miR-125-3p and miR-276b-3p Regulate the Spermatogenesis of Bactrocera dorsalis by Targeting the orb2 Gene.

Bactrocera dorsalis is considered a major threat to horticultural crops. It has evolved resistance against insecticides. It is believed that development of new methods is highly desirable to control this destructive agricultural pest. Sterile insect technique is emerging as a potential tool to control this insect pest by reducing their reproductive ability. Here we report that orb2 has high expression in the testis of B. dorsalis which is the target of miR-125-3p and miR-276b-3p and plays a critical role in the spermatogenesis. Dual luciferase reporter assay using HEKT293 cells demonstrates that orb2 gene is downregulated by miR-125-3p and miR-276b-3p and is a common target of these miRNAs. Dietary treatment of adult male flies separately and in combination of agomir-125-3p (Ago-125-3p) and agomir-276b-3p (Ago-276b-3p) significantly downregulated the mRNA of orb2. The combined treatments of agomirs suppressed the level of mRNA of orb2 significantly more than any single treatment. Altered expression of miR-125-3p and miR-276b-3p significantly decreased the total and live spermatozoa in the testis which ultimately caused reduction in male fertility. Furthermore, we demonstrate that miR-125-3p, miR-276b-3p, and orb2 dsRNA are the novel agents that could be used in a genetic-based sterile insect technique (SIT) to control the B. dorsalis.

Lethal and sublethal effects of emamectin benzoate on life-table and physiological parameters of citrus red mite, Panonychus citri.

The citrus red mite (Panonychus citri) is a challenge to manage in citrus orchards due to resistance against several pesticides. There is a necessity therefore to find new pesticides for effective control of P. citri. This study was designed to evaluate the lethal and sublethal effects of emamectin benzoate against P. citri. The results showed that the LC50 of emamectin benzoate to adults of P. citri was 0.35 (0.26-0.43) mg a.i. L-1 and the LC90 was 1.44 (1.16-1.96) mg a.i. L-1. The sublethal concentration exposures (LC10 and LC30) had a significant negative impact on the larval, protonymph, and deutonymph developmental periods. Male longevity was much lower in LC30 treatments than in the controls. Although female longevity was unaffected, the fecundity (eggs per female) was decreased in the sublethal concentration treatments. Results revealed that the adult pre-oviposition period (APOP) and total pre-oviposition period (TPOP) were increased. Other growth parameters r, λ, and R0 decreased, whereas mean generation time (T) increased due to pesticide exposure. The survival rate (Sxj), age-specific fecundity and net maternity, life expectancy (Exj), and reproduction (Vxj) was reduced by LC10 and LC30 exposure. An increase in malondialdehyde (MDA) contents with increasing emamectin benzoate concentration demonstrates that emamectin benzoate induces oxidative stress in P. citri. The activity of antioxidant enzymes (superoxide dismutase, SOD and catalase, CAT) was decreased due to LC30 and LC10 treatments compared to the control. Detoxification enzyme activity (cytochrome P450, glutathione-S-transferases, GST and acetylcholinesterase, AChE) was increased in treated mites compared to the control. This study demonstrates that emamectin benzoate has both a lethal effect on citrus red mite and sublethal effects on its biology and physiology. It is, therefore, potentially an effective pesticide for management of P. citri.

Emamectin benzoate induced enzymatic and transcriptional alternation in detoxification mechanism of predatory beetle Paederus fuscipes (Coleoptera: Staphylinidae) at the sublethal concentration.

In this study, the detoxification enzyme activity and the transcriptional profile changes in the second instar through RNA-sequencing technology due to emamectin benzoate (EB) were assessed. The cytochrome P450 monooxygenases (P450) enzyme activity was not altered by EB due to the change in concentration and exposure time in all treatments. The glutathione S-transferase (GST) enzyme was not considerably varying in all treatments, while exposure time significantly changed the enzyme activity. Results showed that the esterase (Ests) activity was elevated with the increasing concentrations and exposure time. Two libraries were generated, containing 107,767,542 and 108,142,289 clean reads for the samples treated with LC30 of EB and control. These reads were grouped into 218,070 transcripts and 38,097 unigenes. A total of 2257 differentially expressed genes (DEGs) were identified from these unigenes, of which 599 up-regulated and 1658 were down-regulated. The majority of these DEGs related to pesticides resistance were identified in numerous Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, e.g., steroid hormone biosynthesis, glutathione metabolism, drug metabolism-other enzymes, chemical carcinogenesis, pathways of cancer, metabolism of xenobiotics by cytochrome P450, drug metabolism of cytochrome P450, linoleic acid metabolism, retinol metabolism, and insect hormone biosynthesis. These pathways also shared the same genes as cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), Esterase (Ests), UDP-glucosyltransferases (UGTs), and ATP-binding cassettes (ABCs). A heatmap analysis also showed that regulation of genes in a pathway causes a series of gene expression regulation in subsequent pathways. Our quantitative reverse transcription-PCR (qRT-PCR) results were consistent with the DEG's data of transcriptome analysis. The comprehensive transcriptome sequence resource attained through this study evidence that the EB induces significant modification in enzyme activity and transcriptome profile of Paederus fuscipes, which may enable more significant molecular underpinnings behind the insecticide-resistance mechanism for further investigations.

