The development of silk glands and transcriptome aberration induced by cyantraniliprole in Bombyx mori.

Bombyx mori is an insect species of great economic importance, and its silk gland is a vital organ for the synthesis and secretion of silk protein. However, long-term artificial domestication of B. mori has resulted in high sensitivity to chemical toxins, especially insecticides. Cyantraniliprole (Cya), a second-generation ryanodine receptor modulator insecticide, is widely utilized in agriculture for pest control. In this study, the impact of Cya toxicity on the development of silk glands in the 5th instar larvae of B. mori was assessed using Cya LC5, LC10 and LC20, as well as a starvation treatment group for comparison. Short-term exposure (24 h) to different concentrations of Cya resulted in delayed development of silk glands in B. mori. Meanwhile, the body weight, silk gland weight, silk gland index and cocoon quality were significantly reduced in a concentration-dependent manner, except for the Cya LC5 treatment. Histopathological and ultrastructural analysis revealed that Cya LC10 induced disruption of the nuclear membrane and endoplasmic reticulum in the posterior silk gland (PSG) cells, leading to the formation of intracellular vacuoles. Transcriptome sequencing of PSGs identified 2152 genes that were differentially expressed after exposure to Cya LC10, with 1153 down-regulated genes and 999 up-regulated genes. All differentially expressed genes were subjected to functional annotation using gene ontology and Kyoto encyclopedia of genes and genomes database, and it was found that protein synthesis-related pathways were significantly enriched, with the majority of genes being down-regulated. Furthermore, the transcription levels of genes involved in "protein processing in endoplasmic reticulum", "protein export", "proteasome" and "DNA replication" were quantified using qRT-PCR. Our findings suggested that short-term exposure to Cya LC10 resulted in disruption of DNA replication, as well as protein transport, processing and hydrolysis in the PSG cells of B. mori. The results of this study provide a theoretical foundation for the safe utilization of Cya in sericulture production.

Indoxacarb triggers autophagy and apoptosis through ROS accumulation mediated by oxidative phosphorylation in the midgut of Bombyx mori.

Indoxacarb has been widely utilized in agricultural pest management, posing a significant ecological threat to Bombyx mori, a non-target economic insect. In the present study, short-term exposure to low concentration of indoxacarb significantly suppressed the oxidative phosphorylation pathway, and resulted in an accumulation of reactive oxygen species (ROS) in the midgut of B. mori. While, the ATP content exhibited a declining trend but there was no significant change. Moreover, indoxacarb also significantly altered the transcription levels of six autophagy-related genes, and the transcription levels of ATG2, ATG8 and ATG9 were significantly up-regulated by 2.56-, 1.90-, and 3.36-fold, respectively. The protein levels of ATG8-I and ATG8-II and MDC-stained frozen sections further suggested an increase in autophagy. Furthermore, the protein level and enzyme activity of CASP4 showed a significant increase in accordance with the transcription levels of apoptosis-related genes, indicating the activation of the apoptotic signaling pathway. Meanwhile, the induction of apoptosis signals in the midgut cells triggered by indoxacarb was confirmed through TUNEL staining. These findings suggest that indoxacarb can promote the accumulation of ROS by inhibiting the oxidative phosphorylation pathway, thereby inducing autophagy and apoptosis in the midgut cells of B. mori.

Sublethal chlorantraniliprole exposure induces autophagy and apoptosis through disrupting calcium homeostasis in the silkworm Bombyx mori.

The intensive application of chlorantraniliprole (CAP) leaves residues in the environment, posing a potential threat to non-target organisms. In the present study, we investigated the adverse effects of sublethal CAP exposure on Bombyx mori. Sublethal CAP (0.02 mg/L) was shown to induce the release of intracellular Ca2+ in BmN cells. Meanwhile, Ca2+ -dependent genes were induced in the midgut at 72 h after CAP (0.01 mg/L) exposure, and damaged mitochondria, autophagosomes, nuclear membrane rupture and condensed chromatin were observed. Moreover, the key genes in the oxidative phosphorylation pathway were significantly down-regulated. The transcript levels of autophagy-related genes ATG6 and ATG8 were significantly up-regulated, and the protein levels of LC3-II and ATG7 were significantly increased by 3.72- and 3.33-fold, respectively. Additionally, the transcript levels of the upstream genes in the apoptosis pathway (calpain and Apaf-1) were significantly up-regulated, the protein levels of the downstream gene caspase 3 and its cleaved form were significantly up-regulated by 1.97- and 4.55-fold, respectively, consistent with the elevated caspase 3 activity at 72 h. Collectively, these findings demonstrate that intracellular Ca2+ release induced by sublethal CAP inhibits oxidative phosphorylation pathway, which causes mitochondrial dysfunction, leading to autophagy and apoptosis in the midgut of B. mori.

