Interference Efficiency and Effects of Bacterium-mediated RNAi in the Fall Armyworm (Lepidoptera: Noctuidae).

RNAi is an effective tool for gene function analysis and a promising strategy to provide environmentally friendly control approaches for pathogens and pests. Recent studies support the utility of bacterium-mediated RNAi as a cost-effective method for gene function study and a suitable externally applied delivery mechanism for pest control. Here, we developed a bacterium-mediated RNAi system in Spodoptera frugiperda based on four target genes, specifically, Chitinase (Sf-CHI), Chitin synthase B (Sf-CHSB), Sugar transporter SWEET1 (Sf-ST), and Hemolin (Sf-HEM). RNAi conducted by feeding larvae with bacteria expressing dsRNAs of target genes or injecting pupae and adults with bacterially synthesized dsRNA induced silencing of target genes and resulted in significant negative effects on growth and survival of S. frugiperda. However, RNAi efficiency and effects were variable among different target genes and dsRNA delivery methods. Injection of pupae with dsCHI and dsCHSB induced a significant increase in wing malformation in adults, suggesting that precise regulation of chitin digestion and synthesis is crucial during wing formation. Injection of female moths with dsHEM resulted in lower mating, fecundity, and egg hatching, signifying a critical role of Sf-HEM in the process of egg production and/or embryo development. Our collective results demonstrate that bacterium-mediated RNAi presents an alternative technique for gene function study in S. frugiperda and a potentially effective strategy for control of this pest, and that Sf-CHI, Sf-CHSB, Sf-ST, and Sf-HEM encoding genes can be potent targets.

Social communication activates the circadian gene Tctimeless in Tribolium castaneum.

Chemical communication via pheromones is an integral component in insect behavior, particularly for mate searching and reproduction. Aggregation pheromones, that attract conspecifics of both sexes, are particularly common and have been identified for hundreds of species. These pheromones are among the most ecologically selective pest suppression agents. In this study, we identified an activating effect of the aggregation pheromone of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenibroidae) on a highly conserved circadian clock gene (Tctimeless). Tribolium castaneum is one of the most damaging cosmopolitan pest of flour and other stored food products. Its male produced aggregation pheromone, 4,8-dimethyldecanal (DMD), attracts both conspecific males and females and is used for pest management via monitoring and mating disruption. The Tctimeless gene is an essential component for daily expression patterns of the circadian clock and plays vital roles in eclosion, egg production, and embryonic development. In this study, we demonstrate that constant exposure to the species-specific aggregation pheromone led to Tctimeless up-regulation and a different pattern of rhythmic locomotive behavior. We propose that changing the well-adapted "alarm clock", using DMD is liable to reduce fitness and can be highly useful for pest management.

Juvenile hormone regulates the reproductive diapause through Methoprene-tolerant gene in Galeruca daurica.

Juvenile hormone (JH) signalling plays an important role in regulation of reproductive diapause in insects. However, its underlying molecular mechanism has been unclear. Methoprene-tolerant (Met), as a universal JH receptor, is involved in JH action. To gain some insight into its function in the reproductive diapause of Galeruca daurica, a serious pest on the Inner Mongolia grasslands undergoing obligatory summer diapause at the adult stage, we cloned the complete open-reading frame (ORF) sequences of Met and other 7 JH signalling-related genes, including JH acid methyltransferase (JHAMT), JH esterase (JHE), JH epoxide hydrolase (JHEH), Krüppel homologue 1 (Kr-h1), vitellogenin (Vg), forkhead box O (FOXO) and fatty acid synthase 2 (FAS2), from this species. GdMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family. Expression patterns of these eight genes were developmentally regulated during adult development. Topical application of JH analogue (JHA) methoprene into the 3-day-old and 5-day-old adults induced the expression of GdMet. Silencing GdMet by RNAi inhibited the expression of JHBP, JHE, Kr-h1 and Vg, whereas promoted the FAS2 expression, which enhanced lipid accumulation and fat body development, and finally induced the adults into diapause ahead. Combining with our previous results, we conclude that JH may regulate reproductive diapause through a conserved Met-dependent pathway in G. daurica.

Peroxiredoxin alleviates the fitness costs of imidacloprid resistance in an insect pest of rice.

