Integration analysis of PacBio SMRT- and Illumina RNA-seq reveals P450 genes involved in thiamethoxam detoxification in Bradysia odoriphaga.

The sciarid fly Bradysia odoriphaga is a serious pest of Chinese chive (Liliaceae). Neonicotinoid insecticides including thiamethoxam have been used for B. odoriphaga control. However, thiamethoxam resistance in B. odoriphaga has developed in recent years. To identify potential genes involved in detoxification metabolism of thiamethoxam in B. odoriphaga, a PacBio single-molecule real-time (SMRT) transcriptome sequencing and Illumina RNA-seq analysis on thiamethoxam treated B. odoriphaga were performed to explore differentially expressed genes in B. odoriphaga. After SMRT sequencing, analysis of Illumina RNA-Seq data showed a total of 172 differentially expressed genes (DEGs) after thiamethoxam treatment, among which eight upregulated DEGs were P450 genes that may be related to thiamethoxam metabolism. The qRT-PCR results of the eight up-regulated P450 unigenes after thiamethoxam treatment were consistent with RNA-Seq data. Furthermore, oral delivery mediated RNA interference of the eight upregulated P450 transcripts followed by insecticide bioassay was conducted, and three P450 unigenes were verified to be related to thiamethoxam detoxification in B. odoriphaga. This study provides new information about the P450 genes involved in thiamethoxam detoxification in B. odoriphaga.

The overexpression of three cytochrome P450 genes CYP6CY14, CYP6CY22 and CYP6UN1 contributed to metabolic resistance to dinotefuran in melon/cotton aphid, Aphis gossypii Glover.

Dinotefuran, the third-generation neonicotinoid, has been applied against melon/cotton aphid Aphis gossypii Glover in China. The risk of resistance development, cross-resistance pattern and potential resistance mechanism of dinotefuran in A. gossypii were investigated. A dinotefuran-resistant strain of A. gossypii (DinR) with 74.7-fold resistance was established by continuous selection using dinotefuran. The DinR strain showed a medium level of cross resistance to thiamethoxam (15.2-fold), but no cross resistance to imidacloprid. The synergism assay indicated that piperonyl butoxide and triphenyl phosphate showed synergistic effects on dinotefuran toxicity to the DinR strain with a synergistic ratio of 8.3 and 2.5, respectively, while diethyl maleate showed no synergistic effect. The activities of cytochrome P450 monooxygenase and carboxylesterase were significantly higher in DinR strain than in susceptible strain (SS). Moreover, the gene expression results showed that CYP6CY14, CYP6CY22 and CYP6UN1 were significantly overexpressed in DinR strain compared with SS strain. The expression of CYP6CY14 was 5.8-fold higher in DinR strain than in SS strain. Additionally, the transcription of CYP6CY14, CYP6CY22 and CYP6UN1 in A. gossypii showed dose- and time-dependent responses to dinotefuran exposure. Furthermore, knockdown of CYP6CY14, CYP6CY22 and CYP6UN1 via RNA interference (RNAi) significantly increased mortality of A. gossypii, when A. gossypii was treated with dinotefuran. These results demonstrated the overexpression of CYP6CY14, CYP6CY22 and CYP6UN1 contributed to dinotefuran resistance in A. gossypii.

Molecular characterization and expression profiles of nicotinic acetylcholine receptors in Bradysia odoriphaga.

