In general, hemipteran insects transmit either obligate or facultative symbionts to their offspring using various transmission mechanisms. Riptortus pedestris (Hemiptera: Alydidae) also have facultative symbiosis with Burkholderia sp. which is acquired from the soil every generation especially during 2nd instar nymphal period. This environmental symbiont transmission makes R. pedestris face a great risk of missing their symbionts, however, previous studies showed that they have intimate associations with their symbionts. Therefore, we suspected that R. pedestris 2nd instar nymphs may have a preference toward Burkholderia sp. to achieve an efficient symbiont acquisition during the limited time of window. In order to identify the preference of 2nd instar nymph, we first conducted dual-choice experiments varying with the number of 2nd instar nymphs (a nymph and 100 nymphs) in plastic cages. Furthermore, Y-tube olfactometer experiments varying with the presence of soil as medium of Burkholderia sp. were performed to determine if the insects respond to bacterial volatile induced from the symbiont. Based on our results, we observed that R. pedestris 2nd instar nymphs may not show any specific behavioral preference or response to chemical volatile from Burkholderia sp. in laboratory conditions (P >0.24). Therefore, we will further conduct a dual-choice experiment in a semi-field condition to investigate if such patterns can be detected in a larger arena simulating a more natural environment.

Vertical transmission of symbiont is known as the most effective way to deliver their offspring. However, it has beenreported that the bean bug, Riptortus pedestris, acquires its gut symbiont, Burkholderia sp., from its environment (e.g.soil) during the 2nd instar nymphal stage. Nevertheless, it is not unknown how they locate their symbiont. For this reason,dual-choice experiments were conducted in both solitary (a nymph) and group conditions (100 nymphs) to investigatewhether or not R. pedestris has preference on Burkholderia sp. treated or untreated solution in laboratory conditions. Inthese experiments, there was no significant difference in the preference between the two treatments. We are testing inthe Y-tube olfactometer system to evaluate the response of R. pedestris on possible chemical cues from Burkholderia sp..

The bean bug, Riptortus pedestris, is a pest of leguminous crops. Recent studies have revealed an interesting relationship between R. pedestris and a gut symbiont Burkholderia sp.. R. pedestris mainly acquires Burkholderia sp. during 2nd instar period, which leads to enhancement of growth and reproductive rate of the host. Nevertheless, how R. pedestris acquires its symbiont is not yet clearly described. Therefore, we investigated whether 2nd instar nymphs have preference on Burkholderia sp. compared to untreated control in laboratory settings. In the choice test, no significant difference was found in the preference by R. pedestris between the two treatments. To further investigate single individual’s behaviors (its first choice, approaching frequency to each treatment, and residence time on the treatment). We video-recorded single individuals in the choice setting for 12 hours. Again, there was no significant difference in any of the behavioral traits recorded. Further choice tests will be conducted in the Y-tube olfactometer systems and semi-field conditions.

Recent studies suggested that gut symbionts modulate insect development and reproduction. However, how gut symbionts modulate host physiologies and what types of molecules are involved in these changes are still unclear. When we analyzed hemolymph proteins and transcriptional levels of host insects, hexamerin-α (Hex-α), hexamerin-β (Hex-β) and vitellogenin-1 (Vg-1) were highly expressed in symbiotic insects (Sym) compared to aposymbiotic insects (Apo). Depletion of Hex-β by RNA interference in 2nd Sym-nymphs delayed adult emergence, whereas Hex-α and Vg-1 RNA interference in 5th nymphs decreased reproduction of female insects and caused loss of color of laid eggs. Also, the levels of JHSBIII of Riptortus host were 3-fold higher in the Sym-female insects compared to the Apo-insects. These results demonstrate that the Burkholderia gut symbiont modulates host development and egg production through regulating the expression of three host storage proteins by controlling of brain hormone.

Five phaP family genes and one phaR gene have been identified in the genome of Burkholderia gut symbiont. PhaP proteins function as surface proteins of polyhydroxyalkanoate (PHA) granules, and PhaR protein acts as a negative regulator of PhaP biosynthesis. To address the biological roles of four phaP family genes (phaP1, phaP2, phaP3, and phaP4) and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second instar nymph. The ΔphaR mutant decreased the colonization ability in the host midgut compared to wild-type Burkholderia cells and negatively affected the host insect’s fitness compared with wild-type infected host. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects’ midgut

