The ethylene response factor gene, ThDRE1A, is involved in abscisic acid- and ethylene-mediated cadmium accumulation in Tamarix hispida.

Tamarix hispida is highly tolerant to salt, drought and heavy metal stress and is a potential material for the remediation of cadmium (Cd)-contaminated soil under harsh conditions. In this study, T. hispida growth and chlorophyll content decreased, whereas flavonoid and carotenoid contents increased under long-term Cd stress (25 d). The aboveground components of T. hispida were collected for RNA-seq to investigate the mechanism of Cd accumulation. GO and KEGG enrichment analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in plant hormone-related pathways. Exogenous hormone treatment and determination of Cd2+ levels showed that ethylene (ETH) and abscisic acid (ABA) antagonists regulate Cd accumulation in T. hispida. Twenty-five transcription factors were identified as upstream regulators of hormone-related pathways. ThDRE1A, which was previously identified as an important regulatory factor, was selected for further analysis. The results indicated that ThABAH2.5 and ThACCO3.1 were direct target genes of ThDRE1A. The determination of Cd2+, ABA, and ETH levels indicated that ThDRE1A plays an important role in Cd accumulation through the antagonistic regulation of ABA and ETH. In conclusion, these results reveal the molecular mechanism underlying Cd accumulation in plants and identify candidate genes for further research.

RNAi-based functional analysis of bursicon genes related to wing expansion in gypsy moths.

Bursicon is a heterodimeric neuropeptide composed of Burs-α and Burs-ß subunits that plays an important role in cuticle tanning and wing expansion in insects. In this study, full-length cDNAs of Burs-α (LdBurs-α) and Burs-ß (LdBurs-ß) genes were identified in gypsy moth (Lymantria dispar) and cloned. The 480 bp and 420 bp open reading frames (ORFs) encode 159 and 129 amino acid polypeptides, respectively. LdBurs-α and LdBurs-ß have 11 conserved cysteine residues, and LdBurs-α and LdBurs-ß genes were expressed during all developmental stages according to quantitative reverse transcription PCR (qRT-PCR), with highest expression in the egg stage. High expression levels were also detected in the haemolymph, cuticle and head. To explore the physiological functions of LdBurs-α and LdBurs-ß, the genes were knocked down in larvae and pupae using RNA interference (RNAi), and expression levels of LdBurs-α and LdBurs-ß were decreased by 42.26-80.09%. Wing defects were observed in L. dispar pupae following Ldbursion silencing, with a phenotypic percentage ranging from 10.17% to 15.00%. RNAi-mediated knockdown of Ldbursicon prevented the expansion of male and female L. dispar adult wings, with malformation rates ranging from 6.38% and 30.00% to 57.69% and 69.23%, but no cuticle tanning defects were observed in pupae or adults. The results indicate that bursicon plays a key role in wing expansion in L. dispar adults, making it a potentially novel molecular target for insecticide-based control of this pest species.

Silencing Br-C impairs larval development and chitin synthesis in Lymantria dispar larvae.

In insects, 20-hydroxyecdysone (20E) mediates developmental transitions and regulates molting processes through activation of a series of transcription factors. Broad-Complex (Br-C), a vital gene in the 20E signalling pathway, plays crucial roles during insect growth processes. However, whether Br-C affects chitin synthesis in insects remains unclear. In the present study, the Br-C gene from Lymantria dispar, a notorious defoliator of forestry, was identified based on transcriptome data, and subjected to bioinformatic analysis. The regulatory functions of LdBr-C in chitin synthesis and metabolism in L. dispar larvae were analysed by RNA interference (RNAi). The full-length LdBr-C gene (1431 bp) encodes a 477 amino acid (aa) polypeptide containing a common BRcore region (391 aa) at the N-terminus and a C-terminal Zinc finger domain (56 aa) harbouring two characteristic C2H2 motifs (CXXC and HXXXXH). Phylogenetic analyses showed that LdBr-C shares highest homology and identity with Br-C isoform 7 (83.12%) of Helicoverpa armigera. Expression profiles indicate that LdBr-C was expressed throughout larval and pupal stages, and highly expressed in prepupal and pupal stages. Furthermore, LdBr-C expression was strongly induced by exogenous 20E, and suppressed dramatically after application of dsLdBr-C. Bioassay results showed that knockdown of LdBr-C caused larval developmental deformity, significant weight loss, and a mortality rate of 67.18%. Knockdown of LdBr-C significantly down-regulated transcription levels of eight critical genes (LdTre1, LdTre2, LdG6PI, LdUAP, LdCHS1, LdCHS2, LdTPS and LdCHT) related to chitin synthesis and metabolism, thereby lowering the chitin content in the midgut and epidermis. Our findings demonstrate that Br-C knockdown impairs larval development and chitin synthesis in L. dispar.