Chemical insecticides have been heavily employed as the most effective measure for control of agricultural and medical pests, but evolution of resistance by pests threatens the sustainability of this approach. Resistance-conferring mutations sometimes impose fitness costs, which may drive subsequent evolution of compensatory modifier mutations alleviating the costs of resistance. However, how modifier mutations evolve and function to overcome the fitness cost of resistance still remains unknown. Here we show that overexpression of P450s not only confers imidacloprid resistance in the brown planthopper, Nilaparvata lugens, the most voracious pest of rice, but also leads to elevated production of reactive oxygen species (ROS) through metabolism of imidacloprid and host plant compounds. The inevitable production of ROS incurs a fitness cost to the pest, which drives the increase or fixation of the compensatory modifier allele T65549 within the promoter region of N. lugens peroxiredoxin (NlPrx) in the pest populations. T65549 allele in turn upregulates the expression of NlPrx and thus increases resistant individuals' ability to clear the cost-incurring ROS of any source. The frequent involvement of P450s in insecticide resistance and their capacity to produce ROS while metabolizing their substrates suggest that peroxiredoxin or other ROS-scavenging genes may be among the common modifier genes for alleviating the fitness cost of insecticide resistance.

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.

Effect of diflubenzuron on the chitin biosynthesis pathway in Conopomorpha sinensis eggs.

Conopomorpha sinensis is the dominant borer pest of Litchi chinensis (litchi) and Euphoria longan (longan) in China. Control of C. sinensis is difficult because of its cryptic life habit; thus, an effective ovicide could be beneficial. The larvicidal effects of diflubenzuron (DFB) have been documented in many insect pest species. Therefore, DFB might be a useful ovicide to control C. sinensis. However, the detailed mode of action of DFB interference with insect molting and egg hatching is unclear. Thus, we studied alterations in expression of all genes potentially affected by DFB treatment using a transcriptome approach in 2-d-old C. sinensis eggs. Clean reads were assembled to generate 203 455 unigenes and 440 558 transcripts. A total of 4625 differently expressed genes, which included 2670 up-regulated and 1955 down-regulated unigenes, were identified. Chitin binding and chitin metabolic processes were among the most significant enriched pathways according to Gene Ontology analyses. Most of the genes that encode enzymes involved in the chitin biosynthesis pathway were unaffected, whereas genes that presumably encode cuticle proteins were up-regulated. Furthermore, altered expression patterns of 10 genes involved in the chitin biosynthesis pathway of C. sinensis embryos were observed in response to DFB treatment at different time points by quantitative reverse transcription polymerase chain reaction. We also observed abnormal development; there was reduced chitin content and modulated chitin distribution of newly hatched larvae, and altered egg hatching. Our findings illustrate an ovicidal effect of DFB on C. sinensis, and reveal more molecular consequences of DFB treatment on insects.

Transcriptional responses of soybean aphids to sublethal insecticide exposure.

Insecticides are a key tool in the management of many insect pests of agriculture, including soybean aphids. The selection imposed by insecticide use has often lead to the evolution of resistance by the target pest through enhanced detoxification mechanisms. We hypothesised that exposure of insecticide-susceptible aphids to sublethal doses of insecticides would result in the up-regulation of genes involved in detoxification of insecticides, revealing the genes upon which selection might act in the field. We used the soybean aphid biotype 1 reference genome, version 6.0 as a reference to analyze RNA-Seq data. We identified multiple genes with potential detoxification roles that were up-regulated 12 h after sublethal exposure to esfenvalerate or thiamethoxam. However, these genes were part of a dramatic burst of differential gene expression in which thousands of genes were up- or down-regulated, rather than a defined response to insecticides. Interestingly, the transcriptional burst observed at 12 h s declined dramatically by 24-hrs post-exposure, suggesting a general stress response that may become fine-tuned over time.

Colorado potato beetle chymotrypsin genes are differentially regulated in larval midgut in response to the plant defense inducer hexanoic acid or the Bacillus thuringiensis Cry3Aa toxin.

When Colorado potato beetle larvae ingested potato plants treated with the plant defense inducer compound hexanoic acid, midgut chymotrypsin enzyme activity increased, and the corresponding chymotrypsin genes were differentially expressed, evidence of the larval digestive proteolytic system's plasticity. We previously reported increased susceptibility to Cry3Aa toxin in larvae fed hexanoic acid treated plants. Here we show that the most expressed chymotrypsin gene in larvae fed hexanoic acid treated plants, CTR6, was dramatically downregulated in Cry3Aa intoxicated larvae. lde-miR-965-5p and lde-miR-9a-5p microRNAs, predicted to target CTR6, might be involved in regulating the response to hexanoic acid but not to Cry3Aa toxin.

