Combined effects of azoxystrobin and oxytetracycline on rhizosphere microbiota of Arabidopsis thaliana.

The rhizosphere is one of the key determinants of plant health and productivity. Mixtures of pesticides are commonly used in intensified agriculture. However, the combined mechanisms underlying their impacts on soil microbiota remain unknown. The present study revealed that the rhizosphere microbiota was more sensitive to azoxystrobin and oxytetracycline, two commonly used pesticides, than was the microbiota present in bulk soil. Moreover, the rhizosphere microbiota enhanced network complexity and stability and increased carbohydrate metabolism and xenobiotic biodegradation as well as the expression of metabolic genes involved in defence against pesticide stress. Co-exposure to azoxystrobin and oxytetracycline had antagonistic effects on Arabidopsis thaliana growth and soil microbial variation by recruiting organic-degrading bacteria and regulating ABC transporters to reduce pesticide uptake. Our study explored the composition and function of soil microorganisms through amplicon sequencing and metagenomic approaches, providing comprehensive insights into the synergistic effect of plants and rhizosphere microbiota on pesticides and contributing to our understanding of the ecological risks associated with pesticide use.

DEAD-box helicase 1 inhibited CD8+ T cell antitumor activity by inducing PD-L1 expression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) does not respond well to current treatments, even immune checkpoint inhibitors. PD-L1 (programmed cell death ligand 1 or CD274 molecule)-mediated immune escape of tumor cells may be a key factor affecting the efficacy of immune checkpoint inhibitor (ICI) therapy. However, the regulatory mechanisms of PD-L1 expression and immune escape require further exploration. Here, we observed that DDX1 (DEAD-box helicase 1) was overexpressed in HCC tissues and associated with poor prognosis in patients with HCC. Additionally, DDX1 expression correlated negatively with CD8+ T cell frequency. DDX1 overexpression significantly increased interferon gamma (IFN-γ)-mediated PD-L1 expression in HCC cell lines. DDX1 overexpression decreased IFN-γ and granzyme B production in CD8+ T cells and inhibited CD8+ T cell cytotoxic function in vitro and in vivo. In conclusion, DDX1 plays an essential role in developing the immune escape microenvironment, rendering it a potential predictor of ICI therapy efficacy in HCC.

Pesticide interference and additional effects on plant microbiomes.

Pesticides are essential to modern human production activities, particularly those increasing global food production and quality; however, corresponding pesticide contamination is becoming more prominent. Plant microbiomes, containing different assemblages of microbial communities in the rhizosphere, endosphere, and phyllosphere, in addition to the mycorrhizal microbiome, substantially impact plant health and productivity. Therefore, the relationships among pesticides, plant microbiomes, and plant communities are important to evaluate the ecological safety of pesticides. To date, the majority of research efforts aimed at understanding the effects of pesticides on microbial communities have focused on single niche microbiomes. However, a comprehensive review of the effects of pesticides on microbial communities and co-occurrence patterns in different ecological niches is still lacking. This review fills this gap by providing an overview of the effects of pesticides on plant microbial communities across ecological niches. Specifically, we discuss the potential feedback and risks associated with these effects on plant health. Through a thorough examination of the available literature, we provide a comprehensive perspective of the impacts of pesticides on plant microbiomes, which may facilitate the development of effective strategies to mitigate these effects.

Lipophorin receptor is required for the accumulations of cuticular hydrocarbons and ovarian neutral lipids in Locusta migratoria.

Lipophorin is the most abundant lipoprotein particle in insect hemolymph. Lipophorin receptor (LPR) is a glycoprotein that binds to the lipophorin and mediates cellular uptake and metabolism of lipids by endocytosis. However, the roles of LPR in uptake of lipids in the integument and ovary remain unknown in the migratory locust (Locusta migratoria). In present study, we characterized the molecular properties and biological roles of LmLPR in L. migratoria. The LmLPR transcript level was high in the first 2 days of the adults after eclosion, then gradually declined. LmLPR was predominately expressed in fat body, ovary and integument. Using immuno-detection methods, we revealed that LmLPR was mainly localized in the membrane of oenocytes, epidermal cells, fat body cells and follicular cells. RNAi-mediated silencing of LmLPR led to a slight decrease of the cuticle hydrocarbon contents but with little effect on the cuticular permeability. However, the neutral lipid content was significantly decreased in the ovary after RNAi against LmLPR, which led to a retarded ovarian development. Taken together, our results indicated that LmLPR is involved in the uptake and accumulation of lipids in the ovary and plays a crucial role in ovarian development in L. migratoria. Therefore, LmLPR could be a promising RNAi target for insect pest management by disrupting insect ovarian development.

