Life table analysis and RNA-Seq reveal hormesis and transgenerational effects of deltamethrin on Aphis gossypii.

BACKGROUND: Deltamethrin, as a highly effective and broad-spectrum insecticide, has been widely used for agricultural pest control such as Aphis gossypii worldwide. Increasing evidence has shown that despite great economic benefits brought by it, deltamethrin has also non-negligible side effects. However, the potential risks and related molecular mechanisms remain largely unclear. RESULTS: Herein, the life table parameters and transcriptome sequencing analyses of the four successive aphid generations were performed to investigate the hormesis and transgenerational effects of deltamethrin on A. gossypii. The life table analysis showed that although the exposure of G0 aphid to 30% lethal concentration (LC30) deltamethrin significantly reduced the net reproduction rate (R0), intrinsic rate of increase (r), and fecundity of G0, but it significantly enhanced the R0 and fecundity of subsequent two generations (G1 and G2) of A. gossypii. Moreover, transcriptomic analyses showed that the signaling pathways related to posttranscriptional regulation (spliceosome), protein processing, longevity regulating, and cell proliferation (DNA replication, homologous recombination and non-homologous end-joining) were significantly up-regulated in G1 or G2 under LC30 deltamethrin treatment. Additionally, we also found that the deltamethrin-sulfoxaflor rotation of G0 and G1 still induced reproductive stimulation, but the reproductive stimulation induced by insecticides rotation treatment was significantly lower than that in the deltamethrin exposure alone. CONCLUSION: Our study demonstrates that sublethal concentrations of deltamethrin significantly enhanced the offspring fecundity of cotton aphid. In addition, our study also reveals the transcriptional response mechanism of hormesis-induced fecundity increase, providing valuable reference for optimizing the application of deltamethrin in integrated pest management. © 2024 Society of Chemical Industry.

Antioxidant Enzyme, Transcriptomic, and Metabolomic Changes in Lily (Lilium spp.) Leaves Induced by Aphis gossypii Glover.

Cotton aphids (Aphis gossypii Glover) cause harm by feeding on phloem sap and spreading plant viruses to lily. Understanding the mechanisms by which aphids infest lily plants is crucial for effective aphid management and control. In this study, we investigated the activity of antioxidants, integrated nontargeted metabolomes and transcriptomes of lilies infested by cotton aphids to explore the changes in lily leaves. Overall, the results indicated that the catalase (CAT) activity in the leaves of the lily plants was greater than that in the leaves of the control plants. A comprehensive identification of 604 substances was conducted in the leaves. Furthermore, the differentially abundant metabolite analysis revealed the enrichment of phenylalanine metabolism and α-linolenic acid metabolism. Moreover, 3574 differentially expressed genes (DEGs), whose expression tended to increase, were linked to glutathione metabolism and phenylpropanoid biosynthesis. In addition, the integrated analysis revealed that the defensive response of lily leaves to aphids is manifested through antioxidant reactions, phenylpropane and flavonoid biosynthesis, and α-linolenic acid metabolism. Finally, the key metabolites were CAT, glutathione, coumaric acid, and jasmonic acid, along with the key genes chalcone synthase (CHS), phenylalanine ammonia-lyase (PAL), and 12-oxo-phytodienoic acid reductase (OPR). Accordingly, the findings of this research elucidate the molecular and metabolic reactions of A. gossypii in lily plants, offering valuable insights for developing aphid resistance strategies in lily farming.

Hormetic dose response induced by sublethal-dose sulfoxaflor leads to reproductive stimulation of Aphis gossypii.

