Silencing of CYP6AS160 in Solenopsis invicta Buren by RNA interference enhances worker susceptibility to fipronil.

Cytochrome P450 monooxygenases play a key role in pest resistance to insecticides by detoxification. Four new P450 genes, CYP6AS160, CYP6AS161, CYP4AB73 and CYP4G232 were identified from Solenopsis invicta. CYP6AS160 was highly expressed in the abdomen and its expression could be induced significantly with exposure to fipronil, whereas CYP4AB73 was not highly expressed in the abdomen and its expression could not be significantly induced following exposure to fipronil. Expression levels of CYP6AS160 and CYP4AB73 in workers were significantly higher than that in queens. RNA interference-mediated gene silencing by feeding on double-stranded RNA (dsRNA) found that the levels of this transcript decreased (by maximum to 64.6%) when they fed on CYP6AS160-specific dsRNA. Workers fed dsCYP6AS160 had significantly higher mortality after 24 h of exposure to fipronil compared to controls. Workers fed dsCYP6AS160 had reduced total P450 activity of microsomal preparations toward model substrates p-nitroanisole. However, the knockdown of a non-overexpressed P450 gene, CYP4AB73 did not lead to an increase of mortality or a decrease of total P450 activity. The knockdown effects of CYP6AS160 on worker susceptibility to fipronil, combined with our other findings, indicate that CYP6AS160 is responsible for detoxification of fipronil. Feeding insects dsRNA may be a general strategy to trigger RNA interference and may find applications in entomological research and in the control of insect pests in the field.

Plant volatile compound methyl benzoate is highly effective against Spodoptera frugiperda and safe to non-target organisms as an eco-friendly botanical-insecticide.

Recent studies have indicated that the plant volatile methyl benzoate (MB) exhibits significant insecticidal bioactivity against several common insects. However, the potential environmental hazards of MB and its safety to non-target organisms is poorly understood. In the present study, these characteristics were investigated through laboratory experiments and field investigations. The results revealed that MB was highly toxic to the agricultural pest, fall armyworm Spodoptera frugiperda. Compared with the commercial pesticide lambda-cyhalothrin, the toxicities of MB against S. frugiperda larvae and adults were comparable and 3.41 times higher, respectively. Behavioral bioassays showed that the percentage repellency of MB to S. frugiperda larvae was 56.72 %, and MB induced 69.40 % oviposition deterrence rate in S. frugiperda female adults. Furthermore, in terms of median lethal concentration (LC50) and median lethal doses (LD50), MB exhibited non-toxic effects on non-target animals with 3-d LC50 of > 1 % to natural predators (Coccinella septempunctata and Harmonia axyridis), 3-d LD50 of 467.86 µg/bee to the bumblebee Bombus terrestris, 14-d LC50 of 971.09 mg/kg to the earthworm Eisenia fetida, and 4-d LC50 of 47.30 mg/L to the zebrafish Brachydanio rerio. The accumulation of MB in the soil and earthworms was found to be extremely limited. Our comparative study clearly demonstrated that MB is effective as a selective botanical pesticide against S. frugiperda and it is safe to use in the tested environment, with no toxic effects on non-target animals and natural predators.

MicroRNA-263b confers imidacloprid resistance in Sitobion miscanthi (Takahashi) by regulating the expression of the nAChRß1 subunit.

The Chinese wheat aphid Sitobion miscanthi (CWA) is an important harmful pest in wheat fields. Imidacloprid plays a critical role in controlling pests with sucking mouthparts. However, imidacloprid-resistant pests have been observed after insecticide overuse. Point mutations and low expression levels of the nicotinic acetylcholine receptor ß1 (nAchRß1) subunit are the main imidacloprid-resistant mechanisms. However, the regulatory mechanism underlying nAChRß1 subunit expression is poorly understood. In this study, a target of miR-263b was isolated from the 5'UTR of the nAchRß1 subunit in the CWA. Low expression levels were found in the imidacloprid-resistant strain CWA. Luciferase reporter assays showed that miR-263b could combine with the 5'UTR of the nAChRß1 subunit and suppress its expression by binding to a site in the CWA. Aphids treated with the miR-263b agomir exhibited a significantly reduced abundance of the nAchRß1 subunit and increased imidacloprid resistance. In contrast, aphids treated with the miR-263b antagomir exhibited significantly increased nAchRß1 subunit abundance and decreased imidacloprid resistance. These results provide a basis for an improved understanding of the posttranscriptional regulatory mechanism of the nAChRß1 subunit and further elucidate the function of miRNAs in regulating susceptibility to imidacloprid in the CWA. These results provide a better understanding of the mechanisms of posttranscriptional regulation of nAChRß1 and will be helpful for further studies on the role of miRNAs in the regulation of nAChRß1 subunit resistance in homopteran pests.

