed to have evolved relatively conserved types of bacteriome development, suggesting repeated evolution of host symbiotic cells and organs from multiple independent origins., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Recent studies have provided molecular evidence that gut symbiotic bacteria modulate host insect development, fitness and reproduction. However, the molecular mechanisms through which gut symbionts regulate these aspects of host physiology remain elusive. To address these questions, we prepared two different Riptortus-Burkholderia insect models, Burkholderia gut symbiont-colonized (Sym) Riptortus pedestris insects and gut symbiont-noncolonized (Apo) insects. Upon LC-MS analyses, juvenile hormone III skipped bisepoxide (JHSB 3 ) was newly identified from Riptortus Apo- and Sym-female and male adults' insect hemolymph and JHSB 3 titer in the Apo- and Sym-female insects were measured because JH is important for regulating reproduction in adult insects. The JHSB 3 titer in the Sym-females were consistently higher compared to those of Apo-females. Since previous studies reported that Riptortus hexamerin-α and vitellogenin proteins were upregulated by the topical abdominal application of a JH-analog, chemically synthesized JHSB 3 was administered to Apo-females. As expected, the hexamerin-α and vitellogenin proteins were dramatically increased in the hemolymph of JHSB 3 -treated Apo-females, resulting in increased egg production compared to that in Sym-females. Taken together, these results demonstrate that colonization of Burkholderia gut symbiont in the host insect stimulates biosynthesis of the heteroptera-specific JHSB 3 , leading to larger number of eggs produced and enhanced fitness in Riptortus host insects., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Insects of the heteropteran superfamilies Coreoidea and Lygaeoidea are consistently associated with symbionts of a specific group of the genus Burkholderia, called the "stinkbug-associated beneficial and environmental (SBE)" group. The symbiosis is maintained by the environmental transmission of symbionts. We investigated European and Japanese populations of the dock bug Coreus marginatus (Coreoidea: Coreidae). High nymphal mortality in reared aposymbiotic insects suggested an obligate host-symbiont association in this species. Molecular phylogenetic analyses based on 16S rRNA gene sequences revealed that all 173 individuals investigated were colonized by Burkholderia, which were further assigned to different subgroups of the SBE in a region-dependent pattern.

Symbioses with microorganisms are ubiquitous in nature and confer important ecological traits to animal hosts but also require control mechanisms to ensure homeostasis of the symbiotic interactions. In addition to protecting hosts against pathogens, animal immune systems recognize, respond to, and regulate mutualists. The gut bacterial symbionts of the cotton stainer bug, Dysdercus fasciatus , elicit an immune response characterized by the upregulation of c-type lysozyme and the antimicrobial peptide pyrrhocoricin in bugs with their native gut microbiota compared to that in dysbiotic insects. In this study, we investigated the impact of the elicited antimicrobial immune response on the established cotton stainer gut bacterial symbiont populations. To this end, we used RNA interference (RNAi) to knock down immunity-related genes hypothesized to regulate the symbionts, and we subsequently measured the effect of this silencing on host fitness and on the abundance of the major gut bacterial symbionts. Despite successful downregulation of target genes by both ingestion and injection of double-stranded RNA (dsRNA), the silencing of immunity-related genes had no effect on either host fitness or the qualitative and quantitative composition of established gut bacterial symbionts, indicating that the host immune responses are not actively involved in the regulation of the nutritional and defensive gut bacterial mutualists. These results suggest that close associations of bacterial symbionts with their hosts can result in the evolution of mechanisms ensuring that symbionts remain insensitive to host immunological responses, which may be important for the evolutionary stability of animal-microbe symbiotic associations. IMPORTANCE Animal immune systems are central for the protection of hosts against enemies by preventing or eliminating successful infections. However, in the presence of beneficial bacterial mutualists, the immune system must strike a balance of not killing the beneficial symbionts while at the same time preventing enemy attacks. Here, using the cotton stainer bug, we reveal that its long-term associated bacterial symbionts are insensitive to the host's immune effectors, suggesting adaptation to the host's defenses, thereby strengthening the stability of the symbiotic relationship. The ability of the symbionts to elicit host immune responses but remain insensitive themselves may be a mechanism by which the symbionts prime hosts to fight future pathogenic infections., (Copyright © 2019 American Society for Microbiology.)