Aphis gossypii Glover is one of the most agriculturally important phloem-feeding economic pests, causing tremendous loss in crop yield annually. The hormesis is an important cause of A. gossypii resistance formation, population resurgence, and re-outbreak. However, whether the hormesises induced by different insecticides interact mutually remain largely unclear. In the study, four-generation A. gossypii experiment found that the 24-h sublethal-dose (LC20) sulfoxaflor treatment on G0 significantly increased the net reproductive rate (R0) and fecundity of G1 and G2 generation A. gossypii, but it did not significantly affect the fecundity of G3 and G4 individuals. Transcriptomic analyses revealed that the insecticide-induced significant up-regulation of pathways ribosome, energy metabolism, and the DNA replication and reparation might be responsible for the enhancement of fecundity in G1 and G2 A. gossypii. Notably, G0 exposure to LC20 sulfoxaflor followed by G1 exposure to LC30 deltamethrin resulted in a stronger reproductive stimulation than sulfoxaflor or deltamethrin exposure alone. Our findings provide valuable reference for optimizing sulfoxaflor application in integrated pest management strategies.

Functional analysis of AgJHAMT gene related to developmental period in Aphis gossypii Glover.

Aphis gossypii is one of the most economically important agricultural pests that cause serious crop losses worldwide, and the indiscriminate chemical application causes resistance development in A. gossypii, a major obstacle to successful control. In this study, we selected the up-regulated expression gene AgJHAMT, which was enriched into juvenile hormone pathway though transcriptome sequencing analysis of the cotton aphids that fed on transgenic cotton lines expressing dsAgCYP6CY3 (the TG cotton). The AgJHAMT gene was overexpressed in cotton aphids which fed on the TG cotton, and its expression profile during the nymphs was clarified. Then, silencing AgJHAMT could advance the developmental period of cotton aphids by 0.5 days compared with control groups. The T and t values of cotton aphids in the dsJHAMT treatment group (6.88 ± 0.15, 1.65 ± 0.06) were significantly shorter than that of the sprayed H2O control group (7.6 ± 0.14, 1.97 ± 0.09) (P < 0.05), respectively. The fast growth caused by AgJHAMT silencing was rescued by applying the JH analogue, methoprene. Overall, these findings clarified the function of AgJHAMT in the developmental period of A. gossypii. This study contributes to further clarify the molecular mechanisms of delaying the growth and development of cotton aphids by the transgenic cotton lines expressing dsAgCYP6CY3.

A tiny sample rapid visual detection technology for imidacloprid resistance in Aphis gossypii by CRISPR/Cas12a.

Insecticide resistance monitoring is essential for guiding chemical pest control and resistance management policies. Currently, rapid and effective technology for monitoring the resistance of tiny insects in the field is absent. Aphis gossypii Glover is a typical tiny insect, and one of the most frequently reported insecticide-resistant pests. In this study, we established a novel CRISPR/Cas12a-based rapid visual detection approach for detecting the V62I and R81T mutations in the ß1 subunit of the nAChR in A. gossypii, to reflect target-site resistance to imidacloprid. Based on the nAChR ß1 subunit gene in A. gossypii, the V62I/R81T-specific RPA primers and crRNAs were designed, and the ratio of 10 µM/2 µM/10 µM for ssDNA/Cas12a/crRNA was selected as the optimal dosage for the CRISPR reaction, ensuring that Cas12a only accurately recognizes imidacloprid-resistance templates. Our data show that the field populations of resistant insects possessing V62I and R81T mutations to imidacloprid can be accurately identified within one hour using the RPA-CRISPR/Cas12a detection approach under visible blue light at 440-460 nm. The protocol for RPA-CRISPR detection necessitates a single less than 2 mm specimen of A. gossypii tissues to perform RPA-CRISPR detection, and the process only requires a container at 37 °C and a portable blue light at 440-460 nm. Our research represents the first application of RPA-CRISPR technology in insecticide resistance detection, offers a new method for the resistance monitoring of A. gossypii or other tiny insects, helps delay the development of resistance to imidacloprid, improves the sustainability of chemical control, and provides theoretical guidance for managing pest resistance.

Pyridine Derivatives as Insecticides. Part 5. New Thieno[2,3-b]pyridines and Pyrazolo[3,4-b]pyridines Containing Mainly Ethyl Nicotinate Scaffold and Their Insecticidal Activity toward Aphis gossypii (Glover,1887).

