Knockdown of the ecdysone receptor disrupts development and causes mortality in the melon fly, Zeugodacus cucurbitae.

Cucurbits are important economic plants that are attacked by numerous pests, among which the melon fly Zeugodacus cucurbitae is extremely problematic. New sustainable pest control strategies are necessary to replace chemical insecticides that are harmful to the environment, human health and nontarget species. The RNA interference (RNAi) technology is one of the most promising tools due to high efficiency and species specificity. We developed an RNAi strategy targeting the ecdysone receptor (ECR) of Z. cucurbitae, which plays an important role in moulting and reproduction. We identified, described and isolated the ECR gene of Z. cucurbitae and measured its expression pattern across developmental stages and tissues. ZcECR knockdown via dsZcECR ingestion caused a significant larval mortality and abnormal phenotypes in pupae and adults. About 68% of larvae fed with a dsZcECR-treated diet failed to enter the pupal stage and died. In addition, ZcECR knockdown dramatically reduced pupal weight (by 3.24 mg on average) and fecundity (by about 23%). RNAi targeting the ECR gene is therefore a promising method to control Z. cucurbitae, paving the way for the development of novel sustainable and highly specific control strategies.

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

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.

Argonaute1 and Gawky Are Required for the Development and Reproduction of Melon fly, Zeugodacus cucurbitae.

Argonaute family genes encode a highly conserved group of proteins that have been associated with RNA silencing in both animals and plants. This study investigates the importance of microRNA biogenesis key regulators Argonaute1 (Ago1) and Gawky genes in the post-embryonic and ovarian development of the melon fly, Zeugodacus cucurbitae. The expression levels of these genes were mapped in all developmental stages and different adult tissues. Their roles in development were investigated using RNA interference (RNAi) via two different dsRNA delivery techniques. Embryo microinjection and oral feeding of third instar larvae successfully knocked down and greatly reduced the expression level of the target genes. Additionally, ex vivo essays revealed the stability of dsRNA in food was sufficient for gene silencing, although its integrity was affected in midgut. A wide range of phenotypes were observed on pupation, segmentation, pigmentation, and ovarian development. RNAi-mediated silencing of Gawky caused high mortality and loss of body segmentation, while Ago1 knockdown affected ovarian development and pigmentation. Developmental abnormalities and ovarian malformations caused by silencing these genes suggest that these genes are crucial for viability and reproductive capacity of Z. cucurbitae, and may be used as potential target genes in pest management.

Efficacy of lure mixtures in baited traps to attract different fruit fly species in guava and vegetable fields.

Fruit flies (Diptera: Tephritidae) are major pests of fruits and vegetables worldwide. We measured the efficacy of attractive lure mixtures in baited traps on naturally-occurring fruit flies in commercial mosaic guava and vegetables fields in Pakistan. We tested three mixtures (methyl-eugenol [ME] and cue lure [CL]; GF-120 and methyl eugenol; and GF-120 and cue lure) in eleven ratios: 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0. We recorded three fruit fly species: Bactrocera zonata was the most abundant in baited traps, followed by Bactrocera dorsalis, while Zeugodacus cucurbitae was significantly less attracted to baited traps. We also found that the most attractive mixture and ratio varied among species: B. dorsalis was most attracted by 40CL:60ME, while B. zonata was most and equally attracted by 100ME, 10CL:90ME, 20CL:80ME, 30CL:70ME, and 40CL:60ME. Finally, Z. cucurbitae was most attracted by 10CL:90ME, which resulted in the highest total number of flies counted in 10CL:90ME-baited traps. Mixtures with GF-120 were less attractive to all three species. Our results suggest that lure mixtures in baited traps influence the attraction of fruit flies in a species-specific way. This needs to be considered in the integrated pest management of multiple species of fruit flies simultaneously. If Bactrocera species are most damaging and abundant, a 40CL:60ME mixture in baited traps will likely be most effective to reduce pest abundance and crop damage. However, if Z. cucurbitae is the main pest target causing most crop damage and yield loss, 10CL:90ME-baited traps will be a more effective in their monitoring and management.

60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression.

Radiation is considered as a promising insect pest control strategy for minimizing postharvest yield losses. Among various techniques, irradiation is a method of choice as it induces lethal biochemical or molecular changes that cause a downstream cascade of abrupt physiological abnormalities at the cellular level. In this study, we evaluated the effect of 60Co-γ radiation on various developmental stages of Zeugodacus cucurbitae Coquillett and subsequent carry-over effects on the progeny. For this purpose, we treated eggs with 30- and 50-Gy radiation doses of 60Co-γ. We found that radiation significantly affected cellular antioxidants, insect morphology, and gene expression profiles. Our results indicate that in response to various doses of irradiation reactive oxygen species, catalase, peroxidase, and superoxide dismutase activities were increased along with a significant increase in the malondialdehyde (MDA) content. We observed higher mortality rates during the pupal stage of the insects that hatched from irradiated eggs (50 Gy). Furthermore, the life span of the adults was reduced in response to 50 Gy radiation. The negative effects carried over to the next generation were marked by significantly lower fecundity in the F1 generation of the irradiation groups as compared to control. The radiation induced morphological abnormalities at the pupal, as well as the adult, stages. Furthermore, variations in the gene expression following irradiation are discussed. Taken together, our results signify the utility of 60Co-γ radiation for fruit fly postharvest management.

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

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.