Low concentration of indoxacarb interferes with the growth and development of silkworm by damaging the structure of midgut cells.

As an important economic insect, Bombyx mori plays an essential role in the development of the agricultural economy. Indoxacarb, a novel sodium channel blocker insecticide, has been widely used for the control of various pests in agriculture and forestry, and its environmental pollution caused by flight control operations has seriously affected the safe production of sericulture in recent years. However, the lethal toxicity and adverse effects of indoxacarb on silkworm remain largely unknown. In this study, the toxicity of indoxacarb on the 5th instar larvae of silkworm was determined, with an LC50 (72 h) of 2.07 mg/L. Short-term exposure (24 h) to a low concentration of indoxacarb (1/2 LC50) showed significantly reduced body weight and survival rate of silkworm larvae. In addition, indoxacarb also led to decreased cocoon weight and cocoon shell weight, but had no significant effects on pupation, adult eclosion, and oviposition. Histopathological and ultrastructural analysis indicated that indoxacarb could severely damage the structure of the midgut epithelial cells, and lead to physiological impairment of the midgut. A total of 3883 differentially expressed genes (DEGs) were identified by midgut transcriptome sequencing and functionally annotated using GO and KEGG. Furthermore, the transcription level and enzyme activity of the detoxification related genes were determined, and our results suggested that esterases (ESTs) might play a major role in metabolism of indoxacarb in the midgut of B. mori. Future studies to examine the detoxification or biotransformation function of candidate genes will greatly enhance our understanding of indoxacarb metabolism in B. mori. The results of this study provide a theoretical basis for elucidating the mechanism of toxic effects of indoxacarb on silkworm by interfering with the normal physiological functions of the midgut.

Disruption of peritrophic matrix chitin metabolism and gut immune by chlorantraniliprole results in pathogenic bacterial infection in Bombyx mori.

Chlorantraniliprole (CAP) is widely used in pest control, and its environmental residues affect the disease resistance of non-target insect silkworms. Studies have demonstrated that changes in gut microbial communities of insects are associated with susceptibility to pathogens. In the present study, we examined the effects of CAP exposure on the immune system and gut microbial community structure of silkworms. The results showed that after 96 h of exposure to low-concentration CAP, the peritrophic matrix (PM) of silkworm larvae was disrupted, and pathogenic bacteria invaded hemolymph. The trehalase activity in the midgut was significantly decreased, while the activities of chitinase, ß-N-acetylglucosaminidase, and chitin deacetylase were increased considerably, resulting in decreased chitin content in PM. In addition, exposure to CAP reduced the expressions of key genes in the Toll, IMD, and JAK/STAT pathways, ultimately leading to the downregulation of antimicrobial peptides (AMPs) genes and alterations in the structure of the gut microbial community. Therefore, after infection with the conditional pathogen Enterobacter cloacae (E. cloacae), CAP-exposed individuals exhibited significantly lower body weight and higher mortality. These findings showed that exposure to low-concentration CAP impacted the biological defense system of silkworms, changed the gut microbial community structure, and increased silkworms' susceptibility to bacterial diseases. Collectively, these findings provided a new perspective for the safety evaluation of low-concentration CAP exposure in sericulture.

Characterization of the sublethal toxicity and transcriptome-wide biological changes induced by λ-cyhalothrin in Bombyx mori.