Ethyl 5-cyano-1,6-dihydro-2-methyl-4-(2'-thienyl)-6-thioxonicotinate (A) was synthesized and reacted with ethyl chloroacetate in the presence of sodium acetate or sodium carbonate to give ethyl 5-cyano-6-((2-ethoxy-2-oxoethyl)thio)-2-methyl-4-(2'-thienyl)nicotinate (1a) or its isomeric thieno[2,3-b]pyridine 2a. 3-Aminothieno[2,3-b]pyridine-2-carboxamide 2b was also synthesized by the reaction of A with 2-chloroacetamide. The reaction of 1a with hydrazine hydrate in boiling ethanol gave acethydrazide 3. Heating ester 1a with hydrazine hydrate under neat conditions afforded 3-amino-1H-pyrazolo[3,4-b]pyridine 10. Compounds 2b, 3, and 10 were used as precursors for synthesizing other new thieno[2,3-b]pyridines and pyrazolo[3,4-b]pyridines containing mainly the ethyl nicotinate scaffold. Structures of all new compounds were confirmed by elemental and spectral analyses. Most of the obtained compounds were evaluated for their insecticidal activity toward the nymphs and adults of Aphis gossypii (Glover,1887). Some compounds such as 4, 9b, and 9c showed promising results. The effect of some sublethal concentrations, less than LC50, of compounds 4, 9b, and 9c on the examined Aphis was subjected to a further study. The results demonstrated that exposure of A. gossypii nymphs to sublethal concentrations of compounds 4, 9b, and 9c had noticeable effects on their biological parameters, i.e., nymphal instar duration, generation time, and adult longevity. The highest concentration C1 of all three compounds increased the nymphal instar duration and generation time and decreased adult longevity and vice versa.

Ethyl Formate Fumigation for Controlling Two Major Aphid Pests, Aphis spiraecola and Aphis gossypii, on Passion Fruit, from Cultivation to Post-Harvest Storage.

Tropical and subtropical crops are being increasingly cultivated in South Korea, leading to an increase in damage by exotic insect pests. Consequently, ethyl formate (EF) is currently being considered for quarantine and pre-shipment fumigation. In this study, we evaluated the effectiveness of EF fumigation for controlling Aphis spiraecola Patch and Aphis gossypii Glover, two representative quarantine pests on passion fruit ("Pink Bourbon") during greenhouse cultivation and post-harvest storage. The efficacy of EF against both aphids in terms of the lethal concentration causing 50% mortality (LCt50%) and LCt99% was 1.36-2.61 g h/m3 and 3.73-7.55 g h/m3 under greenhouse conditions (23 °C), and 1.37-2.02 g h/m3 and 3.80-14.59 g h/m3 post-harvest (5 °C), respectively. EF at 4 g/m3 for 4 h resulted in 100% mortality of A. spiraecola, which was more resistant to EF, without causing phytotoxic damage to the trees in a 340 m3 greenhouse. Post-harvest fruit fumigation at 10 g/m3 for 4 h in a mid-size (0.8 m3) fumigation chamber resulted in complete disinfection. Moreover, the EF level decreased below the EF threshold within 10 min after natural ventilation in the greenhouse. Therefore, our results suggest EF fumigation as an effective method for controlling A. spiraecola and A. gossypii.

UDP-Glycosyltransferases UGT350C3 and UGT344L7 Confer Tolerance to Neonicotinoids in Field Populations of Aphis gossypii.

The cotton aphid, Aphis gossypii, is a polyphagous pest that stunts host plant growth via direct feeding or transmitting plant virus. Due to the long-term application of insecticides, A. gossypii has developed different levels of resistance to numerous insecticides. We found that five field populations had evolved multiple resistances to neonicotinoids. To explore the resistance mechanism mediated by uridine diphosphate glycosyltransferases (UGTs), two upregulated UGT genes in these five strains, UGT350C3 and UGT344L7, were selected for functional analysis of their roles in neonicotinoid detoxification. Transgenic Drosophila bioassay results indicated that compared with the control lines, the UGT350C3 and UGT344L7 overexpression lines were more tolerant to thiamethoxam, imidacloprid, and dinotefuran. Knockdown of UGT350C3 and UGT344L7 significantly increased A. gossypii sensitivity to thiamethoxam, imidacloprid, and dinotefuran. Molecular docking analysis demonstrated that these neonicotinoids could bind to the active pockets of UGT350C3 and UGT344L7. This study provides functional evidence of neonicotinoid detoxification mediated by UGTs and will facilitate further work to identify strategies for preventing the development of neonicotinoid resistance in insects.