RNA Interference-Based Silencing of the Chitin Synthase 1 Gene for Reproductive and Developmental Disruptions in Panonychus citri.

Chitin synthase 1 (CHS1) is an essential gene regulating chitin during different developmental stages of arthropods. In the current study, we explored for the first time the role of CHS1 gene regulation in the citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), by silencing its expression using (RNA interference) RNAi-based strategies. The results reveal that P. citri tested in different developmental stages, including larvae, protonymphs, deutonymphs, and adults fed on sweet orange leaves dipped in various concentrations (200, 400, 600, and 800 ng/µL) of dsRNA-PcCHS1, resulted in a continuous reduction in their gene expression, and the extent of transcript knockdown was positively correlated with the concentration of dsRNA. Concentration-mortality response assays revealed a mortality of more than 50% among all the studied developmental stages, except for adulthood. Furthermore, the target gene dsRNA-PcCHS1 treatment of larvae, protonymphs, deutonymphs, and females at a treatment rate of 800 ng/mL of dsRNA significantly decreased the egg-laying rates by 48.50%, 43.79%, 54%, and 39%, respectively, and the hatching rates were also considerably reduced by 64.70%, 70%, 64%, and 52.90%, respectively. Moreover, using the leaf dip method, we found that the RNA interference effectively reduced the PcCHS1 transcript levels by 42.50% and 42.06% in the eggs and adults, respectively. The results of this study demonstrate that the RNAi of PcCHS1 can dramatically reduce the survival and fecundity of P. citri, but the dsRNA concentrations and developmental stages can significantly influence the RNAi effects. These findings indicate the potential utility of the PcCHS1 gene in causing developmental irregularities, which could aid in the development of effective and novel RNAi-based strategies for controlling P. citri.

Insights into the Gryllus bimaculatus Immune-Related Transcriptomic Profiling to Combat Naturally Invading Pathogens.

Natural pathogen pressure is an important factor that shapes the host immune defense mechanism. The current study primarily aimed to explore the molecular basis of the natural immune defense mechanism of a sporadic pest, Gryllus bimaculatus, during swarming by constructing cDNA libraries of the female mid-gut, male mid-gut, testes, and ovaries. The Illumina HiSeq platform generated an average of 7.9 G, 11.77 G, 10.07 G, and 10.07 G bases of outputs from the male mid-gut, female mid-gut, testes, and ovaries and libraries, respectively. The transcriptome of two-spotted field crickets was assembled into 233,172 UniGenes, which yielded approximately 163.58 million reads. On the other hand, there were 43,055 genes in common that were shared among all the biological samples. Gene Ontology analysis successfully annotated 492 immune-related genes, which comprised mainly Pattern Recognition Receptors (62 genes), Signal modulators (57 genes), Signal transduction (214 genes), Effectors (36 genes), and another immune-related 123 genes. In summary, the identified wide range of immune-related genes from G. bimaculatus indicates the existence of a sophisticated and specialized broad spectrum immune mechanism against invading pathogens, which provides, for the first time, insights into the molecular mechanism of disease resistance among two-spotted field crickets.

A Shift Pattern of Bacterial Communities Across the Life Stages of the Citrus Red Mite, Panonychus citri.