Shaping effects of rice, wheat, maize, and soybean seedlings on their rhizosphere microbial community.

The rhizosphere microbiome plays critical roles in plant growth and is an important interface for resource exchange between plants and the soil environment. Crops at various growing stages, especially the seedling stage, have strong shaping effects on the rhizosphere microbial community, and such community reconstruction will positively feed back to the plant growth. In the present study, we analyzed the variations of bacterial and fungal communities in the rhizosphere of four crop species: rice, soybean, maize, and wheat during successive cultivations (three repeats for the seedling stages) using 16S rRNA gene and internal transcribed spacer (ITS) high-throughput sequencing. We found that the relative abundances of specific microorganisms decreased after different cultivation times, e.g., Sphingomonas, Pseudomonas, Rhodanobacter, and Caulobacter, which have been reported as plant-growth beneficial bacteria. The relative abundances of potential plant pathogenic fungi Myrothecium and Ascochyta increased with the successive cultivation times. The co-occurrence network analysis showed that the bacterial and fungal communities under maize were much more stable than those under rice, soybean, and wheat. The present study explored the characteristics of bacteria and fungi in crop seedling rhizosphere and indicated that the characteristics of indigenous soil flora might determine the plant growth status. Further study will focus on the use of the critical microorganisms to control the growth and yield of specific crops.

Factors affecting dissociative tendency among middle school students / 预防医学

Abstract@#Objective To investigate the prevalence of dissociative tendency and its influencing factors among middle school students, so as to provide insights into prevention and early interventions of adolescent dissociative tendency.@* Methods@# Students at ages of 13 to 18 years that studied in 9 high schools were sampled using a multistage, stratified, cluster sampling method from 3 counties (districts) of Hangzhou City from January, 2021 and March, 2022. Participants' demographics and social, school, family environment and psychological factors were collected using self-designed questionnaires and standardized scales. The dissociative tendency was initially screened using the Adolescent Dissociative Experience Scale (A-DES), and the diagnosed by three psychiatrists according to Diagnostic and Statistical Manual of Mental Disorders (5th Edition). Factors affecting ADT were identified using a multivariable logistic regression model. @*Results@# Totally 3 240 students were recruited, and 2 841 students were qualified, with a response rate of 87.69%. The respondents included 1 389 men (48.89%) and 1 452 women (51.11%), and had a mean age of (15.69±1.58) years. The prevalence of dissociative tendency was 24.04%, and the detection of dissociative tendency was higher among men than among women (29.23% vs. 19.08%; χ2=24.529, P<0.001). Multivariable logistic regression analysis identified negative teacher-student relationship (OR=1.055, 95%CI: 1.010-1.103), family emotional expressiveness (OR=0.872, 95%CI: 0.777-0.978), family conflict (OR=1.152, 95%CI: 1.045-1.271), family organization (OR=0.880, 95%CI: 0.780-0.992) and family cohesion (OR=0.871, 95%CI: 0.766-0.987) as factors affecting dissociative tendency among men, and somatization (OR=1.041, 95%CI: 1.002-1.082) and paranoid ideation (OR=1.094, 95%CI: 1.006-1.190) as factors affecting dissociative tendency among women. @*Conclusions@# The prevalence of dissociative tendency was 24.04% among middle school students. Negative teacher-student relationship, family emotional expressiveness, family conflict, family organization, and family cohesion may affect dissociative tendency among men, while somatization and paranoid ideation may affect dissociative tendency among women.

A gyroscope-free visual-inertial flight control and wind sensing system for 10-mg robots.