Identification of microRNAs and their response to the stress of plant allelochemicals in Aphis gossypii (Hemiptera: Aphididae).

BACKGROUND: MicroRNAs (miRNAs) are a group of short non-coding RNAs involved in the inhibition of protein translation or in mRNA degradation. Although the regulatory roles of miRNAs in various biological processes have been investigated, there is as yet an absence of studies about the regulatory roles of miRNAs involved in the metabolism of plant allelochemicals in insects. RESULTS: We constructed five small RNA libraries from apterous Aphis gossypii adults that had fed on an artificial diet containing various allelochemicals. Using Illumina sequencing, a total of 73.27 million clean reads was obtained, and 292 miRNAs were identified from A. gossypii. Comparative analysis of read counts indicated that both conserved and novel miRNAs were differently expressed among the five libraries, and the differential expression was validated via qRT-PCR. We found that the transcript levels of several miRNAs were increased or decreased in all of the allelochemical treatment libraries compared to the control. The putative target genes of the miRNAs were predicted using in silico tools, and the target genes of several miRNAs were presumed to be involved in the metabolism of xenobiotic compounds. Furthermore, the target prediction results were confirmed using dual luciferase reporter assay, and Ago-miR-656a-3p was demonstrated to regulate the expression of CYP6J1 post-transcriptionally through binding to the 3' UTR of CYP6J1. CONCLUSION: Our research results indicate that miRNAs may be involved in the metabolism of plant allelochemicals in A. gossypii, and these results also represent an important new small RNA genomics resource for further studies on this topic.

RNAi-Induced Electrophysiological and Behavioral Changes Reveal two Pheromone Binding Proteins of Helicoverpa armigera Involved in the Perception of the Main Sex Pheromone Component Z11-16:Ald.

Pheromone binding proteins (PBPs) are thought to play key roles in insect sex pheromone recognition; however, there is little in vivo evidence to support this viewpoint in comparison to abundant biochemical data in vitro. In the present study, two noctuid PBP genes HarmPBP1 and HarmPBP2 of the serious agricultural pest, Helicoverpa armigera were selected to be knocked down by RNA interference, and then the changes in electrophysiological and behavioral responses of male mutants to their major sex pheromone component (Z)-11-hexadecenal (Z11-16:Ald) were recorded. There were no significant electrophysiological or behavioral changes of tested male moths in response to Z11-16:Ald when either single PBP gene was knocked down. However, decreased sensitivity of male moths in response to Z11-16:Ald was observed when both HarmPBP1 and HarmPBP2 genes were silenced. These results reveal that both HarmPBP1 and HarmPBP2 are required for the recognition of the main sex pheromone component Z11-16:Ald in H. armigera. Furthermore, these findings may help clarify physiological roles of moth PBPs in the sex pheromone recognition pathway, which in turn could facilitate pest control by exploring sex pheromone blocking agents.

Monitoring insecticide resistance and diagnostics of resistance mechanisms in the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) in China.

Myzus persicae (Sulzer) is one of the most serious agricultural pests in China, and management strategies mainly rely on insecticidal treatment. To evaluate the resistance of field populations of M. persicae to seven insecticides, we assessed the susceptibility of 11 field populations collected from eight provinces in China using leaf-dip bioassays. Toxicity assays showed that M. persicae field populations have developed several levels of resistance to each tested insecticide. For pyrethroids, the field populations have developed a high level of resistance to ß-cypermethrin and cypermethrin, while the resistance to bifenthrin is still low. The resistance ratios of field populations to imidacloprid ranged from 1.48 to 52.36, and eight populations have developed moderate to high resistance. Resistance to acetamiprid is low, and only two populations have a moderate level of resistance. Most of the field populations of M. persicae developed moderate to high resistance to methomyl and omethoate. To investigate potential resistance mechanisms, we analyzed the enzyme activity of carboxylesterases, the type of amplified esterase genes, as well as the kdr (L1014F) mutation. All of the field populations exhibited a higher esterase activity compared to the laboratory susceptible strain. An amplified FE4, as well as the L1014F mutation, were also found in all of our experimental field populations. These results provide valuable insight into the current status of insecticide resistance and will prove to be a valuable resource in designing appropriate resistance management strategies for M. persicae in China.

