RNAi-mediated knockdown of gut receptor-like genes prohibitin and α-amylase altered the susceptibility of Galleria mellonella to Cry1AcF toxin.

BACKGROUND: Due to the prolonged usage of Bt-based biopesticides and Bt-transgenic crops worldwide, insects are continually developing resistance against Cry toxins. This resistance may occur if any mechanistic step in the insecticidal process is disrupted possibly because of the alteration in Cry-receptor binding affinity due to mutation in receptor genes. Compared to other lepidopteran insects, Cry receptor-related research has made asymmetric progress in the model insect Galleria mellonella. RESULTS: Present study describes the molecular characterization and functional analysis of five Cry toxin receptor-related genes (prohibitin, GLTP, α-amylase, ADAM and UDP-GT) and a gut repair gene (arylphorin) from the gut tissues of G. mellonella. Protein-protein docking analysis revealed that Cry1AcF putatively binds with all the five candidate proteins, suggesting their receptor-like function. These receptor-like genes were significantly overexpressed in the gut tissues of fourth-instar G. mellonella larvae upon early exposure to a sub-lethal dose of Cry1AcF toxin. However, targeted knockdown (by using bacterially-expressed dsRNAs) of these genes led to variable effect on insect susceptibility to Cry1AcF toxin. Insects pre-treated with prohibitin and α-amylase dsRNA exhibited significant reduction in Cry1AcF-induced mortality, suggesting their probable role as Cry receptor. By contrast, insects pre-treated with GLTP, ADAM and UDP-GT dsRNA exhibited no significant decline in mortality. This maybe explained by the possibility of RNAi feedback regulation (as few of the receptors belong to multigene family) or redundant role of GLTP, ADAM and UDP-GT in Cry intoxication process. CONCLUSION: Since the laboratory culture of G. mellonella develop Bt resistance quite rapidly, findings of the current investigation may provide some useful information for future Cry receptor-related research in the model insect.

Molecular characterization and functional analysis of Cry toxin receptor-like genes from the model insect Galleria mellonella.

Crystal (Cry) toxins produced from the soil bacterium, Bacillus thuringiensis (Bt), have gained worldwide attention for long due to their insecticidal potential. A number of receptor proteins located on the epithelial cells of the larval midgut were shown to be crucial for Cry intoxication in different insect pests belonging to order Lepidoptera, Diptera and Coleoptera. A beehive pest, Galleria mellonella, serves as an excellent insect model for biochemical research. However, information on the Cry receptor-like genes in G. mellonella is limited. In the present study, the full-length sequences of four putative Cry receptor genes (ABC transporter, alkaline phosphatase, aminopeptidase N and cadherin) were cloned from G. mellonella. All these receptor genes were substantially upregulated in the midgut tissue of fourth-instar G. mellonella larvae upon early exposure (6 h) to a sub-lethal dose of Cry1AcF toxin. Oral and independent delivery of bacterially-expressed dsRNAs corresponding to four receptor genes in G. mellonella suppressed the transcription of target receptors which in turn significantly reduced the larval sensitivity to Cry1AcF toxin. As the laboratory populations of G. mellonella develop Bt resistance in a relatively short time, molecular characterization of Cry receptor genes in G. mellonella performed in the present study may provide some useful information for future research related to the genetic basis of Bt resistance in the model insect.

RNAi-based knockdown of candidate gut receptor genes altered the susceptibility of Spodoptera frugiperda and S. litura larvae to a chimeric toxin Cry1AcF.

Background: A multitude of Cry toxins (secreted by Bacillus thuringiensis or Bt) has been deployed globally either via transgenic mean or bio-pesticidal formulations in order to manage insect pests. However, Bt resistance development in insects is emerging as a major concern. To avoid this problem, multiple gene pyramiding or protein-engineered chimeric toxin-based strategy has been analyzed. Methods: In the present study, one such chimeric toxin Cry1AcF (contain the swapped domains of Cry1Ac and Cry1F) was used to investigate its in vivo pathogenesis process in lepidopteran pests Spodoptera frugiperda and S. litura. A number of biochemical and molecular analysis were performed. Results: Oral ingestion of Cry1AcF caused greater toxicity in S. frugiperda than S. litura with larvae displaying increased hemolymph melanization. Histopathology of the midgut transverse sections exhibited Cry1AcF-induced extensive gut damage in both the test insects followed by cytotoxicity in terms of reduced hemocyte numbers and viability. Elevated hemolymph phenoloxidase activity indicated the immune-stimulatory nature of Cry1AcF. In order to analyze the role of gut receptor proteins in Cry1AcF intoxication in test insects, we performed RNAi-mediated silencing using bacterially-expressed dsRNAs of individual receptor-encoding genes including CAD, ABCC2, ALP1 and APN. Target-specific induced downregulation of receptor mRNAs differentially altered the insect susceptibility to Cry1AcF toxin in our study. The susceptibility of ALP1 and APN dsRNA pre-treated S. frugiperda was considerably decreased when treated with Cry1AcF in LD50 and LD90 doses, whereas susceptibility of CAD and ABCC2 dsRNA pre-treated S. litura was significantly reduced when ingested with Cry1AcF in different doses. CAD/ABCC2-silenced S. frugiperda and ALP1/APN-silenced S. litura were vulnerable to Cry1AcF alike of control larvae. In conclusion, our results indicate ALP1/APN and CAD/ABCC2 as the functional receptor for Cry1AcF toxicity in S. frugiperda and S. litura, respectively.

The induced knockdown of GmCAD receptor protein encoding gene in Galleria mellonella decreased the insect susceptibility to a Photorhabdus akhurstii oral toxin.

Photorhabdus bacteria secrete a repertoire of protein toxins that can kill the host insect. Among them, toxin complex (Tc) proteins have gained significant attention due to their wider conservation across the different bacterial genera. In our laboratory, a C-terminal domain of TcaB protein was characterized from P. akhurstii bacterium that conferred the potent oral insecticidal effect on Galleria mellonella. However, the role of insect gut receptors in the TcaB intoxication process was yet to be investigated. In the current study, we examined the transcription of candidate midgut receptors in TcaB-infected larvae and subsequently cloned a cadherin-like gene, GmCAD, from G. mellonella. GmCAD was highly transcribed in the fourth-instar larval stage and specifically in the midgut tissues. Our ligand blot and binding ELISA assays indicated that TcaB binds to the truncated peptides from the GmCAD transmembrane-proximal region with greater affinity than that from the transmembrane-distal region. Oral administration of bacterially expressed GmCAD dsRNA in G. mellonella severely attenuated the expression of target mRNA, which in turn alleviated the negative effect of TcaB on insect survival (TcaB-induced mortality in CAD dsRNA pretreated larvae reduced by 72-83% compared to control), implying the association of GmCAD in the TcaB intoxication process. Present findings form a basis of future research related to the insect gut receptor interactions with Photorhabdus toxins.