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Plant viruses can facilitate their transmission by modulating the sex ratios of their insect vectors. Previously, we found that exposure to tomato spotted wilt orthotospovirus (TSWV) in the western flower thrips, Frankliniella occidentalis, led to a male-biased sex ratio in the offspring. TSWV, a generalist pathogen with a broad host range, is transmitted primarily by F. occidentalis in a circulative-propagative manner. Here, we integrated proteomic tools with RNAi to comprehensively investigate the genetic basis underlying the shift in vector sex ratio induced by the virus.Proteomic analysis exhibited 104 differentially expressed proteins between F. occidentalis adult males with and without TSWV. The expression of the fiber sheath CABYR-binding-like (FSCB) protein, namely FoFSCB-like, a sperm-specific protein associated with sperm capacitation and motility, was decreased by 46%. The predicted FoFSCB-like protein includes 10 classic Pro-X-X-Pro motifs and 42 phosphorylation sites, which are key features for sperm capacitation. FoFSCB-like expression was gradually increased during the development and peaked at the pupal stage. After exposure to TSWV, FoFSCB-like expression was substantially down-regulated. Nanoparticle-mediated RNAi substantially suppressed FoFSCB-like expression and led to a significant male bias in the offspring.These combined results suggest that down-regulation of FoFSCB-like in virus-exposed thrips leads to a male-biased sex ratio in the offspring. This study not only advances our understanding of virus-vector interactions, but also identifies a potential target for the genetic management of F. occidentalis, the primary vector of TSWV, by manipulating male fertility. © 2022 Society of Chemical Industry.

Cytochrome P450 monooxygenases (P450s) are well-known for their crucial roles in the detoxification of xenobiotics. However, whether CYP6CX2 and CYP6CX3, 2 genes from our Bemisia tabaci (B. tabaci) MED/Q genome data were associated with detoxification metabolism and confer resistance to thiamethoxam is unclear. In this study, we investigated the role of CYP6CX2 and CYP6CX3 in mediating whitefly thiamethoxam resistance. Our results showed that mRNA levels of CYP6CX2 and CYP6CX3 were up-regulated after exposure to thiamethoxam. Transcriptional levels of 2 genes were overexpressed in laboratory and field thiamethoxam resistant strains by RT-qPCR. These results indicate that the enhanced expression of CYP6CX2 and CYP6CX3 appears to confer thiamethoxam resistance in B. tabaci. Moreover, linear regression analysis showed that the expression levels of CYP6CX2 and CYP6CX3 were positively correlated with thiamethoxam resistance levels among populations. The susceptibility of whitefly adults was markedly increased after silencing 2 genes by RNA interference (RNAi) which further confirming their major role in thiamethoxam resistance. Our findings provide information to better understand the roles of P450s in resistance to neonicotinoids and suggest that these genes may be applied to develop target genes for sustainable management tactic of agricultural pests such as B. tabaci.

Orius sauteri (Poppius) (Hemiptera: Anthocoridae) is often used for biological control of small arthropod pests in greenhouse vegetable production systems in Asia. In addition to feeding on arthropod prey, O. sauteri consumes small quantities of plant material. Previous studies demonstrated that tomato plant chemistry confers antixenosis resistance to phloem-feeding whiteflies, but the potential nontarget effects of phytochemicals on the beneficial predator O. sauteri are unknown. Comparison of O. sauteri confined to near-isogenic lines (NILs) of tomatoes producing high levels of flavonoids (NIL-purple hypocotyl; resistant to whiteflies) and low levels of flavonoids (NIL-green hypocotyl; susceptible to whiteflies) revealed that O. sauteri had reduced oviposition, nymphal survival, and development on resistant plants, even if they were also provided with prey that did not feed on the host plant. Moreover, O. sauteri showed a significant ovipositional preference in choice assays, laying significantly more eggs on susceptible than on resistant plants. Molecular gut content analysis using the specific chloroplast trnL gene from tomato confirmed that adult and immature O. sauteri feed on both resistant and susceptible genotypes, and feeding behavior assays revealed that resistance did not affect plant feeding or prey acceptance by O. sauteri adults. These results demonstrate a direct negative effect of phytochemicals on a nontarget beneficial species and indicate that resistance mediated by phytochemicals can affect organisms that do not solely feed on phloem sap. The results also indicate that the mode of action and the potential ecological effects of phytochemical-mediated resistance are broader than previously recognized.

The sweetpotato whitefly, Bemisia tabaci species complex, comprises at least 44 morphologically indistinguishable cryptic species, whose endosymbiont infection patterns often varied at the spatial and temporal dimension. However, the effects of ecological factors (e.g., climatic or geographical factors) on the distribution of whitefly and the infection frequencies of their endosymbionts have not been fully elucidated. We, here, analyzed the associations between ecological factors and the distribution of whitefly and their three facultative endosymbionts (Candidatus Cardinium hertigii, Candidatus Hamiltonella defensa, and Rickettsia sp.) by screening 665 individuals collected from 29 geographical localities across China. The study identified eight B. tabaci species via mitochondrial cytochrome oxidase I (mtCOI) gene sequence alignment: two invasive species, MED (66.9%) and MEAM1 (12.2%), and six native cryptic species (20.9%), which differed in distribution patterns, ecological niches, and high suitability areas. The infection frequencies of the three endosymbionts in different cryptic species were distinct and multiple infections were relatively common in B. tabaci MED populations. Furthermore, the annual mean temperature positively affected Cardinium sp. and Rickettsia sp. infection frequencies in B. tabaci MED but negatively affected the quantitative distribution of B. tabaci MED, which indicates that Cardinium sp. and Rickettsia sp. maybe play a crucial role in the thermotolerance of B. tabaci MED, although the host whitefly per se exhibits no resistance to high temperature. Our findings revealed the complex effects of ecological factors on the expansion of the invasive whitefly.

