Phenylethylamides derived from bacterial secondary metabolites specifically inhibit an insect serotonin receptor.

Serotonin (5-hydroxytryptamine: 5-HT) is a biogenic monoamine that mediates immune responses and modulates nerve signal in insects. Se-5HTR, a specific receptor of serotonin, has been identified in the beet armyworm, Spodoptera exigua. It is classified into subtype 7 among known 5HTRs. Se-5HTR was expressed in all developmental stages of S. exigua. It was expressed in all tested tissues of larval stage. Its expression was up-regulated in hemocytes and fat body in response to immune challenge. RNA interference (RNAi) of Se-5HTR exhibited significant immunosuppression by preventing cellular immune responses such as phagocytosis and nodulation. Treatment with an inhibitor (SB-269970) specific to 5HTR subtype 7 resulted in significant immunosuppression. Furthermore, knockout mutant of Se-5HTR by CRISPR-Cas9 led to significant reduction of phagocytotic activity of S. exigua hemocytes. Such immunosuppression was also induced by bacterial secondary metabolites derived from Xenorhabdus and Photorhabdus. To determine specific bacterial metabolites inhibiting Se-5HTR, this study screened 37 bacterial secondary metabolites with respect to cellular immune responses associated with Se-5HTR and selected 10 potent inhibitors. These 10 selected compounds competitively inhibited cellular immune responses against 5-HT and shared phenylethylamide (PEA) chemical skeleton. Subsequently, 46 PEA derivatives were screened and resulting potent chemicals were used to design a compound to be highly inhibitory against Se-5HTR. The designed compound was chemically synthesized. It showed high immunosuppressive activities along with specific and competitive inhibition activity for Se-5HTR. This study reports the first 5HT receptor from S. exigua and provides its specific inhibitor designed from bacterial metabolites and their derivatives.

Overexpression of PGE2 synthase by in vivo transient expression enhances immunocompetency along with fitness cost in a lepidopteran insect.

Prostaglandins (PGs) mediate various physiological functions in insects. Specifically, PGE2 is known to mediate immunity and egg-laying behavior in the beet armyworm, Spodoptera exigua A PGE2 synthase 2 (Se-PGES2) has been identified to catalyze the final step to produce PGE2 in S. exigua Its expression is inducible in response to immune challenge. Inhibition of the gene expression results in immunosuppression. In contrast, any physiological alteration induced by its uncontrolled overexpression was not recognized in insects. This study used the in vivo transient expression (IVTE) technique to induce overexpression and assessed subsequent physiological alteration in S. exiguaSe-PGES2 was cloned into a eukaryotic expression vector and transfected to Sf9 cells to monitor its heterologous expression. The Sf9 cells expressed the recombinant Se-PGES2 (rSe-PGES2) at an expected size (∼47 kDa), which was localized in the cytoplasm. The recombinant expression vector was then used to transfect larvae of S. exigua Hemocytes collected from the larvae treated with IVTE expressed the rSe-PGES2 gene for at least 48 h. The larvae treated with IVTE exhibited an enhanced competency in cellular immune response measured by hemocyte nodule formation. In addition, IVTE treatment of Se-PGES2 induced gene expression of antimicrobial peptides without any immune challenge. The larvae treated with IVTE became significantly resistant to infection of an entomopathogenic nematode, Steinernema monticolum, or to infection to its symbiotic bacterium, Xenorhabdus hominickii However, IVTE-treated S. exigua larvae suffered from reduced pupal size and fecundity.

Identification of an entomopathogenic bacterium, Xenorhabdus ehlersii KSY, from Steinernema longicaudum GNUS101 and its immunosuppressive activity against insect host by inhibiting eicosanoid biosynthesis.

Steinernema longicaudum GNUS101, an entomopathogenic nematode, was isolated from soils in Korea. Its internal transcribed space sequence was highly similar to the known S. longicaudum species. Infective juveniles (IJs) of S. longicaudum were highly virulent to lepidopteran and coleopteran insects. Two different bacteria were isolated from the hemolymph of lepidopteran larvae infected with S. longicaudum. They exhibited blue and red colonies on nutrient bromothymol blue agar. The red-colored bacterium was identified as Enterococcus mundtii KHY while the blue-colored bacterium was identified as Xenorhabdus ehlersii KSY based on 16S rRNA sequencing and biochemical characters. The bacterial species showed different growth rates, with X. ehlersii KSY growing more slowly than E. mundtii KHY. Both bacteria were entomopathogenic, but showed differences in suppressing host immune responses. X. ehlersii KSY, but not E. mundtii KHY, showed inhibitory activity against cellular immune responses of Spodoptera exigua larvae including hemocyte-spreading behavior and nodule formation in bacteria-cultured broth. Its immunosuppressive activity was reversed by adding arachidonic acid, an eicosanoid biosynthesis precursor. Furthermore, organic extracts of X. ehlersii KSY using hexane or ethyl acetate showed inhibitory activity against cellular immune responses of S. exigua larvae. Arachidonic acid addition to S. exigua larvae infected with X. ehlersii significantly rescued the survival rate of target insect. Of the two bacteria isolated from S. longicaudum GNUS101, only X. ehlersii induced immunosuppression of target insect by inhibiting eicosanoid biosynthesis.

Molecular phylogeography of Culex quinquefasciatus mosquitoes in central Bangladesh.

Mosquitoes in the Culex pipiens complex are a major vector of numerous parasitic and arboviral diseases. Here we report the phylogeography of a prevalent Culex mosquito, Cx. quinquefasciatus, from three locations in Bangladesh: Dhaka, Savar and Mymensingh. Sequence analysis of the genes encoding mitochondrial cytochrome oxidase subunit II, nuclear elongation factor-1 alpha, and acetylcholinesterase-2 revealed the lack of a population genetic structure among the three locations. Moreover, the highly divergent ribosomal internal transcribed spacer 2 suggests that this locus has not evolved in concert. The results further show evidence of historical introgression of internal transcribed spacer 2 from Cx. pipiens to Cx. quinquefasciatus of Bangladesh, and that the introgression occurred before Cx. quinquefasciatus had dispersed within this region. The study also reveals historical population expansion in this region, followed by a post-expansion Wolbachia sweep.