Identification and Genome Characterization of a Novel Nege-like Virus Isolated from Aphids (Aphis gossypii) in Yunnan Province.

Negeviruses are insect-specific enveloped RNA viruses that exhibit a wide geographic distribution. A novel nege-like virus, tentatively named Aphis gossypii nege-like virus (AGNLV, GenBank: OR880429.1), was isolated from aphids (Aphis gossypii) in Lijiang City, Yunnan, China. AGNLV has a genome sequence of 9258 nt (excluding the polyA tail) encoding three open reading frames (ORFs). ORF1 (7149 nt) encodes a viral methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase. ORF2 (1422 nt) encodes a DiSB-ORF2_chro domain and ORF3 encodes an SP24 domain. The genome sequence of AGNLV shares the highest nucleotide identity of 60.0% and 59.5% with Wuhan house centipede virus 1 (WHCV1) and Astegopteryx formosana nege-like virus (AFNLV), respectively. Phylogenetic analysis based on the RNA-dependent RNA polymerase shows that AGNLV is clustered with other negeviruses and nege-like viruses discovered in aphids, forming a distinct "unclassified clade". Interestingly, AGNLV only encodes three ORFs, whereas AFNLV and WHCV1 have four ORFs. Structure and transmembrane domain predictions show the presence of eight alpha helices and five transmembrane helices in the AGNLV ORF3. Translational enhancement of the AGNLV 5' UTR was similar to that of the 5' UTR of plant viruses. Our findings provide evidence of the diversity and structure of nege-like viruses and are the first record of such a virus from a member of the genus Aphis.

Salivary gland transcriptomics of the cotton aphid Aphis gossypii and comparative analysis with other sap-sucking insects.

Aphids are sap-sucking insects responsible for crop losses and a severe threat to crop production. Proteins in the aphid saliva are integral in establishing an interaction between aphids and plants and are responsible for host plant adaptation. The cotton aphid, Aphis gossypii (Hemiptera: Aphididae) is a major pest of Gossypium hirsutum. Despite extensive studies of the salivary proteins of various aphid species, the components of A. gossypii salivary glands are unknown. In this study, we identified 123,008 transcripts from the salivary gland of A. gossypii. Among those, 2933 proteins have signal peptides with no transmembrane domain known to be secreted from the cell upon feeding. The transcriptome includes proteins with more comprehensive functions such as digestion, detoxification, regulating host defenses, regulation of salivary glands, and a large set of uncharacterized proteins. Comparative analysis of salivary proteins of different aphids and other insects with A. gossypii revealed that 183 and 88 orthologous clusters were common in the Aphididae and non-Aphididae groups, respectively. The structure prediction for highly expressed salivary proteins indicated that most possess an intrinsically disordered region. These results provide valuable reference data for exploring novel functions of salivary proteins in A. gossypii with their host interactions. The identified proteins may help develop a sustainable way to manage aphid pests.

Beneficial rhizosphere bacteria provides active assistance in resisting Aphis gossypiis in Ageratina adenophora.

Ageratina adenophora can enhance its invasive ability by using beneficial rhizosphere bacteria. Bacillus cereus is able to promote plant growth and provide a positive feedback effect to A. adenophora. However, the interaction between A. adenophora and B. cereus under the influence of native polyphagous insect feeding is still unclear. In this study, Eupatorium lindleyanum, a local species closely related to A. adenophora, was used as a control, aimed to compare the content of B. cereus in the roots of A. adenophora and rhizosphere soil after different densities of Aphis gossypii feeding, and then investigated the variations in the population of A. gossypii and soil characteristics after the addition of B. cereus. The result showed that B. cereus content in the rhizosphere soil and root of A. adenophora increased significantly under A. gossypii feeding compared with local plants, which also led to the change of α-diversity and ß-diversity of the bacterial community, as well as the increase in nitrate nitrogen (NO3 -N) content. The addition of B.cereus in the soil could also inhibit the population growth of A. gossypii on A. adenophora and increase the content of ammonium nitrogen (NH4 +-N) in the soil. Our research demonstrated that B. cereus enhances the ability of A. adenophora to resist natural enemy by increasing soil ammonium nitrogen (NH4 +-N) and accumulating other beneficial bacteria, which means that rhizosphere microorganisms help invasive plants defend themselves against local natural enemies by regulating the soil environment.