Identification and Validation of Reference Genes for the Normalization of Gene Expression Data in qRT-PCR Analysis in Aphis gossypii (Hemiptera: Aphididae).

To obtain accurate and reliable results from quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, it is necessary to select suitable reference genes as standards for normalizing target gene expression data. QRT-PCR is a popular analytical methodology for studying gene expression and it has been used widely in studies of Aphis gossypii Glover in recent years. However, there is absence of study on the stability of the expression of reference genes in A. gossypii. In this study, eight commonly used candidate reference genes, including 18S, 28S, ß-ACT, GAPDH, EF1α, RPL7, α-TUB, and TBP, were evaluated under various experimental conditions to assess their suitability for use in the normalization of qRT-PCR data. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated by performing normalizations of expression data for the HSP70 gene. The results showed the most suitable combinations of reference genes for the different experimental conditions. For experiments based on divergent developmental stages, EF1α, ß-ACT, and RPL7 are the optimal reference gene combination, both EF1α and ß-ACT are the optimal combination used in the experiments of different geographical populations, whereas for experiments of the temperature changes, the combination of GAPDH and RPL7 is optimal, both 18S and ß-ACT are an optimal combination for feeding assay experiments. These research results should be useful for the selection of the suitable reference genes to obtain reliable qRT-PCR data in the gene expression study of A. gossypii.

Comparison of Life Tables of Cheilomenes sexmaculata (Coleoptera: Coccinellidae) Under Laboratory and Greenhouse Conditions.

The ladybird Cheilomenes sexmaculata (F.) is an important aphidophagous predator in Asia. In order to mass rear predators for biological control, it is valuable to identify the features of populations that are affected by variations in field conditions. Life tables can provide comprehensive descriptions of the development, survival, and fecundity of a population. However, there are few life table studies of C. sexmaculata. Studies of life history have been carried out in many arthropods using the traditional female age-specific life table, which takes only female individuals into consideration, while the variations in developmental rates amongst individuals are ignored. In this paper, we constructed life tables for C. sexmaculata fed on Myzus persicae (Sulzer) both at constant temperature in the laboratory and fluctuating temperature in the greenhouse, and analyzed the data using the age-stage, two-sex life table. The bootstrap technique was used to estimate the standard errors of the population parameters. The results showed that preadult C. sexmaculata developed more slowly and had lower survival and reproductive rates under greenhouse conditions, as indicated by the curves of age-stage survival rate (s(xj)), age-stage-specific fecundity (f(x j)) of the female stage, age-specific fecundity (m(x)), and age-specific maternity (l(x)m(x)). Our results also showed that the intrinsic rate of increase (r), net reproductive rate (R(0)), and finite rate of increase (λ) under laboratory and greenhouse conditions were 0.1668 d(-1) and 0.1027 d(-1), 192.1 and 53.0, and 1.1815 d(-1) and 1.1082 d(-1), respectively. Our results revealed significantly different life table parameters for C. sexmaculata under laboratory and greenhouse conditions. This information will be useful for developing a successful mass-rearing program for C. sexmaculata for use in biological control.

Assessment of physiological sublethal effects of imidacloprid on the mirid bug Apolygus lucorum (Meyer-Dür).

Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) is currently one of major mirid pests in the Yangtze River and the Yellow River regions in China. Imidacloprid (neonicotinoid) is widely used against pierce-sucking pest insects, including against A. lucorum. In addition to its direct lethal effect, multiple negative sublethal effects may also occur in exposed insects. We assessed potential sublethal effects of imidacloprid on some biological characteristics of A. lucorum with the aim of increasing rational use of imidacloprid against that cotton pest. The lethal toxicity of imidacloprid on adults of A. lucorum was determined in laboratory conditions by a topical application bioassay (LD(50) = 6.70 ng a.i. [active ingredient]/A. lucorum adult). We also estimated a sublethal dose, LD(5) (0.38 ng a.i./adult), a low lethal dose, LD(25) (1.96 ng a.i./adult), and moderate lethal dose, LD(40) (3.97 ng a.i./adult). The sublethal dose of imidacloprid (LD(5)) shortened the pre-oviposition period of females but increased the time required for eggs to develop (i.e. longer embryogenesis). The low lethal dose (LD(25)) also reduced the pre-oviposition period. Females exposed to the LD(40) laid eggs that developed faster but overall percentage of eggs hatching was reduced. LD(25) and LD(40) reduced longevity of males but not of females. In addition, the susceptibility to seven insecticides generally used on Chinese crops was not modified in A. lucorum previously exposed to the LD(25) of imidacloprid. Our results demonstrate sublethal effects of low doses of imidacloprid on A. lucorum (notably on pre-oviposition period and egg development) which may have an impact on population dynamics of this pest.