Tiny "gnat robots," weighing just a few milligrams, were first conjectured in the 1980s. How to stabilize one if it were to hover like a small insect has not been answered. Challenges include the requirement that sensors be both low mass and high bandwidth and that silicon-micromachined rate gyroscopes are too heavy. The smallest robot to perform controlled hovering uses a sensor suite weighing hundreds of milligrams. Here, we demonstrate that an accelerometer represents perhaps the most direct way to stabilize flight while satisfying the extreme size, speed, weight, and power constraints of a flying robot even as it scales down to just a few milligrams. As aircraft scale reduces, scaling physics dictates that the ratio of aerodynamic drag to mass increases. This results in reduced noise in an accelerometer's airspeed measurement. We show through simulation and experiment on a 30-gram robot that a 2-milligram off-the-shelf accelerometer is able in principle to stabilize a 10-milligram robot despite high noise in the sensor itself. Inspired by wind-vision sensory fusion in the flight controller of the fruit fly Drosophila melanogaster, we then added a tiny camera and efficient, fly-inspired autocorrelation-based visual processing to allow the robot to estimate and reject wind as well as control its attitude and flight velocity using a Kalman filter. Our biology-inspired approach, validated on a small flying helicopter, has a wind gust response comparable to the fruit fly and is small and efficient enough for a 10-milligram flying vehicle (weighing less than a grain of rice).

Development of a Potentially New Algaecide for Controlling Harmful Cyanobacteria Blooms Which is Ecologically Safe and Selective.

Harmful cyanobacterial blooms (HCBs) caused by Microcystis aeruginosa are of great concern as they negatively affect the aquatic environment and human health. Chemical methods could rapidly eradicate HCBs and have been used for many decades. However, many chemical reagents are not recommended to eliminate HCBs in the long term, given the possible destructive and toxic effects of the chemicals employed on non-target aquatic organisms. We developed a new algaecide, 2-((1,3,4-thiadiazol-2-yl)thio)-N-(4-chlorophenyl) acetamide (Q2), to control harmful cyanobacteria while being environmentally friendly and selective. In our study, Q2 effectively inhibited cyanobacterial growth, especially of M. aeruginosa, but did not affect eukaryotic algae in test concentrations. A critical mechanism was revealed by transcriptome and metagenomic results showing that Q2 affects multiple cellular targets of cyanobacteria for HCB control, including the destruction of organelles, damage in the photosynthesis center, as well as inhibition of gas vesicle growth, and these changes can be highly relevant to the decrease of quorum-sensing functional KEGG pathways. Furthermore, Q2 did not affect the microbial composition and could recover the disrupted aquatic functional pathways in a short period. This is different from the impact on ecosystem functioning of the traditionally used harmful algaecide diuron. All these results verified that Q2 could be friendly to the aquatic environment, providing a new directional choice in managing HCBs in the future.

Effects of nicotinic acetylcholine receptor subunit deletion mutants on insecticide susceptibility and fitness in Drosophila melanogaster.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in insects and also the target site for many insecticides. Unfortunately, the effectiveness of these insecticides is diminishing as a consequence of the evolution of insecticide resistance. Further exploration of insecticide targets is important to sustainable pest management. RESULTS: In order to validate the role of nAChR subunits in insecticide susceptibility and test whether the subunit's absence imposes the fitness cost on insects, we determined the susceptibility of eight nAChR subunit deletion mutants of Drosophila melanogaster to nine insecticides. These findings highlighted the specific resistance of the Dα6 deletion mutant to spinosyns. Although triflumezopyrim, dinotefuran and imidacloprid are competitive modulators of nAChRs, differences in susceptibility of the insect with different deletion mutants suggested that the target sites of these three insecticides do not overlap completely. Mutants showed decreased susceptibility to insecticides, accompanied by a reduction in fitness. The number of eggs produced by Dα1attP , Dα2attP , Dß2attP and Dß3attP females was significantly lesser than that of the vas-Cas9 strain as the control. In addition, adults of Dα2attP , Dα3attP and Dα7attP strains showed lower climbing performance. Meanwhile, males of Dα3attP , Dα5attP , Dß2attP and Dß3attP , and females of Dß2attP showed significantly shorter longevity than those of the vas-Cas9 strain. CONCLUSION: This study provides new insights into the interactions of different insecticides with different nAChRs subunit in D. melanogaster as a research model, it could help better understand such interaction in agricultural pests whose genetic manipulations for toxicological research are often challenging. © 2022 Society of Chemical Industry.

Overexpression of ATP-binding cassette transporter Mdr49-like confers resistance to imidacloprid in the field populations of brown planthopper, Nilaparvata lugens.