Midgut de novo transcriptome analysis and gene expression profiling of Achaea janata larvae exposed with Bacillus thuringiensis (Bt)-based biopesticide formulation.

India is the major producer and exporter of castor oil in the world. Castor semilooper, Achaea janata is one of the main castor crop pests, which causes serious economic loss of crop, hence management and control of the pest are important. Currently, Bacillus thuringiensis (Bt) based biopesticides are being used for their control. However, the insects are known to develop resistance not only against chemical pesticides but also to Bt based biopesticides. In the present study, de novo transcriptome analysis was conducted to monitor the expression pattern of larval midgut genes in Achaea janata exposed to sublethal dose of Bt formulation. A total of 34,612 and 41,109 transcripts were identified in control and toxin-exposed larval midgut samples out of which 18,836 in control and 21,046 in toxin-exposed samples are annotated. Microarray data analysis employed to monitor the gene expression upon Cry toxin exposure revealed that 375 genes were upregulated and 579 genes were downregulated during all the time points (12-60 h) of toxin exposure. The differentially expressed transcripts include i.e. Cry toxin receptors, gut proteases, arylphorin, REPATs, detoxification enzymes and aquaporins. Validation of microarray data was performed by real-time quantitative PCR using few randomly selected genes and the results obtained were in corroboration. This is the first study on transcriptome data from the castor semilooper and the results would provide valuable resources for the characterization of Bt toxin response in the pest.

Molecular characterization of the insecticidal activity of double-stranded RNA targeting the smooth septate junction of western corn rootworm (Diabrotica virgifera virgifera).

The western corn rootworm (WCR, Diabrotica virgifera virgifera) gene, dvssj1, is a putative homolog of the Drosophila melanogaster gene, snakeskin (ssk). This gene encodes a membrane protein associated with the smooth septate junction (SSJ) which is required for the proper barrier function of the epithelial lining of insect intestines. Disruption of DVSSJ integrity by RNAi technique has been shown previously to be an effective approach for corn rootworm control, by apparent suppression of production of DVSSJ1 protein leading to growth inhibition and mortality. To understand the mechanism that leads to the death of WCR larvae by dvssj1 double-stranded RNA, we examined the molecular characteristics associated with SSJ functions during larval development. Dvssj1 dsRNA diet feeding results in dose-dependent suppression of mRNA and protein; this impairs SSJ formation and barrier function of the midgut and results in larval mortality. These findings suggest that the malfunctioning of the SSJ complex in midgut triggered by dvssj1 silencing is the principal cause of WCR death. This study also illustrates that dvssj1 is a midgut-specific gene in WCR and its functions are consistent with biological functions described for ssk.

Dyeing but not dying: Colourful dyes as a non-lethal method of food labelling for in vitro-reared honey bee (Apis mellifera) larvae.

Several environmental factors (e.g. food source, pesticides, toxins, parasites and pathogens) influence development and maturation of honey bees (Apis mellifera). Therefore, controlled experimental conditions are mandatory when studying the impact of environmental factors: particularly food quality and nutrient consumption. In vitro larval rearing is a standard approach for monitoring food intake of larvae and the labelling of food is necessary to quantify intake in controlled feeding experiments. Here, we tested the suitability of two food dyes, Allura Red and Brilliant Blue, in an experimental set up using in vitro reared honey bee larvae and freshly hatched adult workers. Absorbance of both dyes was measured, in food and dye-fed larvae, to determine the optimal dye concentrations for accurate detection and quantification. By quantifying relative dye concentrations in dye mixtures, relative concentrations of mixed dyes can be estimated independent of the total food consumed by the larvae. Survival assays were conducted to test the impact of both dyes on larval and worker bee survival. Worker bees showed no increase in adult mortality, when fed with dyed honey. Larval survival was not significantly different until the late pupal stage. The physiological impact of dye feeding was tested by measuring larval immune response. No changes in innate immune gene expression were detectable for larvae fed with dyed and non-dyed food. In conclusion, we established a non-invasive food labelling protocol for food intake quantification in in vitro reared honey bee larvae, using non-toxic, inexpensive, and easy to apply food dyes.

Response of detoxification and immune genes and of transcriptome expression in Mythimna separata following chlorantraniliprole exposure.