Bradysia odoriphaga (Diptera: Sciaridae) is a major insect pest of seven plant families including 30 commercial crops in Asia. The long-term use of chemical pesticides leads to problems such as insect resistance, environmental issues, and food contamination. Against this background, a novel pest control method should be developed. In insects, odorant-binding proteins (OBPs) transport odor molecules, including pheromones and plant volatiles, to olfactory receptors. Here, we expressed and characterized the recombinant B. odoriphaga OBP BodoOBP10, observing that it could bind the sulfur-containing compounds diallyl disulfide and methyl allyl disulfide with Ki values of 8.01 μM and 7.00 μM, respectively. Homology modeling showed that the BodoOBP10 3D structure was similar to that of a typical OBP. Both diallyl disulfide and methyl allyl disulfide bound to the same site on BodoOBP10, mediated by interactions with six hydrophobic residues Met70, Ile75, Thr89, Met90, Leu93, and Leu94, and one aromatic residue, Phe143. Furthermore, silencing BodoOBP10 expression via RNAi significantly reduced the electroantennogram (EAG) response to diallyl disulfide and methyl allyl disulfide. These findings suggest that BodoOBP10 should be involved in the recognition and localization of host plants.

Thermal stress usually leads to excessive production of reactive oxygen species (ROS) in all aerobic organisms. Catalases (CAT) are the key antioxidant enzymes, which act as the first line of defense against ROS in the antioxidant pathway. The highly invasive and widely distributed whitefly Bemisia tabaci MED damages plants by feeding as well as by transmitting many plant viruses. Previous studies have shown that strong adaptability to high temperature helps explain the spread of MED around the world. However, the mechanism underlying high temperature adaptation of this pest is not well understood. In this study, six CAT genes were identified from the MED genome and transcriptome dataset, among which BtCAT1, BtCAT2, and BtCAT3 were found to be highly expressed in adults. The expression of BtCAT1, BtCAT2, or BtCAT3 increased with induction temperature and induction time. The MED was exposed with mean high temperature (30°C or 35°C) and a short-term extremely high temperature (39°C or 41°C) after the silencing of BtCAT1, BtCAT2, or BtCAT3 to significantly increased ROS levels by at least 0.5 times and significantly decreased survival rate and fecundity of MED adults. The ROS level in the treated specimens gradually returned to a normal level after 24 h at 25°C, but the survival rate still declined significantly. Taken together, our results demonstrate that CAT could help B. tabaci adapt to long-term mean high temperatures and short-term extremely high temperatures by eliminating excessive ROS. This article is protected by copyright. All rights reserved.

Detoxification plays a crucial role in agricultural pests to withstand the pesticides, and cytochrome P450s, carboxyl/cholinesterases (CCEs), and glutathione-S-transferases are the main proteins responsible for their detoxification ability. The activity of CCEs can be up-regulated, down-regulated, or modified by mutation. However, few studies have examined the role of alternative splicing in altering the properties of CCEs. We identified two variants of TcCCE23 in Tetranychus cinnabarinus: a long version (CCE23-V1) and a short version that is 18 nucleotides shorter than CCE23-V1 (CCE23-V2). Whether splicing affects the activity of TcCCE23 remains unclear. Overexpression of CCE23-V2 in fenpropathrin resistant T. cinnabarinus revealed that splicing affected the detoxification of fenpropathrin by CCE23-V2. The mortality of mites was significantly higher when the expression of CCE23-V2 was knocked down (43.2% ± 3.3%) via injection of CCE23-dsRNA compared with the control group injected with GFP-dsRNA under fenpropathrin exposure; however, the down-regulation of CCE23-V1 (61.3 ± 6.3%) by CCE23-siRNA had no such effect, indicating CCE23-V2 plays a greater role in xenobiotic metabolism than CCE23-V1. The tolerance of flies overexpressing CCE23-V2 to fenpropathrin (LD

Tomato spotted wilt orthotospovirus (TSWV) causes substantial economic losses to vegetables and other crops. TSWV is mainly transmitted by thrips in a persistent and proliferative manner, and its most efficient vector is the western flower thrips, Frankliniella occidentalis (Pergande). In moving from the thrips midgut to the salivary glands in preparation for transmission, the virions must overcome multiple barriers. Although several proteins that interact with TSWV in thrips have been characterized, we hypothesized that additional thrips proteins interact with TSWV and facilitate its transmission. In the current study, 67 F. occidentalis proteins that interact with G

BACKGROUND Use of RNA interference (RNAi) technology in effective pest management has been explored for decades. Henosepilachna vigintioctopunctata is a major solanaceous crop pest in Asia. In this study, the effects of the RNAi-mediated silencing of clathrin heavy chain in H. vigintioctopunctata were investigated. RESULTS Feeding either the in vitro-synthesized or the bacterially expressed double-stranded RNAs (dsRNAs) significantly impaired the normal physiology of H. vigintioctopunctata instars and adults. However, the bacterially expressed dsHvChc caused higher mortality than the in vitro-synthesized ones in the larvae and adults. Moreover, on evaluating the potential risk of dsHvChc on Propylea japonica, significant transcriptional effects of dsHvChc1 were observed, while the organismal level effects were not significant. On the contrary, dsHvChc2 did not affect P. japonica at either level. A similar test revealed significant transcriptional effects of dsPjChc1 on H. vigintioctopunctata, while staying ineffective at the organismal levels. Conversely, dsPjChc2 did not affect H. vigintioctopunctata at either level. Importantly, no effect of dsPjChc1 exposure on H. vigintioctopunctata suggested that other factors besides the 21-nucleotide (nt) matches between sequences were responsible. Finally, ingestion of dsHvmChc1 derived from H. vigintioctomaculata, containing 265-nt matches with dsHvChc1, caused 100% mortality in H. vigintioctopunctata. CONCLUSIONS We conclude that (i) species with numerous 21-nt matches in homologous genes are more likely to be susceptible to dsRNA; (ii) dsRNA can be safely designed to avoid negative effects on non-target organisms at both transcriptional and organismal levels; (iii) HvChc can be used as an efficient RNAi target gene to effectively manage H. vigintioctopunctata.

In most organisms, various physiological and behavioral functions are expressed rhythmically. Previous studies have shown that thermoperiod is an important factor affecting circadian clock-related genes that regulate insect locomotor activity. Bradysia odoriphaga Yang & Zhang is an underground pest that attacks more than 30 crops but is especially damaging to Chinese chives. In this study, we analyzed the adult eclosion time and period (Boper) gene expression in B. odoriphaga as affected by temperature (cycling vs constant temperature), insect stage, and tissue specific. We found that the eclosion time and expression of the Boper gene changed during the temperature cycle but not under a constant temperature. Silencing of Boper expression significantly decreased the adult eclosion rate and significantly increased adult mortality and malformation. The findings indicate that thermoperiod alters Boper expression and regulates the eclosion rhythm.