Bradysia odoriphaga is a destructive insect pest, damaging more than 30 crop species. Nicotinic acetylcholine receptors (nAChRs) mediating fast excitatory transmission in the central nervous system in insects are the molecular targets of some economically important insecticides including imidacloprid, which has been widely used to control B. odoriphaga in China since 2013. However, the clear characterization about nAChRs in B. odoriphaga is still unknown. Hence, our objective is to identify and characterize the nAChR gene family in B. odoriphaga based on the transcriptome database and sequence, phylogenetic and expression profiles analysis. In this study, we cloned seven nAChR subunit genes from B. odoriphaga, including Boα1, Boα2, Boα3, Boα7, Boα8, Boß1 and Boß3. Sequence analysis revealed that the seven nAChR subunits of B. odoriphaga shared the typical structural features with Drosophila melanogaster nAChR α1 subunit, including an extracellular N-terminal domain containing six functional loops (loop A-F), a signature Cys-loop with two disulfide bond-forming cysteines separated by 13 amino acid residues, and four typical transmembrane helices (TM1-TM4) in the C-terminal region. Phylogenetic analysis suggested that seven nAChR subunit genes in B. odoriphaga are evolutionarily conserved among four model insects, including D. melanogaster, Bombyx mori, Apis mellifera and Tribolium castaneum. Meanwhile, nAChR α4, α5, α6 and ß2 subunit genes may potentially exist in B. odoriphaga, which need further study. Furthermore, quantitative real-time PCR analysis revealed the specific expression pattern of nAChR subunits in three body parts including head, thorax and abdomen, and developmental expression pattern of nAChR subunits throughout the B. odoriphaga life cycle. These results provided necessary information for further investigating the diverse functions of nAChRs in B. odoriphaga.

Identification and functional analysis of a cytochrome P450 gene involved in imidacloprid resistance in Bradysia odoriphaga Yang et Zhang.

Insect cytochrome P450 monooxygenases played an important role in detoxifying insecticides which potentially contributed to the metabolic resistance to insecticides. Bradysia odoriphaga, as a major pest of Chinese chive, was reported to be highly tolerant to neonicotinoid insecticides imidacloprid. In this study, a novel P450 gene, CYP6FV12, was cloned from B. odoriphaga. The full-length cDNA sequence of CYP6FV12 is 2520 bp long and its open reading frame (ORF) encodes 519 amino acids. Quantitative real-time PCR showed that the highest expression of CYP6FV12 was observed in fourth-instar larvae, which is 154.32-fold higher than that of eggs. Highest expression of CYP6FV12 was observed in the midgut, followed by fat body, which was 13.67 and 5.42-fold higher than that in cuticle, respectively. The expression of CYP6FV12 was significantly up-regulated in B. odoriphaga larvae after exposed to imidacloprid at the concentrations of 10, 30, 50, and 70 mg/L. Moreover, RNAi mediated silencing of CYP6FV12 increased mortality by 28.62% when the fourth-instar larvae were treated with imidacloprid. This is the first systematic study on isolated P450s gene involved in imidacloprid resistance in B. odoriphaga and increased our understanding of the molecular mechanisms of insecticide detoxification in this pest insect.

Identification of immunity-related genes distinctly regulated by Manduca sexta Spӓtzle-1/2 and Escherichia coli peptidoglycan.

The immune system of Manduca sexta has been well studied to understand molecular mechanisms of insect antimicrobial responses. While evidence supports the existence of major immune signaling pathways in this species, it is unclear how induced production of defense proteins is specifically regulated by the Toll and Imd pathways. Our previous studies suggested that diaminopimelic acid-type peptidoglycans (DAP-PG) from Gram-negative and some Gram-positive bacteria, more than Lys-type peptidoglycans (Lys-PG) from other Gram-positive bacteria, triggers both pathways through membrane-bound receptors orthologous to Drosophila Toll and PGRP-LC. In this study, we produced M. sexta proSpätzle-1 and proSpätzle-2 in Sf9 cells, identified their processing enzymes, and used prophenoloxidase activating protease-3 to activate the cytokine precursors. After Spätzle-1 and -2 were isolated from the reaction mixtures, we separately injected the purified cytokines into larval hemocoel to induce gene transcription in fat body through the Toll pathway solely. On the other hand, we treated a M. sexta cell line with E. coli DAP-PG to only induce the Imd pathway and target gene expression. RNA-Seq analysis of the fat body and cultured cells collected at 0, 6, and 24 h after treatment indicated that expression of diapausin-4, -10, -12, -13, cecropin-2, -4, -5, attacin-5, -11, and lebocin D is up-regulated predominantly via Toll signaling, whereas transcription of cecropin-6, gloverin, lysozyme-1, and gallerimycin-2 is mostly induced by DAP-PG via Imd signaling. Other antimicrobial peptides are expressed in response to both pathways. Transcripts of most Toll-specific genes (e.g., lebocin D) peaked at 6 h, contrasting the gradual increase and plateauing of drosomycin mRNA level at 24-48 h in Drosophila. We also used T (oll)-I (md) ratios to estimate relative contributions of the two pathways to transcriptional regulation of other components of the immune system. The differences in pathway specificity and time course of transcriptional regulation call for further investigations in M. sexta and other insects.