BACKGROUND: The brown planthopper (BPH), Nilaparvata lugens (Stål), is the most severe pest attacking rice crops using sucking mouthparts. It causes significant damages to rice growth and food production worldwide. With the long-term and wide use of insecticides, field populations of BPH have developed resistance to many insecticides. RESULTS: Here, we showed that upregulation of an ATP-binding cassette transporter gene NlMdr49-like contributes to imidacloprid resistance in field populations of BPH. A comparative transcriptome analysis was performed to evaluate the gene expression in two field populations (JXSG18 and YNTC18). Compared with a susceptible strain (Sus), 202 upregulated genes and 170 downregulated genes were identified in both field populations. Functional enrichment analysis revealed that the differentially expressed genes (DEGs) are mainly linked to metabolic process and transmembrane transport. Among the candidate DEGs, NlMdr49-like was significantly upregulated in both field populations. Based on the genome and transcriptome of BPH, the full-length complementary DNA of NlMdr49-like was sequenced and its molecular characteristics were analyzed. Expression pattern analysis of various tissues showed that NlMdr49-like was predominantly expressed in midgut and Malpighian tubules which are important excretion organs. Knocking down NlMdr49-like reduced BPH resistance to imidacloprid, but did not affect its resistance to the other nine insecticides (chlorpyrifos, thiamethoxam, nitenpyram, dinotefuran, sulfoxaflor, triflumezopyrim, ethiprole, buprofezin and pymetrozine). Furthermore, a transgenic strain of Drosophila melanogaster overexpressing NlMdr49-like was less susceptible to imidacloprid. CONCLUSIONS: Our findings indicate that upregulation of NlMdr49-like is another mechanism contributing to imidacloprid resistance in N. lugens. This result is helpful to further understand the resistance mechanism of N. lugens to imidacloprid. © 2021 Society of Chemical Industry.

RNA m6A methyltransferase METTL3 promotes colorectal cancer cell proliferation and invasion by regulating Snail expression.

Nitrogen 6-methyladenosine (m6A) is the result of methylation of nitrogen-6 on adenosine, and is the most abundant chemical modification of eukaryotic mRNA. Dysregulation of m6A methylation has been implicated in cancer development and progression through various mechanisms. This type of methylation is primarily regulated by methyltransferase-like 3 (METTL3). However, the molecular mechanisms underlying the role of METTL3 in colorectal cancer (CRC) have not been extensively elucidated. The present study explored m6A modification and the underlying mechanism of m6A, which serve regulatory roles in the development of CRC. It was found that METTL3 is upregulated in CRC cell lines and tissues, and its expression positively correlated with poor overall survival (OS). Mechanistically, the present study demonstrated that METTL3 methylates Snail mRNA, thus stabilizing it to promote CRC malignancy. The present findings indicate that m6A modification is involved in CRC tumorigenesis, and highlight its potential as a therapeutic target against CRC.

Molecular characterization and RNA interference responses of the lethal giant larvae gene in Diabrotica virgifera virgifera adults.

High specificity for silencing target genes and single-copy target genes that yield clear phenotypes are two important factors for the success of RNA interference (RNAi). The lethal giant larvae (Lgl) gene appears to be an ideal gene for RNAi because RNAi can effectively suppress its expression and results in molting defects and mortality in Tribolium castaneum. To investigate the suitability of this gene for RNAi in other insects, we identified and characterized DvLgl from the western corn rootworm, Diabrotica virgifera virgifera, a species exhibiting high RNAi efficiency. DvLgl was expressed in all developmental stages and tissues investigated. The deduced DvLgl protein showed high amino-acid sequence identities and similar domain architecture to Lgls from other insect species. Despite many similarities among insect Lgls, RNAi-mediated suppression of DvLgl failed to produce a phenotype in D. v. virgifera adults. The difference in developing phenotypes could be attributed greatly to the level of gene suppression and the insect developmental stages for RNAi. These results highlight the variability in RNAi response among insects and showcase the importance of screening multiple target genes when conducting RNAi studies. Our findings are expected to help the design of future RNAi studies and future investigations of Lgl in insects.

Comparison of strategies for enhancing RNA interference efficiency in Ostrinia nubilalis.

