Comparative transcriptomics of the irradiated melon fly (Zeugodacus cucurbitae) reveal key developmental genes

Abstract

Irradiation can be used as an insect pest management technique to reduce post-harvest yield losses. It causes major physiological changes, impairing insect development and leading to mortality. This technique is used to control the melon fly Zeugodacus cucurbitae, a major pest of Cucurbitaceae in Asia. Here, we applied irradiation to melon fly eggs, and the larvae emerged from irradiated eggs were used to conduct comparative transcriptomics and thereby identify key genes involved in the development and survival. We found 561 upregulated and 532 downregulated genes in irradiated flies compared to non-irradiated flies. We also observed abnormal small-body phenotypes in irradiated flies. By screening the 532 downregulated genes, we selected eight candidate genes putatively involved in development based in described functions in public databases and in the literature. We first established the expression profile of each candidate gene. Using RNA interference (RNAi), we individually knocked down each gene in third instar larvae and measured the effects on development. The knockdown of ImpE2 ecdysone-inducible gene controlling life stage transitions-led to major body size reductions in both pupae and adults. The knockdown of the tyrosine-protein kinase-like tok (Tpk-tok) caused severe body damage to larvae, characterized by swollen and black body parts. Adults subject to knockdown of the eclosion hormone (Eh_1) failed to shed their old cuticle which remained attached to their bodies. However, no obvious developmental defects were observed following the knockdown of the heat shock protein 67B1-like (Hsp67), the insulin receptor (Insr), the serine/threonine-protein kinase Nek4 (Nek4), the tyrosine-protein kinase transmembrane receptor Ror (Ror_1) and the probable insulin-like peptide 1 (Insp_1). We argue that irradiation can be successfully used not only as a pest management technique but also for the screening of essential developmental genes in insects via comparative transcriptomics. Our results demonstrate that ImpE2 and Eh_1 are essential for the development of melon fly and could therefore be promising candidates for the development of RNAi-based pest control strategies.

Keywords: RNA interference (RNAi); Zeugodacus cucurbitae; abiotic stress; developmental defects; gene expression; insect physiology.

FIGURE 1

Gene ontology enrichment analysis: Ratio of the number of up- and downregulated differentially expressed genes (DEGs) over the total number of genes in each pathway, for the 20 significantly enriched GO pathways (p < .05). “BP”, biological process; “CC”, cellular component; “MF”, molecular function; “up", upregulated; “down”, downregulated (* oxidoreductase activity acting on paired donors, with incorporation or reduction of molecular oxygen).

FIGURE 2

Expression profiles of the eight target genes Insr, Hsp67, Tpk-tok, Nek4, ImpE2, Ror_1, Eh_1, and Insp_1 in different developmental stages of Z. cucurbitae. Eggs, 1st instar (1L), 2nd instar (2L), 3rd instar (3L), Pupae (P) and Adults (A). Values represent means ± SD. Different letters above the bars indicate significant differences at p < .05 (Tukey’s tests). EFα1 and Actin were used as internal controls and the 2^−ΔΔCT method was used to calculate the gene relative expression level.

FIGURE 3

Relative expression profiles (mean ± SD; N = 3 replicates per treatment) of Insr, Hsp67, Tpk-tok, Nek4, ImpE2, Ror_1, Eh_1 and Insp_1 in Z. cucurbitae larvae at different times after feeding on gene-specific dsRNA (treatment; blue) compared to dsGFP (control; purple). *p < .05; **p < .01; ***p < .001; ns, no significant difference. EFα1 and Actin were used as internal controls and the 2^−ΔΔCT method was used to calculate the gene relative expression level.

FIGURE 4

Developmental defects observed in Z. cucurbitae flies after the knockdown of (B) Tpk-tok, (D) Eh_1 and (E) ImpE2 genes. (A) 3rd instar of Z. cucurbitae after feeding on dsGFP-enriched food (control flies). (C) Normal pupae from the dsGFP control treatment. Red lines and circles represent the insect body’s damage, blockage, and swell portion in (B) and reduced body size in (E), while red arrows show the old insect cuticle still attached to their body in (D).

FIGURE 5

Abnormal phenotypes and Mortality observed in different developmental stages of Z. cucurbitae. (A) Percentage of observed abnormalities after dsRNA delivery of target genes and dsGFP (Control). (B) Percentage of insect mortality post feeding dsRNA of target genes compared to the control group. Values represent means % ± SD. Data were analysed using Duncan’s test.*p < .05; **p < .01; ***p < .001; ns no statistically significant difference compared to control.