No abstract

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.

No abstract

Prostaglandins (PGs) mediate various physiological functions in insects. Specifically, PGE 2 is known to mediate immunity and egglaying behavior in the beet armyworm, Spodoptera exigua. A PGE 2 synthase 2 (Se-PGES2) has been identified to catalyze the final step to produce PGE 2 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. exigua. Se-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.

21Serotonin (5-hydroxytryptamine: 5-HT) is a biogenic monoamine that mediates immune 22 responses and modulates nerve signal in insects. Se-5HTR, a specific receptor of serotonin, has 23 been identified in the beet armyworm, Spodoptera exigua. It is classified into subtype 7 among 24 known 5HTRs. Se-5HTR was expressed in all developmental stages of S. exigua. It was 25 expressed in all tested tissues of larval stage. Its expression was up-regulated in hemocytes and 26 fat body in response to immune challenge. RNA interference (RNAi) of Se-5HTR exhibited 27 significant immunosuppression by preventing cellular immune responses such as phagocytosis 28 and nodulation. Treatment with an inhibitor (SB-269970) specific to 5HTR subtype 7 resulted in 29 significant immunosuppression. Such immunosuppression was also induced by bacterial 30 secondary metabolites derived from Xenorhabdus and Photorhabdus. To determine specific 31 bacterial metabolites inhibiting Se-5HTR, this study screened 37 bacterial secondary metabolites 32 with respect to cellular immune responses associated with Se-5HTR and selected 10 potent 33 inhibitors. These 10 selected compounds competitively inhibited cellular immune responses 34 against 5-HT and shared phenylethylamide (PEA) chemical skeleton. Subsequently, 46 PEA 35 derivatives were screened and resulting potent chemicals were used to design a compound to be 36 highly inhibitory against Se-5HTR. The designed compound was chemically synthesized. It 37 showed high immunosuppressive activities along with specific and competitive inhibition 38 activity for Se-5HTR. This study reports the first 5HT receptor from S. exigua and provides its 39 specific inhibitor designed from bacterial metabolites and their derivatives. 40 3 41 Author Summary 42 Serotonin (5-hydroxytryptamine: 5-HT) plays a crucial role in mediating nerve and immune 43 signals in insects. Interruption of 5-HT signal leads to malfunctioning of various insect 44 physiological processes. Se-5HTR, a 5-HT receptor of beet armyworm, Spodoptera exigua, was 45 identified and classified as subtype 7 (5-HT 7 ) of 5-HT receptors. A specific inhibitor (SB-46 269970) for 5-HT 7 highly inhibited immune responses such as phagocytosis and nodulation 47 mediated by Se-5HTR. Two entomopathogenic bacteria, Xenorhabdus and Photorhabdus, could 48 secrete potent inhibitors against immune responses mediated by 5-HTR. Bacterial secondary 49 metabolites were screened against Se-5HTR-mediating immune responses. Most of resulting 50 compounds shared phenylethylamide (PEA) chemical skeleton. Subsequent screening using PEA 51 derivatives supported the importance of this chemical skeleton. Based on their relative inhibitory 52 activities, a compound was designed and synthesized. This novel compound possessed high 53 inhibitory activities against Se-5HTR-mediating immune responses and exhibited competitive 54 inhibition with 5-HT. 55 Serotonin or 5-hydroxytryptamine (5-HT) is a biogenic monoamine found across most phyla of 58 life [1]. This indolamine compound ...