Identification of insect cuticular protein genes LCP17 and SgAbd5 from Helicoverpa armigera and evaluation their roles in fenvalerate resistance.

Insect cuticular protein (ICP) plays an important role in insect growth and development. However, research on the role of ICP in insecticide resistance is very limited. In this study, insect cuticular protein genes LCP17 and SgAbd5 were cloned and characterized in Helicoverpa armigera based on previous transcriptome data. The functions of LCP17 and SgAbd5 genes in fenvalerate resistance were assessed by RNA interference (RNAi), and their response to fenvalerate was further detected. The results showed that LCP17 and SgAbd5 were overexpressed in the fenvalerate-resistant strain comparing with a susceptible strain. The open reading frames of LCP17 and SgAbd5 genes were 423 bp and 369 bp, encoding 141 and 123 amino acids, respectively. LCP17 and SgAbd5 genes were highly expressed in the larval stage, but less expressed in the adult and pupal stages. The expression level of LCP17 and SgAbd5 genes increased significantly after fenvalerate treatment at 24 h. When the cotton bollworms larvae were exposed to fenvalerate at LD50 level, RNAi-mediated silencing of LCP17 and SgAbd5 genes increased the mortality from 50.68% to 68.67% and 63.89%, respectively; the mortality increased to even higher level, which was 73.61%, when these two genes were co-silenced. Moreover, silencing of these two genes caused the cuticle lamellar structure to become loose, which led to increased penetration of fenvalerate into the larvae. The results suggested that LCP17 and SgAbd5 may be involved in the resistance of cotton bollworm to fenvalerate, and LCP17 and SgAbd5 could serve as potential targets for H. armigera control.

Identification and characterization of both cis- and trans-regulators mediating fenvalerate-induced expression of CYP6B7 in Helicoverpa armigera.

As we known, inducibility is an important feature of P450 genes. Previous studies indicated that CYP6B7 could be induced and involved in fenvalerate detoxification in Helicoverpa armigera. However, the regulatory mechanism of CYP6B7 induced by fenvalerate is still unclear. In this study, CYP6B7 promoter of H. armigera was cloned and the cis-acting element of fenvalerate was identified to be located between -84 and - 55 bp of CYP6B7 promoter. Subsequently, 33 candidate transcription factors (CYP6B7-fenvalerate association proteins, CAPs) that may bind to the cis-acting element were screened and verified by yeast one-hybrid. Among them, the expression levels of several CAPs were significantly induced by fenvalerate. Knockdown of juvenile hormone-binding protein-like (JHBP), RNA polymerase II-associated protein 3 (RPAP3), fatty acid synthase-like (FAS) and peptidoglycan recognition protein LB-like (PGRP) resulted in significant expression inhibition of CYP6B7, and increased sensitivity of H. armigera to fenvalerate. Co-transfection of reporter gene p (-84/-55) with pFast-CAP showed that JHBP, RPAP3 and PGRP could significantly increase the activity of CYP6B7 promoter. These results suggested that trans-acting factors JHBP, RPAP3 and PGRP may bind with cis-acting elements to regulate the expression of CYP6B7 induced by fenvalerate, and play an important role in the detoxification of H. armigera to fenvalerate.

Regulation of CncC in insecticide-induced expression of cytochrome P450 CYP9A14 and CYP6AE11 in Helicoverpa armigera.

The expression of many detoxification genes can be regulated by CncC pathway and contributes to insecticide tolerance in insects. Our previous study has demonstrated that the transcripts of CncC and cytochrome P450s (CYP9A14, CYP6AE11) were significantly up-regulated after different insecticides treatment in Helicoverpa armigera. Further study indicated that H2O2, GSH, and MDA contents and antioxidant enzyme activities of H. armigera were enhanced after chlorantraniliprole, cyantraniliprole, indoxacarb, and spinosad exposure. Silencing CncC by RNA interference significantly down-regulated the expression levels of CYP9A14 and CYP6AE11, and increased the susceptibility of dsRNA-injected larvae to λ-cyhalothrin, chlorantraniliprole, and cyantraniliprole. On the contrary, applying CncC agonist curcumin on H. armigera induced the expression of CYP9A14 and CYP6AE11, and enhanced the tolerance of H. armigera to insecticides. Treatment of ROS scavenger N-acetylcysteine on H. armigera reduced the H2O2 content and antioxidant enzyme activities, suppressed the transcripts of CncC, CYP9A14, and CYP6AE11, and decreased the larval tolerance to insecticides. These results demonstrated that the induced-expression of CYP9A14 and CYP6AE11 related with insecticides tolerance in H. armigera was regulated by CncC, which may be activated by ROS generated by insecticides. This study will help to better understand the underlying regulation mechanisms of CncC pathway in H. armigera tolerance to insecticides.