As one of the most detrimental citrus pests worldwide, the citrus red mite, Panonychus citri (McGregor), shows extraordinary fecundity, polyphagia, and acaricide resistance, which may be influenced by microbes as other arthropod pests. However, the community structure and physiological function of microbes in P. citri are still largely unknown. Here, the high-throughput sequencing of 16S rDNA amplicons was employed to identify and compare the profile of bacterial communities across the larva, protonymph, deutonymph, and adult stages of P. citri. We observed a dominance of phylums Proteobacteria and Firmicutes, and classes α-, γ-, ß-Proteobacteria and Bacilli in the bacterial communities across the host lifespan. Based on the dynamic analysis of the bacterial community structure, a significant shift pattern between the immature (larva, protonymph, and deutonymph) and adult stages was observed. Accordingly, among the major families (and corresponding genera), although the relative abundances of Pseudomonadaceae (Pseudomonas), Moraxellaceae (Acinetobacter), and Sphingobacteriaceae (Sphingobacterium) were consistent in larva to deutonymph stages, they were significantly increased to 30.18 ± 8.76% (30.16 ± 8.75%), 20.78 ± 10.86% (18.80 ± 10.84%), and 11.71 ± 5.49% (11.68 ± 5.48%), respectively, in adult stage, which implied the important function of these bacteria on the adults' physiology. Actually, the functional prediction of bacterial communities and Spearman correlation analysis further confirm that these bacteria had positively correlations with the pathway of "lipid metabolism" (including eight sublevel pathways) and "metabolism of cofactors and vitamins" (including five sublevel pathways), which all only increased in adult stages. In addition, the bacterial communities were eliminated by using broad-spectrum antibiotics, streptomycin, which significantly suppressed the survival and oviposition of P. citri. Overall, we not only confirmed the physiological effects of bacteria community on the vitality and fecundity of adult hosts, but also revealed the shift pattern of bacterial community structures across the life stages and demonstrated the co-enhancements of specific bacterial groups and bacterial functions in nutritional metabolism in P. citri. This study sheds light on basic information about the mutualism between spider mites and bacteria, which may be useful in shaping the next generation of control strategies for spider mite pests, especially P. citri.

RNAi-Mediated Knockdown of Tssk1 and Tektin1 Genes Impair Male Fertility in Bactrocera dorsalis.

The genetic-based sterile insect technique (SIT) is an effective and environmentally safe strategy to diminish populations of agricultural and horticultural insect pests. Functional characterization of genes related to male fertility can enhance the genetic-based SIT. Tssk1 has been involved to control male fertility in both mammals and insects. Moreover, Tektin1 has also been revealed to influence male fertility in both human and mammals. These findings suggested that Tssk1 and Tektin1 identified from Bactrocera dorsalis could be required for male fertility in B. dorsalis. In this study, expression profiles of these two genes were studied at different developmental stages and in various tissues of adult males. Remarkably, it was found that Tssk1 and Tektin1 were highly expressed in the testis of mature adult males of B. dorsalis. Furthermore, Tssk1 and Tektin1 genes were downregulated by using the RNA interference (RNAi) method. Fertility assays including egg laying, hatching, and spermatozoa count were also performed to investigate male fertility of B. dorsalis. Results showed that knockdown of Tssk1 and Tektin1 caused male sterility up to 58.99% and 64.49%, respectively. As expected, the total numbers of spermatozoa were also significantly reduced by 65.83% and 73.9%, respectively. These results suggested that male sterility was happened wing to the low number of spermatozoa. In conclusion, we demonstrate that Tssk1 and Tektin1 are the novel agents that could be used to enhance the genetic-based SIT, or their double-stranded RNA (dsRNA) can be used as biopesticides to control the population of B. dorsalis.

The critical role of p16/Rb pathway in the inhibition of GH3 cell cycle induced by T-2 toxin.

T-2 toxin is a worldwide trichothecenetoxin and can cause various toxicities.T-2 toxin is involved in G1 phase arrest in several cell lines but molecular mechanism is still not clear. In present study, we used rat pituitary GH3 cells to investigate the mechanism involved in cell cycle arrest against T-2 toxin (40 nM) for 12, 24, 36 and 48 h as compared to control cells. GH3 cells showed a considerable increase in reactive oxygen species (ROS) as well as loss in mitochondrial membrane potential (△Ym) upon exposure to the T-2 toxin. Flow cytometry showed a significant time-dependent increase in percentage of apoptotic cells and gel electrophoresis showed the hallmark of apoptosis oligonucleosomal DNA fragmentation. Additionally, T-2 toxin-induced oxidative stress and DNA damage with a time-dependent significant increased expression of p53 favors the apoptotic process by the activation of caspase-3 in T-2 toxin treated cells. Cell cycle analysis by flow cytometry revealed a time-dependent increase ofG1 cell population along with the significant time-dependent up-regulation of mRNA and protein expression of p16 and p21 and significant down-regulation of cyclin D1, CDK4, and p-RB levels further verify the G1 phase arrest in GH3 cells. Morphology of GH3 cells by TEM clearly showed the damage and dysfunction to mitochondria and the cell nucleus. These findings for the first time demonstrate that T-2 toxin induces G1 phase cell cycle arrest by the involvement of p16/Rb pathway, along with ROS mediated oxidative stress and DNA damage with p53 and caspase cascade interaction, resulting in apoptosis in GH3 cells.