Short-term and transgenerational effects of the neonicotinoid nitenpyram on susceptibility to insecticides in two whitefly species.

The cosmopolitan silverleaf whitefly, Bemisia tabaci which had coexisted with Trialeurodes vaporariorum in Northern China for many years, has become the dominant species in the last years. Recent reports show that it is gradually displacing the other greenhouse whitefly species. Neonicotinoid, which includes nitenpyram, is a major group of insecticides used against whiteflies in various crops. When exposed to low doses of insecticides, insects may develop resistance by adapting physiologically. The short- and long-term effects of nitenpyram on insecticide sensitivity in B. tabaci biotype B and T. vaporariorum adult populations have been compared in the present study. After being exposed to LC(25) of nitenpyram for 24 h, the B. tabaci biotype B adults showed no significant change in susceptibility to nitenpyram or to five other insecticides: imidacloprid, acetamiprid, abamectin, chlorpyrifos and beta-cypermethrin. By contrast, exposure to the LC(25) of nitenpyram for 24 h led to a significant increase in the susceptibility of T. vaporariorum to nitenpyram and imidacloprid, by 1.8- and 2-fold, respectively. When exposed for seven generations to the LC(25) of nitenpyram, B. tabaci developed 6-fold resistance to nitenpyram, and 3.1- and 5-fold cross-resistance to imidacloprid and acetamiprid, respectively, whereas T. vaporariorum developed lower resistance (3.7-fold) to the nitenpyram and very low cross-resistance to imidacloprid (2.5-fold). The higher adaptable nature of B. tabaci (demonstrated here in the case of nitenpyram) when exposed to low doses of insecticides may provide a selective advantage when competing with T. vaporariorum in crops.

Overexpression of PxαE14 Contributing to Detoxification of Multiple Insecticides in Plutella xylostella (L.).

The diamondback moth, Plutella xylostella (L.), has evolved with varying degrees of resistance to almost all major classes of insecticides and has become the most resistant pest worldwide. The multiresistance to different types of insecticides has been frequently reported in P. xylostella, but little is known about the mechanism. In this study, a carboxylesterase (CarE) gene, PxαE14, was found significantly overexpressed in a field-evolved multiresistant P. xylostella population and can be dramatically induced by eight of nine tested insecticides. Results of the real-time quantitative polymerase chain reaction (RT-qPCR) showed that PxαE14 was predominantly expressed in the midgut and malpighian tubule of larvae. Knockdown of PxαE14 dramatically increased the susceptibility of the larvae to ß-cypermethrin, bifenthrin, chlorpyrifos, fenvalerate, malathion, and phoxim, while overexpression of PxαE14 in Drosophila melanogaster increased the tolerance of the fruit flies to these insecticides obviously. More importantly, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay showed that the recombinant PxαE14 expressed in Escherichia coli exhibited metabolic activity against the six insecticides. The homology modeling, molecular docking, and molecular dynamics simulation analyses showed that these six insecticides could stably bind to PxαE14. Taken together, these results demonstrate that constitutive and inductive overexpression of PxαE14 contributes to detoxification of multiple insecticides involved in multiresistance in P. xylostella. Our findings provide evidence for understanding the molecular mechanisms underlying the multiresistance in insect pests.

Detection of Chitin Synthase Mutations in Lufenuron-Resistant Spodoptera frugiperda in China.