Effects of different insecticides on transcripts of key genes in CncC pathway and detoxification genes in Helicoverpa armigera.

The CncC pathway regulates the expression of multiple detoxification genes and contributes to the detoxification and antioxidation in insects. Many studies have focused on the impacts of plant allelochemicals on the CncC pathway, whereas studies on the effects of pesticides on key genes involved in this pathway are very limited. In this study, the effects of different types of commonly used insecticides on the transcripts of CncC, Keap1, and Maf and multiple detoxification genes of Helicoverpa armigera were evaluated using real-time quantitative polymerase chain reaction. The results showed that 8 insecticides (bifenthrin, λ-cyhalothrin, chlorantraniliprole, cyantraniliprole, spinosad, indoxacarb, chlorfenapyr, tolfenpyrad, and thiacloprid) significantly induced the expression of CncC and 4 insecticides (cypermethrin, acetamiprid, thiacloprid, and indoxacarb) suppressed the expression of Keap1 both at 24 h and 48 h; meanwhile, the expression levels of Maf were induced by 5 insecticides (fenvalerate, chlorantraniliprole, cyantraniliprole, lufenuron, and tolfenpyrad) at 24 h or 48 h. Multiple detoxification genes, especially cytochrome P450s genes, showed different up-regulation after bifenthrin, λ-cyhalothrin, chlorantraniliprole, cyantraniliprole, indoxacarb, and spinosad treatment for 48 h. Our results suggest that the CncC pathway and detoxification genes can be activated by different insecticides in H. armigera. These results establish a foundation for further studies on the relationship between the CncC pathway and the detoxification genes in H. armigera.

A Comparative Study of Transcriptional Regulation Mechanism of Cytochrome P450 CYP6B7 between Resistant and Susceptible Strains of Helicoverpa armigera.

Cytochrome P450 CYP6B7 has previously been proved to be associated with fenvalerate-resistance in Helicoverpa armigera. Here, how CYP6B7 is regulated and involved in the resistance of H. armigera is studied. Seven base differences (M1-M7) were found in CYP6B7 promoter between a fenvalerate-resistant (HDTJFR) and a susceptible (HDTJ) strain of H. armigera. M1-M7 sites in HDTJFR were mutated into the corresponding base in HDTJ, and pGL3-CYP6B7 reporter genes with different mutation sites were constructed. Fenvalerate-induced activities of reporter genes mutated at M3, M4, and M7 sites were significantly reduced. Transcription factors Ubx and Br, whose binding sites contain M3 and M7, respectively, were overexpressed in HDTJFR. Knockdown of Ubx and Br results in significant expression inhibition of CYP6B7 and other resistance-related P450 genes, and increase of sensitivity of H. armigera to fenvalerate. These results indicate that Ubx and Br regulate the expression of CYP6B7 to mediate the fenvalerate-resistance in H. armigera.

Identification of cis-acting elements in response to fenvalerate in the CYP6B7 promoter of Helicoverpa armigera.

Cytochrome P450-mediated detoxification plays an important role in the development of insecticide resistance. Previous studies have shown that cytochrome P450 CYP6B7 was induced by fenvalerate and involved in fenvalerate detoxification in Helicoverpa armigera. However, the transcriptional regulation of CYP6B7 induced by fenvalerate remains unclear. Here, a series of progressive 5' deletions of CYP6B7 promoter reporter genes were constructed, and the relative luciferase activities were detected. The results revealed that the relative luciferase activity of plasmid p (-655/-1) was significantly induced by fenvalerate. Further deletion of the region between -655 and -486 bp showed that the highest luciferase activity induced by fenvalerate was observed in plasmid p (-528/-1), while p (-485/-1) had the lowest fenvalerate-induced luciferase activity. Moreover, internal deletion and mutation in the region between -508 and -486 bp resulted in a significant reduction in fenvalerate-induced CYP6B7 promoter activity, suggesting that the cis-acting element responsible for fenvalerate in the CYP6B7 promoter was located between -508 and -486 bp. These results promote an understanding of the expression regulation mechanism of P450 genes that conferring resistance to insecticides.

