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

Abstract

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.

Keywords: Cry receptor-encoding genes; Gene silencing; Hemocyte viability; Histopathology; Insect mortality; PO activity.

Figure 1. Insecticidal activity of Cry1AcF toxin in S. frugiperda and S. litura.

(A) Oral ingestion of the toxin using hypodermic needle in starved S. frugiperda fourth-instar larvae. (B) At 24 h after inoculation, toxin (150 ng)-treated moribund (M) larvae attained dead-like posture with extended prolegs and hemolymph melanization compared to normal phenotypes of PBS-ingested control (L) larvae (scale bar—0.5 cm). Dose-response curves depict the percent survival of S. frugiperda (C) and S. litura (D) at 24 h after toxin ingestion. TcaB and PBS were used as the positive and negative control, respectively. X- and Y-axis represent toxin dose and percent larval survival, respectively. Treatments (mean ± SE, n = 50) with different letters are significantly different at P < 0.01, Tukey’s HSD test.

Figure 2. Cry1AcF negatively altered the gut homeostasis of S. frugiperda and S. litura fourth-instar larvae.

Histopathology of the midgut transverse sections of S. frugiperda treated with PBS (A), Cry1AcF (B) and S. litura treated with PBS (C), Cry1AcF (D) at 24 h after inoculation. Cry1AcF was administered with LD₅₀ doses. Asterisks (*) indicate disintegration of epithelial cells (EC) followed by sloughing off into the gut lumen (GL). VM, visceral muscle; Nu, Nucleus. Scale bar = 50 μm.

Figure 3. Effect of Cry1AcF toxin on the total circulatory hemocytes of S. frugiperda and S. litura fourth-instar larvae at LD₅₀ and LD₉₀ doses.

(A) Y-axis indicates hemocyte counts (×10⁵) per ml of hemocoel extracted at 24 h after toxin ingestion. (B) Y-axis indicates percent viable cells (determined by Trypan blue staining assay) at 24 h after toxin ingestion. PBS was used as the control. Treatments (mean ± SE, n = 10) with different letters are significantly different at P < 0.01, Tukey’s HSD test. Comparative photomicrographs show live cells (prevent dye invasion) in PBS ingested S. frugiperda larvae and dead cells (stain dark blue because of disintegrated membrane) in Cry1AcF ingested S. frugiperda larvae. Scale bar = 10 μm.

Figure 4. Hemolymph phenoloxidase (PO) enzyme activity in S. frugiperda and S. litura fourth-instar larvae upon ingestion of Cry1AcF in LD₅₀ and LD₉₀ doses.

PO activity was measured at 12 (A) and 24 (B) h after toxin administration. PBS was used as the control. PO activity was measured using L-DOPA as substrate and change in absorbance (due to melanin synthesis) after 30 min was recorded at 490 nm. PO activity was expressed as OD₄₉₀ per minute per mg protein. PBS + L-DOPA was used as blank and its absorbance reading (at 490 nm) was subtracted from each sample. Treatments (mean ± SE, n = 10) with different letters are significantly different at P < 0.01, Tukey’s HSD test.

Figure 5. Relative expression of receptor protein encoding genes in the midgut of S. frugiperda.

(A) and S. litura (B) fourth-instar larvae at 12 and 24 h after Cry1AcF ingestion (in LD₅₀ doses). The asterisk (*P < 0.01, **P < 0.001; Tukey’s HSD test) is indicative of significant differential expression of target gene mRNAs compared to their baseline expression (fold change values set at 1) in insects ingested with PBS. Gene expression was normalized using endogenous reference genes of S. frugiperda (β-actin and GAPDH) and S. litura (rps3). Each bar represents the mean fold change value ± SE of RT-qPCR runs in five biological and three technical replicates.

Figure 6. The dsRNA binding sites (indicated by arrows and perpendicular lines) in the coding sequences of S. frugiperda (A) and S. litura (B) CAD, ABCC2, ALP1 and APN are shown.

Numbers indicate the sequence coordinates. The homologous transcripts (indicated by grey boxes) that were aligned with the target receptors are shown. Perpendicular lines indicate percent identity in the dsRNA target region. NCBI Genebank identifiers for different transcripts are listed at the end.

Figure 7. RNAi-mediated silencing (si) of Cry receptor encoding genes differentially altered the susceptibility of S. frugiperda (A) and S. litura (B) to Cry1AcF toxin.

Initially, fourth-instar larvae were orally ingested with target receptor dsRNA-expressing E. coli HT115 cells. At 24 h of dsRNA ingestion, LD₅₀ (30 and 40 ng per larva for S. frugiperda and S. litura, respectively) and LD₉₀ (100 and 120 ng per larva for S. frugiperda and S. litura, respectively) doses of Cry1AcF were orally administered. After another 24 h percent mortality data was recorded. Different letters indicate treatments (mean ± SE) are significantly different at P < 0.01, Tukey’s HSD test, n = 50. GFP dsRNA and PBS were used as the non-native and negative control, respectively.