Backgroud Cardinium symbiont is a maternally inherited bacterial endosymbiont and widely spreads in arthropods including Bemisia tabaci (Hemiptera: Aleyrodidae). However, the potential role of Cardinium played in the biology of their hosts is largely unknown. In two genetic lines (i.e. LS and SG lines) of B. tabaci MED, collected from different locations in China, we tested the effects of Cardinium on the performance of the host whitefly under a constant high temperature (31 °C) using the age-stage two-sex life table method, and explored the genes influenced by Cardinium-infection by RNA-sequencing. Results We found that Cardinium did provide protection of B. tabaci against heat stress under 31 °C. However, there was a significant connection between Cardinium-infection and whitefly genetic backgrounds. Performance revealed that Cardinium infection can increase the longevity of both female and male adults and oviposition periods in both lines, but it also conferred benefits of fecundity and pre-adult period to LS line. Additionally, the population parameters such as intrinsic rate of increase (r), finite rate of increase (λ) and mean generation time (T) demonstrated that Cardinium infection conferred fitness benefits to LS line but not to SG line. Transcriptome analysis indicated that several genes related to homeostasis and metamorphosis such as ubiquitin-related genes were highly expressed in Cardinium-infected B. tabaci. Conclusion The research provided the first evidence that Cardinium can increase the thermal tolerance of whitefly, which may be associated with host genetic background.

The western flower thrips Frankliniella occidentalis, a worldwide agricultural pest, has developed resistance to an array of insecticides. Spinosad resistance confers an apparent fitness cost in F. occidentalis. In the present study, we compared the reproductive capacities, ovary development, and the expression of the vitellogenin (Vg) gene in spinosad-susceptible (Ivf03) and -resistant (NIL-R) near isogenetic lines of F. occidentalis in order to clarify the reason for the fitness cost in spinosad resistance.The NIL-R strain exhibited a 17.9% decrease in fecundity (eggs laid per female) as compared to the Ivf03 strain, and the ovariole was significantly shortened by 2.8% in the NIL-R strain relative to the Ivf03 strain. Compared to the Ivf03 strain, the expression levels of Vg mRNA and protein were downregulated by 33.7% and 32.9% in the NIL-R strain, respectively. Moreover, interference with the Vg gene significantly reduced the expression levels of Vg mRNA and protein, and decreased ovariole length, survival rates and the fecundity of both strains.The results indicate that the downregulated expression of Vg may contribute to the reduction of ovariole length and consequently to a fitness cost in spinosad-resistant F. occidentalis. The results not only increase our understanding of the evolution of insecticide resistance, but also could contribute to the formulation of control strategy of F. occidentalis. © 2022 Society of Chemical Industry.

Bacillus thuringiensis (Bt), a naturally occurring entomopathogenic soil bacterium, has been the active ingredient of sprayable Bt biopesticides for over a century. Insecticidal Bt Cry proteins are particularly well suited for use as plant-incorporated protectants in transgenic crops (Bt crops) due to their specificity against insect pests and safety for non-target organisms. The sustainability of these Bt products, however, has been challenged by the development of resistance in the field. RNA interference (RNAi), a species-specific control alternative that has been deregulated and commercialized in the USA and Canada, provides a new mode of action to complement the existing Bt products. Based on our preliminary research, we hypothesized that pyramiding/integrating Bt with RNAi can address/manage resistance issues related to Bt traits. To examine this overarching hypothesis, we (1) cloned and characterized a serine/threonine kinase gene (fused) of the Hedgehog (Hh) signaling pathway in the diamondback moth, Plutella xylostella (L.), a global superpest; (2) profiled Pxfused expression in Bt-resistant P. xylostella strains; and (3) investigated the involvement of Pxfused in Bt Cry1Ac resistance in P. xylostella. Pxfused expression was elevated ubiquitously in all Bt-resistant strains, and silencing of Pxfused led to larval and pupal mortality in both Cry1Ac-susceptible and -resistant strains, suggesting that Pxfused is a potential target for RNAi-based resistance management. Taken together, our results not only identify a molecular target to control a devastating lepidopteran pest, but also shed light on a novel resistance management strategy through the integration of two biotechnological techniques with distinct modes of action.

Tyrosine hydroxylase (TH), a melanin synthesis pathway enzyme hydroxylating tyrosine into 3,4-dihydroxyphenylalanine, is involved in the pigmentation and sclerotization of insect cuticles. However, the role of TH in 28-spotted potato ladybeetle (Henosepilachna vigintioctopunctata), an emerging pest of the solanaceous crops has been explored to a limited extent. In this study, we integrated dietary RNA interference (RNAi) and hematoxylin and eosin (HE) staining with various bioassays to analyze the role of tyrosine hydroxylase (HvTH) throughout the developmental processes of Henosepilachna vigintioctopunctata.The results revealed that ingestion of dsHvTH led to cuticle tanning impairment, arrested larval feeding in the first and second instars of Henosepilachna vigintioctopunctata, and subsequently resulted in 100% mortality. The HE staining assays revealed that dsHvTH prevented new abdominal cuticle formation. A pharmacological study using 3-iodo-tyrosine (3-IT), a HvTH inhibitor, disrupted larval-larval-pupal cuticle tanning during the third-fourth instar larval development and eventually failed to pupate. Similarly, dsHvTH fed to fourth instars hindered larval-pupal-adult cuticle tanning, and the eclose adults were 100% malformed. Ingestion of dsHvTH or 3-IT significantly down-regulated HvTH, HvDDC, Hvebony, and Hvlaccase2 expression and reduced dopamine levels. Finally, HvTH silencing in adult females substantially reduced the offspring hatching rates.The collective results of the study suggested that HvTH plays conserved roles in larval-pupal-adult cuticle melanization and sclerotization while exhibiting a novel function in Henosepilachna vigintioctopunctata reproduction. © 2022 Society of Chemical Industry.

