CRISPR/Cas9-mediated knockout of a male accessory glands-specific gene takeout1 decreases the fecundity of Zeugodacus cucurbitae female.

BACKGROUND: The melon fly, Zeugodacus cucurbitae (Coquillett), is an invasive Tephritidae pest with robust fertility. The male accessory glands (MAGs) form a vital organ that ensures insect reproductive efficiency. Most of the secreted proteins by MAGs exhibit a male bias expression. Takeout, one of these proteins, is abundantly present in the MAGs of many insects. RESULTS: In this study, we identified 32 takeout genes in Z. cucurbitae. The phylogenetic analysis and multiple sequence alignment results showed that Zctakeout1 is the most related homolog to the MAGs-specific takeout in Tephritidae. The real-time quantitative PCR results showed that Zctakeout1 was exclusively expressed in the male adult stage, and its expression level gradually increased with the increase in age and then remained stable at the sexually matured stage. The distribution among tissues demonstrated the specific expression of Zctakeout1 in the MAGs, and fluorescence immunohistochemical results confirmed the presence of Zctakeout1 in close proximity to binuclear cells of the mesoderm epidermal MAGs. In continuation, CRISPR/Cas9-mediated genome editing was employed, resulting in successfully generating a homozygous strain with an +8 bp insertion. The mating experiments with the Zctakeout1-/- males resulted in significant reductions in both the mating rate and egg production of females. CONCLUSION: These findings prove that the MAGs-specific Zctakeout1 is essential in regulating fecundity in female Z. cucurbitae fruit flies. Our data suggests its utilization in future essential insect-specific gene-directed sterility insect technique (SIT) by the genetic manipulation to keep these important Tephritidae populations under control. © 2024 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.

Molecular Characterization and Transcriptional Expression Analysis of ABC Transporter H Subfamily Genes in the Oriental Fruit Fly.

The oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephretidae), is a serious pest of fruits and vegetables and has developed high levels of insecticide resistance. ATP-binding cassette transporter genes (ABC transporters) are involved in mediating the energy-driven transport of many substances across membranes and are closely associated with development and insecticide detoxification. In this study, three ABC transporters in the H subfamily were identified, and the possible roles of these genes in B. dorsalis are discussed. Bioinformatics analysis revealed that those genes are conserved, typical of half-transporters. The expression profiles of BdABCH genes (BdABCHs) in the developmental stages, tissues, and following insecticide exposure, extreme temperature, warm- and cold-acclimated strain, starvation, and desiccation stress were determined by quantitative real-time PCR. Expression of BdABCHs can be detected in various tissues and in different developmental stages. They were most highly expressed in the hindgut and in newly emerged adults. The mRNA levels of BdABCHs in males (including most tissues and body segments) were higher than in females. The expression of BdABCH1 was significantly upregulated 3.8-fold in the cold-acclimated strain, and was significantly upregulated by 1.9-, 3.8- and 4.1-fold in the 0°C, starvation, and desiccation treatments, respectively. Treatment with malathion and avermectin at LD20 and LD30 concentrations produced no obvious changes in the levels of BdABCHs. BdABCHs may be involved in the transport of related hormones during eclosion, as well as water and inorganic salts. BdABCH1 also demonstrated that it is related to the ability to cope with adverse environments.

Identification and Expression Characterization of ATP-Binding Cassette (ABC) Transporter Genes in Melon Fly.

The ATP-binding cassette (ABC) transporter is a protein superfamily that transports specific substrate molecules across lipid membranes in all living species. In insects, ABC transporter is one of the major transmembrane protein families involved in the development of xenobiotic resistance. Here, we report 49 ABC transporter genes divided into eight subfamilies (ABCA-ABCH), including seven ABCAs, seven ABCBs, 10 ABCCs, two ABCDs, one ABCE, three ABCFs, 16 ABCGs, and three ABCHs according to phylogenetic analysis in Zeugodacus cucurbitae, a highly destructive insect pest of cucurbitaceous and other related crops. The expressions level of 49 ABC transporters throughout various developmental stages and within different tissues were evaluated by quantitative transcriptomic analysis, and their expressions in response to three different insecticides were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). These ABC transporter genes were widely expressed at developmental stages but most highly expressed in tissues of the midgut, fat body and Malpighian tube. When challenged by exposure to three insecticides, abamectin, ß-cypermethrin, and dinotefuran, the expressions of ZcABCB7 and ZcABCC2 were significantly up-regulated. ZcABCB1, ZcABCB6, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 were significantly up-regulated in the fat body at 24 h after ß-cypermethrin exposure. These data suggest that ZcABCB7 and ZcABCC2 might play key roles in xenobiotic metabolism in Z. cucurbitae. Collectively, these data provide a foundation for further analysis of ABCs in Z. cucurbitae.

Genome-wide analysis of long non-coding RNAs in adult tissues of the melon fly, Zeugodacus cucurbitae (Coquillett).