An evolutionarily conserved serine protease network mediates melanization and Toll activation in Drosophila.

Melanization and Toll pathway activation are essential innate immune mechanisms in insects, which result in the generation of reactive compounds and antimicrobial peptides, respectively, to kill pathogens. These two processes are mediated by phenoloxidase (PO) and Spätzle (Spz) through an extracellular network of serine proteases. While some proteases have been identified in Drosophila melanogaster in genetic studies, the exact order of proteolytic activation events remains controversial. Here, we reconstituted the serine protease framework in Drosophila by biochemical methods. This system comprises 10 proteases, i.e., ModSP, cSP48, Grass, Psh, Hayan-PA, Hayan-PB, Sp7, MP1, SPE and Ser7, which form cascade pathways that recognize microbial molecular patterns and virulence factors, and generate PO1, PO2, and Spz from their precursors. Furthermore, the serpin Necrotic negatively regulates the immune response progression by inhibiting ModSP and Grass. The biochemical approach, when combined with genetic analysis, is crucial for addressing problems that long stand in this important research field.

Genome-wide identification, classification, and expression profiling of serine esterases and other esterase-related proteins in the tobacco hornworm, Manduca sexta.

Serine esterases (SEs) are hydrolases that catalyze the conversion of carboxylic esters into acids and alcohols. Lipases and carboxylesterases constitute two major groups of SEs. Although over a hundred of insect genomes are known, systematic identification and classification of SEs are rarely performed, likely due to large size and complex composition of the gene family in each species. Considering their key roles in lipid metabolism and other physiological processes, we have categorized 144 M. sexta SEs and SE homologs (SEHs), 114 of which contain a motif of GXSXG. Multiple sequence alignment and phylogenetic tree analysis have revealed 39 neutral lipases (NLs), 3 neutral lipase homologs (NLHs), 11 acidic lipases (ALs), 3 acidic lipase homologs (ALHs), a lipase-3, a triglyceride lipase, a monoglyceride lipase, a hormone-sensitive lipase, and a GDSL lipase. Eighty-three carboxylesterase genes encode 29 α-esterases (AEs), 12 AEHs (e.g., SEH4-1-3), 20 feruloyl esterases (FEs), 2 FEHs, 2 ß-esterases (BEs), 2 integument esterases (IEs), 1 IEH, 4 juvenile hormone esterases, 2 acetylcholinesterases, gliotactin, 6 neuroligins, neurotactin, and an uncharacteristic esterase homolog. In addition to these GXSXG proteins, we have identified 26 phospholipases and 13 thioesterases. Expression profiling of these genes in specific tissues and stages has provided insights into their functions including digestion, detoxification, hormone processing, neurotransmission, reproduction, and developmental regulation. In summary, we have established a framework of information on SEs and related proteins in M. sexta to stimulate their research in the model species and comparative investigations in agricultural pests or disease vectors.

Serine Protease Networks Mediate Immune Responses in Extra-Embryonic Tissues of Eggs in the Tobacco Hornworm, Manduca sexta.

The melanization and Toll pathways, regulated by a network of serine proteases and noncatalytic serine protease homologs (SPHs), have been investigated mostly in adult and larval insects. However, how these innate immune reactions are regulated in insect eggs remains unclear. Here we present evidence from transcriptome and proteome analyses that extra-embryonic tissues (yolk and serosa) of early-stage Manduca sexta eggs are immune competent, with expression of immune effector genes including prophenoloxidase and antimicrobial peptides. We identified gene products of the melanization and Toll pathways in M. sexta eggs. Through in vitro reconstitution experiments, we demonstrated that constitutive and infection-induced serine protease cascade modules that stimulate immune responses exist in the extra-embryonic tissues of M. sexta eggs. The constitutive module (HP14b-SP144-GP6) may promote rapid early immune signaling by forming a cascade activating the cytokine Spätzle and regulating melanization by activating prophenoloxidase (proPO). The inducible module (HP14a-HP21-HP5) may trigger enhanced activation of Spätzle and proPO at a later phase of infection. Crosstalk between the two modules may occur in transition from the constitutive to the induced response in eggs inoculated with bacteria. Examination of data from two other well-studied insect species, Tribolium castaneum and Drosophila melanogaster, supports a role for a serosa-dependent constitutive protease cascade in protecting early embryos against invading pathogens.

