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Harmonia axyridis (Coleoptera: Coccinellidae) is a polyphagous insect that is an important biological agent used to control agricultural and forestry pests. The role of functional genes in H. axyridis based on quantitative real-time PCR (qRT-PCR) is increasingly well understood to investigate biology, physiology, feeding behavior and the role of important genes in physiological processes. Quantitative real-time PCR (qRT-PCR) is a powerful and reliable technique to quantify gene expression. Using qRT-PCR, expression levels of target genes are determined based on the levels of internal reference genes; therefore, reference genes need to be stably expressed under specific experimental conditions. However, there have been no studies on the stability of reference genes used in H. axyridis. In this study, we systematically investigated expression profiles of nine candidate reference genes from H. axyridis, including β-actin (ACTIN); elongation factor 1 α (EF1A); ribosomal proteins L10, L18, L28, S13, and S15 (RPL10, RPL18, RPL28, RPS13 and RPS15); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); and superoxide dismutase (SOD). Four analytical methods (geNorm, BestKeeper, NormFinder, and the ΔCt method) were used to evaluate the suitability of these genes as internal reference genes for three biotic factors (developmental stage, tissue, and sex) and two abiotic treatments (temperature and photoperiod). RefFinder, a comprehensive evaluation platform integrating the four analytical methods, was used to rank the overall stability of these reference genes. Among the nine candidate genes, different reference genes were identified as having the most stable expression across biotic and abiotic factors. Genes encoding ribosomal proteins typically had the most stable expression, though EF1A was the most stable across developmental stages and photoperiods. To validate the suitability of these reference genes, heat shock protein 90 (HSP90) was chosen as a target. Significant up-regulation in HSP90 expression level in response to both low and high temperature was observed when using the most suitable reference genes but not when using an arbitrarily selected reference gene. The reference genes identified in this study will provide the basis for future functional genomics research in H. axyridis and will also facilitate the establishment of a standardized qRT-PCR program for other related insects.

Abamectin, produced by the soil-dwelling actinomycete Streptomyces avermitilis, belongs to the macrocyclic lactones class of pesticides, has nematocidal, acaricidal, and insecticidal activity, and is highly effective when used against targeted species. Bemisia tabaci, the tobacco whitefly, is a highly destructive insect to agricultural production worldwide, and various insecticide-resistant strains have been identified in China. Here, we monitored levels of resistance to abamectin in twelve field-collected B. tabaci populations from northern China, and confirmed that, compared with the lab reference strain, six field populations exhibited strong abamectin resistance, while the other six exhibited low-to-medium resistance. Among these, the Xinzheng (XZ) population displayed about a 40-fold increased resistance to abamectin, and experienced significant cross-resistance to chlorpyrifos and imidacloprid. The abamectin resistance of XZ was found to be autosomal and incompletely dominant. Metabolic enzyme and synergism tests were conducted, and two metabolic enzymes, glutathione S-transferase and P450 monooxygenase, were found to be conducive to the field-developed abamectin resistance of the XZ population. The above results provide valuable information that can be used in identifying new pest control strategies and delaying the evolution of resistance to abamectin in field populations of whiteflies.

Afidopyropen, a novel biopesticide, is derived from Aspergillus fumigatus, a fungus, and shows promise as a novel insecticidal agent for the management of the whitefly pest Bemisia tabaci in horticultural and economical crop production. In the present work, we monitored the susceptibilities of B. tabaci to afidopyropen in 18 field populations, sampled from 9 provinces of China, and found that, in comparison with the susceptible strain (MED-S), B. tabaci from most field populations were highly susceptible, except for the Haidian population (HD) which exhibited an approximately 40-fold increase in resistance. The HD population also displayed significant cross-resistance to sulfoxaflor (14.5-fold) but little cross-resistance to cyantraniliprole, flonicamid, imidacloprid, pymetrozine, and thiamethoxam. Afidopyropen resistance of the HD population was determined to be incomplete dominant and autosomal, and synergism assays demonstrated that P450 monooxygenases could contribute to the field-evolved afidopyropen resistance observed in the HD population. These results will further our understanding of the molecular underpinnings of insecticide resistance in B. tabaci and can inform the development of field-based pest control tactics to slow the development of afidopyropen resistance and to control whiteflies more sustainably.

Bemisia tabaci , the tobacco whitefly, is one of the most notorious agricultural sucking insect pests that severely damage a series of crops worldwide. Throughout China, B. tabaci threatens agricultural production with increasing cases of resistance to commonly used insecticides, prompting the widespread use of cyantraniliprole as an alternative to control hemipteran pests. Here, we found overexpression of the CYP4G68 gene conferring cyantraniliprole resistance using quantitative real-time PCR (qPCR) and RNA interference (RNAi) in one lab-selected resistant strain CYAN-R (to about 80-fold higher than control). Furthermore, we measured levels of resistance to cyantraniliprole in whiteflies with 18 field-sampled populations across China and then confirmed that, among them, 14 field-sampled populations showed low-to-high resistance to cyantraniliprole compared with the susceptible strain. We measured CYP4G68 expression in the 14 field populations, and the results of qPCR and RNAi indicated that in two of these populations, Haikou and Wuhan, significant overexpression of CYP4G68 contributed to the development of field-evolved resistance to cyantraniliprole. These results indicate the need to facilitate strategies of management to delay the evolution of resistance to cyantraniliprole and control of whiteflies more sustainably, and to prevent overuse of insecticides in the environment through rational application practices.