The oriental armyworm Mythimna separata is a serious polyphagous pest in China and there are major efforts to control this pest. In the present study, an RNA-Seq method was used to explore transcriptome data of M. separata and identify the responses of genes to chlorantraniliprole. Sequencing and de novo assembly yielded 134,533 transcripts that were further assembled into 77,628 unigenes with an N50 length of 2165 bp. A total of 76 unigenes encoding insecticide targets were identified. Furthermore, 62 cytochrome P450s, 34 glutathione S-transferase (GSTs)and 64 carboxylesterase (CCEs) were curated to construct phylogenetic trees. In addition, we identified 647 the differentially expressed genes following treatment with chlorantraniliprole. The pathways of calcium signaling was identified as response to the pesticide The transcriptome data we generated represents a comprehensive genomic resource for further studies focused on control of M. separata. The response of genes to chlorantraniliprole treatment will elucidate the molecular mechanisms of insecticide resistance and allow for the development of new chemical pesticides to control this pest.

Development of a rapid field-applicable molecular diagnostic for knockdown resistance (kdr) markers in An. gambiae.

BACKGROUND: The spread of insecticide resistance (IR) is a major threat to vector control programmes for mosquito-borne diseases. Early detection of IR using diagnostic markers could help inform these programmes, especially in remote locations where gathering reliable bioassay data is challenging. Most current molecular tests for genetic IR markers are only suitable for use in well-equipped laboratory settings. There is an unmet need for field-applicable diagnostics. METHODS: A single-cartridge test was designed to detect key IR mutations in the major African vector of malaria, Anopheles gambiae. Developed on the portable, rapid, point-of-care compatible PCR platform - Genedrive® (genedrive® plc), the test comprises two assays which target single nucleotide polymorphisms (SNPs) in the voltage gated sodium channel (VGSC) gene that exert interactive effects on knockdown resistance (kdr): L1014F, L1014S and N1575Y. RESULTS: Distinct melt peaks were observed for each allele at each locus. Preliminary validation of these assays using a test panel of 70 An. gambiae samples showed complete agreement of our assays with the widely-used TaqMan assays, achieving a sensitivity and specificity of 100%. CONCLUSION: Here we show the development of an insecticide resistance detection assay for use on the Genedrive® platform that has the potential to be the first field-applicable diagnostic for kdr.

A pharmacological screen for compounds that rescue the developmental lethality of a Drosophila ATM mutant.

Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, we performed a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. We carried out further studies of Ronnel because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T.

Expression Differences of Resistance-Related Genes Induced by Cycloxaprid Using qRT-PCR in the Female Adult of Sogatella furcifera (Hemiptera: Delphacidae).

As a newer cis-nitromethylene neonicotinoid pesticide at present, cycloxaprid has good industrialization prospects, including the management of imidacloprid-resistant populations, because this chemical have an excellent efficiency against rice planthoppers. Sogatella furcifera (Horváth) is the most economically important pest of rice worldwide and has developed resistance to many insecticides. This study focused on the expression change of these resistance genes, induced by cycloxaprid, involved in metabolic detoxification and receptor protein. Twenty-two differentially expressed genes (DEGs) that may be related with the insecticide resistance were found in the transcriptome of S. furcifera, including 2 cytochrome P450 genes, 2 glutathione S-transferase (GST) genes, 1 acid phosphatase (ACP) gene, 12 decarboxylase genes, 2 glycolipid genes, 1 cadherin gene, and 2 glycosyltransferase genes, which were up- or downregulated in response to an exposure of cycloxaprid. Furthermore, two P450 genes (CYP4 and CYP6 family, respectively), two decarboxylase genes, and one glycosyltransferase gene were validated by qRT-PCR. Expression differences of these genes verified successfully by qRT-PCR in response to different concentrations and times treated with cycloxaprid could explain the insecticide resistance mechanism under cycloxaprid stress in S. furcifera.

Multiple Modes of Action of the Squamocin in the Midgut Cells of Aedes aegypti Larvae.

Annonaceous acetogenins are botanical compounds with good potential for use as insecticides. In the vector, Aedes aegypti (L.) (Diptera: Culicidae), squamocin (acetogenin) has been reported to be a larvicide and cytotoxic, but the modes of action of this molecule are still poorly understood. This study evaluated the changes in the cell morphology, and in the expression of genes, for autophagy (Atg1 and Atg8), for membrane ion transporter V-ATPase, and for water channel aquaporin-4 (Aqp4) in the midgut of A. aegypti larvae exposed to squamocin from Annona mucosa Jacq. (Annonaceae). Squamocin showed cytotoxic action with changes in the midgut epithelium and digestive cells of A. aegypti larvae, increase in the expression for autophagy gene Atg1 and Atg8, decrease in the expression of V-ATPase, decrease in the expression of Aqp4 gene in LC20 and inhibition of Apq4 genes in the midgut of this vector in LC50. These multiple modes of action for squamocin are described for the first time in insects, and they are important because different sites of action of squamocin from A. mucosa may reduce the possibility of resistance of A. aegypti to this molecule.