Preferential binding of DAP-PGs by major peptidoglycan recognition proteins found in cell-free hemolymph of Manduca sexta.

Peptidoglycan recognition proteins (PGRPs) detect invading bacteria to trigger or modulate immune responses in insects. While these roles are established in Drosophila, functional studies are not yet achieved at the PGRP family level in other insects. To attain this goal, we selected Manduca sexta PGRP12 and five of the nine secreted PGRPs for recombinant expression and biochemical characterization. We cloned PGRP2-5, 12 and 13 cDNAs, produced the proteins in full (PGRP2-5, 13) or in part (PGRP3s, 12e, 13N, 13C) in Sf9 cells, and tested their bindings of two muramyl pentapeptides by surface plasmon resonance, two soluble peptidoglycans by competitive ELISA, and four insoluble peptidoglycans and eight whole bacteria by a pull-down assay. Preferential binding of meso-diaminopimelic acid-peptidoglycans (DAP-PGs) was observed in all the proteins containing a peptidoglycan binding domain and, since PGRP6, 7 and 9 proteins were hardly detected in cell-free hemolymph, the reportoire of PGRPs (including PGRP1 published previously) in M. sexta hemolymph is likely adapted to mainly detect Gram-negative bacteria and certain Gram-positive bacteria with DAP-PGs located on their surface. After incubation with plasma from naïve larvae, PGRP2, 3f, 4, 5, 13f and 13N considerably stimulated prophenoloxidase activation in the absence of a bacterial elicitor. PGRP3s and 12e had much smaller effects. Inclusion of the full-length PGRPs and their regions in the plasma also led to proHP8 activation, supporting their connections to the Toll pathway, since HP8 is a Spӓtzle-1 processing enzyme in M. sexta. Together, these findings raised concerns on the common belief that the Toll-pathway is specific for Gram-positive bacteria in insects.

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success.

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.

Cross-resistance and Fitness Cost Analysis of Resistance to Thiamethoxam in Melon and Cotton Aphid (Hemiptera: Aphididae).

The melon/cotton aphid, Aphis gossypii Glover, is a notorious pest in many crops. The neonicotinoid insecticide thiamethoxam is widely used for A. gossypii control. To evaluate thiamethoxam resistance risk, a melon/cotton aphid strain with an extremely high level of resistance to thiamethoxam (>2,325.6-fold) was established after selection with thiamethoxam for 24 generations. Additionally, the cross-resistance pattern to other neonicotinoids and fitness were analyzed. The cross-resistance results showed the thiamethoxam-resistant strain had extremely high levels of cross-resistance against clothianidin (>311.7-fold) and nitenpyram (299.9-fold), high levels of cross-resistance against dinotefuran (142.3-fold) and acetamiprid (76.6-fold), and low cross-resistance against imidacloprid (9.3-fold). Compared with the life table of susceptible strain, the thiamethoxam-resistant strain had a relative fitness of 0.950, with significant decreases in oviposition days and fecundity and prolonged developmental duration. The molecular mechanism for fitness costs was studied by comparing the mRNA expression levels of juvenile hormone acid O-methyltransferase (JHAMT), juvenile hormone-binding protein (JHBP), juvenile hormone epoxide hydrolase (JHEH), ecdysone receptor (EcR), ultraspiracle protein (USP), and Vitellogenin (Vg) in the susceptible and thiamethoxam-resistant strains. Significant overexpression of JHEH and JHBP and downregulation of EcR and Vg expression were found in the thiamethoxam-resistant strain. These results indicate that A. gossypii has the potential to develop extremely high resistance to thiamethoxam after continuous exposure, with a considerable fitness cost and cross-resistance to other neonicotinoids.