λ-Cyhalothrin (λ-cyh) is widely used in agricultural production and has been reported to cause damages to numerous nontarget insects. As an important economic and model insect of Lepidoptera, Bombyx mori was extremely sensitive to λ-cyh, and pesticide drift often leads to silkworm poisoning. However, little is known about the persistence of sublethal effects or the potential recovery from short-term exposure to sublethal doses of pesticides. In this study, we estimated the sublethal effects caused by short-term exposure (24 h) of λ-cyh LC1 , LC10 , LC25 , and LC50 , respectively, and investigated the persistent negative effects on the growth, survival, and pupal metamorphosis of silkworm larvae. Silkworm growth was mostly retarded after λ-cyh exposure, with dose-dependent recovery observed at delayed time points. Relative to the control, the treatment groups showed significantly higher larval mortalities and abnormal pupa rates. Additionally, transcriptome sequencing was conducted to investigate the effects of λ-cyh LC10 on the normal physiological functions in the midgut of B. mori. A total of 2697 differentially expressed genes were identified, and 57.1% of DEGs were down-regulated. Gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis further revealed that energy and nutrient metabolisms were negatively affected. Moreover, we demonstrated that sublethal λ-cyh inhibited the oxidative phosphorylation pathway by reducing the expression of mitochondrial electron transport chain complex genes and consequently the synthesis of ATP. This study has provided useful transcriptome-wide expression resources to facilitate the overall knowledge of the molecular basis of sublethal toxicity caused by λ-cyh in the midgut of B. mori.

Effects of glyphosate on the growth, development, and physiological functions of silkworm, Bombyx mori.

Glyphosate is an herbicide widely used worldwide, but whether it is safe to nontarget organisms is controversial. In this study, the lepidopteran model insect silkworm was used to investigate the effects of glyphosate residues. The LC50 (72 h) of glyphosate on silkworm was determined to be 14875.98 mg/L, and after exposure to glyphosate at 2975.20 mg/L (a concentration comparable to that used for weed control in mulberry fields), silkworm growth was inhibited by 9.00%, total cocoon weight was lowered by 10.53%, feed digestibility was decreased by 7.56%, and the activities of alpha-amylase and trypsin were reduced by 10.41% and 21.32%, respectively. Pathological analysis revealed that glyphosate exposure led to significantly damaged midgut, along with thinner basal layer, shedding microvilli, blurred cytoplasmic membrane, and appearance of vacuoles. Exposure to glyphosate also led to accumulation of peroxides in the intestinal tissue; the messenger RNA transcription of SOD, Cu/Zn-SOD, and Mn-SOD was all significantly upregulated by glyphosate treatment for 24 h, while CAT transcription was increased at 24, 48, and 72 h. The activity of SOD was increased significantly at 24 h, while significant activity changes were observed for CAT at 72 and 96 h. These results indicated that exposure to glyphosate caused oxidative stress in the midgut of silkworm and affected the midgut's physiological function. This study provides important insights in evaluating the impact of glyphosate residues in the environment on nontarget organisms.

Evaluation of tolerance to λ-cyhalothrin and response of detoxification enzymes in silkworms reared on artificial diet.

A representative silkworm rearing mode of Ⅰ-Ⅲ instars reared on artificial diet and Ⅳ-Ⅴ instars reared on fresh mulberry leaves has been recognized in some sericultural areas of China. Under this rearing mode, silkworms are prone to be poisoned by pesticide residues on mulberry leaves at the Ⅳ and Ⅴ instar stages. As one of the most widely applied insecticides, λ-cyhalothrin was used to study the insecticide tolerance of silkworm reared on artificial diet (referred as the AD group). Our results showed that the newly ecdysized Ⅳ instar larvae in the AD group were less tolerant to λ-cyhalothrin compared to the mulberry leaves reared group (referred as the ML group). After continuous exposure to trace λ-cyhalothrin, the weight gain and the survival rate of silkworms were significantly lower in the AD group than those in the ML group, even though compensatory growth was observed in the control of the AD group. Histopathology and ultrastructure of fat body showed that λ-cyhalothrin induced more severe cell injuries in the AD group, such as shrunken nucleus, dilatation of endoplasmic reticulum, and mitochondrial swelling. The transcription levels of detoxification related genes (CYP4M5, CYP6AB4, CarE2, CarE5, GSTe1 and GSTe3) and the enzyme activities of P450s, CarEs and GSTs were inducible by trace λ-cyhalothrin in a time-specific manner, and the data showed that the response of P450 enzyme activity was retarded in the AD group, indicating a potential reason for a higher sensitivity to λ-cyhalothrin. Our results provided a new clue for the study of the relationship between feed nutrition and detoxification ability, and also provided an important reference for the development of modern silkworm rearing mode.