Spodoptera frugiperda (J. E. Smith), is commonly known as fall armyworm, native to tropical and subtropical regions of America, is an important migratory agricultural pest. It is important to understand the resistance and internal mechanism of action of S. frugiperda against lufenuron in China. Lufenuron is one of the main insecticides recommended for field use in China and has a broad prospect in the future. We conducted a bioassay using the diet-overlay method and found that the current S. frugiperda in China are still at a low level of resistance to lufenuron. Secondly, we examined whether the mutation I1040M (I1042M in Plutella xylostella), associated with lufenuron resistance, was produced in the field. And then we tested the expression of chitin synthase SfCHSA and SfCHSB in different tissues, and the changes of these two genes after lufenuron induction. The results showed that there is still no mutation generation in China and there is a significant change in the expression of SfCHSA under the effect of lufenuron. In conclusion, our study suggests that field S. frugiperda populations in 2019 and 2020 were less resistant to lufenuron. In fall armyworm, chitin synthases included SfCHSA and SfCHSB genes, and after induction treatment with lufenuron, the expression of the SfCHSA gene was significantly increased. In SfCHSA, no mutation has been detected in the site associated with lufenuron resistance. Secondly, in S. frugiperda larvae, the SfCHSA gene was the highest in the head of the larvae, followed by the integument; while the SfCHSB gene was mainly concentrated in the midgut. Therefore, we believe that the SfCHSA gene plays a greater role in the resistance of S. frugiperda to lufenuron than the SfCHSB gene. It is worth noting that understanding the level of resistance to lufenuron in China, the main mechanism of action of lufenuron on larvae, and the mechanism of resistance to lufenuron in S. frugiperda will help in crop protection as well as in extending the life span of this insecticide.

Binding characterization of recombinant odorant-binding proteins from the parasitic wasp, Microplitis mediator (Hymenoptera: Braconidae).

Chemoreception in insects is mediated by small odorant-binding proteins (OBPs) that are believed to carry lipophilic stimuli to the olfactory receptor cells through the aqueous sensillar lymph. Binding experiments and recent structural studies of OBPs have illustrated their versatility and ability to accommodate ligands of different shapes and chemical structures. We expressed and purified seven recombinant OBPs (MmedOBP1-MmedOBP7) from the parasitic wasp, Microplitis mediator (Hymenoptera: Braconidae) in a prokaryotic expression system. With 4,4'-dianilino-1,1'-binaphthyl-5,5'-sulfonic acid (bis-ANS) as a fluorescent probe, the ligand-binding specificities of these seven MmedOBPs with 50 small organic compounds were investigated in vitro. The results revealed that all of the M. mediator OBPs can bind a wide variety of odorant molecules with different binding affinities. The best ligand for all seven MmedOBPs was ß-ionone. MmedOBP2 showed affinity for some aromatic compounds, whereas MmedOBP4 and MmedOBP6 bound several terpenoids. MmedOBP5 bound ß-ionone, but did not bind any of the other potential ligands that we tested.

Detection of ryanodine receptor target-site mutations in diamide insecticide-resistant Spodoptera frugiperda in China.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a widely distributed pest of corn. Since it invaded China in 2018, it has caused serious damage to local corn production. Chlorantraniliprole, an anthranilic diamide insecticide, has been widely used to control lepidopteran pests. Tetrachloropyramid is a new allosteric modulator insecticide developed based on chlorantraniliprole, so it has a similar mechanism and insecticidal effect. In this study, we investigated resistance levels to chlorantraniliprole and tetrachloropyramid in S. frugiperda from 13 populations in China. Among the populations tested, the relative highest resistance to chlorantraniliprole occurred in the Guangzhou population, and the most susceptible to chlorantraniliprole was found in the Wuhan population. The lethal dosage LD50 value of the Guangzhou population against chlorantraniliprole was 27.8-fold higher than that of the Wuhan population. Minimal differences were observed among S. frugiperda populations in terms of sensitivity to tetrachloropyramid. Heterozygous mutations at the I4734 site of the ryanodine receptor (RyR) were found, while no mutations were found in the G4891 site. The mutations were detected in only two of the 786 individuals analyzed, one from the Qinzhou population and other from the Anshun population (frequency below 2% in both cases). There were no significant differences in the expression levels of RyR between Guangzhou and Wuhan populations. In summary, our results indicate that: (i) S. frugiperda has low resistance levels to diamide insecticides in China; and (ii) the differences in relative resistance among the 13 populations analyzed are not caused by the mutations in RyR or the expression of RyR.

Combined Transcriptomic and Proteomic Analysis of Myzus persicae, the Green Peach Aphid, Infected with Cucumber Mosaic Virus.