The diamondback moth, Plutella xylostella L., is a worldwide crop pest that is difficult to control because of its ability to develop resistance to many insecticides. To provide a reference for resistance management of P. xylostella in China, the present study used a leaf-dip bioassay to monitor the resistance of P. xylostella to nine insecticides in eight regions of China. The results showed that P. xylostella had developed a high level of resistance to beta-cypermethrin (resistance ratio [RR] > 112), and moderate (RR < 40) to high levels of resistance to indoxacarb, abamectin, and chlorfluazuron. For chlorantraniliprole, RRs > 100 were found in Midu (Yunnan Province) and Jinghai (Tianjin). In most regions, the resistance to spinetoram and chlorfenapyr and Bacillus thuringiensis (Bt) was low. No resistance was detected to diafenthiuron. Overall, P. xylostella resistance to insecticides was higher in Midu than in other regions. The data in this study should help guide the selection of insecticides for management of P. xylostella in China.

Background Over recent decades, many efficacious insecticides have been applied for control of Bemisia tabaci, one of the most notorious insect pests worldwide. Field-evolved insecticide resistance in B. tabaci has developed globally, but remains poorly understood in China. Results In this study, a total of 30 field samples of the whitefly Bemisia tabaci from eight provinces of China were collected in 2015 to 2018. Twenty-four of the populations were identified as Mediterranean, 'Q' type (MED), three were Middle East-Asia Minor 1, 'B' type (MEAM1), and three were mixtures of MED/ MEAM1. After identifying whether they belong to MED or MEAM1, the selected individuals were used in bioassays assessing insecticide resistance to abamectin, thiamethoxam, spirotetramat, cyantraniliprole, and pyriproxyfen. Our results showed that all populations in the eight regions had little or no resistance to abamectin; abamectin resistance was highest in the Hunan (Changsha) and Hubei (Wuhan) regions and was lowest in the island region of Hainan (Sanya). The resistance of B. tabaci to spirotetramat, cyantraniliprole, and pyriproxyfen increased each year. The resistance to thiamethoxam remained low because of the high LC50 value for the laboratory strain. Conclusion These findings suggest that a rotation system using efficacious B. tabaci insecticides with differing mode of actions ought to be implemented for sustainable control to reduce the potential of resistance development. This study provides important data to support the integrated pest management and insecticide resistance management of B. tabaci in China. © 2021 Society of Chemical Industry.

RNA interference (RNAi) has emerged as a powerful tool for developing novel management strategies for controlling insect pests. The 28-spotted ladybeetle, Henosepilachna vigintioctopunctata is one of the most important pests attacking solanaceous plants in Asia. In this study, the potential of dietary RNAi to manage H. vigintioctopunctata was investigated using both in vitro synthesized and bacterially expressed double-stranded RNAs (dsRNAs) of HvvATPase A and HvvATPase E. The expression levels of HvvATPase A and HvvATPase E were higher in Malpighian tubules than in other tissue types. The silencing of HvvATPase A and HvvATPase E led to significant mortality in H. vigintioctopunctata larvae. In addition, the ingestion of HvvATPase A and HvvATPase E significantly deterred feeding behavior and subsequently arrested the development of H. vigintioctopunctata. Notably, the bacterially expressed dsRNAs consistently caused higher mortality in larvae and adults. Finally, the nontarget effects of the dsRNAs of H. vigintioctopunctata on the predatory ladybeetle Propylaea japonica were evaluated. P. japonica 1st instar larvae were administered vATPase A and vATPase E dsRNAs from H. vigintioctopunctata and P. japonica under the worst-case scenario, in which dsGFP served as negative control. There were significant effects of dsHvvATPase A on P. japonica at the transcriptional level but not at the organismal level, whereas dsHvvATPase E did not effect P. japonica at either the transcriptional or the organismal level. Collectively, the results of the study suggest that HvvATPase A and HvvATPase E can act as novel molecular targets for the control of H. vigintioctopunctata.

The invasive pest whitefly (Bemisia tabaci) is a complex species, of which Middle East-Minor Asia 1 (MEAM1) and Mediterranean (MED) are the two most damaging members. Previous research showed that cabbage is frequently infested with MEAM1 but seldomly with MED, and this difference in performance is associated with glucosinolate (GS) content. Some insects can modify GS using glucosinolate sulfatase (SULF), the activity of which is regulated by sulfatase modifying factor 1 (SUMF1); therefore, to increase our understanding of different performances of MEAM1 and MED on cabbage plants, we identified and compared nine putative SULFs and one SUMF in MEAM1 and MED. We found that the lengths of two genes, BtSulf2 and BtSulf4, differed between MEAM1 and MED. The messenger RNA levels of BtSulf4 increased more than 20-fold after MEAM1 and MED adults were exposed to GS, but BtSulf2 expression was only induced by GS in MEAM1. Knockdown of BtSulf2 and BtSulf4 in MEAM1 resulted in a substantial increase in the mortality of GS-treated adults but not in MED. These results indicate that differences in BtSulf2 and BtSulf4 sequences and/or expression may explain why MEAM1 performs better than MED on cabbage. Our results provide a basis for future functional research on SULF and SUMF in B. tabaci.

Species displacement by the outcome of interspecific competition is of particular importance to pest management. Over the past decade, spinetoram has been extensively applied in control of the two closely related thrips Megalurothrips usitatus and Frankliniella intonsa worldwide, while whether its resistance is implicated in mediating interspecific interplay of the two thrips remains elusive to date.Field population dynamics (from 2017 to 2019) demonstrated a trend toward displacement of F. intonsa by M. usitatus on cowpea crops, supporting an existing interspecific competition. Following exposure to spinetoram, M. usitatus became the predominate species, which suggests the use of spinetoram appears to be responsible for mediating interspecific interactions of the two thrips. Further annual and seasonal analysis (from 2016 to 2020) of field-evolved resistance dynamics revealed that M. usitatus developed remarkably higher resistance to spinetoram compared to that of F. intonsa, implying a close relationship between evolution of spinetoram resistance and their competitive interactions. After 12 generations of laboratory selection, resistance to spinetoram in M. usitatus and F. intonsa increased up to 64.50-fold and 28.33-fold, and the average realized heritability (hCollectively, our results uncover the involvement of insecticide resistance in conferring displacement mechanism behind interspecific competition, providing a framework for understanding the significance of the evolutionary relationships among insects under ongoing changing environments. These findings also can be invaluable in proposing the most appropriate strategies for sustainable thrips control programs. © 2022 Society of Chemical Industry.

