Two heat shock cognate 70 genes involved in spermatogenesis regulate the male fertility of Zeugodacus cucurbitae, as potential targets for pest control.

The melon fly Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae) is an agricultural quarantine pest threatening fruit and vegetable production. Heat shock cognate 70 (Hsc70), which is a homolog of the heat shock protein 70 (Hsp70), was first discovered in mice testes and plays an important role in spermatogenesis. In this study, we identified and cloned five Hsc70 genes from melon fly, namely ZcHsc70_1/2/3/4/5. Phylogenetic analysis showed that these proteins are closely related to Hsc70s from other Diptera insects. Spatiotemporal expression analysis showed that ZcHsc70_1 and ZcHsc70_2 are highly expressed in Z. cucurbitae testes. Fluorescence in situ hybridization further demonstrated that ZcHsc70_1 and ZcHsc70_2 are expressed in the transformation and maturation regions of testes, respectively. Moreover, RNA interference-based suppression of ZcHsc70_1 or ZcHsc70_2 resulted in a significant decrease of 74.61% and 63.28% in egg hatchability, respectively. Suppression of ZcHsc70_1 expression delayed the transformation of sperm cells to mature sperms. Meanwhile, suppression of ZcHsc70_2 expression decreased both sperm cells and mature sperms by inhibiting the meiosis of spermatocytes. Our findings show that ZcHsc70_1/2 regulates spermatogenesis and further affects the male fertility in the melon fly, showing potential as targets for pest control in sterile insect technique by genetic manipulation of males.

Overexpression of miR-927-5p suppresses stalky expression and negatively reduces the spermatid production in Zeugodacus cucurbitae.

BACKGROUND: The melon fly, Zeugodacus cucurbitae Coquillett, is one of the major pests attacking Cucurbitaceae crops. Identifying critical genes or proteins regulating fertility is essential for sustainable pest control and a research hotspot in insect physiology. MicroRNAs (miRNAs) are short RNAs that do not directly participate in protein translation, but instead function in post-transcriptional regulation of gene expression involved in male fertility. RESULTS: We found that miR-927-5p is highly expressed in the testes and investigated its function in spermatogenesis in Z. cucurbitae. Fluorescence in situ hybridization (FISH) showed miR-927-5p in the transformation and maturation region of the testis, and overexpression of miR-927-5p reduced the number of sperms by 53%. In continuation, we predicted 12 target genes of miR-927-5p using bioinformatics combined with transcriptome sequencing data, and found that miR-927-5p targets the new gene Stalky in insects, which was validated by quantitative real-time PCR, RNA pull-down and dual luciferase reporter assays. FISH also confirmed the co-localization of miR-927-5p and the transcript Stalky_1 in the testis. Moreover, silencing of Stalky_1 by RNA interference reduced the number of sperms by 32% and reduced sperm viability by 39% in physiologically mature male adults. Meanwhile, the silencing of Stalky_1 also resulted in low hatchability. CONCLUSION: Our work not only presents a new, so far unreported mechanism regulating spermatogenesis by miR-927-5p targeting a new unknown target, Stalky, which is providing new knowledge on the regulatory network of insect spermatogenesis, but also lays a foundation for the development of SIT against important tephritid fly pests. © 2024 Society of Chemical Industry.

Suppressing the expression of glutathione S-transferase gene GSTd10 increases the sensitivity of Zeugodacus cucurbitae against ß-cypermethrin.

Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae) is an agriculturally and economically important pest worldwide that has developed resistance to ß-cypermethrin. Glutathione S-transferases (GSTs) have been reported to be involved in the detoxification of insecticides in insects. We have found that both ZcGSTd6 and ZcGSTd10 were up-regulated by ß-cypermethrin induction in our previous study, so we aimed to explore their potential relationship with ß-cypermethrin tolerance in this study. The heterologous expression of ZcGSTd6 and ZcGSTd10 in Escherichia coli showed significantly high activities against 1-chloro-2,4-dinitrobenzene (CDNB). The kinetic parameters of ZcGSTd6 and ZcGSTd10 were determined by Lineweaver-Burk. The Vmax and Km of ZcGSTd6 were 0.50 µmol/min·mg and 0.3 mM, respectively. The Vmax and Km of ZcGSTd10 were 1.82 µmol/min·mg and 0.53 mM. The 3D modelling and molecular docking results revealed that ß-cypermethrin exhibited a stronger bounding to the active site SER-9 of ZcGSTd10. The sensitivity to ß-cypermethrin was significantly increased by 18.73% and 27.21%, respectively, after the knockdown of ZcGSTd6 and ZcGSTd10 by using RNA interference. In addition, the inhibition of CDNB at 50% (IC50) and the inhibition constants (Ki) of ß-cypermethrin against ZcGSTd10 were determined as 0.41 and 0.33 mM, respectively. The Ki and IC50 of ß-cypermethrin against ZcSGTd6 were not analysed. These results suggested that ZcGSTd10 could be an essential regulator involved in the tolerance of Z. cucurbitae to ß-cypermethrin.

Comparative multi-tissue analyses identify testis-specific serine/threonine protein kinase (TSSK) genes involved in male fertility in the melon fly Zeugodacus cucurbitae.

BACKGROUND: Zeugodacus cucurbitae is an agricultural pest species with robust reproductive capabilities capable of causing extensive damage. The advent of novel male fertility-related pest control strategies has been an area of active entomological research focused on the sterile insect technique (SIT) strategy. RESULTS: RNA-sequencing analyses were conducted using 16 tissue samples from adult male Z. cucurbitae, leading to the identification of 5338 genes that were differentially expressed between the testes and three other analyzed tissue types. Of these genes, 808 exhibited high levels of testis expression. A quantitative polymerase chain reaction (qPCR) approach was used to validate the expression of ten of these genes selected at random, including ZcTSSK1 and ZcTSSK3, which are testis-specific serine/threonine protein kinase (TSSK) genes. Evaluation via a loss-of-function-based knockdown assay showed that the down-regulation of either of these two genes in males was associated with significantly decreased egg hatching rates. In situ hybridization analyses revealed the expression of both of these transcripts in the transformation zone, and significant decreases in Z. cucurbitae sperm numbers were observed following double-stranded RNA treatment. Together, these results suggested that inhibiting ZcTSSK1 and ZcTSSK3 expression was sufficient to alter male fertility in Z. cucurbitae. CONCLUSION: These transcriptional sequencing results provide a foundation for further efforts to clarify the regulators of Z. cucurbitae male fertility. These preliminary analyses of the functions of ZcTSSK family genes as regulators of spermatogenesis underscore their importance in the processes integral to male fecundity and their potential as targets for pest control efforts centered on the genetic manipulation of males. © 2023 Society of Chemical Industry.

Genome-wide screening and expression of glutathione S-transferase genes reveal that GSTe4 contributes to sensitivity against ß-cypermethrin in Zeugodacus cucurbitae.

Glutathione S-transferases (GSTs) are an essential multifunctional protein family with common detoxifying enzymes. In this study, 34 GST genes were identified from the melon fly, Zeugodacus cucurbitae, one of the most destructive pests worldwide. These GSTs include 32 cytosolic genes and two microsomal genes. Furthermore, these cytosolic GSTs were classified into six classes: 11 delta, 13 epsilon, three theta, one sigma, two zeta, and two omega. Most of these showed dynamic expression during the developmental stage, some of which showed stage-specific expression. The expression in various adult tissues showed that most of them were expressed in anti-stress-related tissues. The transcriptional response of the delta and epsilon families was determined when Z. cucurbitae was exposed to three insecticides, abamectin, dinotefuran, and ß-cypermethrin. Seven genes were significantly up-regulated by abamectin exposure. Moreover, five and four genes were significantly up-regulated with dinotefuran and ß-cypermethrin exposure, respectively, demonstrating their involvement in the detoxification of these such toxic substances in Z. cucurbitae. One example of these genes, ZcGSTe4, was randomly selected to explore its function in response to ß-cypermethrin exposure. Over-expressed ZcGSTe4 in E. coli showed significant tolerance to ß-cypermethrin, and RNAi-mediated suppression of ZcGSTe4 also increased the sensitivity of melon fly to this agent. This study provides a foundation for further studies on the mechanism of detoxification metabolism in the melon fly.