Coccinellid predation efficiency on Aphis gossypii (Hemiptera: Aphididae) under laboratory conditions.

In this study, we investigated the biological aspects and predation efficiency of 3 aphidophagous ladybird beetles, Coccinella novemnotata, Hippodamia variegata, and Coccinella septempunctata, on the cotton aphid, Aphis gossypii, reared on cucumber plants (Cucumis sativus L. cultivar barracuda) under laboratory conditions. The developmental periods of C. novemnotata, H. variegata, and C. septempunctata were observed to be 16.00 ±â€…0.25, 16.00 ±â€…0.25, and 20.58 ±â€…0.40 days, respectively. The larvae of these ladybird beetles consumed an average of 218.93 ±â€…8.86, 254.77 ±â€…8.86, and 537.36 ±â€…10.49 aphids, respectively. Fourth-instar larvae were particularly efficient, consuming 53.68%, 52.68%, and 52.64% of total aphids for C. novemnotata, H. variegata, and C. septempunctata, respectively. Adult emergence rates were promising, with 91.67%, 100.00%, and 92.86%, accompanied by sex ratios of 63.64%, 53.84%, and 61.54%, respectively. Notably, a single female of C. novemnotata, H. variegata, and C. septempunctata consumed an average of 2,215.30, 2,232.00, and 3,364.50 aphids, respectively, over its lifespan. Coccinella septempunctata demonstrated the highest predation efficiency among the 3 species, suggesting its potential for biological control of A. gossypii in both open fields and greenhouses, promoting sustainable agricultural practices.

Effects of Seven Plant Essential Oils on the Growth, Development and Feeding Behavior of the Wingless Aphis gossypii Glover.

Cotton aphid Aphis gossypii Glover damages plants such as cotton directly by feeding on leaves and indirectly by transmitting viruses and excreting honeydew, which interferes with photosynthesis. The control of A. gossypii is still dominated by the frequent use of insecticides, which leads to a gradual increase in pesticide resistance in A. gossypii. Research is therefore needed on non-pesticide controls. In this study, seven plant essential oils (EOs) of Ocimum sanctum L., Ocimum basilicum L., Ocimum gratissimum L., Mentha piperita L., Mentha arvensis L., Tagetes erecta L., and Lavandula angustifolia Mill. were examined as potential controls for A. gossypii. We used life tables and electrical penetration graphs (EPG) to explore the effects of these EOs on the growth, development, and feeding behavior of A. gossypii, followed by a study of effects of the EOs on honeydew secretion by A. gossypii as a measure of their antifeedant activity. We found that the EOs of O. sanctum, M. piperita, M. arvensis and T. erecta significantly extended the pre-adult developmental period. Also, adult longevity, number of oviposition days, and total fecundity of A. gossypii treated with the EOs of M. arvensis or T. erecta were all significantly reduced. Aphids treated with the EOs of O. sanctum, M. piperita, or L. angustifolia showed significant reductions in their net reproductive rate (R0), intrinsic rate of increase (rm), and finite rate of increase (λ), and significant increases in mean generation time (T). In terms of their effects on the feeding behavior of A. gossypii, all seven EOs significantly reduced the total duration of phloem feeding (E2 waves), the number of phloem-feeding bouts, and the proportion of time spent in secretion of saliva into phloem sieve elements (E1 waves) and phloem feeding (E2). The total duration and number of E1 waves (saliva secretion) were significantly reduced by the EOs of O. sanctum, O. gratissimum, and M. arvensis. For C waves (probing in non-vascular tissues), the total duration spent in this behavior was significantly increased by the EOs of O. gratissimum, M. piperita, and L. angustifolia, but the number of such probing events was increased only by L. angustifolia EO. The EOs of O. basilicum, M. arvensis, and T. erecta significantly increased the total duration of ingestion of xylem sap (G waves), while the total time of mechanical difficulty in stylet penetration (F waves) was increased by M. arvensis. The total duration and number of the non-probing events (Np waves) were significantly increased by EOs of O. sanctum and O. basilicum. After treatment with all seven of these EOs, the area covered by honeydew was significantly reduced compared with the control. Studies have analyzed that EOs of O. sanctum, M. piperita, and T. erecta were most effective, followed by the EOs of M. arvensis and L. angustifolia, and finally the EOs of O. basilicum and O. gratissimum. In the present study, the EOs of O. sanctum, M. piperita, and T. erecta were found to have potential for the development as antifeedants of A. gossypii, and these data provide a basis for future research on non-pesticide chemical control of A. gossypii.