RNA interference (RNAi) techniques have emerged as powerful tools that facilitate development of novel management strategies for insect pests, such as Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae), which is a major pest of solanaceous plants in Asia. In this study, the potential of oral delivery of in vitro-synthesized and bacterially expressed double-stranded H. vigintioctopunctata lesswright (lwr) gene (dsHvlwr) to manage of H. vigintioctopunctata was investigated. Our results showed that the gene Hvlwr had a 480-bp open reading frame and encoded a 160-amino acid protein. Hvlwr expression levels were greater in the fat body than other tissue types. Hvlwr silencing led to greater H. vigintioctopunctata mortality rates and appeared to be time- and partially dose-dependent, likely as a result of the number of hemocytes increasing with dsRNA concentration, but decreasing with time. Bacterially expressed dsHvlwr that was applied to leaf discs caused 88%, 66%, and 36% mortality in 1st instars, 3rd instars, and adults after 10, 10, and 14 d, respectively; when applied to living plants, there was greater mortality in 1st and 3rd instars, but there was no effect on adults. Furthermore, dsHvlwr led to improved plant protection against H. vigintioctopunctata. Our study shows an effective dietary RNAi response in H. vigintioctopunctata and that Hvlwr is a promising RNAi target gene for control of this pest species.

The production of Chinese chives is reduced throughout China due to a root-feeding dipteran pest Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae), therefore deciphering the conditions influencing its growth and development are important in developing ecological control strategies. A study was conducted from 2014 to 2017 to determine the relationship between the abundance of B. odoriphaga and temperature (atmospheric and soil), soil water content, and atmospheric humidity in a Chinese chive field in Beijing City, China. Numbers of adults peaked in March and October to November and were lowest in July to August and December to next February; numbers of larvae were highest in December to next February and lowest in July to August. From 2014 to 2017, the numbers of adults and larvae were significantly correlated with monthly mean atmospheric temperatures and soil temperatures, but were not significantly correlated with monthly mean atmospheric relative humidity and soil water content. However, for both adults and larvae, numbers were significantly greater with high soil water contents compared with drought treatment. The results of this study suggest that the very low soil water contents, high atmospheric temperatures, and high soil temperatures were critical for regulating field populations of B. odoriphaga.

MEAM1 (Middle East-Asia Minor 1, "B" biotype) and MED (Mediterranean, "Q" biotype) are the two most destructive cryptic species of the Bemisia tabaci complex on the planet. Our previous studies have shown that MEAM1 outcompetes MED on cabbage; the underlying mechanism is unknown. In the Brassicaceae family, the glucosinolate-myrosinase defense system plays a crucial role in deterring feeding, inhibiting growth, and causing acute toxicity against a wide range of generalist herbivores. In the present study, we first compared the survival of MEAM1 and MED exposed to sinigrin (a glucosinolate) and myrosinase (an enzyme that degrades glucosinolates); we found that survival of both species was high in response to sinigrin alone but was near zero in response to sinigrin + myrosinase. We then used electropenetrography (electrical penetration graphs, EPG) to assess the feeding behaviors of MEAM1 and MED whiteflies on cabbage. The EPG results revealed that the mean duration of each potential drop (pd, indicating an intracellular puncture) was substantially longer for MED than MEAM1 on cabbage, indicating that the exposure to the toxic hydrolysates of glucosinolate and myrosinase is greater for MED than for MEAM1. We therefore conclude that differences in penetrating behaviors may help explain the different effects of cabbage on MEAM1 and MED whitefly species.

Background RNA interference (RNAi) is a potential tool for plant protection against insect pests. The great challenge for effective pest control using RNAi in the field is the development of efficient and reliable methods for the production and delivery of double-stranded RNA (dsRNA). Results In the present study, we investigated the potential of feeding in vitro synthesized or bacterially expressed dsRNA to populations of the 28-spotted ladybeetle Henosepilachna vigintioctopunctata as a method of biological pest control. Ingestion of in vitro synthesized dsHvRPS18 or dsHvRPL13 led to significant down-regulation of the ribosomal protein-encoding genes HvRPS18 and HvRPL13, respectively, and significantly decreased the survival of H. vigintioctopunctata. Such silencing of HvRPS18 or HvRPL13 expression appeared to be partially dose-dependent and also inhibited the growth of H. vigintioctopunctata and significantly suppressed the expression of digestive enzyme-related genes. Finally, ingestion of bacterially expressed dsHvRPS18 or dsHvRPL13 induced significant mortality in the first and third instars, and in adults. Conclusion The effectiveness of RNAi-based gene silencing in H. vigintioctopunctata provides a powerful reverse genetic tool for the functional annotation of its genes. This study demonstrates that HvRPS18 and HvRPL13 represent candidate genes for RNAi-based biological control of H. vigintioctopunctata. © 2020 Society of Chemical Industry.

Background Nuclear receptors (NRs) play an essential role in diverse biological processes, such as insect metamorphosis. Here, transcriptome analysis and functional studies were used to determine whether NRs are involved in metamorphosis of whitefly Bemisia tabaci Q, a serious pest to crops, and to find some potential insecticide targets. Results Twenty NRs were identified in the Bemisia tabaci Q genome and categorized into the NR0-NR6 subfamilies. The phylogenetic tree of NRs from Bemisia tabaci Q and other representative species was constructed, which provided evolutionary insight into their genetic distances. The results of spatiotemporal gene expression indicated that the majority of NR gene expression was higher in the head than the abdomen and higher in eggs than adults. Further functional analysis using RNA interference (RNAi) showed that NR genes play an important role in Bemisia tabaci Q pupation and eclosion. With respect to high mortality and effects on growth, this was reflected in the unable to become pupa when the third-stage nymph treated with double-stranded RNA (dsRNA) and the developmental time delay (4-7 days) when pupae were treated with dsRNA for the 12 NR genes during molting compared with the development time in the control. Conclusion This study provides insight into NR functions during the metamorphosis stages of Bemisia tabaci Q. Several candidate genes could be potential insecticide targets for whitefly pest control due to their important roles in insect development. © 2020 Society of Chemical Industry.

