Binding properties of chemosensory protein 4 in Riptortus pedestris to aggregation pheromones.

In insects, chemosensory proteins (CSPs) play an important role in the perception of the external environment and have been widely used for protein-binding characterization. Riptortus pedestris has received increased attention as a potential cause of soybean staygreen syndrome in recent years. In this study, we found that RpedCSP4 expression in the antennae of adult R. pedestris increased with age, with no significant difference in expression level observed between males and females, as determined through quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, we investigated the ability of RpedCSP4 to bind various ligands (five aggregated pheromone components and 13 soybean volatiles) using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP4 binds to three aggregated pheromone components of R. pedestris, namely, ((E)-2-hexenyl (Z)-3-hexenoate (E2Z3), (E)-2-hexenyl (E)-2-hexenoate (E2E2), and (E)-2-hexenyl hexenoate (E2HH)), and that its binding capacities are most stable under acidic condition. Finally, the structure and protein-ligand interactions of RpedCSP4 were further analyzed via homology modeling, molecular docking, and targeted mutagenesis experiments. The L29A mutant exhibited a loss of binding ability to these three aggregated pheromone components. Our results show that the olfactory function of RpedCSP4 provides new insights into the binding mechanism of RpedCSPs to aggregation pheromones and contributes to discover new target candidates that will provide a theoretical basis for future population control of R. pedestris.

The Lethal and Sublethal Effects of Lambda-Cyhalothrin and Emamectin Benzoate on the Soybean Pest Riptortus pedestris (Fabricius).

Riptortus pedestris (Fabricius, 1775) (Hemiptera: Alydidae) is a major soybean pest in East Asia that can cause soybean staygreen syndrome. To date, no insecticides have been registered for the control of R. pedestris in China, and these insects are primarily controlled in the field through the application of broad-spectrum insecticides including lambda-cyhalothrin (LCT) and emamectin benzoate (EMB). Here, the lethal and sublethal effects of LCT and EMB on R. pedestris were comprehensively evaluated. LCT and EMB were both found to exhibit high levels of toxicity and concentration-dependent repellent effects for R. pedestris. The exposure of third instar nymphs from the F0 generation to LC30 concentrations of LCT and EMB resulted in a significant increase in the duration of nymph development and adult pre-oviposition period (APOP), together with reductions in fifth instar nymph and adult body weight, longevity, oviposition days, fecundity, vitellarium length, lateral oviduct diameter, and vitellogenin (Vg) gene expression as compared to control treatment. Strikingly, these suppressive effects were transmitted to the F1 generation, which similarly experienced the prolongation of preadult development and the preoviposition period (TPOP). Relative to control-treated populations, the F1 generation for these insecticide-treated groups also exhibited significant decreases in population parameter values. Overall, these data offer new insight into the impact that LCT and EMB treatment can have on R. pedestris, providing a valuable foundation for the application of these pesticides in the context of integrated pest management strategies aimed at soybean crop preservation.

Genome-wide identification of candidate chemosensory receptors in the bean bug Riptortus pedestris (Hemiptera: Alydidae) and the functional verification of its odorant receptor co-receptor (Orco) in recognizing aggregation pheromone.

A sophisticated and sensitive olfactory system plays a vital role in the survival and reproduction of insects. Chemosensory receptors are indispensable for the molecular recognition and discrimination of semiochemicals. Riptortus pedestris is a notorious pest of legume plants, resulting in yield losses and quality decreases in soybeans. It is well accepted that R. pedestris highly relies on its olfactory system in detecting aggregation pheromones, host volatiles, and pesticides; however, little research focused on its chemosensory receptors. In the present study, we identified 237 odorant receptors (ORs), 42 gustatory receptors (GRs), and 31 ionotropic receptors (IRs) from the reported genome of R. pedestris, and analyzed their phylogenetic relationship with other hemipteran species. Through the results of RNA-seq and real-time quantitative PCR (qRT-PCR), we found that RpedORs displayed different expression levels in the antennae of R. pedestris at different development stages. To further verify the function of odorant receptor co-receptor (Orco), an obligate and unique insect OR, we silenced RpedOrco by RNA interference (RNAi) method. The results showed that silencing RpedOrco could significantly impair the response to aggregation pheromone in R. pedestris, indicating that RpedOrco plays an essential role in odorant detection. Our results can provide the theoretical foundations for revealing the olfactory recognition mechanism of R. pedestris and help explore and develop novel olfactory-based agents against this pest.