Sublethal Effects of Spirotetramat, Cyantraniliprole, and Pymetrozine on Aphis gossypii (Hemiptera: Aphididae).

The toxicity and sublethal effects of three insecticides (spirotetramat, cyantraniliprole, and pymetrozine) on Aphis gossypii, a major agricultural pest, were investigated. The nymphal stage showed greater susceptibility than the adult stage to all the insecticides, with a difference of up to 8.9 times at the LC50 of spirotetramat. The effects of sublethal concentrations (LC10, LC30, LC50, and LC70) of the insecticides on the on the developmental period, survival rate, adult longevity, fecundity, and deformity rate were compared with those of the control. Compared with the control, cyantraniliprole and pymetrozine did not significantly affect the developmental period in the parental or F1 generation when applied at the nymphal stage at any concentration. Nonviable nymphs occurred in the F1 generation when both nymphs and adults were treated with spirotetramat and cyantraniliprole but not in the F2 generation. The age-specific maternity (lxmx) of A. gossypii treated with sublethal concentrations (LC10, LC30) decreased with increasing concentration. Spirotetramat at the LC30 resulted in significant differences in all life table parameters (R0, rm, λ, T, DT) compared with those of the control. Similarly, compared with that of the control (43.8), the net reproductive rate (R0) significantly decreased for all the insecticides except cyantraniliprole at the LC10 (37.5). Therefore, this study indicated that sublethal concentrations (over the LC30) of spirotetramat, cyantraniliprole, or pymetrozine might be useful for the density management of A. gossypii.

Aphid gene expression following polerovirus acquisition is host species dependent.

Upon acquisition of persistent circulative viruses such as poleroviruses, the virus particles transcytose through membrane barriers of aphids at the midgut and salivary glands via hemolymph. Such intricate interactions can influence aphid behavior and fitness and induce associated gene expression in viruliferous aphids. Differential gene expression can be evaluated by omics approaches such as transcriptomics. Previously conducted aphid transcriptome studies used only one host species as the source of virus inoculum. Viruses typically have alternate hosts. Hence, it is not clear how alternate hosts infected with the same virus isolate alter gene expression in viruliferous vectors. To address the question, this study conducted a transcriptome analysis of viruliferous aphids that acquired the virus from different host species. A polerovirus, cotton leafroll dwarf virus (CLRDV), which induced gene expression in the cotton aphid, Aphis gossypii Glover, was assessed using four alternate hosts, viz., cotton, hibiscus, okra, and prickly sida. Among a total of 2,942 differentially expressed genes (DEGs), 750, 310, 1,193, and 689 genes were identified in A. gossypii that acquired CLRDV from infected cotton, hibiscus, okra, and prickly sida, respectively, compared with non-viruliferous aphids that developed on non-infected hosts. A higher proportion of aphid genes were overexpressed than underexpressed following CLRDV acquisition from cotton, hibiscus, and prickly sida. In contrast, more aphid genes were underexpressed than overexpressed following CLRDV acquisition from okra plants. Only four common DEGs (heat shock protein, juvenile hormone acid O-methyltransferase, and two unannotated genes) were identified among viruliferous aphids from four alternate hosts. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations indicated that the acquisition of CLRDV induced DEGs in aphids associated with virus infection, signal transduction, immune systems, and fitness. However, these induced changes were not consistent across four alternate hosts. These data indicate that alternate hosts could differentially influence gene expression in aphids and presumably aphid behavior and fitness despite being infected with the same virus isolate.