Impact of sublethal chlorantraniliprole on epidermis of Bombyx mori during prepupal-pupal transition.

The silkworm Bombyx mori, an economically important insect with a long domestication history, exhibits high sensitivity to chemical pesticides. Extensive application of chlorantraniliprole (CAP) in control of pests of agricultural crops and mulberry plants causes residue toxicity to silkworm. We have demonstrated that sublethal concentration of CAP exposure causes defects in the formation of new epidermis and incomplete shedding of old epidermis during prepupal-pupal transition of B. mori. However, the underlying mechanism still remains unclear. Here, we investigated the transcriptional responses of the epidermis of B. mori on day 2 at prepupal stage to sublethal CAP exposure using digital gene expression (DGE) profiling sequencing. We identified 5823 differentially expressed genes (DEGs), with 4830 genes up-regulated and 993 genes down-regulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that CAP exposure induced disruption of energy homeostasis, oxidative stress, autophagy and apoptosis in the epidermis of B. mori. Meanwhile, trehalose content was increased while most of the genes involved in trehalose metabolism were down-regulated. In addition, chitin contents in CAP-exposed silkworms were decreased. Taken together, these results reveal that sublethal concentration of CAP probably targets trehalose metabolism to impair chitin synthesis, leading to perturbation of pupation metamorphosis in B. mori.

An assessment of the reproductive toxicity of GONPs exposure to Bombyx mori.

This study aimed to explore the toxicity of environmental residues of graphene oxide nanoparticles (GONPs) to reproduction of Lepidopteron insects using both ovary cell line (BmN) and individual female Bombyx mori as the research subjects. The results showed that GONPs dose dependently affect BmN cells. At higher concentrations (>25 mg/L), GONPs led to oxidative stress, ROS accumulation and DNA damage in BmN cells and significantly reduced their survival rate (p ≤ 0.05). Moreover, feeding female B. mori larvae with mulberry leaves treated with 25 mg/L GONPs significantly decreased their gonadosomatic index (GSI) by 40.84%, and increased oxidation levels and antioxidant enzyme activity in silkworm ovary tissues. Pathological analysis found that exposure to GONPs decreased the numbers of both oogonia and oocytes in ovarian tissues, increased the formation of peroxisome and vacuoles in follicle cells, reduced the transcription of genes (Vg, Ovo, Sxl-s, Sxl-l, and Otu) related to ovarian development in B. mori by 0.61, 0.65, 0.75, 0.72, and 0.42-fold, respectively, and lowered the amount of spawning by 52.25%. Overall, these results revealed that GONPs exposure is toxic to the reproduction of B. mori. The underlying mechanism is that oxidative stress due to GONPs causes oxidative damage to DNA, damages ovarian tissues, as well as hinders B. mori development and spawning. Thus, this study provides important experimental data for safety evaluation of reproductive toxicity due to GONPs exposure.

Low concentration acetamiprid-induced oxidative stress hinders the growth and development of silkworm posterior silk glands.

Acetamiprid is a new type of nicotinic insecticide that is widely used in pest control. Its environmental residues may cause silkworm cocooning disorder. In this study, silkworms that received continuous feeding of low concentration acetamiprid (0.15 mg/L) showed significantly decreased silk gland index and cocooning rate. Gene expression profiling of posterior silk glands (PSGs) revealed that the differentially expressed genes were significantly enriched in oxidative stress-related signal pathways with significant up-regulation. The contents of both H2O2 and MDA were increased, along with significantly elevated SOD and CAT activities, all of which reached maximal values at 48 h when H2O2 and MDA's contents were 10.46 and 7.98 nmol/mgprot, respectively, and SOD and CAT activities were 5.51 U/mgprot and 33.48 U/gprot, respectively. The transcription levels of antioxidant enzyme-related genes SOD, Mn-SOD, CuZn-SOD, CAT, TPX and GPX were all up-regulated, indicating that exposure to low concentration acetamiprid led to antioxidant response in silkworm PSG. The key genes in the FoxO/CncC/Keap1 signaling pathway that regulates antioxidant enzyme activity, FoxO, CncC, Keap1, NQO1, HO-1 and sMaf were all up-regulated during the whole process of treatment, with maximal values being reached at 72 h with 2.91, 1.46, 1.82, 2.52, 2.32 and 4.01 times of increases, respectively. These results demonstrate that exposure to low concentration acetamiprid causes oxidative stress in silkworm PSG, which may be the cause of cocooning disorder in silkworm. Our study provides a reference for the safety evaluation of environmental residues of acetamiprid on non-target insects.