A G-protein-coupled receptor regulation pathway in cytochrome P450-mediated permethrin-resistance in mosquitoes, Culex quinquefasciatus.

Rhodopsin-like G protein-coupled receptors (GPCRs) are known to be involved in the GPCR signal transduction system and regulate many essential physiological processes in organisms. This study, for the first time, revealed that knockdown of the rhodopsin-like GPCR gene in resistant mosquitoes resulted in a reduction of mosquitoes' resistance to permethrin, simultaneously reducing the expression of two cAMP-dependent protein kinase A genes (PKAs) and four resistance related cytochrome P450 genes. The function of rhodopsin-like GPCR was further confirmed using transgenic lines of Drosophila melanogaster, in which the tolerance to permethrin and the expression of Drosophila resistance P450 genes were both increased. The roles of GPCR signaling pathway second messenger cyclic adenosine monophosphate (cAMP) and downstream effectors PKAs in resistance were investigated using cAMP production inhibitor Bupivacaine HCl and the RNAi technique. Inhibition of cAMP production led to significant decreases in both the expression of four resistance P450 genes and two PKA genes and mosquito resistance to permethrin. Knockdown of the PKA genes had shown the similar effects on permethrin resistance and P450 gene expression. Taken together, our studies revealed, for the first time, the role of the GPCR/cAMP/PKA-mediated regulatory pathway governing P450 gene expression and P450-mediated resistance in Culex mosquitoes.

Purification, characterization, and sensitivity to pesticides of carboxylesterase from Dendrolimus superans (Lepidoptera: Lasiocampidae).

Through a combination of steps including centrifugation, ammonium sulfate gradient precipitation, sephadex G-25 gel chromatography, diethylaminoethyl cellulose 52 ion-exchange chromatography and hydroxyapatite affinity chromatography, carboxylesterase (CarE, EC3.1.1.1) from sixth instar larch caterpillar moth, Dendrolimus superans (Lepidoptera: Lasiocampidae) larvae was purified and its biochemical properties were compared between crude homogenate and purified CarE. The final purified CarE after hydroxyapatite chromatography had a specific activity of 52.019 µmol/(min·mg protein), 138.348-fold of crude homogenate, and the yield of 2.782%. The molecular weight of the purified CarE was approximately 84.78 kDa by SDS-PAGE. Three pesticides (dichlorvos, lambda-cyhalothrin, and avermectins) showed different inhibition to crude CarE and purified CarE, respectively. In vitro median inhibitory concentration indicated that the sensitivity of CarE (both crude homogenate and final purified CarE) to pesticides was in decreasing order of dichlorvos > avermectins > lambda-cyhalothrin. By the kinetic analysis, the substrates alpha-naphthyl acetate (α-NA) and beta-naphthyl acetate (ß-NA) showed lesser affinity to crude extract than purified CarE. The results also indicated that both crude homogenate and purified CarE had more affinity to α-NA than to ß-NA, and the Kcat and Vmax values of crude extract were lower than purified CarE using α-NA or ß-NA as substrate.