Aphids transmit CMV (cucumber mosaic virus) in a non-persistent manner. However, little is known about the mechanism of CMV transmission. In this study, an integrated analysis of the mRNA and protein was performed to identify important putative regulators involved in the transmission of CMV by aphids. At the level of transcription, a total of 20,550 genes (≥2-fold expression difference) were identified as being differentially expressed genes (DEGs) 24 h after healthy aphid transfer to infected tobacco plants using the RNA-seq approach. At the protein level, 744 proteins were classified as being differentially abundant between virus-treated and control M. persicae using iTRAQ (isobaric tags for relative and absolute quantitation) analysis. The combined mRNA and protein analysis enabled the identification of some viral putative regulators, such as cuticle proteins, ribosomal proteins, and cytochrome P450 enzymes. The results show that most of the key putative regulators were highly accumulated at the protein level. Based on those findings, we can speculate that the process by which aphids spread CMV is mainly related to post-translational regulation rather than transcription.

Coordinative mediation of the response to alarm pheromones by three odorant binding proteins in the green peach aphid Myzus persicae.

Odorant binding proteins (OBPs) play an essential role for insect chemosensation in insect peripheral nervous systems of antennae. Each antennal sensilla contains more than one OBP at high concentrations but the interactions and cooperation between co-localized OBPs are rarely reported. In present study, we cloned, expressed and purified eight OBPs of the green peach aphid Myzus persicae. The effects of knocking down the expression of these OBP genes by RNAi on the electrophysiological and behavioural responses of M. persicae to the aphid alarm pheromone, (E)-ß-farnesene (EßF) were investigated. The results showed that the aphids could still be repelled by EßF when the expression of each of three OBP genes was individually knocked down. However, the simultaneous knockdown of MperOBP3/7/9 expression significantly reduced the electrophysiological response and the repellent behaviours of M. persicae to EßF than the single OBP gene knockdown (P < 0.05). Rather than a normal saturation binding curve of individual OBP, the binding curve of MperOBP3/7/9 is bell-shaped with a higher affinity for the fluorescent probe N-phenyl-1-naphthylamine (1-NPN). The competitive binding assays confirmed that MperOBP3, MperOBP7, MperOBP9 and MperOBP3/7/9 mixture exhibited a stronger binding affinity for EßF, than for sex pheromones and plant volatiles with a dissociation constant of 2.5 µM, 1.1 µM, 3.9 µM and 1.0 µM, respectively. The competitive binding curve of MperOBP3/7/9 mixture to EßF is shallow without bottom plateau, suggesting a conformational change and a rapid dissociation after the displacement of all 1-NPN (in vivo after the saturation binding of all OBPs by EßF). The interaction between OBPs and formation of a heterogeneous unit may facilitate the delivery EßF to the OR at electrophysiological and behavioural levels during insect odorant signal transduction thus mediate M. persicae response to the alarm pheromone EßF.

Characterization and target gene analysis of microRNAs in the antennae of the parasitoid wasp Microplitis mediator.

MicroRNAs (miRNAs), a class of non-coding single-strand RNA molecules encoded by endogenous genes, are about 21-24 nucleotides long and are involved in the post-transcriptional regulation of gene expression in plants and animals. Generally, the types and quantities of miRNAs in the different tissues of an organism are diverse, and these divergences may be related to their specific functions. Here we have identified 296 known miRNAs and 46 novel miRNAs in the antennae of the parasitoid wasp Microplitis mediator by high-throughput sequencing. Thirty-three miRNAs were predicted to target olfactory-associated genes, including odorant binding proteins (OBPs), chemosensory proteins, odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors. Among these, 17 miRNAs were significantly highly expressed in the antennae, four miRNAs were highly expressed both in the antennae and head or wings, while the remaining 12 miRNAs were mainly expressed in the head, thorax, abdomen, legs and wings. Notably, miR-9a-5p and miR-2525-3p were highly expressed in male antennae, whereas miR-1000-5p and novel-miR-13 were enriched in female antennae. The 17 miRNAs highly expressed in antennae are likely to be associated with olfaction, and were predicted to target one OBP (targeted by miR-3751-3p), one IR (targeted by miR-7-5p) and 14 ORs (targeted by 15 miRNAs including miR-6-3p, miR-9a-5p, miR-9b-5p, miR-29-5p, miR-71-5p, miR-275-3p, miR-1000-5p, miR-1000-3p, miR-2525-3p, miR-6012-3p, miR-9719-3p, novel-miR-10, novel-miR-13, novel-miR-14 and novel-miR-28). These candidate olfactory-associated miRNAs are all likely to be involved in chemoreception through the regulation of chemosensory gene expression in the antennae of M. mediator.

