Effects of the symbiotic bacteria, Caballeronia insecticola, on the life history parameters of Riptortus pedestris (Hemiptera: Alydidae) and their implications for the host population growth.

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.

Effects of four potent entomopathogenic fungal isolates on the survival and performance of Telenomus remus, an egg parasitoid of fall armyworm.

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 LT50 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 109 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 105 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.

Geographical, Seasonal, and Growth-Related Dynamics of Gut Microbiota in a Grapevine Pest, Apolygus spinolae (Heteroptera: Miridae).

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 ~ 104 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.

Impact of diet and bacterial supplementation regimes on Orius strigicollis microbiota and life history performance.

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.

A strong priority effect in the assembly of a specialized insect-microbe symbiosis.

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.

Rich diversity of RNA viruses in the biological control agent, Orius laevigatus.

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.

Exploring the effects of entomopathogenic nematode symbiotic bacteria and their cell free filtrates on the tomato leafminer Tuta absoluta and its predator Nesidiocoris tenuis.

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).

Host range of naturally and artificially evolved symbiotic bacteria for a specific host insect.

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 ΔcyaA 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.

The plant-sucking insect selects assembly of the gut microbiota from environment to enhance host reproduction.

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.

Microbiota of pest insect Nezara viridula mediate detoxification and plant defense repression.

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.

Paraburkholderia largidicola sp. nov., a gut symbiont of the bordered plant bug Physopelta gutta.

Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacteria, designated strains F2T 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 MWAP64T (99.3 %). The genome of strain F2T 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 F2T and P. caribensis MWAP64T or phylogenetically closely related Paraburkholderia species were 44.3 % or below 49.1 %. The predominant fatty acids of both strains were C16 : 0, C17 : 0 cyclo, summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), and C19 : 0 cyclo ω8c, and their respiratory quinone was ubiquinone 8. Based on the above genotypic and phenotypic characteristics, strains F2T and PGU16 represent a novel species of the genus Paraburkholderia for which the name Paraburkholderia largidicola sp. nov. is proposed. The type strain is F2T (=NBRC 115765T=LMG 32765T).

Ecological drift during colonization drives within-host and between-host heterogeneity in an animal-associated symbiont.

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.

Specialized digestive mechanism for an insect-bacterium gut symbiosis.

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.

Different roles of host and habitat in determining the microbial communities of plant-feeding true bugs.

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.

Changes in the gut bacterial community affect miRNA profiles in Riptortus pedestris under different rearing conditions.

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.

Mechanisms Underpinning Morphogenesis of a Symbiotic Organ Specialized for Hosting an Indispensable Microbial Symbiont in Stinkbugs.

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.

From the dual cyclone harvest performance of single conidium powder to the effect of Metarhizium anisopliae on the management of Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae).

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.

Identification and characterization of gut-associated lactic acid bacteria isolated from the bean bug, Riptortus pedestris (Hemiptera: Alydidae).

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.

Proteolytic Activity of DegP Is Required for the Burkholderia Symbiont To Persist in Its Host Bean Bug.

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 ΔdegP strain showed no symbiotic competitiveness. In vivo monoinfection with the ΔdegP strain revealed a lower symbiont titer in the symbiotic organ than that of the wild-type strain, indicating that the ΔdegP strain failed to persist in the host. In in vitro assays, the ΔdegP 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 DegPS248A exhibiting a defect in protease activity only. The ΔdegP strain complemented with degPS248A showed in vitro characteristics similar to those of the ΔdegP 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.

Obligate Gut Symbiotic Association with Caballeronia in the Mulberry Seed Bug Paradieuches dissimilis (Lygaeoidea: Rhyparochromidae).

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.