Global change and biotic stress, such as tropospheric contamination and virus infection, can individually modify the quality of host plants, thereby altering the palatability of the plant for herbivorous insects. The bottom-up effects of elevated O3 and tomato yellow leaf curl virus (TYLCV) infection on tomato plants and the associated performance of Bemisia tabaci Mediterranean (MED) were determined in open-top chambers. Elevated O3 decreased eight amino acid levels and increased the salicylic acid (SA) and jasmonic acid (JA) content and the gene expression of pathogenesis-related protein (PR1) and proteinase inhibitor (PI1) in both wild-type (CM) and JA defense-deficient tomato genotype (spr2). TYLCV infection and the combination of elevated O3 and TYLCV infection increased eight amino acids levels, SA content and PR1 expression, and decreased JA content and PI1 expression in both tomato genotypes. In uninfected tomato, elevated O3 increased developmental time and decreased fecundity by 6.1 and 18.8% in the CM, respectively, and by 6.8 and 18.9% in the spr2, respectively. In TYLCV-infected tomato, elevated O3 decreased developmental time and increased fecundity by 4.6 and 14.2%, respectively, in the CM and by 4.3 and 16.8%, respectively, in the spr2. These results showed that the interactive effects of elevated O3 and TYLCV infection partially increased the amino acid content and weakened the JA-dependent defense, resulting in increased population fitness of MED on tomato plants. This study suggests that whiteflies would be more successful at TYLCV-infected plants than at uninfected plants in elevated O3 levels.

BACKGROUND Whitefly (Bemisia tabaci) is a typical pest that causes severe damage to hundreds of agricultural crops. The trehalose-6-phosphate synthase (TPS) genes, as the key genes in the insect trehalose synthesis pathway, are important for insect growth and development. The whitefly TPS genes may be a main reason for the severe damage and may represent potential targets for the control of whiteflies. RESULTS In this study, we identified and cloned three TPS genes from B. tabaci MED and found that the BtTPS1 and BtTPS2 genes showed higher expression levels than the BtTPS3 gene. Then, RNA interference (RNAi) of BtTPS1 and BtTPS2 resulted in significant mortality and influenced the expression of related genes involved in energy metabolism and chitin biosynthesis in whitefly adults. Finally, the transgenic tobacco plants showed a significant effect on B. tabaci, and knockdown of BtTPS1 or BtTPS2 led to retarded growth and low hatchability in whitefly nymphs, and caused 90% mortality and decreased the fecundity in whitefly adults. Additionally, the transgenic tobacco with combinatorial RNAi of BtTPS1 and BtTPS2 showed a better efficacy against whiteflies than individual silencing. CONCLUSION Our results suggest that silencing of the BtTPS genes can compromise the growth and development of whiteflies, offering not only a new option for whitefly control but also a secure and environmentally friendly management strategy.

The belowground pest Bradysia odoriphaga (Diptera: Sciaridae) has a sophisticated and sensitive olfactory system to detect semiochemical signals from the surrounding environment. In particular, odorant-binding proteins (OBPs) are crucial in capturing and transporting these semiochemical signals across the sensilla lymph to the corresponding odorant receptors. In this study, we cloned a full-length cDNA sequence of BodoOBP8 from B. odoriphaga. Real-time PCR (qRT-PCR) analysis revealed that BodoOBP8 has the highest expression levels in males, with more pronounced expression in the male antennae than in other tissues. In this study, the recombinant protein BodoOBP8 was successfully expressed by a bacterial system to explore its function. Competitive binding assays with 33 host plant volatiles and one putative sex pheromone (n-heptadecane) revealed that purified BodoOBP8 strongly bound to two sulfur compounds (methyl allyl disulfide and diallyl disulfide) and to n-heptadecane, the corresponding dissolution constants (Ki) were 4.04, 6.73, and 4.04 μM, respectively. Molecular docking indicated that Ile96, Ile103, Ala107, and Leu111, located in the hydrophobic cavity of BodoOBP8, are the key residues mediating the interaction of BodoOBP8 with two sulfur compounds (methyl allyl disulfide and diallyl disulfide) and n-heptadecane. These results show that BodoOBP8 plays a role in the recognition of plant volatiles and sex pheromones, suggesting its application as a molecular target for the screening of B. odoriphaga attractants and repellents and facilitating a new mechanism of B. odoriphaga control.

BACKGROUND Tomato chlorosis virus (ToCV) is mainly vectored by Bemisia tabaci in China, which has a worldwide distribution, and greatly reduces the yields of tomato and other vegetables. At present, control of ToCV has been focused mainly by the use of insecticides to control whitefly populations. Transcriptome sequencing showed high expression of the B. tabaci Bta11975 gene, an α-glucosidase (AGLU) during ToCV acquisition by whitefly Mediterranean (MED) species. To investigate the role of Bta11975 gene in ToCV acquisition and transmission by B. tabaci MED, we used RNA interference (RNAi) to reduce the expression of the Bta11975 gene. RESULTS The relative expression of the Bta11975 gene was correlated with the ToCV content in B. tabaci. The AGLU is highly expressed in primary salivary gland and gut. After the Bta11975 gene was silenced, the gene expression of B. tabaci was reduced and B. tabaci mortality was increased. Besides, ToCV acquisition by B. tabaci at 48 and 72 h AAP was reduced, and ToCV transmission was significantly reduced by 25 or 50 of B. tabaci. CONCLUSIONS These results indicate that suppression of expression of the Bta11975 gene in B. tabaci MED by RNAi can reduce acquisition and transmission of ToCV by B. tabaci MED.

