In Korea, the orchid species Cymbidium goeringii and C. kanran are very important as natural genetic resources and floriculture crops. With respect to nutrient uptake, both species mixotrophic, and can shift to autotrophy with the development of photosynthetic organs. In this study, microscope observation was performed to examine the existing symbiotic fungi and their pelotons in root cortical tissues. Molecular identification was performed to identify orchid mycorrhizal fungi (OMF) and evaluate phylogenetic relationships with other OMF taxa in the database. Essential molecular data encompassing ribosomal internal transcribed spacers (ITS) were amplified from the extracted genomic DNA of root tissues. The hyphae grown into cortical root tissues of two individual cymbidiums were observed and revealed to form typical coiled structures called pelotons. OMF sequences obtained from C. goeringii root tissues were determined to be 673 bp nucleotide sequences (MF101371), while those obtained from C. kanran root tissues were 788 bp nucleotide sequences (MF101372). The resulting ITS rDNA sequence was analyzed using the UPGMA cluster analysis method and a dendrogram was generated. According to the phylogenetic tree, the ITS rDNA sequence from C. goeringii was positioned among other Tulasnella species, and that from C. kanran was grouped with Russula species. Producing elementary, but fundamental, data about the nutritionally helpful symbiotic mycorrhizal fungi is very important to facilitate orchid-fungal microbe interactions and for cultivation and conservation programs for native orchids.

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.

Many insects possess symbiotic microorganisms in their bodies, wherein host-symbiont intimate interactions occur. Despite recent advances in omics technology, the molecular bases of the symbiotic associations remain unclear. The bean bug Riptortus pedestris harbors an environmentally acquired Burkholderia symbiont in their midgut crypts. Unlike other insect symbionts, the Burkholderia symbiont is easily culturable and genetically manipulatable outside the host insect. In conjunction with the experimental advantages of the bean bug, the Riptortus–Burkholderia association is an ideal model system for approaching the molecular bases underpinning insect-microbe symbioses. In this presentation, the current knowledge of this model system is summarized.

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

It is not uncommon in the nature for insects to have symbiotic relationships with microorganisms. The bean bug, Riptortus pedestris, which harbors symbiont Burkholderia sp., is a serious agricultural pest in South Korea and Japan. It is reported that the symbiont has positive effects on the fitness of R. pedestris. However, virtually no information is available for how this insect finds the symbiont in the nature and how the behavior of R. pedestris is affected by the symbiont. To investigate the symbiont acquisition mechanism, the frequencies of R. pedestris approaching to symbiont or untreated control treatment were compared in a plastic cage (465×290×260mm) (n=20). In the experiment, there was no detectable difference between the two treatments. Further study using video tracking systems will help us to better understand detailed behaviors for symbiont acquisition.

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.

Symbiotic bacteria are common in insects. Because symbiotic bacteria are known to intimately affect the various aspects of insect host biology, ideally insects can be controlled by manipulating their symbiont. However, the attempts to control insects through their symbiont have been very limited. The paucity of the insect pest control using their symbiont is most likely due to the poor understanding of the symbiotic interactions between host insect and symbiont, which is attributed to the difficulty in cultivation of insect symbionts. However, the recently established bean bug, Riptortus pedestris, symbiotic system provides good opportunities to study insect’s symbiont in molecular level through their cultivable symbionts. Bean bugs acquire genus Burkholderia cells from environment and harbor them as their gut symbionts in the specialized posterior midgut. The genome of the Burkholderia symbiont was sequenced, and the genomic information has been used to generate the genetically manipulated Burkholderia symbiont strains. After orally administering the mutant Burkholderia symbionts into bean bugs for symbiotic association, the bacterial colonization levels in the host gut and host phenotypes were analyzed. As a result, we have identified novel symbiotic factors necessary for establishing successful association with host. Our recent understandings on the bacterial symbiotic factors demonstrate a great possibility to control the bean bug pest using genetically modified Burkholderia 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.