BACKGROUND: Long non-coding RNAs (lncRNAs) are involved in many fundamental biological processes, such as transcription regulation, protein degradation, and cell differentiation. Information on lncRNA in the melon fly, Zeugodacus cucurbitae (Coquillett) is currently limited. RESULTS: We constructed 24 RNA-seq libraries from eight tissues (midgut, Malpighian tubules, fat body, ovary, and testis) of Z. cucurbitae adults. A total of 3124 lncRNA transcripts were identified. Among those, 1464 were lincRNAs, 1037 were intronic lncRNAs, 301 were anti-sense lncRNAs, and 322 were sense lncRNAs. The majority of lncRNAs contained two exons and one isoform. Differentially expressed lncRNAs were analyzed between tissues, and Malpighian tubules versus testis had the largest number. Some lncRNAs exhibited strong tissue specificity. Specifically expressed lncRNAs were identified and filtered in tissues of female and male Z. cucurbitae based on their expression levels. Four midgut-specific lncRNAs were validated by quantitative real-time polymerase chain reaction (RT-qPCR), and the data were consistent with RNA-seq data. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of targets of midgut-specific lncRNAs indicated an enrichment of the metabolic process. CONCLUSIONS: This was the first systematic identification of lncRNA in the melon fly. Expressions of lncRNAs in multiple adult tissues were evaluated by quantitative transcriptomic analysis. These qualitative and quantitative analyses of lncRNAs, especially the tissue-specific lncRNAs in Z. cucurbitae, provide useful data for further functional studies.

Genome-wide gene expression profiling of the melon fly, Zeugodacus cucurbitae, during thirteen life stages.

The melon fly, Zeugodacus cucurbitae (Coquillett), is an important destructive pest worldwide. Functional studies of the genes associated with development and reproduction during different life stages are limited in Z. cucurbitae. There have yet to be comprehensive transcriptomic resources for genetic and functional genomic studies to identify the molecular mechanisms related to its development and reproduction. In this study, we comprehensively sequenced the transcriptomes of four different developmental stages: egg, larva, pupa, and adults. Using the Illumina RNA-Seq technology, we constructed 52 libraries from 13 stages with four biological replicates in each and generated 435.61 Gb clean reads. We comprehensively characterized the transcriptomes with high-coverage mapping to the reference genome. A total of 13,760 genes were mapped to the reference genome, and another 4481 genes were characterized as new genes. Finally, 14,931 genes (81.85%) were functionally annotated against six annotation databases. This study provides the first comprehensive transcriptome data of all developmental stages of Z. cucurbitae, and will serve as a valuable resource for future genetic and functional studies.

[Progress of researches on prevention and treatment of sports fatigue with moxibustion therapy].

Sports fatigue belongs to the category of functional deficiency-syndrome according to the theory of traditional Chinese medicine. The moxibustion therapy has a long history and possesses a definite therapeutic effect in the prevention and treatment of sports fatigue. In the present paper, the authors reviewed development of researches on the effects of moxibustion intervention in the prevention and treatment of sports fatigue in recent 5 years. Results of researches showed that moxibustion intervention can 1) eliminate free radicals and reduce oxidative damage; 2) increase energy (glycogen) supply to delay the production of fatigue; 3) raise serum testosterone level (relieve exercise-induced neuroendocrine disorder) and reduce post-sports fatigue; 4) raise the anaerobic exercise ability, reduce the accumulation of metabolic products in the body and strengthen the endurance capacity of the skeletal muscle; and 5) improve ischemic cardiac function, and suppress cardiomyocyte apopotosis, etc. However, we should further strengthen our investigations on the moxibustion therapy in the ancient classical literature and sum up academic thoughts of different academic schools in the successive dynasties, put emphasis on the large sample randomized controlled clinical trails, establish united treatment standards, etc., and provide much evidence for effectively treating sports fatigue in the future.

[Progress of researches on protective effect of moxibustion on myocardial cells during exhaustive exercise ].

Moxibustion is a side-effect-free external therapy for relieving different clinical disorders. Its effects in eliminating sports fatigue, preventing myocardial injury in people undergeing exhausted exercise and its underlying mechanisms have been induced great concern by the sports medicine community. In the present paper, the authors make a review on the development of researches on the underlying mechanism of moxibustion in the prevention and treatment of sports myocardial injury in re- cent 10 years from 1) regulating myocardial endocrine function, 2) scavenging free radicals, 3) resisting oxidative damage, and 4) modulating blood rheology. On the basis of results mentioned above, the authors raise a new potential mechanism underlying protective effect of moxibustion on myocardial cells during exhaustive exercise from the cellular molecular level, i.e., mitochondrial telomerase-mediated anti-aging. This mitochondrial telomerase-mediated anti-aging may be a new target of moxibustion intervention for protecting cardiomyocytes from injury under exhaustive exercise condition.