RNAi-Mediated Knockdown of Imaginal Disc Growth Factors (IDGFs) Genes Causes Developmental Malformation and Mortality in Melon Fly, Zeugodacus cucurbitae

Abstract

This study reports the first successful use of oral feeding dsRNA technique for functional characterization of imaginal disc growth factors (IDGFs) genes (IDGF1, IDGF3_1, IDGF4_0, IDGF4_1, and IDGF6) in melon fly Zeugodacus cucurbitae. Phylogenetic and domain analysis indicates that these genes had high similarity with other Tephritidae fruit flies homolog and contain only one conserved domain among these five genes, which is glyco-18 domain (glyco-hydro-18 domain). Gene expression analysis at different developmental stages revealed that these genes were expressed at larval, pupal, and adult stages. To understand their role in different developmental stages, larvae were fed dsRNA-corresponding to each of the five IDGFs, in an artificial diet. RNAi-mediated knockdown of IDGF1 shows no phenotypic effects but caused mortality (10.4%), while IDGF4_0 caused malformed pharate at the adult stage where insects failed to shed their old cuticle and remained attached with their body, highest mortality (49.2%) was recorded compared to dsRNA-green fluorescent protein (GFP) or DEPC. Silencing of IDGF3_1 and IDGF4_1 cause lethal phenotype in larvae, (17.2%) and (40%) mortality was indexed in Z. cucurbitae. IDGF6 was mainly expressed in pupae and adult stages, and its silencing caused a malformation in adult wings. The developmental defects such as malformation in wings, larval-larval lethality, pupal-adult malformation, and small body size show that IDGFs are key developmental genes in the melon fly. Our results provide a baseline for the melon fly management and understanding of IDGFs specific functions in Z. cucurbitae.

Keywords: RNA interference; Tephritidae; chitinase; mortality; wings malformation.

FIGURE 1

Phylogenetic analysis of IDGFs genes with model family Tephritidae (taxid: 7211) and Drosophilidae (taxid: 7214) are shown in the tree. The highlighted part indicates our target genes. Tree indicates relationship between IDGFs gene and species tree. Maximum likelihood method was used to construct insects IDGFs coding sequences phylogenetic tree. Complete details of all IDGFs are listed in Supplementary Table 1.

FIGURE 2

(A) Imaginal disc growth factors gene, also their domain architecture and motif in Z. cucurbitae. The deduced amino acid sequences were used to predict the domain architectures of the five IDGFs genes using online conserved domain database (CDD) and presented through TBTool software. (B) Amino acid sequence alignment of IDGFs was performed using ClustalW alignment method in MEGA7. In GeneDoc program, ClustalW alignment was used to shade the identical and similar amino acids in the alignment. The conserved regions among five IDGFs sequences are tinted with red box. Dark shade indicates identical amino acids and gray shade represents similar amino acids.

FIGURE 3

Temporal expression of eight developmental stages of Z. cucurbitae was determined, RNA was extracted from the whole body of flies in different developmental stages including 2nd instar larvae (L2), 3rd early-instar larvae (L3-1), Third late instar larvae (L3-3), 1–2 days mixed pupae as early pupae (P-E), 5–6 days as mid pupae (P-M), and 7–9 days as of late pupae (P-L), 1–2 days adults as (A-E), and 10-day adults as (A-M). We had presented our results after normalization against reference gene EFα1 as the relative expression. All IDGFs gene expression is relative to the gene expression of each gene in 2nd instar larvae. One-way ANOVA with post hoc Tukey test was used to test the statistical significance *p < 0.05; **p < 0.01; ***p ≤ 0.001, ns: not significant.

FIGURE 4

RNAi suppresses only the target transcripts. (A) Larvae fed with dsIDGF1 and the other four genes are non-target transcript. (B) Larvae fed with dsIDGF3_1. (C) Larvae fed with dsIDGF4_0. (D) Larvae fed with dsIDGF4_1. (E) Larvae fed with dsIDGF6. No effects observed on non-target transcript.

FIGURE 5

Relative expression pattern of IDGFs in different time intervals post feeding to dsRNA or dsGFP or DEPC were determined as mean (±SE) of the three biological replicates, and two flies were used per pooled RNA sample with control as the calibrator, i.e., cDNA from non-RNAi flies (only fed on artificial diet with DEPC-water and dsGFP). EF1α is used as the internal control. One-way ANOVA with post hoc Tukey test was used to test the statistical significance *p < 0.05; **p < 0.01; ***p ≤ 0.001, ns: not significant.

FIGURE 6

Phenotypes, abnormalities after feeding dsRNA of IDGFs compared to control group dsGFP or DEPC in different developmental stages of Z. cucurbitae. All Pictures were taken with a scale bar 200 μm. The Control group represents either dsGFP or DEPC, and the Phenotype group represents abnormalities post feeding dsRNA for each gene. In phenotypes groups IDGF6 represents wings malformation in Z. cucurbitae, IDGF3_1 and IDGF4_1 represents larval lethal phenotypes and IDGF4_0 represents phenotype at pupal–adult stage where flies fail to shed their old cuticle.

FIGURE 7

Mortality rate (%) of Z. cucurbitae at different developmental stages after being artificially fed with dsGFP or DEPC or dsRNA of IDGFs. The letters (A–E) represents IDGF1, IDGF3_1, IDGF4_0, IDGF4_1, and IDGF6. The white portion represent larval stages, light gray indicates pupal stage, and dark gray indicates adult stage of Z. cucurbitae. The values are presented as the mean (±SE) of five biological replications (50 insects were used per replicate). Treatments were compared using one-way ANOVA (Turkey’s test, p < 0.05).