Overexpression of UDP-glycosyltransferase potentially involved in insecticide resistance in Aphis gossypii Glover collected from Bt cotton fields in China.

BACKGROUND: The cotton aphid Aphis gossypii Glover is one of the most destructive insect pests. It has evolved resistance to numerous insecticides around the world due to the application of insecticides. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) have been reported to potentially facilitate the detoxification process of imidacloprid and thiamethoxam in A. gossypii. RESULTS: In this study, the field populations of A. gossypii developed different levels of resistance to multiple insecticides. A UGT inhibitor, 5-nitrouracil, dramatically increased the toxicity of acetamiprid in resistant populations, moderately increased the toxicity of sulfoxaflor in the imidacloprid susceptible (IMI_S) population, and populations from Yuncheng in Shanxi Province (SXYC) and Jingzhou in Hubei Province (HBJZ), and increased the toxicity of bifenthrin in the IMI_S and HBJZ populations, but there was no synergism on omethoate or carbosulfan. Quantitative real-time PCR analysis revealed that UGT344B4 and UGT344C7 were overexpressed in all field populations, and UGT344N4 was overexpressed in the SDBZ and HBZJ populations. Furthermore, the suppression of UGT344B4 or UGT344C7 by RNA interference significantly increased the susceptibility to bifenthrin in the IMI_S population and the susceptibility to sulfoxaflor in the SXYC population. CONCLUSION: These results suggested that UGTs are potentially involved in the detoxification of neonicotinoid, sulfoximine, and pyrethroid insecticides in A. gossypii. Furthermore, the overexpression of UGTs could be associated with insecticide resistance in field populations of A. gossypii. The results might be helpful for the management of insecticide resistance in field populations of A. gossypii. © 2019 Society of Chemical Industry.

Molecular characterization and expression of sensory neuron membrane proteins in the parasitoid Microplitis mediator (Hymenoptera: Braconidae).

Sensory neuron membrane proteins (SNMPs), homologs of the human fatty acid transport protein CD36 family, are observed to play a significant role in chemoreception, especially in detecting sex pheromone in Drosophila and some lepidopteran species. In the current study, two full-length SNMP transcripts, MmedSNMP1 and MmedSNMP2, were identified in the parasitoid Microplitis mediator (Hymenoptera: Braconidae). Quantitative real-time polymerase chain reaction analysis showed that the expression of MmedSNMP1 was significantly higher in antennae than in other tissues of both sexes. In addition, the MmedSNMP1 transcript was increased dramatically in newly emerged adults and there were no significant differences between adults with or without mating and parasitic experiences. However, compared with MmedSNMP1, the expression of MmedSNMP2 was widely found in various tissues, significantly increased at half-pigmented pupae stage and remained at a relatively constant level during the following developmental stages. It was found that MmedSNMP1 contained eight exons and seven introns, which was highly conserved compared with other insect species. In situ hybridization assay demonstrated that MmedSNMP1 transcript was distributed widely in antennal flagella. Among selected chemosensory genes (odorant binding protein, odorant receptor, and ionotropic receptor genes), MmedSNMP1 only partially overlapped with MmedORco in olfactory sensory neurons of antennae. Subsequent immunolocalization results further indicated that MmedSNMP1 was mainly expressed in sensilla placodea of antennae and possibly involved in perceiving plant volatiles and sex pheromones. These findings lay a foundation for further investigating the roles of SNMPs in the chemosensation of parasitoids.

Identification and expression pattern of putative odorant-binding proteins and chemosensory proteins in antennae of the Microplitis mediator (Hymenoptera: Braconidae).

The parasitoids of Cotton Bollworm Microplitis mediator (Hymenoptera: Braconidae) find their hosts through the odor released by stressed plants. In this study, preliminary characterization and isolation of cDNAs from male M. mediator antennal libraries identified 8 putative odorant-binding proteins (OBPs). Real-time polymerase chain reaction method was used to study the expression pattern of these isolated genes. Their gene expression profiles under a wide range of conditions indicated that only 4 OBP genes in M. mediator were antenna specific. The remaining 4 genes are either expressed ubiquitously or strictly regulated in specialized tissues or during different developmental stages. Some OBP genes were gender specific. These findings support that OBPs play dynamic roles during development of M. mediator and are likely to be involved in broader physiological functions.