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

Abstract

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.

Keywords: ADAM; Cry1AcF; GLTP; Prohibitin; RNAi; UDP-GT; α-amylase.

Fig. 1

Differential expression of Cry receptor-like genes in the midgut of G. mellonella fourth-instar larvae at 6 and 12 h after oral administration of Cry1AcF toxin (dose: 27 ng per larva). Asterisk indicates significant (*P < 0.01, **P < 0.001; Tukey’s HSD test) difference in fold change value of the target gene compared to its baseline expression (fold change value set at 1) in control insect. G. mellonella housekeeping genes 18S rRNA and EF-1α were used as internal reference. Each bar represents the mean fold change value ± SE of qPCR runs in five biological and three technical replicates

Fig. 2

Protein–protein interaction between Cry1AcF ligand and putative gut receptors prohibitin, GLTP, α-amylase, ADAM and UDP-GT. Cry1AcF bound with these receptors via a number of Pi interactions, hydrogen bonds and salt bridges. The ZDock scores (greater value indicates greater contact surface area between ligand and receptor) for Cry-prohibitin, Cry-GLTP, Cry-α-amylase, Cry-ADAM and Cry-UDP-GT complexes were 1510, 1961, 2041, 1916 and 1836 Ų, respectively. Domain I, II and III of Cry1AcF are highlighted in magenta, ochre yellow and green color, respectively. Exposed α helices and β sheets (with default colors) do not encompass any domain of Cry1AcF

Fig. 3

Predicted secondary structures of five Cry receptor-like proteins and a gut repair protein. Membrane anchored prohibitin contains SPFH (wheatish box) and coiled-coil (black box) domains. Intracellular GLTP contains a characteristic GLTP domain (light green box). α-amylase contains an intracellular, extracellular and a transmembrane (dark green box) domain. Extracellular ADAM includes N-terminal signal peptide, a prodomain (blue box), metalloprotease catalytic site (yellow box), cysteine-rich region (red box), C-terminal disintegrin domain (green box) and numerous N- and O-glycosylated residues. Extracellular UDP-GT includes N-terminal signal peptide, C-terminal UGT signature motif (magenta box) and few N/ O-glycosylated residues. Extracellular arylphorin includes a signal peptide and three Hexamerin domains (blue boxes). Solid and dotted perpendicular lines indicate N- and O-glycosylated residues, respectively

Fig. 4

Stage-specific expression patterns of Cry receptor-related genes and a gut repair gene in different life stages of G. mellonella. Fold change in expression of a candidate gene in different developmental (second-, third-, fourth- and fifth-instar) stages was quantified in relation to the gene’s expression in first-instar stage (value set at 1). Significant differential expression is indicated by different letters (P < 0.01, Tukey’s HSD test). G. mellonella 18S rRNA and EF-1α genes were used as the internal reference. Each bar represents the mean fold change value ± SE of qPCR runs in five biological and three technical replicates

Fig. 5

Tissue-specific expression patterns of Cry receptor-related genes and a gut repair gene in different tissues of G. mellonella fourth-instar larvae. Fold change in expression of a candidate gene in different tissues (fat body (FB), foregut (FG), midgut (MG), hindgut (HG) and Malpighian tubules (MT)) was quantified in relation to the gene’s expression in head (H) tissue (value set at 1). Significant differential expression is indicated by different letters (P < 0.01, Tukey’s HSD test). G. mellonella 18S rRNA and EF-1α genes were used as the internal reference. Each bar represents the mean fold change value ± SE of qPCR runs in five biological and three technical replicates

Fig. 6

RNAi knockdown of five receptor-related genes and a gut repair gene in G. mellonella midgut led to target-specific silencing. Abundance of transcripts corresponding to prohibitin, GLTP, α-amylase, ADAM, UDP-GT and arylphorin in insects silenced (si) with these target genes were analyzed by qPCR. Fourth-instar larvae were force-fed with dsRNA-expressing E. coli HT115 cells and inoculated for 24 h. Insects orally ingested with GFP dsRNA and PBS were used as the non-native and negative control, respectively. G. mellonella 18S rRNA and EF-1α genes were used as the internal reference. Each bar represents the mean fold change value ± SE of qPCR runs in five biological and three technical replicates. Significant differential expression is indicated by different letters (P < 0.01, Tukey’s HSD test)

Fig. 7

The dsRNA binding sites (indicated by perpendicular lines) in the coding sequences of G. mellonella prohibitin, GLTP, α-amylase, ADAM, UDP-GT and arylphorin are shown. Additionally, the homologous transcripts (indicated by grey boxes) that were aligned with the target receptors are shown. Numbers indicate the sequence coordinates

Fig. 8

RNAi-induced downregulation of Cry receptor-related genes differentially altered the insect susceptibility to Cry1AcF toxin. A Percent pupation and adult emergence data of dsRNA-treated G. mellonella during 7 to 10 days after ingestion. Each bar with same letter is indicative of no significant difference between treatments (P > 0.01, Tukey’s HSD test, n = 30). B Percent mortality data of Cry1AcF force-fed larvae, which were pre-inoculated with dsRNAs corresponding to five Cry receptor-related genes and one gut repair gene. Post 24 h of dsRNA treatment, LD₅₀ (27 ng per larva) and LD₉₀ (90 ng per larva) doses of Cry1AcF were orally delivered. After another 24 h mortality data was recorded. Different letters are indicate significant difference between treatments (P < 0.01, Tukey’s HSD test, n = 50)