In silico identification and expression analyses of Defensin genes in the mealworm beetle Tenebrio molitor

Abstract

Defensins are a major family of antimicrobial peptides that serve as the innate immune defense of both vertebrates and invertebrates. Due to their antimicrobial, chemotactic, and regulatory activities, Defensins have been exploited for their therapeutic potential. Insect Defensins are cysteine-rich and contain an N-terminal loop, α-helix, and antiparallel β-sheet, forming a "cysteine-stabilized alpha beta (CSαβ)" or "loop-helix-sheet" structure. In this study, we identified the full-length open reading frame (ORF) sequences of Defensin (TmDef) and Defensin-like (TmDef-like) genes from the mealworm beetle Tenebrio molitor using in silico methods. TmDef and TmDef-like genes encode the peptides of 72 and 71 amino acid residues, respectively. TmDefensin is comprised of a Defensin domain and the TmDefensin-like is comprised of a signal peptide of 21 amino acid residues. Phylogenetic analysis revealed close similarities of TmDefensin with the Defensin of Acalolepta luxuriosa of the longhorn beetle family. The expression of TmDef mRNA was found to be greater than that of TmDef-like mRNA and was mostly expressed in the pupal and adult stages. Tissue distribution showed high expression of TmDef-like mRNA in larval hemocytes, gut, integument, and fat body, while in adults, the expression was high in gut and hemocytes. Following bacterial and fungal stimulation in vivo, TmDef was upregulated at 24 h post-infection in whole body, fat body, and hemocytes of the larvae. Even TmDef-like mRNA was upregulated in the gut and hemocytes at 12 and 9 h post-infection respectively. These results suggest that TmDef and TmDef-like genes play important roles in protecting T. molitor from microbial contact.

Innate Immune Response of TmToll-3 Following Systemic Microbial Infection in Tenebrio molitor

“…Consistent with previous findings, the decreased survival rates of TmToll-3 -silenced T. molitor larvae following E. coli infection observed herein suggested that humoral innate immunity could occur via TmToll-3 . TmToll3 silencing in larvae rendered larvae more susceptible to E. coli infection and suppressed certain AMP genes induced by E. coli challenge, including TmTenesin-1, -4, TmDefensin, TmDefensin-like, TmColeoptericin-A, -B, -C , and TmAttacin-1a, -1b, -2 , all four of which belong to AMP families known to exhibit antibacterial activity against Gram-negative bacteria [ 49 , 50 , 51 , 52 ]. Tm Toll-7 and Tm Toll-2 have been reported to activate their downstream NF-kB transcription factor, which leads to the induction of immune response genes including AMP genes [ 30 , 37 ].…”

Tenebrio molitor Spätzle 1b Is Required to Confer Antibacterial Defense Against Gram-Negative Bacteria by Regulation of Antimicrobial Peptides

“…Expression patterns of 14 AMP genes including TmTenecin1, 2, 3 , and 4 ( TmTene1 , − 2 , − 3 , and − 4 ) ( Kim et al, 1998 ; Chae et al, 2012 ; Yang et al, 2017 ), TmDefensin and TmDefensin-like ( TmDef and TmDef-like ) ( Jang et al, 2020b ), TmColeoptericin-A and -B ( TmColeA and − B ) ( Zhu et al, 2014 ; Jang et al, 2020a ), TmAttacin-1a, −1b and -2 ( TmAtt1a , − 1b and − 2 ) ( Jo et al, 2018 ), TmCecropin-2 ( TmCec2 ) ( Ali Mohammadie Kojour et al, 2021 ), and TmThaumatin - like protein-1 and − 2 ( TmTLP1 and − 2 ) ( Noh and Jo, 2016 ; Kim et al, 2017 ), were examined by qRT-PCR with the AMP gene-specific primers ( Table 1 ). A relative quantitative PCR was performed as detailed above in an AMP-specific primer.…”

Available for millions of years but discovered through the last decade: Insects as a source of nutrients and energy in animal diets

“… Keshavarz et al. (2019) Jang et al. (2020b) Defensin 2 Full sequences not presented in references, Authors indicated defensins region: MPHEDVEVFEEAVHRVERGFFCNPGLCHRQCKQSGHRRASCSGDECVCLG – The highest expression after E. coli immunization of larvae; the AMP gene expression observed in fat body, gut, and Malpighian tubules of young larvae.…”

“… Keshavarz et al. (2019) Jang et al. (2020b) Thaumatin-like proteins 1 and 2 Immunized larvae of T. molitor with E. coli ATCC K12, S. aureus ATCC RN4220, C. albicans ATCC/the fat body, hemocytes, gut, and Malpighian tubule Thaumatin-like protein, GenBank (XP_012568089.2): MSDSCLMNIGGHQVTFYIHNKCPFPIWPATPSNTGQPIIAYGGFCLASSQTKKIQAPWSWSGRIWASTGCNFDSNSWKPS CETGDSDGKLACNGLIGTPP ATLDEITLQGDKGKTNFYGVSLVDGYIVPVSITPSKNINSKCNIEGCLKDVKSLCPNELQ SLESVKKKEEEEAKKKEVQL KAVKADGKSAEEVKDNGAIY Analysis of thaumatin-like-proteins 1 and 2 gene expression (qRT–PCR) and antimicrobial activity Antifungal activity, C. albicans – The highest expression was observed in larvae infected with C. albicans.…”