Toxic effects of boscalid in adult zebrafish (Danio rerio) on carbohydrate and lipid metabolism.

Boscalid as one of the most widely used succinate dehydrogenase inhibitor (SDHI) fungicides has been frequently detected in both freshwater and estuarine environments. Its acute toxic effects on zebrafish and freshwater algae have been reported in our previous studies. To further investigate its chronic toxic effects to aquatic organisms, adult zebrafish were exposed for 28 days to a series of environmentally relevant boscalid concentrations in this study. Growth indicators and histopathology were determined in this study. Results indicated that boscalid inhibited the growth of zebrafish and induced damage in the kidneys and liver. Carbohydrate and lipid metabolism as the key pathways of energy metabolism in growth of zebrafish were also investigated. Results showed boscalid caused an increase in the activity of hexokinase (HK), the content of glycogen, glucose-6-phosphatase (G6Pase), and insulin (INS) in liver and a decrease in blood glucose content and succinate dehydrogenase (SDH) activity. Boscalid reduced the total content of triacylglyceride (TG) and cholesterol (TC) and the activity of fatty acid synthase (FAS) and acetyl coenzyme A carboxylase (ACC) in the liver. Correspondingly, expression of the genes related to carbohydrate and lipid metabolism in liver and intestine was affected by boscalid, especially in the significant upregulation of G6Pase and pparα and downregulation of SGLT-1 and AMY. Results suggested that boscalid could affect carbohydrate metabolism of adult zebrafish via regulation of gluconeogenesis and glycolysis at 0.1 mg/L. Moreover, boscalid might induce an increase in ß-oxidation and a decrease in lipid synthesis at 0.01 mg/L. In conclusion, our study identified that carbohydrate and lipid metabolism are the possible biological pathways that mediate boscalid-induced developmental effects.

Long-Term Exposure to Environmental Concentrations of Azoxystrobin Delays Sexual Development and Alters Reproduction in Zebrafish ( Danio rerio).

The strobilurin fungicide azoxystrobin (AZO) can induce adverse effects in aquatic organisms, but data are lacking on endpoints associated with sexual development and reproduction following chronic exposure to AZO. In this study, zebrafish embryos (F0) at 2-4 h postfertilization (hpf) were exposed to 0.2, 2.0, and 20.0 µg/L AZO until 120 d postfertilization (dpf). Decreased male ratio and increased intersex ratio were observed by 20.0 µg/L AZO at 42 and 60 dpf, but this effect disappeared at 120 dpf. AZO at 20.0 µg/L inhibited growth, retarded gonadal development, and disrupted sex hormone and vitellogenin in females at 60 and 120 dpf and in males at 42, 60, and 120 dpf. These effects were associated with altered expression of cyp19a, cyp19b, hsd3b, hsd17b, vtg1, and vtg2. Exposure to 2.0 µg/L AZO altered mRNA levels of these transcripts in females at 120 dpf and in males at 60 and 120 dpf. Reproduction ability was reduced by 20.0 µg/L AZO at 120 dpf. Developmental defects were observed after F1 embryos from exposed parents of 20.0 µg/L were reared in AZO-free water at 96 hpf. Overall, these data provide new understanding of fish sexual development and reproduction following chronic exposures to AZO.

Parental exposure to azoxystrobin causes developmental effects and disrupts gene expression in F1 embryonic zebrafish (Danio rerio).