BACKGROUND: Targeting insect-specific genes through post-transcriptional gene silencing with RNA interference (RNAi) is a new strategy for insect pest management. However, lepidopterans are recalcitrant to RNAi, which prevents application of novel RNAi technology to many notorious pests, including Ostrinia nubilalis (ECB). Strategies for enhancing RNAi efficiency, including large doses of double-stranded RNA (dsRNA), nuclease inhibitors, transfection reagents, and nanoparticles, have proved useful in other insects exhibiting substantial dsRNA degradation, a major mechanism limiting RNAi efficacy. To determine if similar strategies can enhance RNAi efficiency in ECB, various reagents were tested for their ability to enhance dsRNA stability in ECB tissues, then compared for their effectiveness in whole ECB. RESULTS: Ex vivo incubation experiments revealed that Meta dsRNA lipoplexes, EDTA, chitosan-based dsRNA nanoparticles, and Zn2+ enhanced dsRNA stability in ECB hemolymph and gut content extracts, compared with uncoated dsRNA. Despite these positive results, the reagents used in this study were ineffective at enhancing RNAi efficiency in ECB in vivo. To reduce assay time and required dsRNA, midguts were dissected and incubated in tissue culture medium containing dsRNA with and without reagents. These experiments showed that RNAi efficiency varied between target genes, and nuclease inhibitors improved RNAi efficiency for only a portion of the refractory target genes investigated ex vivo. CONCLUSION: These results indicate that enhancing dsRNA stability is insufficient to improve RNAi efficiency in ECB and suggests the existence of additional, complex mechanisms contributing to low RNAi efficiency in ECB.

Characterization, expression patterns, and transcriptional responses of three core RNA interference pathway genes from Ostrinia nubilalis.

RNA interference (RNAi) is commonly used in the laboratory to analyze gene function, and RNAi-based pest management strategies are now being employed. Unfortunately, RNAi is hindered by inefficient and highly-variable results when different insects are targeted, especially lepidopterans, such as the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae). Previous efforts to achieve RNAi-mediated gene suppression in ECB revealed low RNAi efficiency with both double-stranded RNA (dsRNA) injection and ingestion. One mechanism that can affect RNAi efficiency in insects is the expression and function of core RNAi pathway genes, such as those encoding Argonaut 2 (Ago2), Dicer 2 (Dcr2), and a dsRNA binding protein (R2D2). To determine if deficiencies in these core RNAi pathway genes contribute to low RNAi efficiency in ECB, full-length complementary DNAs encoding OnAgo2, OnDcr2, and OnR2D2 were cloned, sequenced, and characterized. A comparison of domain architecture suggested that all three predicted proteins contained the necessary domains to function. However, a comparison of evolutionary distances revealed potentially important variations in the first RNase III domain of OnDcr2, the double-stranded RNA binding domains of OnR2D2, and both the PAZ and PIWI domains of OnAgo2, which may indicate functional differences in enzymatic activity between species. Expression analysis indicated that transcripts for all three genes were expressed in all developmental stages and tissues investigated. Interestingly, the introduction of non-target dsRNA into ECB second-instar larvae via microinjection did not affect OnAgo2, OnDcr2, or OnR2D2 expression. In contrast, ingestion of the same dsRNAs resulted in upregulation of OnDcr2 but downregulation of OnR2D2. The unexpected transcriptional responses of the core machinery and the divergence in amino-acid sequence between specific domains in each core RNAi protein may possibly contribute to low RNAi efficiency in ECB. Understanding the contributions of different RNAi pathway components is critical to adapting this technology for use in controlling lepidopteran pests that exhibit low RNAi efficiency.

Molecular Characterizations of Double-Stranded RNA Degrading Nuclease Genes from Ostrinia nubilalis.