Background Rapid evolution of pest resistance has seriously threatened the sustainable use of Bacillus thuringiensis (Bt). The diamondback moth, Plutella xylostella (L.), is the first pest to develop resistance to Bt biopesticides in the open field, which renders it an excellent model to explore the molecular basis of Bt resistance in insects. Our previous midgut transcriptome and RNA-Seq profiles showed that the P-glycoprotein gene PxABCB1 was down-regulated in two Cry1Ac-resistant P. xylostella strains, suggesting its potential involvement in Cry1Ac resistance in P. xylostella. Results In this study, the bona fide full-length cDNA sequence of the PxABCB1 gene was cloned and analyzed, and the expression of the PxABCB1 gene was detected in all tissues and developmental stages, with the highest expression in midgut tissue and the female adult stage. Although no consistent non-synonymous mutations were identified between the susceptible and resistant strains, PxABCB1 gene expression was remarkably decreased in all resistant strains, and the association was further validated by Cry1Ac selection in the moderately resistant SZ-R strain. Moreover, knockdown of the PxABCB1 gene expression resulted in significantly reduced larval susceptibility to Cry1Ac toxin in the DBM1Ac-S strain, and decreased expression of the PxABCB1 gene was tightly linked to Cry1Ac resistance in P. xylostella. Conclusion Our results demonstrated that down-regulation of the PxABCB1 gene is associated with both laboratory-selected and field-evolved Cry1Ac resistance in P. xylostella. This knowledge will be conducive to further elucidating the complicated molecular basis of Bt resistance and developing new insect resistance management tactics. © 2019 Society of Chemical Industry.

Bradysia odoriphaga Yang & Zhang (Diptera: Sciaridae) is an important pest of Chinese chives. Information on the effects of biotic and abiotic factors on the flight performance of B. odoriphaga is crucial for understanding the pest’s ability to disperse and migrate. In this study, the effects of sex and air temperature on the flight performance of B. odoriphaga imagoes were assessed by tethering individual imagoes to computerized flight mills for a 10-h experiment. The results showed that the percentage of imagoes that flew a particular distance gradually decreased as flight distance increased. The percentage of imagoes was significantly higher for males than females when the flight distance was

Background As a method of RNA-mediated gene silencing, RNA interference (RNAi) is a useful reverse genetic tool with which to study gene function, and holds great promise for pest management. Bemisia tabaci is a cosmopolitan pest that causes extensive damage to crops. The mechanism underlying RNAi efficiency in B. tabaci is not well known. We identified and analyzed candidate genes in the RNAi pathway to understand the RNAi mechanism and provide a basis for the application of RNAi in pest management. Results We identified 33 genes putatively involved in the RNAi pathway from the B. tabaci Q genome. Phylogenetic and structural analyses confirmed the characteristics of these genes. Furthermore, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and transcriptomic analysis profiled gene expression patterns during different developmental stages. Gene expression levels estimated by qRT-PCR and RNA-seq analyses were significantly correlated. Moreover, gene functions were verified by RNAi. When accompanied by knockdown of AGO2, Dicer2 and Sid1, the efficiency of CYP6DB3 RNAi decreased correspondingly. Conclusion In this study, we annotated and validated genes involved in B. tabaci RNAi. A better understanding of the building blocks of the RNAi process in B. tabaci facilitates integration of this novel biotechnology into the management of this emerging pest, either directly or indirectly. © 2019 Society of Chemical Industry.

Bradysia odoriphaga Yang et Zhang is a serious belowground pest of Chinese chives (Allium tuberosum). Our previous studies have indicated that B. odoriphaga females prefer to oviposit near the roots of Chinese chives rather than the roots of other plants, and that the performance (longevity and fecundity) of B. odoriphaga offspring was better on Chinese chives than on Lettuce (var. ramosa Hort.), Onion (Allium cepa) and Potato (Solanum tuberosum) but little is known about how the volatiles released by Chinese chives affect the host-finding and oviposition behaviors of B. odoriphaga. Here, we used gas chromatography-mass spectrometry and determined that Chinese chives releases the following volatiles: methyl allyl disulfide, β-myrcene, cis-ocimene, diallyl disulfide, nonane, n-dodecane, n-tetradecane, and n-hexadecane; quantities released were highest for methyl allyl disulfide and diallyl disulfide. In addition to eliciting strong responses in females in electroantennography assays, the latter two sulfur compounds and their mixtures attracted females in Y-tube olfactometer assays. The addition of methyl allyl disulfide, diallyl disulfide, or a mixture of the two compounds at a 1:5 ratio to chive plants increased oviposition when compared to control plants. These results indicate that methyl allyl disulfide and diallyl disulfide, either alone or in combination, influence the host-seeking behavior of B. odoriphaga.

Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play essential roles in insect chemosensory recognition. Here, we identified nine OBPs and nine CSPs from the Myzus persicae transcriptome and genome. Genomic structure analysis showed that the number and length of the introns are much higher, and this appears to be a unique feature of aphid OBP genes. Three M. persicae OBP genes (OBP3/7/8) as well as CSP1/4/6, CSP2/9 and CSP5/8 are tandem arrayed in the genome. Phylogenetic analyses of five different aphid species suggest that aphid OBPs and CSPs are conserved in single copy across all aphids (with occasional losses), indicating that each OBP and CSP class evolved from a single gene in the common ancestor of aphids without subsequent duplication. Motif pattern analysis revealed that aphid OBP and CSP motifs are highly conserved, and this could suggest the conserved functions of aphid OBPs and CSPs. Three OBPs (MperOBP6/7/10) are expressed antennae specifically, and five OBPs (MperOBP2/4/5/8/9) are expressed antennae enriched, consistent with their putative olfactory roles. M. persicae CSPs showed much broader expression profiles in nonsensory organs than OBPs. None of the nine MperCSPs were found to be antennae specific, but five of them (MperCSP1/2/4/5/6) showed higher expression levels in the legs than in other tissues. MperCSP10 mainly expressed in the antennae and legs. The broad and diverse expression patterns of M. persicae CSPs suggest their multifunctions in olfactory perception, development and other processes.

Many damaging agricultural pests can, in addition to their direct feeding damage, acquire and transmit plant pathogens. Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is considered a ‘supervector’ of disease-causing plant pathogens and viruses. One of the most damaging of these is Tomato yellow leaf curl virus (TYLCV), a circulatively transmitted begomovirus than can extensively damage field and greenhouse crops. Because sustained feeding periods are necessary to acquire and transmit circulatively transmitted viruses, pesticides that, in addition to their direct lethality, suppress feeding in surviving individuals may be particularly effective in decreasing viral transmission. We assessed the impact of sulfoxaflor, a sulfoximine insecticide, on the settling preference, feeding, and viral transmission of TYLCV-carrying B. tabaci on tomato. We found that viruliferous B. tabaci avoided both settling and feeding on sulfoxaflor-treated plants, and that sulfoxaflor virtually eliminated the transmission of TYLCV by B. tabaci. The antifeedant properties of sulfoxaflor have previously been reported in other pest systems; our results document similar effects on viruliferous B. tabaci and demonstrate that this pesticide can reduce TYLCV transmission by surviving individuals.