Complete mitochondrial genome of Spilosoma lubricipedum (Noctuoidea: Erebidae) and implications for phylogeny of noctuid insects.

Mitochondrial genomes (mitogenomes) have been widely used for studies on phylogenetic relationships and molecular evolutionary biology. Here, the complete mitogenome sequence of Spilosoma lubricipedum (Noctuoidea: Erebidae: Arctiinae) was determined (total length 15,375 bp) and phylogenetic analyses S. lubricipedum were inferred from available noctuid sequence data. The mitogenome of S. lubricipedum was found to be highly A + T-biased (81.39%) and exhibited negative AT- and GC-skews. All 13 protein-coding genes (PCGs) were initiated by ATN codons, except for cox1 with CGA. All tRNAs exhibited typical clover-leaf secondary structures, except for trnS1. The gene order of the S. lubricipedum mitogenome was trnM-trnI-trnQ-nad2. The A + T-rich region of S. lubricipedum contained several conservative features common to noctuid insects. Phylogenetic analysis within Noctuoidea was carried out based on mitochondrial data. Results showed that S. lubricipedum belonged to Erebidae and the Noctuoidea insects could be divided into five well-supported families (Notodontidae + (Erebidae + (Nolidae + (Euteliidae + Noctuidae)))).

The mechanism of damage to the posterior silk gland by trace amounts of acetamiprid in the silkworm, Bombyx mori.

Acetamiprid is a new neonicotinoid insecticide widely used in the prevention and control of pests in agriculture. However, its residues in the environment affect the cocooning of the silkworm, Bombyx mori (B. mori), a non-target insect. To investigate the mechanism of damage, B. mori larvae were fed with trace amounts of acetamiprid (0.15 mg/L). At 96 h after exposure, the larvae showed signs of poisoning and decreased body weight, resulting in reduced survival and ratio of cocoon shell. At 48 h and 96 h after exposure, the residues in the posterior silk gland (PSG), which is responsible for synthesizing silk fibroin, were 0.72 µg/mg and 1.21 µg/mg, respectively, as measured by high performance liquid chromatography, indicating that acetamiprid can accumulate in the PSG. Moreover, pathological sections and transmission electron microscopy also demonstrate the damage of the PSG by acetamiprid. Digital gene expression (DGE) and KEGG pathway enrichment analysis revealed that genes related to metabolism, stress responses and inflammation were significantly up-regulated after exposure. Quantitative RT-PCR analysis showed that the transcript levels of FMBP-1 and FTZ-F1 (transcription factors for synthesizing silk protein) were up-regulated by 2.55-and 1.56-fold, respectively, and the transcript levels of fibroin heavy chain (Fib-H), fibroin light chain (Fib-L), P25, Bmsage and Bmdimm were down-regulated by 0.75-, 0.76-, 0.65-, 0.44- and 0.40-fold, respectively. The results indicate that accumulated acetamiprid causes damage to the PSG and leads to reduced expression of genes responsible for synthesizing silk fibroin. Our data provide reference for evaluating the safety of acetamiprid residues in the environment for non-target insects.

CYP-mediated resistance and cross-resistance to pyrethroids and organophosphates in Aedes aegypti in the presence and absence of kdr.