Low expression levels of nicotinic acetylcholine receptor subunits Boα1 and Boß1 are associated with imidacloprid resistance in Bradysia odoriphaga.

BACKGROUND: Neonicotinoid insecticide imidacloprid acts on insect nicotinic acetylcholine receptors (nAChRs). The mechanisms of insect resistance to imidacloprid include target-site alteration and increased detoxification metabolism. In Bradysia odoriphaga, cytochrome P450 monooxygenase has been found involved in metabolic resistance to imidacloprid. However, the situation of target-site related resistance to imidacloprid in B. odoriphaga is still unknown. RESULTS: Nine field-collected B. odoriphaga populations showed various sensitivities to imidacloprid compared with the susceptible (SS) strain, including susceptibility, decreased susceptibility, low resistance, moderate resistance and high resistance. Seven nAChR subunit genes including α1, α2, α3, α7, α8, ß1 and ß3, were examined for site mutation and changes in transcription levels in field populations. No nAChR polymorphism potentially related to the resistant phenotypes was found. However, differential expression of nAChR subunit genes was found in imidacloprid resistant field population. In high imidacloprid resistant population LC-2 (93.14-fold resistance), the transcription levels of α1, α2 and ß1 subunits were significantly down-regulated, while the transcription levels of α3 and α8 subunits were significantly up-regulated, compared with that in SS strain. In addition, imidacloprid acute exposure induced differential expression of nAChR subunit genes in B. odoriphaga. Furthermore, RNA interference (RNAi) suppressed the transcriptional expression of Boα1 and Boß1, and decreased mortality of B. odoriphaga by 23.03% and 18.69%, respectively, when treated with imidacloprid. CONCLUSION: These results indicated that, although no target-site mutation was found in imidacloprid resistant B. odoriphaga population, the reduced expression of α1 and ß1 subunits contributed to B. odoriphaga resistance to imidacloprid. © 2020 Society of Chemical Industry.

Identification of a novel cytochrome P450 CYP3356A1 linked with insecticide detoxification in Bradysia odoriphaga.

BACKGROUND: Cytochrome P450 monooxygenases play an important role in the metabolic detoxification of insecticides in insect pests. However, little is known about the role of a specific P450 gene and its responses to insecticide exposure in Bradysia odoriphaga, a major pest in Chinese chive production. RESULTS: In this study, a novel P450 gene, CYP3356A1, was cloned from Bradysia odoriphaga. The full-length cDNA sequence of CYP3356A1 is 2153 bp and its open reading frame (ORF) encodes 508 amino acids. Quantitative real time PCR(qRT-PCR) analyses in different tissues showed that CYP3356A1 expression was the highest in the Malpighian tubule. Moreover, among the different developmental stages of the insect, the highest expression of CYP3356A1 was found in fourth-instar larvae. Expression of CYP3356A1 was upregulated by treatment with imidacloprid, thiamethoxam, and ß-cypermethrin at median lethal concentrations (LC50 ). RNA interference (RNAi)-mediated silencing of CYP3356A1 significantly increased mortality by 36.90%, 25.17%, and 36.73 when fourth-instar B. odoriphaga larvae were exposed to imidacloprid, thiamethoxam, and ß-cypermethrin, respectively, at the LC50 dose. CONCLUSION: These results demonstrate that CYP3356A1 is related to the detoxification of imidacloprid, thiamethoxam, and ß-cypermethrin in B. odoriphaga. Moreover, the study also increased our understanding of the molecular mechanisms of insecticide detoxification in this pest insect. © 2018 Society of Chemical Industry.

Modeling of chiral gas chromatographic separation of alkyl and cycloalkyl 2-bromopropionates using cyclodextrin derivatives as stationary phases.