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.

Binding properties of odorant-binding protein 4 from bean bug Riptortus pedestris to soybean volatiles.

The bean bug Riptortus pedestris is a notorious insect pest that can damage various crops, especially soybean, in East Asia. In insects, the olfactory system plays a crucial role in host finding and feeding behaviour in which the odorant-binding proteins (OBPs) are believed to be involved in initial step in this system. In this study, we produced the R. pedestris adult antennae-expressed RpedOBP4 protein using a recombinant expression system in E. coli. Fluorescence competitive binding confirmed that RpedOBP4 has binding affinities to 7 of 20 soybean volatiles (ligands), and that a neutral condition is the best environment for it. The binding property of RpedOBP4 to these ligands was further revealed by integrating data from molecular docking, site-directed mutagenesis and ligand binding assays. This demonstrated that five amino acid residues (I30, L33, Y47, I57 and Y121) are involved in the binding process of RpedOBP4 to corresponding ligands. These findings will not only help us to more thoroughly explore the olfactory mechanism of R. pedestris during feeding on soybean, but also lead to the identification of key candidate targets for developing environmental and efficient behaviour inhibitors to prevent population expansion of R. pedestris in the future.

Symbiont coordinates stem cell proliferation, apoptosis, and morphogenesis of gut symbiotic organ in the stinkbug-Caballeronia symbiosis.

The bean bug Riptortus pedestris obtains a specific bacterial symbiont, Caballeronia insecticola (Burkholderia insecticola), from the environmental soil and harbors it in the posterior midgut region that is composed of hundreds of crypts. While newly hatched aposymbiotic insects possess primordial midgut crypts with little or no lumen, colonization of C. insecticola triggers swift development of the symbiotic organ, forming enlarged and opened crypts, and the symbiont subsequently fills the luminal cavities of those mature crypts. The cellular processes of crypt development triggered by C. insecticola colonization are poorly understood. Here we identified a fundamental mechanism of the symbiont-mediated midgut development by investigating cell cycles of intestinal epithelial cells. Intestinal stem cells of the bean bug are located and proliferate at the crypt base. Differentiated enterocytes migrate upward along the epithelial cell layer of the crypt as the midgut develops, induction of apoptosis in enterocytes primarily occurred on the tip side of the crypts, and apoptotic cells then eventually were shed from the crypts into the hemolymph. The proliferation rate of the stem cells at the base of the crypts was low while a high apoptotic rate was observed at the crypt tip in aposymbiotic insects, resulting in undeveloped short crypts. On the contrary, the gut-colonizing C. insecticola promoted the proliferation of the stem cells at the base of crypts and simultaneously inhibited apoptosis at the tip of crypts, resulting in a net growth of the crypts and the generation of a crypt lumen that becomes colonized by the bacterial symbiont. These results demonstrated that the Caballeronia symbiont colonization induces the development of the midgut crypts via finely regulating the enterocyte cell cycles, enabling it to stably and abundantly colonize the generated spacious crypts of the bean bug host.

Behavioral and Electrophysiological Effects of Ajowan (Trachyspermum ammi Sprague) (Apiales: Apiaceae) Essential Oil and Its Constituents on Nymphal and Adult Bean Bugs, Riptortus clavatus (Thunberg) (Hemiptera: Alydidae).

We investigated the repellent effect of 12 Apiaceae plant essential oils on nymphal and adult (male and female) forms of the bean bug, Riptortus clavatus (Thunberg) (Hemiptera: Alydidae), using a four-arm olfactometer. Among the essential oils tested, ajowan (Trachyspermum ammi Sprague) essential oil showed the strongest repellent activity against the nymphal and adult bean bugs. For female adults, the repellent activity was significantly different between an ajowan oil-treated chamber and an untreated chamber down to a concentration of 14.15 µg/cm2. We also investigated the repellent activity of individual ajowan essential oil constituents. Of the compounds examined, carvacrol and thymol showed the most potent repellent activity against the nymphal and adult bean bugs. Carvacrol and thymol exhibited 73.08% and 70.0% repellent activity for the bean bug nymph at 0.71 and 2.83 µg/cm2, respectively, and 82.6% and 80.7% at 5.66 and 11.32 µg/cm2, respectively, for male adults. Carvacrol and thymol exhibited strong repellent activity against female adult bean bugs down to a concentration of 2.83 µg/cm2. Ajowan essential oil, thymol and carvacrol elicited a negative electroantennogram (EAG) response from adult bean bugs. This could explain the repellent activity of ajowan essential oil and its constituents. Our results indicate that ajowan essential oil and its constituents carvacrol and thymol can be potential candidates as the 'push' component in a 'push-pull' strategy for bean bug control.