Cultivable anaerobic and aerobic bacterial communities in the fermentation chambers of Holotrichia parallela (coleoptera: scarabaeidae) larvae.

As important pests, scarab beetle larvae survive on plant biomass and the microbiota of the fermentation chamber play an important role in the digestion of lignocellulose-rich diets. However, the cultivable microbes, especially the anaerobic cultivable microbes, are still largely unknown. Here, both cultivable anaerobic and aerobic bacterial communities associated with the fermentation chamber of Holotrichia parallela larvae were investigated. In total bacteria cells directly enumerated by the 4', 6-diamidino-2-phenylindole (DAPI) staining method, the viable plate counts of cultivable bacteria in the fermentation chamber accounted for 0.92% of proportion. These cultivable bacteria were prone to attach to the fermentation chamber wall (88.41%) compared to the chamber contents. Anaerobic bacteria were dominant in the cultivable bacteria attaching to the fermentation chamber wall (70.20%), while the quantities of anaerobes and aerobes were similar in the chamber contents. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), fingerprinting and sequence analysis of isolated colonies revealed that the cultivable bacteria are affiliated with class γ-Proteobacteria, Bacteroidia, Actinobacteria, Clostridia and ß-Proteobacteria. γ-Proteobacteria was the major type of anaerobic cultivable bacteria and even the only one type of aerobic cultivable bacteria. Taken together, our results suggest, for the first time, that anaerobic microbiota are dominant in cultivable bacteria in the special anoxia niche of the fermentation chamber from H. parallela larvae. These bacterial isolates could be a treasure trove for screening lignocellulytic microbes which are essential for the plant biomass digestion of this scarab species.

Biofunctional analysis of Vitellogenin and Vitellogenin receptor in citrus red mites, Panonychus citri by RNA interference.

Panonychus citri is one of the most damaging pests of horticultural crops. Conventional control of this pest population through pesticides has led to the enhanced pest resistance. Management of P. citri population through RNAi, is still largely unknown. In oviparous organisms, fabrication and development of yolk protein play a vital role in the reproduction. Vitellin (Vn) is the source of eggs storage that helps in proper functioning of Vitellogenin (Vg) and Vitellogenin receptor (VgR). VgR is very compulsory protein for the development of Vg into oocytes. In the current study, Vg (PcVg) and VgR (PcVgR) genes were studied and their expressions at different developmental stages were quantified by RT-qPCR. Females treated with dsRNA of PcVg and PcVgR genes exhibited reduction in gene expression. Down regulation of target genes significantly effected oviposition and reduced the egg laying capacity up to 48% as compared to control (ds-egfp). Synergistic effect of target gene's dsRNA was also accessed that reduced the egg laying up to 60.42%. Furthermore, combination of target dsRNA on deutonymph and protonymph also resulted in 67% and 70% reduction in eggs, respectively. Synergistic effect of dsRNA at 1000 ng/ul resulted in longer life span as compared to control treatments. This study suggests to develop a new strategy of P. citri population control by reducing its reproduction.

A genetically enhanced sterile insect technique against the fruit fly, Bactrocera dorsalis (Hendel) by feeding adult double-stranded RNAs.

RNAi based sterile insect technique (SIT) is an authentic insect management approach but requires proper target genes. During this study, spermless males were developed by interfering with germ cell differentiation and azoospermia related genes. Data demonstrates significant reductions in the target genes expressions (boul, zpg, dsx M , fzo and gas8) after oral dsRNAs administration. Knock down of target genes significantly affected the reproductive ability of males and reduced egg-hatching as compared to the control group. Furthermore, different combinations of selected gene dsRNAs (boul + zpg, boul + dsx M and zpg + dsx M ) were made, which resulted up to 85.40% of male sterility. The most effective combination was selected to prepare different concentrations of dsRNA, 250, 500, 750 and 1000 ng/µl, that caused 18.97%, 38.68%, 58.02% and 85.40% male sterility, respectively. Subsequently, 1000 ng/µl of the same combination of ds-RNAs was used against differently aged adult flies (1, 5, 7, 10 days) which lead to 85.40%, 31.42%, 21.76% and 9.90% male sterility, respectively. SIT developed in this study showed that, boul + zpg combination of dsRNA feeding for 6 hours significantly reduced the number of spermatozoa and viability of sperm in 1-day-old B. dorsalis flies. In short, this study provides an effective SIT technique for long-term B. dorsalis management.