Chiral 2-bromopropionates are widely used as raw materials or intermediates for synthesis of chiral pesticides and medicines with high biological activities. Enantioseparation of 2-bromopropionates is crucial for evaluating the optical purity of chiral 2-bromopropionates. In this study, the enantioseparation of 8 pairs of alkyl 2-bromopropionates and 5 pairs of cycloalkyl 2-bromopropionates were examined using 7 different cyclodextrin derivatives (CDs). For the enantiomers of methyl, ethyl, bromoethyl and prop-2-yn-1-yl 2-bromopropionates, three different kinds of enantioseparation results including baseline separation, partial separation and no separation, are obtained on the 7 different CDs. Besides the baseline enantioseparation of methyl and ethyl 2-bromopropionates on some CDs, baseline enantioseparation of 2-bromoethyl 2-bromopropionate on 2,3,6-tri-O-methyl-ß-CD with resolution as 1.78, prop-2-yn-1-yl 2-bromopropionate on 2,3,6-tri-O-methyl-ß-CD and 2,3-di-O-pentyl-6-O-propyl-ß-CD with the resolution as 3.45 and 1.77, respectively, are also obtained, and can be used for determination of the optical purity. However, for the enantiomers of isopropyl, isobutyl, tert-butyl and 3-methylbut-2-en-1-yl 2-bromopropionates, only partial enantioseparation are obtained on 5 CDs, meaning that these methods cannot be used for determination of the optical purity. To 5 pairs of cycloalkyl 2-bromopropionates, the 7 CDs exhibit low enantioseparation abilities. While the investigated 5 cycloalkyl-2-bromopropionates don't result in baseline enantioseparation when they are analyzed on the 7 CDs, partial enantioseparation of 4 cycloalkyl 2-bromopropionates, cyclopentyl, cyclohexyl, cyclohexylmethyl and benzyl 2-bromopropionates, are obtained on some CDs, and no any enantioseparation of phenyl 2-bromopropionates are observed on the 7 CDs. The baseline enantioseparation methods for methyl, ethyl, bromoethyl, and prop-2-yn-1-yl 2-bromopropionates have been validated for the parameters linearity, limit of detection, limit of quantification, recovery, intra-day and inter-day precision. Moreover, the validated baseline enantioseparation methods for methyl 2-bromopropionate using columns coated with 4 different CDs have been successfully applied on the determination of optical purity of chiral methyl 2-bromopropionate sample. Furthermore, molecular docking study is performed to investigate the 2,3,6-tri-O-methyl-ß-cyclodextrin (PM-ß-CD) inclusion complexes with various 2-bromopropionates. The docking results show the contribution of differences of geometry and interaction energy between the inclusion complexes of two enantiomers with PM-ß-CD to enantioseparation of some 2-bromopropionates, however the docking results cannot explain the enantioseparation mechanism of other 2-bromopropionates, especially cycloalkyl 2-bromopropionates. This study provides information on the selection of chiral stationary phase CDs for the optimal GC enantioseparation conditions of some new 2-bromopropionates. The methodology of this study can be applied to evaluate the optical purity of some chiral 2-bromopropionates.

Impact of the secondary plant metabolite Cucurbitacin B on the demographical traits of the melon aphid, Aphis gossypii.

Cucurbitacin B is a natural triterpene present in plants of Cucurbitaceae family, which are among the host plants for melon aphid, Aphis gossypii. In present study we characterized the effects of two cucurbitacin B concentrations on the biological parameters of adults (F0) and of juveniles and adults of their progeny (F1). The results showed that cucurbitacin B at 25 ppm significantly reduced the adult longevity and fecundity of both F0 and F1 generation. Exposure of F0 generation to 25 ppm though reduced the demographic traits of F1 including the intrinsic rate of increase r (day-1), generation time T (day), finite rate of increase λ (day-1), however, only net reproductive rate R0 (offspring/individual) decreased significantly. While 100 ppm reduced not only the longevity and fecundity of F0 generation but also the longevity of F1 generation. Fecundity of F1 was not affected by 100 ppm of cucurbitacin B, however, R0 (offspring/individual) and T (day) of F1 generation were lower than the control population. These results support the hypothesis that high contents of cucurbitacin B caused negative impact on melon aphid and could be used as a lead for classical selection of resistant varieties of plants that are main hosts for the melon aphid.