Molecular and functional characterization of heat-shock protein 70 in Aphis gossypii under thermal and xenobiotic stresses.

Aphis gossypii, a globally distributed and economically significant pest of several crops, is known to infest a wide range of host plants. Heat shock proteins (Hsps), acting as molecular chaperones, are essential for the insect's environmental stress responses. The present study investigated the molecular characteristics and expression patterns of AgHsp70, a heat shock protein gene, in Aphis gossypii. Our phylogenetic analysis revealed that AgHsp70 shared high similarity with homologs from other insects, suggesting a conserved function across species. The developmental expression profiles of AgHsp70 in A. gossypii showed that the highest transcript levels were observed in the fourth instar nymphs, while the lowest levels were detected in the third instar nymphs. Heat stress and exposure to four different xenobiotics (2-tridecanone, tannic acid, gossypol, and flupyradifurone (4-[(2,2-difluoroethyl)amino]-2(5H)-furanone)) significantly up-regulated AgHsp70 expression. Knockdown of AgHsp70 using RNAi obviously increased the susceptibility of cotton aphids to 2-tridecanone, gossypol and flupyradifurone. Dual-luciferase reporter assays revealed that gossypol and flupyradifurone significantly enhanced the promoter activity of AgHsp70 at a concentration of 10 mg/L. Furthermore, we identified the transcription factor heat shock factor (HSF) as a regulator of AgHsp70, as silencing AgHSF reduced AgHsp70 expression. Our results shed light on the role of AgHsp70 in xenobiotic adaptation and thermo-tolerance.

Silencing the rhythm gene AgCLK-1 reduced feeding of Aphis gossypii.

The cotton aphid Aphis gossypii Glover is an important cotton pest, and means of controlling this insect is a primary research focus. Although biological rhythm is an important mechanism that regulates numerous insect processes and activities, its role in cotton aphid has not been elucidated. In the present study, four highly-expressed circadian rhythm genes were selected from the cotton aphid genome database and their physicochemical properties and protein structures were analyzed. These genes were in the Takeout, Timeless, and Timeless interacting-related families, and the corresponding proteins contained highly-conserved Swis and TIMELESS domains. Gene expression analysis at multiple developmental stages revealed differing expression patterns between the four genes. AgCLK-1 had the highest relative expression of the four, especially during the nymph period. Silencing AgCLK-1 caused a significant refusal of the cotton aphids to feed at 1, 3, and 5 d of treatment. These results demonstrated that AgCLK-1 played a key role in regulating the feeding behavior of cotton aphid. This new functional understanding provides novel insights into cotton aphid biology and suggests new targeting strategies for agricultural pest control.

Effects of dimpropyridaz on feeding behavior, locomotivity and biological parameters of Aphis gossypii.