The sweetpotato whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), is a broadly invasive pest of great economic significance to agricultural and horticultural systems in the world. Bemisia tabaci contains more than 44 cryptic species, among which B. tabaci Middle East Asia Minor 1 (MEAM1) and Mediterranean (MED) are the most invasive and destructive, and have spread to many countries. Research on B. tabaci cryptic species increased dramatically in China following the introduction of MEAM1 in the middle-late 1990s. Our report discussed significant advances over the past two decades to understand the invasive genetics of B. tabaci in China. We reviewed the main findings including changes in the cryptic species composition and invasion characteristics of B. tabaci, also summarizing the factors that affect the displacement of dominant cryptic species. Finally, the integrated management of B. tabaci in China was discussed. We hope that this review can stimulate interest in and deepen understanding of B. tabaci, which will lay a foundation for further research in this field.

Pesticides primarily affect target organisms via direct toxicity, but may also alter the feeding behaviors of surviving individuals in ways that alter their effect on host plants. The latter impact is especially important when pests can transmit plant pathogens. The Mediterranean (MED) population of the sweetpotato whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) transmits Tomato yellow leaf curl virus (TYLCV), a pathogen that can be economically devastating in field and greenhouse cropping systems. We first assessed the impact of sublethal (LC15) and label concentrations of flupyradifurone, a butenolide-derived insecticide, on the feeding behavior of TYLCV-infected MED on tomato. We next measured the effect of flupyradifurone on plant TYLCV load, vector transmission efficiency, and MED survival. Both the LC15 and label flupyradifurone concentrations dramatically altered MED feeding and caused the near cessation of both salivation and phloem ingestion (necessary for viral transmission and acquisition, respectively). Both concentrations also significantly reduced plant TYLCV load, and the label rate of flupyradifurone sharply decreased TYLCV transmission while killing >99% of MED. As the first report of pesticide-induced changes in the feeding behavior of viruliferous Bemisia, our findings highlight the potential importance of chemically driven feeding cessation in the control of TYLCV and other Bemisia-transmitted plant pathogens.

Mating triggers substantial changes in gene expression and leads to subsequent physiological and behavioral modifications. However, postmating transcriptomic changes responding to mating have not yet been fully understood. Here, we carried out RNA sequencing (RNAseq) analysis in the sweet potato whitefly, Bemisia tabaci MED, to identify genes in females in response to mating. We compared mRNA expression in virgin and mated females at 24 h. As a result, 434 differentially expressed gene transcripts (DEGs) were identified between the mated and unmated groups, including 331 up- and 103 down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that many of these DEGs encode binding-related proteins and genes associated with longevity. An RT-qPCR validation study was consistent with our transcriptomic analysis (14/15). Specifically, expression of P450s (Cyp18a1 and Cyp4g68), ubiquitin-protein ligases (UBR5 and RNF123), Hsps (Hsp68 and Hsf), carboxylase (ACC-2), facilitated trehalose transporters (Tret1-2), transcription factor (phtf), and serine-protein kinase (TLK2) were significantly elevated in mated females throughout seven assay days. These combined results offer a glimpe of postmating molecular modifications to facilitate reproduction in B. tabaci females.

Background The two-spotted spider mite Tetranychus urticae is a polyphagous and cosmopolitan pest that has developed high resistance to abamectin, making it difficult to control. Although 'target resistance' related to glutamate-gated chloride channel mutations was found in T. urticae field populations in China, other resistance mechanisms appear to be involved. Here, we conducted genome-wide transcriptome profiling using RNA-sequencing of two abamectin-resistant populations (NB-ZJ and SY-BJ) and one susceptible strain (Lab-SS) to identify differentially expressed genes that might contribute to the resistance of T. urticae to abamectin in China. Results Our experiments showed that abamectin resistance was synergized by piperonyl butoxide (PBO) and triphenyl phosphate (TPP), with synergistic ratios (SR) of 2.95-fold and 2.21-fold for PBO and 3.55-fold and 2.84-fold for TPP in NB-ZJ and SY-BJ populations, respectively. Transcriptome data and quantitative real-time PCR (qRT-PCR) revealed that seven detoxification enzyme genes were overexpressed in the two resistant populations. Furthermore, functional analysis by RNA interference (RNAi) indicated that the mortality caused by abamectin was significantly increased by the separate silencing of the P450 genes CYP389C10, CYP392D8, CYP392A11, and CYP392A12. Conclusion qRT-PCR expression and RNAi data suggest that the overexpression of P450 genes CYP389C10, CYP392D8, CYP392A11, and CYP392A12 may be involved in the abamectin-resistance of field populations of T. urticae in China. This knowledge could facilitate the elucidation of resistance mechanisms and the development of resistance management of T. urticae field populations. © 2020 Society of Chemical Industry.

Nicotinic acetylcholine receptors (nAChRs), a molecular target for spinosyns and neonicotinoids, mediate rapid cholinergic transmission in insect central nervous system by binding acetylcholine. Previous studies have shown that mutations in nAChRs contribute to the high level of resistance to these two classes of insecticides. In this study, we identified nine nAChR subunits from a transcriptome of the western flower thrips, Frankliniella occidentalis, including α1-7, β1, and β2. Exon 4 of α4 and exons 3 and 8 of α6 each have two splicing variants, respectively. In addition, altered or incorrect splicing leads to truncated forms of α3, α5, and α6 subunits. The abundance of every nAChRs in both spinosad susceptible and resistant strains was highest in the 1st instar nymph. Significantly more truncated forms of α6 subunit were detected in spinosad resistant strains, whereas, hardly any full-length form was found in the two highly resistant F. occidentalis strains (resistance ratio >104-fold). Under laboratory conditions, spinosad resistance was positively correlated with truncated α6 transcripts. The correlation was later confirmed under the field conditions using five field strains. As the molecular target of spinosad, the percentage of truncated nAChR α6 subunits can be used as a diagnostic tool to detect and quantify spinosad resistance in the field.