The fungicide azoxystrobin induces reproductive toxicity in adult zebrafish. However, data are lacking regarding the impact of azoxystrobin in the F1 generation after parental exposure. To address this knowledge gap, parental zebrafish (F0) were exposed to 2, 20 and 200 µg/L azoxystrobin for 21 days. Following this, fertilized F1 embryos from the exposed parents were either exposed to the same concentration as their corresponding exposed parents (F0+/F1+) or were reared in clean water (F0+/F1-) for 96 h ("+", exposed; "-" unexposed). Likewise, F1 embryos from the non-exposed parents were either reared in clean water (F0-/F0-) as the control group or were exposed to 2, 20 and 200 µg/L azoxystrobin (F0-/F1+) for 96 h. Mortality, deformities, hatching rate, body length, and the expression of transcripts related to the endocrine system, oxidative stress, and apoptosis were measured. Increased mortality, higher malformation rate, decreased hatching rate, and a shorter total body length, as well as up-regulated cyp19b, vtg1, vtg2, p53, casp3, and casp9 mRNA and down-regulated sod1 and sod2 mRNA were detected in F1 embryos from the F0 and F1 exposure group at 20 and 200 µg/L azoxystrobin (F0+/F1+) when compared with the group from the F0 exposure alone (F0+/F1-). Interestingly, F1 exposure alone (F0-/F1+) did not induce mortality, developmental impairments, nor morphological deformations compared to the control group, but it did increase expression level of sod1, sod2, cat, p53, and casp9 at 200 µg/L azoxystrobin. Taken together, these data suggest that azoxystrobin affects survivability, development, and genes involved in the endocrine system, oxidative stress, and apoptosis in F1 embryos if their parents are initially exposed to this fungicide compared to embryos from non-exposed parents. Moreover, the effects are more severe if the offspring are continuously exposed to azoxystrobin similar to their parents.

Short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish (Danio rerio).

Previous study indicated that azoxystrobin had high acute toxicity to zebrafish, and larval zebrafish were more sensitive to azoxystrobin than adult zebrafish. The objective of the present study was to investigate short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish. After zebrafish embryos and adults were exposed to 0.01, 0.05 and 0.20 mg/L azoxystrobin (equal to 25, 124 and 496 nM azoxystrobin, respectively) for 8 days, the lethal effect, physiological responses, liver histology, mitochondrial ultrastructure, and expression alteration of genes related to mitochondrial respiration, oxidative stress, cell apoptosis and innate immune response were determined. The results showed that there was no significant effect on larval and adult zebrafish after exposure to 0.01 mg/L azoxystrobin. However, increased ROS, MDA concentration and il1b in larval zebrafish, as well as increased il1b, il8 and cxcl-c1c in adult zebrafish were induced after exposure to 0.05 mg/L azoxystrobin. Reduced mitochondrial complex III activity and ATP concentration, increased SOD activity, ROS and MDA concentration, decreased cytb, as well as increased sod1, sod2, cat, il1b, il8 and cxcl-c1c were observed both in larval and adult zebrafish after exposure to 0.20 mg/L azoxystrobin; meanwhile, increased p53, bax, apaf1 and casp9, alteration of liver histology and mitochondrial ultrastructure in larval zebrafish, and alteration of mitochondrial ultrastructure in adult zebrafish were also induced. The results demonstrated that azoxytrobin induced short-term developmental effects on larval zebrafish and adult zebrafish, including mitochondrial dysfunction, oxidative stress, cell apoptosis and innate immune response. Statistical analysis indicated that azoxystrobin induced more negative effects on larval zebrafish, which might be the reason for the differences of developmental toxicity between larval and adult zebrafish caused by azoxystrobin. These results provided a new insight into potential mechanisms of azoxystrobin in larval zebrafish and adult zebrafish.

Shape memory effect of poly(D,L-lactide)/Fe3O4 nanocomposites by inductive heating of magnetite particles.

In this paper, the preparation of biocompatible poly(D,L-lactide) (PDLLA)/magnetite (Fe(3)O(4)) nanocomposites and their shape memory effect are reported. Fe(3)O(4) nanoparticles with an average size of 20 nm were synthesized by chemical co-precipitation and mixed uniformly with a PDLLA matrix. Scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and mechanical testing were carried out to determine the micro-surface morphology, glass transition temperature (T(g)), functional groups change and mechanical properties of the PDLLA/Fe(3)O(4) nanocomposites. The interesting shape memory behavior of the nanocomposites induced by an ultrasonic alternating magnetic field were also observed. SEM and DSC showed that there was a close interaction between the polymer matrix and the magnetic nanoparticles. Formation of weak hydrogen bonds between the C=O in PDLLA and Fe-OH groups of the surface of nano-crystalline Fe(3)O(4) was examined by FTIR. The PDLLA/Fe(3)O(4) nanocomposites displayed a desirable shape memory effect.