Aphis gossypii is a worldwide agricultural pest insect that has developed resistance to multiple pesticides. Dimpropyridaz is a new chordotonal organ regulator and has been registered for control of sap-sucking insects including A. gossypii. For the aim to effectively apply dimpropyridaz for A. gossypii control, it is necessary to clarify the toxic effects of dimpropyridaz on cotton aphids. In the present study, the effects of dimpropyridaz on feeding behavior, locomotivity and biological parameters of A. gossypii were investigated. The bioassay results showed that dimpropyridaz had good insecticidal activity against A. gossypii, with LC50 as 1.91 mg/L at 72 h post exposure. Moreover, the dimpropyridaz treated A. gossypii showed obvious poisoning symptoms of dehydration and shrivel. Through the gentle-touch experiment and feeding experiment, it was found that dimpropyridaz treatment had significant adverse impacts on the locomotivity and feeding behavior of A. gossypii. Compared with the control group, the coordinated movement ability of the treated A. gossypii attenuated, moreover the feeding behavior of A. gossypii was inhibited. The feeding rate decreased by 62.00%, 64.00% and 71.67% after treatment with 50.33 mg/L dimpropyridaz for 24 h, 48 h and 72 h, respectively. Especially, EPG recordings showed that the number of intracellular stylet puncture and the total duration of phloem sap ingestion and concurrent salivation decreased substantially, while the total duration of non-probing increased after exposure to dimpropyridaz. Furthermore, the treatments with LC10 and LC30 of dimpropyridaz significantly reduced the longevity and fecundity of F0, and led to a decrease of the relative fitness of F0 to 0.48 and 0.32, respectively. The net reproductive rate (R0) and mean generation time (T) of F1 generation were also significantly reduced, moreover the duration of reproduction was significantly shortened. In addition, at 72 h post treatment with LC30 dimpropyridaz, the gene expression levels of JHEH and USP of cotton aphids significantly increased, while the expression of FOXO, INR, EcR and INRS decreased. These results provide basis for clarifying the toxicology of dimpropyridaz to cotton aphids, and also are beneficial for effective control of cotton aphid using dimpropyridaz.

Aphid Resistance Evaluation and Constitutive Resistance Analysis of Eighteen Lilies.

Lilies (Lilium spp.) are famous bulb flowers worldwide, with high ornamental value. Aphid damage has seriously constrained the development of the lily industry. In this study, the aphid resistance of 16 lily cultivars and 2 wild lily species was characterized in the field and greenhouse. Leaf color parameters, stomatal density and size, thickness of leaf layers, leaf waxy content, and leaf water content were determined to explore the constitutive resistance of lilies. The results show that there was a significant positive correlation between the number of aphids in the field and in the greenhouse (p ≤ 0.05, r = 0.47). This indicated that the level of aphid infestation in both the field and the greenhouse is generally consistent across different types of lily plants. Among these 18 lilies, 'Palazzo', 'Nymph', 'Cameleon' and L. lancifolium were resistant to A. gossypii, while 'Black Beauty' and 'Magnefique' had poor resistance. The correlation analysis results showed that the number of aphids was negatively correlated with leaf abaxial surface a*, stomatal size, water content, and thickness of leaf palisade tissue and positively correlated with leaf distal axial surface b*, C*, and waxy content. Among them, the correlation between the number of aphids and the thickness of leaf palisade tissue reached a significant level (p ≤ 0.05, r = -0.521). This indicated that the thickness of the palisade tissue of lily leaves might be an important factor influencing the proliferation of aphids. This study not only screened out aphid-resistant lilies but also established a crucial research foundation for the targeted breeding and molecular breeding of lilies with aphid resistance.

Development of Sustainable Insecticide Candidates for Protecting Pollinators: Insight into the Bioactivities, Selective Mechanism of Action and QSAR of Natural Coumarin Derivatives against Aphids.

Plants employ abundant toxic secondary metabolites to withstand insect attack, while pollinators can tolerate some natural defensive compounds. Coumarins, as promising green alternatives to chemical insecticides, possess wide application prospects in the crop protection field. Herein, the bioactivities of 30 natural coumarin derivatives against Aphis gossypii were assessed and revealed that 6-methylcoumarin exhibited potent aphicidal activity against aphids but displayed no toxicity to honeybees. Additionally, using biochemical, bioinformatic, and molecular assays, we confirmed that the action mode of 6-methylcoumarin against aphids was by inhibiting acetylcholinesterase (AChE). Meanwhile, functional assays revealed that the difference in action site, which located in Lys585 in aphid AChE (equivalent to Val548 in honeybee AChE), was the principal reason for 6-methylcoumarin being toxic to aphids but safe to pollinators. This action site was further validated by mutagenesis data, which uncovered how 6-methylcoumarin was unique selective to the aphid over honeybee or mammalian AChE. Furthermore, a 2D-QSAR model was established, revealing that the central structural feature was H3m, which offers guidance for the future design of more potent coumarin compounds. This work provides a sustainable strategy to take advantage of coumarin analogues for pest management while protecting nontarget pollinators.