Aedes aegypti thrives in urban environments and transmits several debilitating human viral diseases. Thus, our ability to control this mosquito species in endemic areas is of utmost importance. The use of insecticides, mostly pyrethroids and organophosphates (OPs), has long been the primary means of controlling A. aegypti, but widespread insecticide resistance has emerged. The two main mechanisms of pyrethroid resistance in A. aegypti are CYP-mediated detoxification and mutations in the target site, voltage-sensitive sodium channel (Vssc), referred to as knockdown resistance (kdr). Knowledge about the contributions and interactions of these mechanisms to resistance is important for the understanding of the molecular and evolutionary basis of insecticide resistance, and to determine the effectiveness of insecticides. In this study, we address two aims: 1) determine the patterns of CYP-mediated cross-resistance to pyrethroid and OP insecticides, both in the presence and absence of kdr (S989P + V1016G), and 2) determine whether the interaction between the two mechanisms yields a greater than, less than, or additive effect on resistance. We tested seven pyrethroids and four OPs against three congenic strains of A. aegypti: ROCK (susceptible), CYP:ROCK (CR) (resistant due to CYP-mediated detoxification without kdr), and CYP + KDR:ROCK (CKR) (resistant due to both CYPs and kdr), and compared these to the congenic KDR:ROCK strain that was previously reported. We found that resistance ratios (RRs) were variable between pyrethroids and strains, ranging from 6.2- to 42-fold for CR, and 70- to 261-fold for CKR. In general, we found that CYP-mediated resistance alone contributes less to resistance than kdr. The effect of the combined mechanisms on resistance was significantly greater than additive for all pyrethroids except (1R)-trans-fenfluthrin. CYP-mediated pyrethroid resistance conferred cross-resistance to both methyl paraoxon and fenitrothion, and negative cross-resistance to methyl parathion and naled. Based on our results, we recommend that etofenprox and cyfluthrin be avoided for A. aegypti control in areas where these two resistance mechanisms are prevalent.

Self assembly and controlled drug release of a nano-laminated graphite carbon nitride/methotrexate complex.

In this study, g-C3N4/methotrexate (g-C3N4/MTX) nanohybrids were obtained via a self assembly method. XRD and TEM demonstrated that bulk g-C3N4 had been stripped into thin nanosheets with size range of 150-250 nm. FTIR investigation indicated that the self assembly of the hybrid was attributed to the hydrogen bond between g-C3N4 nanosheets and MTX molecules. It is confirmed by the UV-vis spectra that the hybrids can achieve a sustained drug release within long period for 70 h. Furthermore, the drug release mechanism was investigated by kinetic models and a first-order relationship was concluded, which indicated that the drug release is a simple diffusion process cohydroxyapatite/methotrexate complexntrolled by gradient drug concentration. Cell viability tests confirmed that g-C3N4 presented excellent biocompatibility and g-C3N4/MTX hybrids had obvious suppression efficiency on MG63 cells which showed a positive correlation to the drug concentration and incubation time.

Correction to: Self assembly and controlled drug release of a nano-laminated graphite carbon nitride/methotrexate complex.

The original version of this article unfortunately contained a mistake. In the Abstract section the sentence "Furthermore, the drug release mechanism was investigated by kinetic models and a first-order relationship was concluded, which indicated that the drug release is a simple diffusion process cohydroxyapatite/methotrexate complexntrolled by gradient drug concentration." was wrong. It should read as "Furthermore, the drug release mechanism was investigated by kinetic models and a first-order relationship was concluded, which indicated that the drug release is a simple diffusion process controlled by gradient drug concentration."

Morphology effect of nano-hydroxyapatite as a drug carrier of methotrexate.

In this study, morphology effect of nano-hydroxyapatite as a drug carrier was investigated for the first time. Hydroxyapatite/methotrexate (HAp/MTX) hybrids with different morphologies were successfully prepared in situ using polyethylene glycol (PEG) as a template. SEM, TEM, XRD and FTIR results confirmed that the hybrids of different morphologies (laminated, rod-like and spherical) with similar phase composition and functional groups were obtained by changing the preparation parameters. UV-Vis spectroscopy was used to identify the drug loading capacity and drug release mechanism of the three hybrids with different morphologies. It is concluded that the laminated hybrid exhibits a higher drug loading capacity compared to the other two hybrids, and all the three hybrids showed a sustained slow release which were fitted well by Bhaskar equation. Additionally, the result of in vitro bioassay test confirms that the inhibition efficacy of the three hybrids showed a positive correlation to the drug loading capacity.

