A C-type lectin with dual carbohydrate recognition domains functions in innate immune response in Asian corn borer, Ostrinia furnacalis.

C-type lectins (CTLs) act as pattern recognition receptors (PRRs) to initiate the innate immune response in insects. A CTL with dual carbohydrate recognition domains (CRDs) (named immulectin-4 [IML-4]) was selected from the Ostrinia furnacalis transcriptome dataset for functional studies. We cloned the full-length complementary DNA of O. furnacalis IML-4 (OfIML-4). It encodes a 328-residue protein with a Glu-Pro-Asn (EPN) and Gln-Pro-Asp (QPD) motifs in 2 CRDs, respectively. OfIML-4 messenger RNA levels increased significantly upon the bacterial and fungal infection. Recombinant OfIML-4 (rIML-4) and its individual CRDs (rCRD1 and rCRD2) exhibited the binding ability to various microorganisms including Escherichia coli, Micrococcus luteus, Pichia pastoris, and Beauveria bassiana, and the cell wall components including lipopolysaccharide from E. coli, peptidoglycan from M. luteus or Bacillus subtilis, and curdlan from Alcaligenes faecalis. The binding further induced the agglutination of E. coli, M. luteus, and B. bassiana in the presence of calcium, the phagocytosis of Staphylococcus aureus by the hemocytes, in vitro encapsulation and melanization of nickel-nitrilotriacetic acid beads, and a significant increase in phenoloxidase activity of plasma. In addition, rIML-4 significantly enhanced the phagocytosis, nodulation, and resistance of O. furnacalis to B. bassiana. Taken together, our results suggest that OfIML-4 potentially works as a PRR to recognize the invading microorganisms, and functions in the innate immune response in O. furnacalis.

Involvement of Holotrichia parallela odorant-binding protein 3 in the localization of oviposition sites.

Organic fertilizers-derived volatiles attract Holotrichia parallela during oviposition. However, the mechanisms underlying the perception of oviposition cues in H. parallela remain unclear. Here, H. parallela odorant-binding protein 3 (HparOBP3) was identified as a key OBP. Bioinformatics analysis showed that HparOBP3 clustered together with Holotrichia oblita OBP8. HparOBP3 was mainly expressed in the antennae of both sexes. Recombinant HparOBP3 exhibited distinct binding affinities towards 22 compounds released by organic fertilizers. After 48 h of RNA interference (RNAi), the expression of HparOBP3 in male and female antennae was decreased by 90.77 % and 82.30 %, respectively. In addition, silencing of HparOBP3 significantly reduced the electrophysiological responses and tropism of males to cis-3-hexen-1-ol, 1-hexanol, and (Z)-ß-ocimene as well as females to cis-3-hexen-1-ol, 1-hexanol, benzaldehyde, and (Z)-ß-ocimene. Molecular docking indicated that hydrophobic residues Leu-83, Leu-87, Phe-108, and Ile-120 of HparOBP3 were important amino acids for interacting with ligands. Mutation of the key residue, Leu-83, significantly diminished the binding ability of HparOBP3. Furthermore, acrylic plastic arena bioassays showed that the attraction and oviposition indexes of organic fertilizers to H. parallela were reduced by 55.78 % and 60.11 %, respectively, after silencing HparOBP3. These results suggest that HparOBP3 is essential in mediating the oviposition behavior of H. parallela.

PI3K/Akt/CncC signaling pathway mediates the response to EPN-Bt infection in Holotrichia parallela larvae.

BACKGROUND: Combining the entomopathogenic nematode (EPN), Heterorhabditis beicherriana LF strain, and Bacillus thuringiensis (Bt) HBF-18 strain is a practical strategy to manage the larvae of Holotrichia parallela Motschulsky (white grubs). However, the mechanisms underlying the larval defense response to this combined biocontrol strategy are unknown. RESULTS: The activities of some antioxidant enzymes (SOD, POD, CAT) and some detoxifying enzymes (AChE, P-450, CarE, GST) in grubs showed an activation-inhibition trend throughout the EPN-Bt exposure time course. Eight potentially key antioxidant and detoxifying enzyme genes in response to EPN-Bt infection were identified from the midgut of grubs through RNA sequencing. After silencing CAT, CarE18, and GSTs1, the enzyme activities were significantly decreased by 30.29%, 68.80%, and 34.63%, respectively. Meanwhile, the mortality of grubs was increased by 18.40%, 46.30%, and 42.59% after exposure to EPN-Bt for 1 day. Interestingly, the PI3K/Akt signaling pathway was significantly enriched in KEGG enrichment analysis, and the expression levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), cap 'n' collar isoform-C (CncC), kelch-like ECH-associated protein 1 (Keap1), and CarE18 were all up-regulated when exposed to EPN-Bt for 1 day. Furthermore, RNAi-mediated PI3K silencing showed a similar down-regulated trend between PI3K/Akt/CncC and CarE18. Moreover, silencing PI3K rendered grubs more susceptible to EPN-Bt and accelerated symbiotic bacteria multiplication in grubs. CONCLUSION: These results suggest that the PI3K/Akt/CncC pathway mediates the expression of CarE18 and participates in the defense response of H. parallela larvae against EPN-Bt infection. Our data provide valuable insights into the design of appropriate management strategies for this well-known agricultural pest. © 2022 Society of Chemical Industry.

Molecular and functional characterization of odorant-binding protein genes in Holotrichia oblita Faldermann.

Holotrichia oblita is one of the nastiest pests in China. In present research, four full-length cDNA encoding of HoblOBP genes were cloned and sequenced from H. oblita. The mRNA of HoblOBPs were predominantly expressed in antenna. The recombinant HoblOBPs proteins were obtained for fluorescence binding assays. Four of HoblOBPs could mediate the response of H. oblita to organic fertilizers-derived attractants, including HoblOBP5 binding to skatole; HoblOBP8 binding to p-cresol, indole and skatole; HoblOBP9 binding to indole and 4-allylanisole; and HoblOBP24 binding to p-cresol, indole and 4-ethylphenol. Further, RNA interference demonstrated that transcripts of HoblOBP5, 8, 9, and 24 decreased in a time-dependent manner after dsRNA-injection. Knockdown of HoblOBP5, 8, 9, and 24 by injection of dsRNA successfully interfered with behavioral responses towards the target compounds in beetles. Our results showed that HoblOBP5, HoblOBP8, HoblOBP9 and HoblOBP24 are essential in mediating the approach behavior of H. oblita.