Variable RNA interference (RNAi) efficiencies limit RNAi-based pest management strategies for many pests. Previous efforts to understand mechanisms contributing to low RNAi efficiency indicate that double-stranded RNA (dsRNA) is degraded in the European corn borer (ECB), Ostrinia nubilalis, due to nuclease activity. To investigate the contribution of dsRNA-degrading endonucleases (dsRNases) and lepidopteran-specific RNAi efficiency-related nucleases (REases) to dsRNA instability and low RNAi efficiency in ECB, five complementary DNAs putatively encoding four dsRNases (OndsRNase1, 2, 3, and 4) and one REase (OnREase) were sequenced. Characterization of these transcripts revealed that substrate specificity might vary among the four dsRNases due to different amino acid combinations in the substrate-binding sites. Gene expression analysis indicated that OndsRNase2 and OnREase were highly expressed in the larval gut, and OndsRNase1 showed the highest expression in hemolymph, especially in older developmental stages. Transcript level analysis after dsRNA exposure revealed that expression of OnREase rapidly increased upon dsRNA ingestion or injection, whereas OndsRNase4 expression only increased after long-term ingestion of dsRNA. While the biological function of these nucleases remains to be verified, our results suggest that OnREase and OndsRNase2, and OndsRNase1 and OndsRNase4 may be responsible for degradation of dsRNAs in the ECB gut and hemolymph, respectively, thereby contributing to low RNAi efficiency.

Stability of double-stranded RNA in gut contents and hemolymph of Ostrinia nubilalis larvae.

RNA interference (RNAi) is a revolutionary technique for silencing gene expression, but the success of this technique is dependent upon the stability of double-stranded RNA (dsRNA) molecules. In many insects, especially lepidopteran species, RNAi efficiency is limited by high instability of dsRNA in the gut and/or hemolymph, preventing the development of RNAi-based strategies for many serious pests. Previous attempts to perform RNAi on Ostrinia nubilalis (ECB, Lepidoptera: Crambidae) indicate low RNAi efficiency with both dsRNA injection and feeding. To investigate the contribution of dsRNA instability to low RNAi efficiency in ECB, a serious of ex vivo incubation experiments were performed where dsRNA integrity was assessed following incubation in larval gut continents and hemolymph using gel electrophoresis or RT-qPCR. DsRNA was less stable in the gut contents from ECB than in gut contents from Diabrotica virgifera virgifera, a coleopteran exhibiting high RNAi efficiency. Furthermore, characterization of dsRNA stability in ECB gut contents and hemolymph revealed that dsRNA was rapidly degraded under physiologically relevant conditions as a result of enzymatic activity that was neither size- nor sequence-dependent. These findings suggest that instability of dsRNA in ECB tissues is a contributing factor to the poor efficiency of RNAi in this pest. This work advances our understanding of mechanisms impacting RNAi efficiency in ECB and related lepidopteran insects for which novel pest management strategies are needed, and may facilitate the development of strategies for enhancing dsRNA stability in ECB tissues.

Apolipophorin-II/I Contributes to Cuticular Hydrocarbon Transport and Cuticle Barrier Construction in Locusta migratoria.

Apolipophorins are carrier proteins that bind lipids and mediate their transport from tissue to tissue in animals. Apolipophorin I and II (apoLp-II/I) are the major apolipophorins in insects. The implication of apoLp-II/I in cuticle lipid-barrier formation in insects has not been addressed to date. In the present study, we investigated the function of apoLp-II/I in the migratory locust Locusta migratoria (LmapoLp-II/I). During the development of fifth instar nymphs, LmapoLp-II/I transcript levels increased until mid-instar, and then decreased gradually until molting to the adult stage. We found that LmapoLp-II/I was predominately expressed in the fat body and the integument including oenocytes and epidermal cells. Immunodetection experiments revealed that LmapoLp-I mainly localized in the cytoplasm of oenocytes and epidermal cells. Silencing of LmapoLp-II/I caused molting defects in nymphs. Importantly, RNA interference against LmapoLp-II/I resulted in a significant decrease in the content of cuticle surface lipids including alkanes and methyl alkanes. Cuticular permeability was significantly enhanced in these nymphs in Eosin Y penetration assays. By consequence, desiccation resistance and insecticide tolerance of dsLmapoLp-II/I-treated locusts were reduced. Taken together, our results indicate that LmapoLp-II/I is involved in the transport and deposition of surface-cuticular lipids that are crucial for maintaining normal cuticle barrier function in L. migratoria.

Knockdown of LmCYP303A1 alters cuticular hydrocarbon profiles and increases the susceptibility to desiccation and insecticides in Locusta migratoria.

Cytochrome P450 monooxygenases (CYPs) serve many functions in insects, from the regulation of development to xenobiotic detoxification. Several conserved CYPs have been shown to play a role in insect growth and development. CYP303A1 is a highly conserved CYP with a single ortholog in most insects, but its underlying molecular characteristics and specific physiological functions remain poorly understood. In Drosophila melanogaster and Locusta migratoria, CYP303A1 is indispensable for eclosion to adult. Here, we report additional functions of the locust gene LmCYP303A1 in nymphal molts, cuticular lipid deposition and insecticide penetration. RT-qPCR revealed that LmCYP303A1 had a high expression level before ecdysis and was highly expressed in integument, wing pads, foregut and hindgut. Suppression of LmCYP303A1 expression by RNA interference (RNAi) caused a lethal phenotype with molting defect from nymph to nymph. In addition, LmCYP303A1 RNAi resulted in locusts being more susceptible to desiccation and to insecticide toxicity. Furthermore, knockdown of LmCYP303A1 efficiently suppressed the transcript level of key genes (ELO7, FAR15 and CYP4G102) responsible for cuticular hydrocarbon (CHC) synthesis, which led to a decrease in some CHC levels. Taken together, our results suggest that one of the functions of LmCYP303A1 is to regulate the biosynthesis of CHC, which plays critical roles in protecting locusts from water loss and insecticide penetration.

Both LmCYP4G genes function in decreasing cuticular penetration of insecticides in Locusta migratoria.

BACKGROUND: Cuticular hydrocarbons (CHCs) have a critical role in preventing desiccation and penetration of xenobiotics in insects. Previous studies have shown that cytochrome P450 subfamily 4G (CYP4G) enzymes are oxidative decarbonylases, essential for CHC biosynthesis. However, it is unclear whether there are functional differences between the two CYP4G genes in most insects. In Locusta migratoria, we identified two CYP4G genes (LmCYP4G62 and LmCYP4G102). LmCYP4G102 plays a critical role in the synthesis of CHCs, but the function of LmCYP4G62 is unknown. RESULTS: We identified, characterized, and compared two LmCYP4G genes, based on L. migratoria transcriptomic and genomic databases. RT-qPCR showed that both were highly expressed in tissues with which oenocytes are associated, the integument and fat body. Immunostaining indicated that LmCYP4G62 and LmCYP4G102 were highly abundant in oenocytes in these tissues. However, the two enzymes had a different subcellular distribution, with LmCYP4G62 localized on the plasma membrane and LmCYP4G102 dispersed throughout the oenocyte cytoplasm, presumably on the endoplasmic reticulum. RNA interference-mediated gene silencing against each of the two genes resulted in reduced CHC contents, in all classes for LmCYP4G102, but mostly shorter chain CHCs for LmCYP4G62. Silencing of both genes resulted in increased insecticide penetration through the cuticle, and increased locust susceptibility to desiccation and insecticides. CONCLUSION: Our studies suggest that both LmCYP4G62 and LmCYP4G102 contribute to hydrocarbon biosynthesis and play key roles in protecting locusts from water loss and insecticide penetration, but they are not fully redundant. Further, the two LmCYP4G genes might be used as new targets for insect pest management. © 2020 Society of Chemical Industry.

Progress and prospects of arthropod chitin pathways and structures as targets for pest management.

Chitin is a structural component of the arthropod cuticular exoskeleton and the peritrophic matrix of the gut, which play crucial roles in growth and development. In the past few decades, our understanding of the composition, biosynthesis, assembly, degradation, and regulation of chitinous structures has increased. Many chemicals have been developed that target chitin biosynthesis (benzoyphenyl ureas, etoxazole), chitin degradation (allosamidin, psammaplin), and chitin regulation (benzoyl hydrazines), thus resulting in molting deformities and lethality. In addition, proteins that disrupt chitin structures, such as lectins, proteases, and chitinases have been utilized to halt feeding and induce mortality. Chitin-degrading enzymes, such as chitinases are also useful for improving the efficacy of bio-insecticides. Transgenic plants, baculoviruses, fungi, and bacteria have been engineered to express chitinases from a variety of organisms for control of arthropod pests. In addition, RNA interference targeting genes involved in chitin pathways and structures are now being investigated for the development of environmentally friendly pest management strategies. This review describes the chemicals and proteins used to target chitin structures and enzymes for arthropod pest management, as well as pest management strategies based upon these compounds, such as plant-incorporated-protectants and recombinant entomopathogens. Recent advances in RNA interference-based pest management, and how this technology can be used to target chitin pathways and structures are also discussed.