Two novel house fly Vssc mutations, D600N and T929I, give rise to new insecticide resistance alleles.

The house fly, Musca domestica, is a serious pest because it transmits a large diversity of human and veterinary diseases. Insecticides, particularly pyrethroids, are commonly used to control house flies. However, the evolution of pyrethroid resistance has reduced the effectiveness of these insecticides. A major mechanism of resistance to pyrethroids is target site insensitivity caused by the mutations in the voltage-sensitive sodium channel (Vssc) gene (e.g. kdr [L1014F] and super-kdr [M918T+L1014F]). Recently, two novel Vssc alleles, super-kdr+D600N and kdr+T929I were detected in a field collected resistant house fly population in Kansas, USA in 2013. To determine the levels of resistance that these new alleles confer to pyrethroids, we isolated strains having the unique Vssc alleles, but being otherwise congenic to the susceptible strain, aabys. We compared levels of resistance conferred to 14 pyrethroids and determined the inheritance of resistance to 8 pyrethroids. Our results revealed that super-kdr+D600N conferred higher levels of resistance to seven pyrethroids relative to super-kdr, and kdr+T929I showed super-kdr-like levels of resistance in house flies. Our results are compared with previous studies and reveal that addition of T929I to the kdr mutation (L1014F) increased resistance to all pyrethroids (except etofenprox), and enhanced resistance by ~1000-fold to acrinathrin and flumethrin. The implications of these results on the evolution of resistance are discussed.

Genome-wide analysis reveals the expansion of Cytochrome P450 genes associated with xenobiotic metabolism in rice striped stem borer, Chilo suppressalis.

The Cytochrome P450 (CYP) superfamily is a large group of ancient proteins with enzymatic activities involved in various physiological processes. The rice striped stem borer, Chilo suppressalis, is an important insect pest in rice production. Here, we report the identification and characterization of 77 CYP genes from rice striped stem borer (SSB) through genome and transcriptome sequence analyses. All these CYP genes were confirmed by RT-PCR and direct sequencing. Twenty-eight CYP transcripts have full open reading frame (ORF) and four additional transcripts have a nearly full length coding region. The SSB CYP genes were classified into four clans, the mitochondrial, CYP2, CYP3, and CYP4. Phylogenetic analysis indicated that there was an apparent expansion of the CYP3 clan in insects. The CYP6AB subfamily of the CYP3 clan had nine members in SSB. Evolutionary analysis showed that this subfamily was expanded only in lepidopteran insects. In this study, we identified a new P450 subfamily, CYP321F, which is unique to SSB and located in the genome as tandem repeats. Our work provided a foundation for future studies on the functions and mechanism of P450s in the destructive rice pest.

JH degradation pathway participates in hormonal regulation of larval development of Bombyx mori following λ-cyhalothrin exposure.

λ-Cyhalothrin (λ-cyh), a widely utilized pyrethroid insecticide, poses serious threats to non-target organisms due to its persistence nature in the environment. Exposure to low concentrations of λ-cyh has been observed to result in prolonged larval development in Bombyx mori, leading to substantial financial losses in sericulture. The present study was undertaken to elucidate the underlying mechanisms for prolonged development caused by λ-cyh (LC10) exposure. The results showed that the JH Ⅲ titer was significantly increased at 24 h of λ-cyh exposure, and the JH interacting genes Methoprene-tolerant 2, Steroid Receptor Co-activator, Krüppel-homolog 1, and JH binding proteins were also up-regulated. Although the target of rapamycin (Tor) genes were induced by λ-cyh, the biosynthesis of JH in the corpora allata was not promoted. Notably, 13 JH degradation genes were found to be significantly down-regulated in the midgut of B. mori. The mRNA levels and enzyme activity assays indicated that λ-cyh had inhibitory effects on JH esterase, JH epoxide hydrolase, and JH diol kinase (JHDK). Furthermore, the suppression of JHDK (KWMTBOMO01580) was further confirmed by both western blot and immunohistochemistry. This study has offered a comprehensive perspective on the mechanisms underlying the prolonged development caused by insecticides, and our results also hold significant implications for the safe production of sericulture.