Trichogramma dendrolimi is an essential egg parasitic wasp with a broad host range and has been widely used for controlling agricultural and forestry pests. Despite the availability of fragmented genomes of Trichogramma , a high-quality, chromosome-level genome reference is not yet available for this diverse genus. In this study, we assembled a high-quality chromosome-level genome of T. dendrolimi using PacBio CLR long sequencing and further scaffolded it with Hi-C technologies. The genome size was 216.24 Mb, featuring a N50 of 39.07 Mb, and 98.79% of scaffolds were anchored to five chromosomes. In addition, we annotated 12,902 protein-coding genes and 60.74 Mb repeat sequences for this genome assembly. In conclusion, our chromosome-level genome assembly provided important genomic resources that benefit the utilization of Trichogramma parasitoids in pest control.

Broflanilide is a novel pesticide used in agriculture that binds to unique receptors on pests; however, the widespread use of broflanilide has led to toxicity in Daphnia magna . At present, little information on the potential threats broflanilide imposes on D. magna is available. Therefore, the present study examined the chronic toxicity of broflanilide in D. magna by comparing changes in molting, neurotransmitter function, and behavior. The results showed that broflanilide caused chronic toxicity in D. magna at a concentration of 8.45 μg/L, and growth, development, reproduction, and the development of offspring were affected. In addition, broflanilide affected the molting of D. magna by significantly inhibiting the expression of chitinase, ecdysteroid, and related genes. Broflanilide also affected the expression of γ-glutamic acid, glutamine, gamma-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan, dopa, and dopamine. Furthermore, the swimming distance and speed of D. magna were reduced . Taken together, the results demonstrate the chronic toxicity and exposure risk of broflanilide in D. magna .

Bemisia tabaci is a threat to agriculture worldwide because of its potential to cause devastating damage to various crops. β-asarone is a bioactive pesticidal chemical originating from Acorus calamus (or “Sweet Flag”) plants, and it displays significant lethal effects against insect pests. In this study, we established a baseline of susceptibility to β-asarone from China and patterns of cross-resistance to other popular insecticides. We found that all the 12 field-collected B. tabaci populations exhibited high susceptibility to β-asarone, and there was no cross-resistance detected for other tested insecticides. We subsequently evaluated the sublethal effects of β-asarone on physiology and biochemistry via LC25 treatment (4.7 mg/L). LC25 of β-asarone resulted in prolonged developmental duration and decreased survival rates in B. tabaci nymphs, pseudopupae, and adults. Significant reductions in oviposition duration, fecundity, and hatchability were also observed. Additionally, the metabolic enzyme activity and expression profiles of selected cytochrome P450 monooxygenase (P450) genes following the LC25 treatment of β-asarone suggest that enhanced detoxification via P450s could be involved in the observed sublethal effects. These findings demonstrate the strong toxicity and significant sublethal effects of β-asarone on B. tabaci and suggest that the induced overexpression of P450 genes could be associated with the response to β-asarone.

Broflanilide exerted negative impacts on the gill of zebrafish. Thus, in this study, zebrafish gill was used to assess the apoptosis toxicity of broflanilide by determining the levels of reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) and apoptosis-related genes. The results found that the minimum threshold for the content and time of broflanilide affecting enzyme content and gene expression was 0.26 mg/L after 24 h exposure. After 96 h exposure, broflanilide could cause apoptosis and exerted significantly increased contents of ROS and MDA, while inhibiting the activities of SOD, CAT, and GPx at 0.26 and 0.57 mg/L. Broflanilide also had adverse effects on apoptosis-related genes, such as tumor protein p53 ( p53 ), associated × ( Bax) , B-cell lymphama-2 ( Bcl-2 ), caspase-3, caspase-9 , and apoptotic protease activating factor-1( apaf-1 ), at 0.26 mg/L and 0.57 mg/L after 96 h exposure, respectively. These results provide new insight into the potential toxicity mechanisms of broflanilide in zebrafish gills.

Beet armyworm, Spodoptera exigua (Hübner), is a worldwide pest of many crops. Chemical insecticides are heavily used for its control in China, and serious resistance has been evolved in the field to a variety of insecticides including emamectin benzoate. Through repeated backcrossing to a susceptible strain (WH-S) and selection with emamectin benzoate, the trait conferring resistance to emamectin benzoate in a field-collected population of S. exigua (moderately resistant to emamectin benzoate and strongly resistant to pyrethroids and indoxacarb) was introgressed into WH-S to generate a near-isogenic resistant strain (WH-EB). Compared with WH-S, the WH-EB strain developed a 1,110-fold resistance to emamectin benzoate and a high level of cross-resistance to abamectin (202-fold), with low levels of cross-resistance to cypermethrin (10-fold) and chlorfluazuron (7-fold), but no crossresistance to representatives of another six different classes of insecticides (chlorantraniliprole, chlorfenapyr, indoxacarb, spinosad, tebufenozide, and chlorpyrifos). Resistance to emamectin benzoate in WH-EB was autosomal, incompletely dominant, and polygenic. Limited cross-resistance in WH-EB indicates that emamectin benzoate can be rotated with other classes of insecticides to which it does not show cross-resistance to delay the evolution of resistance in S. exigua. The incompletely dominant nature of resistance in S. exigua may explain the rapid evolution of resistance to emamectin benzoate in the field, and careful deployment of this chemical within a resistance management program should be considered.

Imidacloprid is a well-known pesticide and it is timely to evaluate its toxicity to earthworms ( Eisenia fetida ). In the present study, the effect of imidacloprid on reproduction, growth, acetylcholinesterase (AChE) and DNA damage in earthworms was assessed using an artificial soil medium. The median lethal concentration (LC 50 ) and the median number of hatched cocoons (EC 50 ) of imidacloprid to earthworms was 3.05 and 0.92 mg/kg respectively, the lowest observed effect concentration of imidacloprid about hatchability, growth, AChE activity and DNA damage was 0.02, 0.5, 0.1 and 0.5 mg/kg, respectively.