Riptortus pedestris is a major agricultural pest on leguminous plants in South Korea and Japan. Recent studies have revealed that R. pedestris can form beneficial symbiosis with bacteria belonging to genus Burkholderia acquired from soil newly for every generation. Although their physiological interactions are relatively well-understood, infection rate and abundance of the Burkholderia in overwintering natural populations of R. pedestris remain unknown. Therefore, the objective of this study was to characterize Burkholderia infection ratio and clade composition of overwintering R. pedestris populations as well as prevalence and diversity of the genus Burkholderia in soil by conducting a two-year field survey. From the field survey, we found 29 overwintering R. pedestris adults in forested areas nearby soybean fields. Diagnostic PCR analysis revealed that overall infection rate of the symbiotic Burkholderia was 93.1% from overwintering adults. Among the Burkholderia-infected R. pedestris, 70.4% of individuals harbored unclassified Burkholderia clades whereas 22.2% and 7.4% of R. pedestris harbor stinkbug-associated beneficial and environmental (SBE) group and Burkholderia cepacia and complex (BCC), respectively. All R. pedestris were infected with a single clade of Burkholderia. In soil, 56.2% of soil samples were Burkholderia positive, and unlike R. pedestris, multiple Burkholderia clades were detected from 62.2% of those samples. Clade composition of the genus Burkholderia in the samples with the bacteria was 91.1%, 60.0%, 31.1% and 8.8% for plant-associated beneficial and environment (PBE), BCC, SBE and unclassified clade, respectively., Competing Interests: The authors have declared that no competing interests exist.

In the symbiosis of the bean bug Riptortus pedestris with Burkholderia insecticola, the bacteria occupy an exclusive niche in the insect midgut and favor insect development and reproduction. In order to understand how the symbiotic bacteria stably colonize the midgut crypts and which services they provide to the host, we compared the cytology, physiology, and transcriptomics of free-living and midgut-colonizing B. insecticola. The analyses revealed that midgut-colonizing bacteria were smaller in size and had lower DNA content, they had increased stress sensitivity, lost motility, and an altered cell surface. Transcriptomics revealed what kinds of nutrients are provided by the bean bug to the Burkholderia symbiont. Transporters and metabolic pathways of diverse sugars such as rhamnose and ribose, and sulfur compounds like sulfate and taurine were upregulated in the midgut-colonizing symbionts. Moreover, pathways enabling the assimilation of insect nitrogen wastes, i.e. allantoin and urea, were also upregulated. The data further suggested that the midgut-colonizing symbionts produced all essential amino acids and B vitamins, some of which are scarce in the soybean food of the host insect. Together, these findings suggest that the Burkholderia symbiont is fed with specific nutrients and also recycles host metabolic wastes in the insect gut, and in return, the bacterial symbiont provides the host with essential nutrients limited in the insect food, contributing to the rapid growth and enhanced reproduction of the bean bug host.

This study examined the expression and role of vitellogenin (Vg) in the body of the firebug Pyrrhocoris apterus (Heteroptera, Insecta) during infection elicited by two entomopathogenic organisms, the nematode Steinernema carpocapsae and the fungus Isaria fumosorosea Infection by S. carpocapsae significantly upregulated Vg mRNA expression in the male body. The corresponding increase in Vg protein expression was also confirmed by electrophoretic and immunoblotting analyses. Remarkably, in females, the opposite tendency was noted. Nematodal infection significantly reduced both Vg mRNA and Vg protein expression levels in fat body and hemolymph, respectively. We speculate that infection of reproductive females reduces Vg expression to a level that is still sufficient for defense, but is insufficient for reproduction. This circumstance reduces energy expenditure and helps the individual to cope with the infection. Importantly, purified Vg significantly inhibited growth of Xenorhabdus spp., an entomotoxic bacteria isolated from S. carpocapsae. However, the effect of Vg against I. fumosorosea was not so obvious. The fungus significantly stimulated Vg gene expression in males; however, a similar increase was not recapitulated at the protein level. Nevertheless, in females, both mRNA and protein Vg levels were significantly reduced after the fungal infection. The obtained data demonstrate that Vg is probably an important defense protein, possibly with a specific activity. This considerably expands the known spectrum of Vg functions, as its primary role was thought to be limited to regulating egg development in the female body., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)