Evaluating the efficacy of two insect detection methods with Riptortus pedestris (Hemiptera: Alydidae): portable harmonic radar system and fluorescent marking system.

BACKGROUND: Although portable harmonic radar system and fluorescent marking system are two widely used detection methods, their comparative effectiveness has not been studied. Therefore, we first tested the applicability of fluorescent marking system on Riptortus pedestris (Hemiptera: Alydidae). Then, we evaluated the efficacy of the two methods used either alone or combined in a grass field and bean field, varying with complexity, during day and night. RESULTS: Fluorescent marking did not affect the behavior or fitness of Riptortus pedestris except for vertical walking, while allowing the detection from >25 m when paired with a handheld laser. Generally, the portable harmonic radar system and both methods combined were more successful in sample detection, although the fluorescent marking system in the bean field at night was as competitive as the two. Combining both methods made sample retrieval easier at night than the portable harmonic radar system. Nevertheless, the total detection time showed a large variance across the methods. CONCLUSION: The portable harmonic radar system can be an effective detection method in either landscape during both day and night. Furthermore, the fluorescent marking system can be a reliable tool at night as well. Lastly, combining both methods can improve the night detection. © 2018 Society of Chemical Industry.

Relative Toxicity of Spirotetramat to Riptortus pedestris (Hemiptera: Alydidae) and its Egg Parasitoids.

Spirotetramat, a lipid biosynthesis inhibitor, is effective against sucking insect pests but harmless to insect natural enemies. As spirotetramat can be registered for the management of sucking insect pests such as aphids and bugs in soybeans, we evaluated the insecticide against Riptortus pedestris (Fabricius) (Hemiptera: Alydidae), one of the most important soybean pests in Korea, as well as its effect on two of its important egg parasitoids, Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) and Gryon japonicum (Ashmead; Hymenoptera: Platygastridae). Oral toxicities of five concentrations of spirotetramat (1.00, 0.50, 0.25, 0.13, and 0.06 ml/liter) were tested by feeding these test solutions to insects for 24 h after 12 h of starvation. The lethal median concentration (LC50) for second instars of the bean bug was 0.3 ml/liter after 48 h while values for fourth instars and adults were 9.2 and 19.0 ml/liter, respectively. The median lethal time (LT50) for bean bugs when exposed to a concentration of 0.50 ml/liter was 1.2-1.5 times less than that of the control, while in G. japonicum and O. nezarae it was 1.1-1.2 times less than the control. These results show that spirotetramat is less toxic to the egg parasitoids of bean bug than to bean bug itself and would thus be useful in an integrated management program for this pest.

Females of Riptortus pedestris (Hemiptera: Alydidae) in Reproductive Diapause are More Responsive to Synthetic Aggregation Pheromone.

Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) enters reproductive diapause under short-day length (< 13.5 h) conditions. As an apparent increase of field populations of R. pedestris has been observed during the fall when aggregation pheromone traps are placed in soybean fields, we tested the hypothesis that R. pedestris becomes more responsive to the aggregation pheromone during fall as it enters its reproductive diapause. The response of R. pedestris females-either ones collected seasonally from fields or reared in the laboratory under short-day length (10:14 [L:D] h) conditions-to the aggregation pheromone was examined using a Y-tube olfactometer. Riptortus pedestris collected in fall showed a higher response to the aggregation pheromone (74-80%) than those collected in July (40%). Females in which diapause was induced in the laboratory also showed a higher response to the pheromone than those not in diapause. In a wind tunnel assay, female bugs in laboratory-induced diapause also responded positively to the aggregation pheromone. In addition, we assessed the reproduction of females to verify their diapausing status. Diapausing females never mated with either short- or long-day-reared males, nor did they reproduce, but non-diapausing females mated with short-day-reared males at a rate of 13%. Females collected from fields during fall never mated. In a soybean field evaluation, the number of adult female R. pedestris was higher in the presence of an aggregation pheromone trap than in the absence of one. This is the first confirmation of higher response to the aggregation pheromone of both field-collected and laboratory-reared R. pedestris in reproductive diapause.

Insect Gut Symbiont Susceptibility to Host Antimicrobial Peptides Caused by Alteration of the Bacterial Cell Envelope.

The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis.