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Uridine diphosphate (UDP)-glycosyltransferases (UGTs), which are major phase II detoxification enzymes, have been implicated in the glycosylation of lipophilic endobiotics and xenobiotics and thus potentially lead to the evolution of insecticide resistance. In this study, we identified and cloned two putative UGT genes from transcriptome data which are named UGT352A4 and UGT352A5. As demonstrated by qRT-PCR, two UGT genes were over-expressed in the thiamethoxam-resistant (THQR) strain relative to the susceptible (THQS) strain. Moreover, the induction experiment revealed that the expression of the UGT352A5 gene was significantly increased following exposure to thiamethoxam in the THQR strain. Furthermore, the expression of both UGT352A4 and UGT352A5 was downregulated after RNA interference, whereas only the silencing of UGT352A5 resulted in a noticeable increase in the mortality of THQR adults. Our results represent the first line of evidence showing that UGT352A5 might be responsible for conferring thiamethoxam resistance in B. tabaci. The results will be shed new insights for obtaining a better understanding of the role of UGTs in the evolution of insecticide resistance and developing new insect resistance management tactics within the sustainable integrated pest management framework., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: RNA interference (RNAi) is a cellular mechanism used to fight various threats, including transposons, aberrant RNAs, and some types of viruses. This mechanism relies on the detection of dsRNA molecules, which through a pathway involving Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), produces small interfering RNAs (siRNAs) that bind to the complementary RNAs triggering their degradation., Methods: Using the cockroach Blattella germanica as a model, we examined AGO2 activity by depleting its mRNA using RNAi and analyzing the phenotypes produced., Results: Depleting AGO2 expression had no remarkable effect on nymphal development or reproduction. dsRNA treatment triggered an immediate and transitory increase in AGO2 expression, independently of Dcr-2 action. In addition, we analyzed the siRNAs generated after injecting a heterologous dsRNA in control and AGO2-depleted animals. The results revealed that obtained siRNAs mapped non-uniformly along the dsRNA sequence. In AGO2-depleted animals, the proportion of 22 nucleotide reads was higher and accumulations of reads appeared in areas less well-represented in the controls. We also detected a preference for cytosine as the first nucleotide in controls that was significantly attenuated in AGO2-depleted individuals., Conclusions/general Significance: The siRNAs produced from a dsRNA mapped heterogeneously along the length of the dsRNA and this arrangement depends on the dsRNA sequence. AGO2 exerts its role as nuclease on the siRNA duplexes independently of its action on the corresponding mRNA. This study sheds light on an extremely useful process for reverse genetics in laboratories, in addition to the design of more effective, specific, and eco-friendly pest-control strategies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rhodnius prolixus, a vector of Chagas disease, is a hematophagous insect that feeds exclusively on blood. Each blood meal is digested within the first fourteen days after feeding, providing substrates for lipid synthesis for storage and egg production. These events are precisely regulated and emerging evidence points to a key function of insulin-like peptides (ILPs) in this control. Here we investigated the role of insulin receptor in the regulation of nutrient metabolism in fed adult females. The expression of insulin receptor (RhoprIR) gene was determined in adult organs, and it was highest in ovaries and previtellogenic follicles. We generated insects with RNAi-mediated knockdown of RhoprIR to address the physiological role of this receptor. RhoprIR deficiency improved longevity and reduced triacylglycerol storage in the fat body, whereas blood digestion remained unchanged for seven days after blood meal. The lower lipid content was attributable to decreased de novo lipogenesis as well as reduced incorporation of hemolymph-derived fatty acids into newly synthesized lipids within this organ. Consistent with that, fat bodies from RhoprIR-deficient insects exhibited decreased gene expression levels of lipophorin receptor (RhoprLpR), glycerol-3-phosphate acyltransferase 1 and 4 (RhoprGpat1 and RhoprGpat4), and carnitine palmitoyltransferase 1 (RhoprCpt1). Although hemolymph lipid profile was not affected by RhoprIR disruption, the concentration of circulating vitellogenin was increased. In line with these changes, RhoprIR-deficient females exhibited smaller ovaries and a marked reduction in oviposition. Taken together, these findings support a key role of insulin receptor in nutrient homeostasis, lipid synthesis and egg production following a blood meal., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Despite the essentiality for faithful chromosome segregation, centromere architectures are diverse among eukaryotes 1 , 2 and embody two main configurations: mono- and holocentromeres, referring, respectively, to localized or unrestricted distribution of centromeric activity. Of the two, some holocentromeres offer the curious condition of having arisen independently in multiple insects, most of which have lost the otherwise essential centromere-specifying factor CenH3 3 (first described as CENP-A in humans). 4-7 The loss of CenH3 raises intuitive questions about how holocentromeres are organized and regulated in CenH3-lacking insects. Here, we report the first chromatin-level description of CenH3-deficient holocentromeres by leveraging recently identified centromere components 6 , 7 and genomics approaches to map and characterize the holocentromeres of the silk moth Bombyx mori, a representative lepidopteran insect lacking CenH3. This uncovered a robust correlation between the distribution of centromere sites and regions of low chromatin activity along B. mori chromosomes. Transcriptional perturbation experiments recapitulated the exclusion of B. mori centromeres from active chromatin. Based on reciprocal centromere occupancy patterns observed along differentially expressed orthologous genes of Lepidoptera, we further found that holocentromere formation in a manner that is recessive to chromatin dynamics is evolutionarily conserved. Our results help us discuss the plasticity of centromeres in the context of a role for the chromosome-wide chromatin landscape in conferring centromere identity rather than the presence of CenH3. Given the co-occurrence of CenH3 loss and holocentricity in insects, 7 we further propose that the evolutionary establishment of holocentromeres in insects was facilitated through the loss of a CenH3-specified centromere., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Insect metamorphosis originated around the middle Devonian, associated with the innovation of the final molt; this occurs after histolysis of the prothoracic gland (PG; which produces the molting hormone) in the first days of adulthood. We previously hypothesized that transcription factor E93 is crucial in the emergence of metamorphosis, because it triggers metamorphosis in extant insects. This work on the cockroach Blattella germanica reveals that E93 also plays a crucial role in the histolysis of PG, which fits the above hypothesis. Previous studies have shown that the transcription factor FTZ-F1 is essential for PG histolysis. We have found that FTZ-F1 depletion towards the end of the final nymphal instar downregulates the expression of E93 , whereas E93-depleted nymphs molt to adults that retain a functional PG. Interestingly, these adults are able to molt again, which is exceptional in insects. The study of insects able to molt again in the adult stage may reveal clues about how nymphal epidermal cells definitively become adult cells, and whether it is possible to reverse this process., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parental RNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell., (© 2020 Federation of American Societies for Experimental Biology.)

The tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a key cytoplasm signaling adaptor that mediates signals activated by TNFR superfamily and the interleukin-1/Toll-like receptor (IL-1/TLR) superfamily. In the present research, a housefly Musca domestica TRAF6 (MdTRAF6) gene is identified and characterized, with a 51.7-kDa protein possessing a RING domain and a conserved C-terminal TRAF homology MATH domain encoded. MdTRAF6 is widely expressed in diverse tissues with high expression levels in gut and fat body, which is of the highest levels in adult in all growth stages. The expression of MdTRAF6 could be remarkably induced by bacterial challenge, and the silencing MdTRAF6 could alter the expressions of NF-κB-like genes (relish and dorsal) and antimicrobial peptide genes (cecropin, diptericin, attacin, muscin), thus leading elevated mortalities of larvae followed by bacterial infection. Inspiringly. MdTRAF6-depleted adult flies display higher mortality, lower fertility and reduced survival of offspring than the controls. Further investigation reveals that knockdown of MdTRAF6 disturbs the ovarian development and impaires the expressions of vitellogenin and vitellogenin receptor genes in the adult females. All these phenotypes show crucial roles of MdTRAF6 in innate immunity via positive regulation of the Toll pathway and negative regulation of the Imd pathway, and in reproduction by maintaining ovarian development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A recent in vitro characterization of a recombinant pyruvate kinase (PK) from Aedes aegypti mosquitoes demonstrated that the enzyme is uniquely regulated by multiple allosteric effectors. Here, we further explored PK gene and protein expression, and enzymatic activity in key metabolic tissues of mosquitoes maintained under different nutritional conditions. We also studied the metabolic effects of PK depletion using several techniques including RNA interference and mass spectrometry-based stable-isotope tracing. Transcriptional analysis showed a dynamic post-feeding PK mRNA expression pattern within and across mosquito tissues, whereas corresponding protein levels remained stable throughout the time course analyzed. Nevertheless, PK activity significantly differed in the fat body of sucrose-, blood-fed, and starved mosquitoes. Genetic silencing of PK did not alter survival in blood-fed females maintained on sucrose. However, an enhanced survivorship was observed in PK-deficient females maintained under different nutritional regimens. Our results indicate that mosquitoes overcame PK deficiency by up-regulating the expression of genes encoding NADP-malic enzyme-1, phosphoenolpyruvate carboxykinase-1, phosphoglycerate dehydrogenase and glutamate dehydrogenase, and by decreasing glucose oxidation and metabolic pathways associated with ammonia detoxification. Taken together, our data demonstrate that PK confers to A. aegypti a metabolic plasticity to tightly regulate both carbon and nitrogen metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The adoption of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target pests. Previous studies indicate that resistance to Bacillus thuringiensis Cry1A and Cry1F toxins was genetically linked with mutations of ATP-binding cassette (ABC) transporter subfamily C gene ABCC2 in at least seven lepidopteran insects. Several strains selected in the laboratory of the Asian corn borer, Ostrinia furnacalis , a destructive pest of corn in Asian Western Pacific countries, developed high levels of resistance to Cry1A and Cry1F toxins. The causality between the O. furnacalis ABCC2 ( OfABCC2 ) gene and resistance to Cry1A and Cry1F toxins remains unknown. Here, we successfully generated a homozygous strain (OfC2-KO) of O. furnacalis with an 8-bp deletion mutation of ABCC2 by the CRISPR/Cas9 approach. The 8-bp deletion mutation results in a frame shift in the open reading frame of transcripts, which produced a predicted protein truncated in the TM4-TM5 loop region. The knockout strain OfC2-KO showed much more than a 300-fold resistance to Cry1Fa, and low levels of resistance to Cry1Ab and Cry1Ac (<10-fold), but no significant effects on the toxicities of Cry1Aa and two chemical insecticides (abamectin and chlorantraniliprole), compared to the background NJ-S strain. Furthermore, we found that the Cry1Fa resistance was autosomal, recessive, and significantly linked with the 8-bp deletion mutation of OfABCC2 in the OfC2-KO strain. In conclusion, in vivo functional investigation demonstrates the causality of the OfABCC2 truncating mutation with high-level resistance to the Cry1Fa toxin in O. furnacalis . Our results suggest that the OfABCC2 protein might be a functional receptor for Cry1Fa and reinforces the association of this gene to the mode of action of the Cry1Fa toxin.

Transmission from an infected mosquito to a host is an essential process in the life cycle of mosquito-borne flaviviruses. Numerous studies have demonstrated that mosquito saliva facilitates viral transmission. Here we find that a saliva-specific protein, named Aedes aegypti venom allergen-1 (AaVA-1), promotes dengue and Zika virus transmission by activating autophagy in host immune cells of the monocyte lineage. The AG6 mice (ifnar1 -/- ifngr1 -/- ) bitten by the virus-infected AaVA-1-deficient mosquitoes present a lower viremia and prolonged survival. AaVA-1 intracellularly interacts with a dominant negative binder of Beclin-1, known as leucine-rich pentatricopeptide repeat-containing protein (LRPPRC), and releases Beclin-1 from LRPPRC-mediated sequestration, thereby enabling the initialization of downstream autophagic signaling. A deficiency in Beclin-1 reduces viral infection in mice and abolishes AaVA-1-mediated enhancement of ZIKV transmission by mosquitoes. Our study provides a mechanistic insight into saliva-aided viral transmission and could offer a potential prophylactic target for reducing flavivirus transmission.

Holotrichia oblita is one of the nastiest pests in China. In present research, four full-length cDNA encoding of HoblOBP genes were cloned and sequenced from H. oblita. The mRNA of HoblOBPs were predominantly expressed in antenna. The recombinant HoblOBPs proteins were obtained for fluorescence binding assays. Four of HoblOBPs could mediate the response of H. oblita to organic fertilizers-derived attractants, including HoblOBP5 binding to skatole; HoblOBP8 binding to p-cresol, indole and skatole; HoblOBP9 binding to indole and 4-allylanisole; and HoblOBP24 binding to p-cresol, indole and 4-ethylphenol. Further, RNA interference demonstrated that transcripts of HoblOBP5, 8, 9, and 24 decreased in a time-dependent manner after dsRNA-injection. Knockdown of HoblOBP5, 8, 9, and 24 by injection of dsRNA successfully interfered with behavioral responses towards the target compounds in beetles. Our results showed that HoblOBP5, HoblOBP8, HoblOBP9 and HoblOBP24 are essential in mediating the approach behavior of H. oblita., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Insecticides act as toxins, inhibitors of digestion and deterrents, and affect the expression of many genes in insects. To assess key genes associated with the detoxification or regulation of imidacloprid in greenbug, Schizaphis graminum (Rondani), the transcriptome and digital gene expression (DGE) profile were analyzed using Illumina sequencing., Results: In total, 48 763 494 clean reads were obtained by sequencing. Expression profile analysis showed that 2782 unigenes were differently expressed between the imidacloprid treatment and control groups. After exposure to imidacloprid, the expression levels of 1846 unigenes were upregulated and 936 were downregulated in comparison with controls. Expression patterns of the top 20 highly expressed genes show that they could be involved in the detoxification of imidacloprid. Silencing of multidrug resistance-associated gene (MRA), GATA-binding gene (GAT) and takeout-like precursor gene (TLP) resulted in increasing susceptibility to imidacloprid., Conclusions: The differentially expressed genes in S. graminum have potential regulatory or detoxification roles in response to imidacloprid. These results should be useful in understanding the molecular mechanisms of greenbug adaption to imidacloprid. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

As a core member of the Hippo signaling pathway, Hpo plays a critical role in regulating growth and development. Previous studies reported that loss of function of Hpo results in increased proliferation, reduced apoptosis and induction of tissue overgrowth in Drosophila. In this study, we used CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) to study Hpo gene (BmHpo) function in the lepidopteran insect Bombyx mori, known commonly as the silkworm. Sequence analysis of BmHpo revealed an array of deletions in mutants. We found that BmHpo knockout resulted in defects in body size regulation, in developmental defects and pigment accumulation and early death. Our data show that BmHpo is essential for regulation of insect growth and development and that CRISPR/Cas9 technology can serve as a basis for functional analysis of target genes in lepidopteran insects., (© 2018 Institute of Zoology, Chinese Academy of Sciences.)

The cotton bollworm Helicoverpa armigera, is one of the world's major pest of agriculture, feeding on over 300 hosts in 68 plant families. Resistance cases to most insecticide classes have been reported for this insect. Management of this pest in agroecosystems relies on a better understanding of how it copes with phytochemical or synthetic toxins. We have used genome editing to knock out a cluster of nine P450 genes and show that this significantly reduces the survival rate of the insect when exposed to two classes of host plant chemicals and two classes of insecticides. Functional expression of all members of this gene cluster identified the P450 enzymes capable of metabolism of these xenobiotics. The CRISPR-Cas9-based reverse genetics approach in conjunction with in vitro metabolism can rapidly identify the contributions of insect P450s in xenobiotic detoxification and serve to identify candidate genes for insecticide resistance.

Nephotettix cincticeps, a prevalent rice pest, injects gelling and watery saliva into plant tissues during the sucking process. Certain components within the saliva are believed to interact with plant cellular constituents and play important roles in overcoming host plant defense responses. Based on our previous analysis of the salivary gland transcriptome and secreted saliva proteome of N. cincticeps, in this study, we analyzed the biological functions of salivary protein, NcSP75 (N. cincticeps salivary protein 75 kD). NcSP75, a salivary glands-specific gene, showed low similarities to any previously reported sequences. Knockdown of NcSP75 by RNA interference (RNAi) reduced the longevity of treated nymphs to approximately half of the longevity of controls and caused severe developmental retardation. Furthermore, the knockdown of NcSP75 decreased the survival rate of adults, and reduced the number of deposited eggs and hatched nymphs. Thus, the adverse effects caused by the knockdown of NcSP75 were observed throughout the lifetime of N. cincticeps, when feeding on rice plants. In contrast, no reduction was observed in the survival rate of the knockdown of NcSP75 adults fed on an artificial diet. Electrical penetration graph measurements taken from adult females feeding on rice plants showed a significantly shorter duration of phloem ingestion associated with the knockdown of NcSP75 than the knockdown of the enhanced green fluorescent protein (EGFP). Furthermore, the total sugar content of the honeydew was lower when NcSP75 was knocked down. These results suggest that the NcSP75 protein contribute to successful and sustainable ingestion from the sieve elements of rice plants. The NcSP75 protein of N. cincticeps can, accordingly, be considered as a key effector for establishing compatible interaction with rice plants and could be a potential target for controlling this species., Competing Interests: The authors have declared that no competing interests exist.

Among various genome editing tools available for functional genomic studies, reagents based on clustered regularly interspersed palindromic repeats (CRISPR) have gained popularity due to ease and versatility. CRISPR reagents consist of ribonucleoprotein (RNP) complexes formed by combining guide RNA (gRNA) that target specific genomics regions and a CRISPR associated nuclease (Cas). The gRNA targeting specific gene sequences may be delivered as a plasmid construct that needs to be transcribed or as a synthetic RNA. The Cas nuclease can be introduced as a plasmid construct, mRNA, or purified protein. The efficiency of target editing is dependent on intrinsic factors specific to each species, the target gene sequence, and the delivery methods of CRISPR gRNA and the Cas nuclease. Although intrinsic factors affecting genome editing may not be altered in most experiments, the delivery method for CRISPR/Cas reagents can be optimized to produce the best results. In this study, the efficiency of genome editing by CRISPR/Cas system in the bollworm, Helicoverpa zea (Boddie), was evaluated using ribonucleoprotein (RNP) complexes assembled by binding synthetic gRNA with purified Cas9 nuclease engineered with nuclear localization signals to target the vermillion (eye color) gene. Mutation rates of adults emerging from embryos microinjected with 1, 2, or 4 μM RNP complexes were compared using replicated experiments. Embryos injected with 2 or 4 μM RNP complexes displayed significantly higher mutation rates (>88%) in surviving adults compared to those injected with 1 μM. The hatch rate in embryos injected with RNP complexes and with injection buffer only (mock injections) was reduced by 19.8(±5.2)% compared to noninjected control embryos, but did not differ significantly between injected embryos. Evaluation of potential off-target sites in H. zea genome did not identify any mutations. This study demonstrates that in vitro assembled synthetic RNP complexes can be used to obtain high genome editing rates in a reproducible manner in functional genomics or genetic manipulation studies., Competing Interests: The authors have declared that no competing interests exist.

Little is known about the effects of dietary macronutrients on the capacity of insects to ward off a fungal pathogen. Here we tested the hypothesis that Mormon crickets fed restricted protein diets have lower enzymatic assays of generalized immunity, slower rates of encapsulation of foreign bodies, and greater mortality from infection by Beauveria bassiana, a fungal pathogen. Beginning in the last nymphal instar, Mormon crickets were fed a high, intermediate, or low protein diet with correspondingly low, intermediate, or high carbohydrate proportions. After they eclosed to adult, we drew hemolymph, topically applied B. bassiana, maintained them on diet treatments, and measured mortality for 21 days. Mormon crickets fed high protein diets had higher prophenoloxidase titers, greater encapsulation response, and higher survivorship to Beauveria fungal infection than those on low protein diets. We replicated the study adding very high and very low protein diets to the treatments. A high protein diet increased phenoloxidase titers, and those fed the very high protein diet had more circulating prophenoloxidase. Mormon crickets fed the very low protein diet were the most susceptible to B. bassiana infection, but the more concentrated phenoloxidase and prophenoloxidase associated with the highest protein diets did not confer the greatest protection from the fungal pathogen as in the first replicate. We conclude that protein-restricted diets caused Mormon crickets to have lower phenoloxidase titers, slower encapsulation of foreign bodies, and greater mortality from B. bassiana infection than those fed high protein diets. These results support the nutrition-based dichotomy of migrating Mormon crickets, protein-deficient ones are more susceptible to pathogenic fungi whereas carbohydrate-deficient ones are more vulnerable to bacterial challenge., (Published by Elsevier Ltd.)

Lepidopteran midgut aminopeptidases N (APNs) are widely studied for their potential roles as one of the receptors for Bacillus thuringiensis (Bt) crystal toxins. In the present study, a loss of function analyses by RNAi (RNA interference) silencing of the Plutella xylostella APN5 (PxAPN5), a binding protein of Bt crystal toxin Cry2Ab, were performed. The knocking down of PxAPN5 in P. xylostella larvae greatly reduced their susceptibility to Cry2Ab and led to a decrease of Cry2Ab binding to P. xylostella midgut. Four truncated fragments of PxAPN5 were further constructed and expressed in Escherichia coli (E.coli) to find the binding region of PxAPN5 to Cry2Ab. The ligand blot result indicated that D1 domain (residues 1-262) and D3 domain (residues 510-620) of PxAPN5 could specially bind to Cry2Ab., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Asian citrus psyllid, Diaphorina citri Kuwayama, is the most important economic pest of citrus because it transmits Candidatus Liberibacter asiaticus (CLas), the causal agent of huanglongbing (HLB). Silencing genes by RNA interference (RNAi) is a promising approach for controlling D. citri. RNAi-based insect management strategies depend on the selection of suitable target genes., Results: The muscle protein 20 gene DcMP20 was characterized from D. citri in an effort to impair proper muscle development through RNAi. Phylogenetic analysis showed that DcMP20 was more closely related to MP20 from Drosophila compared with its counterpart from other insect species. Developmental expression analysis revealed that transcription of DcMP20 was development dependent and reached a maximum level in the last instar (fourth-fifth) of the nymphal stage. The extent of RNAi in D. citri was dose dependent, with dsRNA-DcMP20 at 75 ng µL -1 being sufficient to knock down endogenous DcMP20 expression, which resulted in significant mortality and reduced body weight that positively correlated with the silencing of DcMP20. No effect was found when dsRNA-GFP or water was used, indicating the specific effect of dsRNA-DcMP20., Conclusion: Our results suggest that dsRNA can be delivered to D. citri through soaking, and DcMP20 is an effective RNAi target to be used in the management of D. citri. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Background: Malaria control in Africa is dependent upon the use insecticides but intensive use of a limited number of chemicals has led to resistance in mosquito populations. Increased production of enzymes that detoxify insecticides is one of the most potent resistance mechanisms. Several metabolic enzymes have been implicated in insecticide resistance but the processes controlling their expression have remained largely elusive., Results: Here, we show that the transcription factor Maf-S regulates expression of multiple detoxification genes, including the key insecticide metabolisers CYP6M2 and GSTD1 in the African malaria vector Anopheles gambiae. Attenuation of this transcription factor through RNAi induced knockdown reduced transcript levels of these effectors and significantly increased mortality after exposure to the pyrethroid insecticides and DDT (permethrin: 9.2% to 19.2% (p = 0.015), deltamethrin: 3.9% to 21.6% (p = 0.036) and DDT: 1% to 11.7% (p = <0.01), whilst dramatically decreasing mortality induced by the organophosphate malathion (79.6% to 8.0% (p = <0.01)). Additional genes regulated by Maf-S were also identified providing new insight into the role of this transcription factor in insects., Conclusion: Maf-S is a key regulator of detoxification genes in Anopheles mosquitoes. Disrupting this transcription factor has opposing effects on the mosquito's response to different insecticide classes providing a mechanistic explanation to the negative cross resistance that has been reported between pyrethroids and organophosphates.

ATP-binding cassette (ABC) transporters constitute a large superfamily of proteins that mediate transport of a diverse number of substrates including nutrients, lipids and xenobiotics across membranes serving a variety of developmental and physiological functions. Here, we report on the molecular properties and biological roles of the ABC transporter LmABCH-9C in the migratory locust Locusta migratoria. LmABCH-9C was expressed continuously during nymphal development in all tissues including the integument. Expression was highest just after molting. Suppression of LmABCH-9C transcript levels by RNA interference (RNAi) in nymphs provoked death during or soon after molting to the next stage. These nymphs lost weight within minutes after molting. Moreover, high humidity rescued the lethality of molted dsLmABCH-9C-injected nymphs. In histological experiments, we find that the amounts of inner-cuticular lipids are reduced in nymphs with suppressed LmABCH-9C expression. These data together indicate that LmABCH-9C is needed for lipid-dependent desiccation resistance, paralleling the function of ABCH-9C in Tribolium castaneum. Hence, the function of this ABC transporter seems to be conserved across insect species ranging from hemimetabolous (L. migratoria) to holometabolous (T. castaneum) species. In addition, we find that cuticle inward impermeability is compromised in nymphs with reduced LmABCH-9C function. In summary, consistent with the model that cuticular lipids are necessary to prevent desiccation and penetration of xenobiotics in insects, we hypothesize that LmABCH-9C is involved in the construction of a lipid-based barrier at the surface of the cuticle especially after molting to protect the animal against uncontrolled water loss and entry. Susceptibility of this ABC transporter to RNAi-mediated knockdown designates it as an excellent target for RNAi-based insect pest control., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines., Results: We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells., Conclusion: The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.

Uric acid accumulates in the epidermis of Bombyx mori larvae and renders the larval integument opaque and white. Yamamoto translucent (oya) is a novel spontaneous mutant with a translucent larval integument and unique phenotypic characteristics, such as male-biased lethality and flaccid larval paralysis. Xanthine dehydrogenase (XDH) that requires a molybdenum cofactor (MoCo) for its activity is a key enzyme for uric acid synthesis. It has been observed that injection of a bovine xanthine oxidase, which corresponds functionally to XDH and contains its own MoCo activity, changes the integuments of oya mutants from translucent to opaque and white. This finding suggests that XDH/MoCo activity might be defective in oya mutants. Our linkage analysis identified an association between the oya locus and chromosome 23. Because XDH is not linked to chromosome 23 in B. mori, MoCo appears to be defective in oya mutants. In eukaryotes, MoCo is synthesized by a conserved biosynthesis pathway governed by four loci (MOCS1, MOCS2, MOCS3, and GEPH). Through a candidate gene approach followed by sequence analysis, a 6-bp deletion was detected in an exon of the B. mori molybdenum cofactor synthesis-step 1 gene (BmMOCS1) in the oya strain. Moreover, recombination was not observed between the oya and BmMOCS1 loci. These results indicate that the BmMOCS1 locus is responsible for the oya locus. Finally, we discuss the potential cause of male-biased lethality and flaccid paralysis observed in the oya mutants., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

RNA interference (RNAi) has been used as a gene silencing strategy by the introduction of long double stranded RNA (dsRNA) for the control of pest insects. The aim of the present study was to examine whether the expression of vg gene which is responsible for wing development, can be repressed by chitosan/dsRNA based nanoparticles in Aedes aegypti. The vestigial gene (vg) was amplified from adult mosquito and cloned in pLitmus28i vector. Genetically engineered recombinant plasmid was transformed into RNase III deficient strain for synthesis of bacterially expressed dsRNA. Nanoparticles were prepared via electrostatic interaction between cationic polymer chitosan and anionic nucleic acids (dsRNA). The formation of chitosan/dsRNAnanoparticles and their size were confirmed by Atomic force microscopy (AFM). Chitosan/dsRNA mediated knockdown of Enhanced Green Fluorescence Protein (EGFP) was demonstrated in Sf21 cells. Further, we tested whether such an approach could be used to target vg gene in Ae. aegypti. The results showed that chitosan/dsRNA caused significant mortality, delayed growth development and caused adult wing-malformation. A qRT-PCR analysis confirmed that the chitosan/dsRNA mediated transcriptional level was downregulated. Our findings suggest that vg gene intervention strategies through RNAi can emerge as viable option for pest control., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3-5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites.

Plant phenolics are a group of important secondary metabolites that are toxic to many animals and insects if ingested at high concentrations. Because most insects consume plant phenolics daily, they have likely evolved the capacity to detoxify these compounds. Here, we used Drosophila melanogaster, Bombyx mori and Helicoverpa armigera as models to study the metabolism of plant phenolics by prophenoloxidases. We found that insect foreguts release prophenoloxidases into the lumen, and that the survival of prophenoloxidase-deletion mutants was impaired when fed several plant phenolics and tea extracts. Using l-DOPA as a model substrate, biochemical assays in large Lepidopteran insects demonstrated that low levels of l-DOPA are rapidly metabolized into intermediates by phenoloxidases. Feeding with excess l-DOPA showed that the metabolic intermediate 5,6-dihydroxyindole reached the hindgut either by passing directly through the midgut, or by transport through the hemolymph. In the hindgut, 5,6-dihydroxyindole was further oxidized by prophenoloxidases. Intermediates exerted no toxicity in the hemocoel or midgut. These results show that plant phenolics are not toxic to insects unless prophenoloxidase genes are lost or the levels of phenolics exceed the catalytic activity of the gut prophenoloxidases.

The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera.

The Omega-class of GSTs (GSTOs) is a class of cytosolic GSTs that have specific structural and functional characteristics that differ from those of other GST groups. In this study, we demonstrated the involvement of the GSTO1 gene from A. cerana cerana in the oxidative stress response and further investigated the effects of three cysteine residues of GSTO1 protein on this response. Real-time quantitative PCR (qPCR) showed that AccGSTO1 was highly expressed in larvae and foragers, primarily in the midgut, epidermis, and flight muscles. The AccGSTO1 mRNA was significantly induced by cold and heat at 1 h and 3 h. The TBA (2-Thiobarbituric acid) method indicated that cold or heat resulted in MDA accumulation, but silencing of AccGSTO1 by RNAi in honeybees increased the concentration of MDA. RNAi also increased the temperature sensitivity of honeybees and markedly reduced their survival. Disc diffusion assay indicated that overexpression of AccGSTO1 in E. coli caused the resistance to long-term oxidative stress. Furthermore, AccGSTO1 was active in an in vitro DNA protection assay. Mutations in Cys-28, Cys-70, and Cys-124 affected the catalytic activity and antioxidant activity of AccGSTO1. The predicted three-dimensional structure of AccGSTO1 was also influenced by the replacement of these cysteine residues. These findings suggest that AccGSTO1 plays a protective role in the response to oxidative stress.

Vitellogenin (Vg) is best known as a yolk protein precursor. Vg also functions to regulate behavioural maturation in adult honey bee workers, but the underlying molecular mechanisms by which it exerts this novel effect are largely unknown. We used abdominal vitellogenin (vg) knockdown with RNA interference (RNAi) and brain transcriptomic profiling to gain insights into how Vg influences honey bee behavioural maturation. We found that vg knockdown caused extensive gene expression changes in the bee brain, with much of this transcriptional response involving changes in central biological functions such as energy metabolism. vg knockdown targeted many of the same genes that show natural, maturation-related differences, but the direction of change for the genes in these two contrasts was not correlated. By contrast, vg knockdown targeted many of the same genes that are regulated by juvenile hormone (JH) and there was a significant correlation for the direction of change for the genes in these two contrasts. These results indicate that the tight coregulatory relationship that exists between JH and Vg in the regulation of honey bee behavioural maturation is manifest at the genomic level and suggest that these two physiological factors act through common pathways to regulate brain gene expression and behaviour., (© 2013 Royal Entomological Society.)

Dietary copper is essential for multicellular organisms. Copper is redox active and required as a cofactor for enzymes such as the antioxidant Superoxide Dismutase 1 (SOD1). Copper dyshomeostasis has been implicated in Alzheimer's disease. Mutations in the presenilin genes encoding PS1 and PS2 are major causes of early-onset familial Alzheimer's disease. PS1 and PS2 are required for efficient copper uptake in mammalian systems. Here we demonstrate a conserved role for presenilin in dietary copper uptake in the fly Drosophila melanogaster. Ubiquitous RNA interference-mediated knockdown of the single Drosophila presenilin (PSN) gene is lethal. However, PSN knockdown in the midgut produces viable flies. These flies have reduced copper levels and are more tolerant to excess dietary copper. Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake. SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat. These data support presenilin being needed for dietary copper uptake in the gut and so impacting on SOD activity and tolerance to oxidative stress. These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.

Background: RNA interference (RNAi) is a powerful tool to study gene function in organisms that are not amenable to classical forward genetics. Hence, together with the ease of comprehensively identifying genes by new generation sequencing, RNAi is expanding the scope of animal species and questions that can be addressed in terms of gene function. In the case of genetic mutants, the genetic background of the strains used is known to influence the phenotype while this has not been described for RNAi experiments., Results: Here we show in the red flour beetle Tribolium castaneum that RNAi against Tc-importin α1 leads to different phenotypes depending on the injected strain. We rule out off target effects and show that sequence divergence does not account for this difference. By quantitatively comparing phenotypes elicited by RNAi knockdown of four different genes we show that there is no general difference in RNAi sensitivity between these strains. Finally, we show that in case of Tc-importin α1 the difference depends on the maternal genotype., Conclusions: These results show that in RNAi experiments strain specific differences have to be considered and that a proper documentation of the injected strain is required. This is especially important for the increasing number of emerging model organisms that are being functionally investigated using RNAi. In addition, our work shows that RNAi is suitable to systematically identify the differences in the gene regulatory networks present in populations of the same species, which will allow novel insights into the evolution of animal diversity.

Background: The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance., Results: We have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H). This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi) screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA) into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport., Conclusions: The results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.

Expression of sterol carrier protein-2 (SCP-2) in Aedes aegypti shows a distinct temporal/spatial pattern throughout the life cycle. In order to identify the transcription factors responsible for the larval temporal/spatial regulation of AeSCP-2 transcription, AeSCP-2 promoter activities were studied in vivo via transient transfection of promoter/reporter gene assays. Regulatory sequences upstream -1.3 kb of the transcription start site of AeSCP-2 were found to be critical for the in vivo temporal/spatial promoter activity. Interestingly, the -1.6 kb promoter sequence efficiently drove the larval midgut-specific siRNA expression, indicating that the -1.6 kb upstream sequence is sufficient for temporal/spatial AeSCP-2 transcriptional activity. Four transcription factors were identified in the midgut nuclear extract from feeding larvae via labeled -1.6/-1.3 kb DNA probe pull-down and proteomic analysis. Co-transfection of the promoter/reporter gene with inducible siRNA expression of each transcription factor was performed to confirm the regulatory function of individual transcription factor on AeSCP-2 transcriptional activities in the larval midgut. The results indicate that two of the identified transcription factors, Thanatos-associated protein (THAP) and activating transcription factor-2 (ATF-2), antagonistically control AeSCP-2 transcriptional activity in the midgut of feeding larvae via the regulatory sequences between -1.6 to -1.3 kb 5' upstream of the transcription start site. In vivo expression knockdown of THAP and ATF-2 resulted in significant changes in developmental progression, which may be partially due to their effects on AeSCP-2 expression.

Background: Plasmodium infection increases the oxidative stress inside the mosquito, leading to a significant alteration on transcription of Anopheles gambiae detoxification genes. Among these detoxification genes several P450 cytochromes and tubulins were differently expressed, suggesting their involvement in the mosquito's response to parasite invasion. P450 cytochromes are usually involved in the metabolism and detoxification of several compounds, but are also regulated by several pathogens, including malaria parasite. Tubulins are extremely important as components of the cytoskeleton, which rearrangement functions as a response to malaria parasite invasion., Methodology/principal Findings: Gene silencing methods were used to uncover the effects of cytochrome P450 reductase, tubulinA and tubulinB silencing on the A. gambiae response to Plasmodium berghei invasion. The role of tubulins in counter infection processes was also investigated by inhibiting their effect. Colchicine, vinblastine and paclitaxel, three different tubulin inhibitors were injected into A. gambiae mosquitoes. Twenty-four hours post injection these mosquitoes were infected with P. berghei through a blood meal from infected CD1 mice. Cytochrome P450 gene expression was measured using RT-qPCR to detect differences in cytochrome expression between silenced, inhibited and control mosquitoes. Results showed that cytochrome P450 reductase silencing, as well as tubulin (A and B) silencing and inhibition affected the efficiency of Plasmodium infection. Silencing and inhibition also affected the expression levels of cytochromes P450., Conclusions: Our results suggest the existence of a relationship between tubulins and P450 cytochromes during A. gambiae immune response to P. berghei invasion. One of the P450 cytochromes in this study, CYP6Z2, stands out as the potential link in this association. Further work is needed to fully understand the role of tubulin genes in the response to Plasmodium infection.

The potato/tomato psyllid, Bactericerca cockerelli (B. cockerelli), and the Asian citrus psyllid, Diaphorina citri (D. citri), are very important plant pests, but they are also vectors of phloem-limited bacteria that are associated with two devastating plant diseases. B. cockerelli is the vector of Candidatus Liberibacter psyllaurous (solanacearum), which is associated with zebra chip disease of potatoes, and D. citri is the vector of Ca. Liberibacter asiaticus, which is associated with the Huanglongbing (citrus greening) disease that currently threatens the entire Florida citrus industry. Here we used EST sequence information from D. citri to identify potential targets for RNA interference in B. cockerelli. We targeted ubiquitously expressed and gut-abundant mRNAs via injection and oral acquisition of double-stranded RNAs and siRNAs and were able to induce mortality in recipient psyllids. We also showed knockdown of target mRNAs, and that oral acquisition resulted primarily in mRNA knockdown in the psyllid gut. Concurrent with gene knockdown was the accumulation of target specific ∼ 21 nucleotide siRNAs for an abundant mRNA for BC-Actin. These results showed that RNAi can be a powerful tool for gene function studies in psyllids, and give support for continued efforts for investigating RNAi approaches as possible tools for psyllid and plant disease control.