The Riptortus pedestris-Burkholderia symbiotic system is a promising model for understanding molecular mechanism of symbiosis. In previous studies, the Burkholderia symbiont has been shown to play important biological roles in the growth and fitness of host R. pedestris. The Burkholderia symbiont, one of bacteria found in the soil, is the only bacterium that can colonize the symbiotic midgut region of R. pedestris. However, the molecular mechanism of host selectivity for the Burkholderia symbiont remains unknown. To determine where the selection occurs, we firstly compared initial infectivity of different mid-gut regions after oral infection of Escherichia coli and Burkholderia. Interestingly, E. coli were not detected in any regions of mid-gut, while Burkholderia could reach to the posterior region of mid-gut. Therefore, we hypothesized that host selectivity for the Burkholderia symbiont is occurred in the salivary gland. To address this hypothesis, we treated E. coli and Burkholderia with lysate of salivary gland and examined their survival by estimation of colony forming unit (CFU) on the plate. We found that E. coli, but not Burkholderia, was susceptible to the lysate of salivary gland. To determine molecular basis of the selective mechanism in the salivary gland, we analyzed antimicrobial proteins (AMPs) from lysate of salivary gland. we identified three AMPs, namely rip-trialysin1, rip-trialysin2 and lysozyme and further purified rip-trialysin1 and rip-trialysin2. When E. coli and Burkholderia were treated with rip-trialysin1 and rip-trialysin2, rip-trialysin1 exhibited little antimicrobial activity, but rip-trialysin2 exhibited antimicrobial activity. Furthermore, we found that E. coli was susceptible, but Burkholderia is resistant to commerciallypurchased egg white lysozyme. Our results suggest that R. pedestris salivary gland provides a chance of selection for the Burkholderia symbiont and lysozyme in salivary gland seems to play an important role for the selection of gut symbiont.

The Riptortus-Burkholderia symbiosis is a newly emerging insect-bacterium symbiotic system. This symbiosis system has a good merit as an experimental model system to produce the non-symbiotic (apo) and symbiotic (sym) host insect. In recent reported papers, the symbionts play important biological roles for the host insects. Meanwhile, juvenile hormone (JH) is one of major hormone synthesized corpora allata(CA) to control many physiology of insect. However, the study for cross-talk mechanism between symbionts and host hormones to control important physiological phenomenon of insects is almost none. In this study, we found that Riptortus speed up adult emerging and increase egg laying on presence of symbiont Burkholderia. Also we found that hexamerin proteins, which were controlled the expression by JH, were accumulated in sym-Riptortus hemolymph compare with apo-Riptortus. According as combined results, we hypothesized that the gut symbiont Burkholderia can control JH titer to conclude out beneficial effects such as development and reproduction of R. pedestris. To verify this hypothesis, we examined measurement of JH titer, expression of hexamerins as JH response genes and RNAi for hexamerin protein during whole Riptortus life on presence or absence of symbiont Burkholderia. All results demonstrated that gut symbiont controlled JH titer of Riptortus. Controlled JH amount by symbiont Burkholderia in host midgut regulated hexamerin protein expression for speeding up adult emerging and increasing egg production.

The Riptortus (stinkbug) has a specialized symbiotic organ, M4 midgut, to harboring symbiont Burkholderia. M4 midgut is located in abdomen and surrounded with insect hemolymph. Recently our group demonstrated that symbiotic Burkholderia showed different physiology after adapting in M4 gut compare with in vitro cultured Burkholderia. And population of symbiotic Burkholderia in the M4 midgut is regulated by special organ. However, the molecular mechanism to prevent spreading and migrating symbiont bacteria to other host tissues from symbiotic organ is not clear. Therefore, we assumed that symbiont Burkholderia are susceptible to host humoral immunity after established infection in M4 midgut to prevent spreading and migrating into the other host tissues through Riptortus hemolymph. To prove this assuming, we tested the susceptibility and survival rate of symbiont Burkholderia in hemolymph of Riptortus in vitro and in vivo. We also examined the susceptibility of symbiont Burkholderia using purified antimicrobial peptides (AMP), pyrrhocoricin-like, thanatin-like and defensin-like AMPs. Finally, we tested inducing ability for AMPs by systemic infection of symbiotic Burkholderia. Gene expression of purified AMPs was not different after systemic infection of both symbiont and in vitro cultured Burkholderia. Surprisingly, in vitro cultured Burkholderia resisted on bacteria injected hemolymph and purified AMPs but symbiont Burkholderia were highly susceptible in bacteria injected hemolymph and purified AMP. These results suggest that symbiont Burkholderia can't survive in the hemolymph after escaping symbiotic organ. Moreover, humoral immunity of host Riptortus is important to prevent spreading and migrating symbiont Burkholderia into the other host tissue or organ from symbiotic organ.