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

ABSTRACT: 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.

SUBMITTER: Jamil M

PROVIDER: S-EPMC9260231 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:BackgroundSymbiotic bacteria contribute to a multitude of important biological functions such as nutrition and reproduction and affect multiple physiological factors like fitness and longevity in their insect hosts. The melon fly, Zeugodacus cucurbitae (Coquillett), is an important agricultural pest that affects a variety of cultivated plants belonging mostly to the Cucurbitaceae family. It is considered invasive and widespread in many parts of the world. Several approaches are currently being considered for the management of its populations including the environmentally friendly and effective sterile insect technique (SIT), as a component of an integrated pest management (IPM) strategy. In the present study, we examined the effect of diet and radiation on the bacterial symbiome of Z. cucurbitae flies with the use of Next Generation Sequencing technologies.ResultsMelon flies were reared on two diets at the larval stage, an artificial bran-based diet and on sweet gourd, which affected significantly the development of the bacterial profiles. Significant differentiation was also observed based on gender. The effect of radiation was mostly diet dependent, with irradiated melon flies reared on the bran diet exhibiting a significant reduction in species diversity and richness compared to their non-irradiated controls. Changes in the bacterial symbiome of the irradiated melon flies included a drastic reduction in the number of sequences affiliated with members of Citrobacter, Raoultella, and Enterobacteriaceae. At the same time, an increase was observed for members of Enterobacter, Providencia and Morganella. Interestingly, the irradiated male melon flies reared on sweet gourd showed a clear differentiation compared to their non-irradiated controls, namely a significant reduction in species richness and minor differences in the relative abundance for members of Enterobacter and Providencia.ConclusionsThe two diets in conjunction with the irradiation affected significantly the formation of the bacterial symbiome. Melon flies reared on the bran-based artificial diet displayed significant changes in the bacterial symbiome upon irradiation, in all aspects, including species richness, diversity and composition. When reared on sweet gourd, significant changes occurred to male samples due to radiation, only in terms of species richness.

Project description:Tephritid fruit flies, such as the melon fly, Zeugodacus cucurbitae, are major horticultural pests worldwide and pose invasion risks due primarily to international trade. Determining movement parameters for fruit flies is critical to effective surveillance and control strategies, from setting quarantine boundaries after incursions to development of agent-based models for management. While mark-release-recapture, flight mills, and visual observations have been used to study tephritid movement, none of these techniques give a full picture of fruit fly movement in nature. Tracking tagged flies offers an alternative method which has the potential to observe individual fly movements in the field, mirroring studies conducted by ecologists on larger animals. In this study, harmonic radar (HR) tags were fabricated using superelastic nitinol wire which is light (tags weighed less than 1 mg), flexible, and does not tangle. Flight tests with wild melon flies showed no obvious adverse effects of HR tag attachment. Subsequent experiments successfully tracked HR tagged flies in large field cages, a papaya field, and open parkland. Unexpectedly, a majority of tagged flies showed strong flight directional biases with these biases varying between flies, similar to what has been observed in the migratory butterfly Pieris brassicae. In field cage experiments, 30 of the 36 flies observed (83%) showed directionally biased flights while similar biases were observed in roughly half the flies tracked in a papaya field. Turning angles from both cage and field experiments were non-random and indicate a strong bias toward continued "forward" movement. At least some of the observed direction bias can be explained by wind direction with a correlation observed between collective melon fly flight directions in field cage, papaya field, and open field experiments. However, individual mean flight directions coincided with the observed wind direction for only 9 out of the 25 flies in the cage experiment and half of the flies in the papaya field, suggesting wind is unlikely to be the only factor affecting flight direction. Individual flight distances (meters per flight) differed between the field cage, papaya field, and open field experiments with longer mean step-distances observed in the open field. Data on flight directionality and step-distances determined in this study might assist in the development of more effective control and better parametrize models of pest tephritid fruit fly movement.

Project description:Simple Summary Melon flies in Hawaii have developed high levels of resistance to a widely used insecticidal bait containing the active ingredient spinosad. Since farmers relied heavily on this product and have few alternatives, we tested three other insectides mixed with a bait spray called Nu-Lure. The three products (Agri-Mek SC, Mustang Maxx, and Malathion 5EC) were all found to be effective in either decreasing melon fly numbers or suppressing the growth of the population in the field. When used one-by-one in rotation for two weeks per product at one-week intervals on a commercial zucchini farm, melon fly populations were dramatically reduced and the yield of zucchinis harvested was substantially increased. Abstract High levels of resistance to the spinosad-based insecticidal protein bait GF-120 have been detected in some populations of melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae), in Hawaii in 2017. To provide cucurbit farmers in Hawaii with alternative insecticides, we field-tested the effectiveness of Agri-Mek SC (a.i., abamectin), Mustang Maxx (a.i., zeta-cypermethrin), and Malathion 5EC (a.i., malathion), added to a protein bait spray (Nu-Lure Insect Bait). The insecticide and protein bait combinations were applied to the roosting plants of Z. cucurbitae around the perimeter of the cucurbit fields at one-week intervals. When individually tested, all three insecticides in combination with protein bait significantly reduced or suppressed the numbers of female flies caught in torula yeast traps. A two-week rotation of weekly applications of the three insecticides and GF-120 significantly reduced Z. cucurbitae numbers on a commercial zucchini farm on Maui. The percentage of marketable fruits harvested increased from 51% to 98% after implementing the insecticide rotation. Our findings will be used to provide cucurbit farmers with additional products to control Z. cucurbitae. The future focus will be on educating cucurbit farmers to use the insecticide rotation strategy to prevent or delay resistance development.

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

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