Effect of exogenous hormones on transcription levels of pyridoxal 5'-phosphate biosynthetic enzymes in the silkworm (Bombyx mori).

Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies.

A transgenic embryonic sexing system for the Australian sheep blow fly Lucilia cuprina.

Genetic approaches, including the sterile insect technique (SIT), have previously been considered for control of the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. In an SIT program, females consume 50% of the diet but are ineffective as control agents and compete with females in the field for mating with sterile males, thereby decreasing the efficiency of the program. Consequently, transgenic sexing strains of L. cuprina were developed that produce 100% males when raised on diet that lacks tetracycline. However, as females die mostly at the pupal stage, rearing costs would not be significantly reduced. Here we report the development of transgenic embryonic sexing strains of L. cuprina. In these strains, the Lsbnk cellularization gene promoter drives high levels of expression of the tetracycline transactivator (tTA) in the early embryo. In the absence of tetracycline, tTA activates expression of the Lshid proapoptotic gene, leading to death of the embryo. Sex-specific RNA splicing of Lshid transcripts ensures that only female embryos die. Embryonic sexing strains were also made by combining the Lsbnk-tTA and tetO-Lshid components into a single gene construct, which will facilitate transfer of the technology to other major calliphorid livestock pests.

Neonicotinoid-Coated Zea mays Seeds Indirectly Affect Honeybee Performance and Pathogen Susceptibility in Field Trials.

Thirty-two honeybee (Apis mellifera) colonies were studied in order to detect and measure potential in vivo effects of neonicotinoid pesticides used in cornfields (Zea mays spp) on honeybee health. Honeybee colonies were randomly split on four different agricultural cornfield areas located near Quebec City, Canada. Two locations contained cornfields treated with a seed-coated systemic neonicotinoid insecticide while the two others were organic cornfields used as control treatments. Hives were extensively monitored for their performance and health traits over a period of two years. Honeybee viruses (brood queen cell virus BQCV, deformed wing virus DWV, and Israeli acute paralysis virus IAPV) and the brain specific expression of a biomarker of host physiological stress, the Acetylcholinesterase gene AChE, were investigated using RT-qPCR. Liquid chromatography-mass spectrometry (LC-MS) was performed to detect pesticide residues in adult bees, honey, pollen, and corn flowers collected from the studied hives in each location. In addition, general hive conditions were assessed by monitoring colony weight and brood development. Neonicotinoids were only identified in corn flowers at low concentrations. However, honeybee colonies located in neonicotinoid treated cornfields expressed significantly higher pathogen infection than those located in untreated cornfields. AChE levels showed elevated levels among honeybees that collected corn pollen from treated fields. Positive correlations were recorded between pathogens and the treated locations. Our data suggests that neonicotinoids indirectly weaken honeybee health by inducing physiological stress and increasing pathogen loads.

Differentially expressed genes in the fat body of Bombyx mori in response to phoxim insecticide.

The silkworm, Bombyx mori, is an economically important insect. However, poisoning of silkworms by organophosphate pesticides causes tremendous loss to the sericulture. The fat body is the major tissue involved in detoxification and produces antimicrobial peptides and regulates hormones. In this study, a microarray system comprising 22,987 oligonucluotide 70-mer probes was employed to examine differentially expressed genes in the fat body of B. mori exposed to phoxim insecticide. The results showed that a total of 774 genes were differentially expressed upon phoxim exposure, including 500 up-regulated genes and 274 down-regulated genes. The expression levels of eight detoxification-related genes were up-regulated upon phoxim exposure, including six cytochrome P450s and two glutathione-S-transferases. It was firstly found that eight antimicrobial peptide genes were down-regulated, which might provide important references for studying the larvae of B. mori become more susceptible to microbial infections after phoxim treatment. In addition, we firstly detected the expression level of metamorphosis-related genes after phoxim exposure, which may lead to impacted reproduction. Our results may facilitate the overall understanding of the molecular mechanism of multiple pathways following exposure to phoxim insecticide in the fat body of B. mori.