Transcriptional regulation of host insulin signaling pathway genes controlling larval development by Microplitis manilae parasitization.

Idiobiont parasitoids using other insects as hosts sabotage the host growth and development to ensure their offspring survival. Numerous studies have discovered that insect development is subtly regulated by the conserved insulin signaling pathway. However, little is known about how wasp parasitization disrupts host development controlled by the insulin signaling pathway. Here we address this study to determine the effect of wasp parasitism on host Spodoptera frugiperda development using the idiobiont parasitoid Microplitis manilae as a model. Upon M. manilae parasitization, the body weight, body length, and food consumption of host insect were dramatically reduced compared to the unparasitized S. frugiperda. We next identified the core genes involved in host insulin signaling pathway and further analyzed the domain organizations of these genes. Phylogenetic reconstruction based on the insulin receptors clustered S. frugiperda together with other noctuidae insects. In the latter study, we profiled the expression patterns of host insulin signaling pathway genes in response to M. manilae parasitization at 2, 24, and 48 h, significant decreases in mRNA levels were recorded in S. frugiperda larvae upon 24 and 48 h parasitization. These current findings substantially add to our understanding of the physiological interaction between parasitoid and host insects, thus contributing to revealing the molecular mechanism of parasitic wasps regulating host development.

Molecular and Pharmacological Characterization of ß-Adrenergic-like Octopamine Receptors in the Endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae).

Octopamine (OA) is structurally and functionally similar to adrenaline/noradrenaline in vertebrates, and OA modulates diverse physiological and behavioral processes in invertebrates. OA exerts its actions by binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been investigated in several insects. However, the literature on OARs is scarce for parasitoids. Here we cloned three ß-adrenergic-like OARs (CcOctßRs) from Cotesia chilonis. CcOctßRs share high similarity with their own orthologous receptors. The transcript levels of CcOctßRs were varied in different tissues. When heterologously expressed in CHO-K1 cells, CcOctßRs induced cAMP production, and were dose-dependently activated by OA, TA and putative octopaminergic agonists. Their activities were inhibited by potential antagonists and were most efficiently blocked by epinastine. Our study offers important information about the molecular and pharmacological properties of ß-adrenergic-like OARs from C. chilonis that will provide the basis to reveal the contribution of individual receptors to the physiological processes and behaviors in parasitoids.

An endoparasitoid uses its egg surface proteins to regulate its host immune response.

With proteomic analysis, we identified 379 egg surface proteins from an endoparasitoid, Cotesia chilonis. Proteins containing conserved enzymatic domains constitute a large proportion of egg surface components. Some proteins, such as superoxidase dismutase, homolog of C. rubecula 32-kDa protein, and immunoevasive protein-2A, are classical parasitism factors that have known functions in host immunity regulation. Melanization assays revealed that a novel egg surface protein, C. chilonis egg surface serpin domain-containing protein had the same function as a C. chilonis venom serpin, as both suppressed host melanization in a dose-dependent manner. C. chilonis egg surface serpin domain-containing protein is mainly transcribed in C. chilonis oocytes with follicular cells, and it is located on both the anterior and posterior sides of the mature egg surface. Additionally, we used LC-MS/MS to identify 586 binding proteins sourced from C. suppressalis plasma located on the eggshell surface of C. chilonis, which included some immunity-related proteins. These results not only indicate that C. chilonis uses its egg surface proteins to reduce the immune response of its host but also imply that endoparasitoid egg surface proteins might be a new parasitism factor involved in host immune regulation.

Cellular and humoral immune interactions between Drosophila and its parasitoids.

The immune interactions occurring between parasitoids and their host insects, especially in Drosophila-wasp models, have long been the research focus of insect immunology and parasitology. Parasitoid infestation in Drosophila is counteracted by its multiple natural immune defense systems, which include cellular and humoral immunity. Occurring in the hemocoel, cellular immune responses involve the proliferation, differentiation, migration and spreading of host hemocytes and parasitoid encapsulation by them. Contrastingly, humoral immune responses rely more heavily on melanization and on the Toll, Imd and Jak/Stat immune pathways associated with antimicrobial peptides along with stress factors. On the wasps' side, successful development is achieved by introducing various virulence factors to counteract immune responses of Drosophila. Some or all of these factors manipulate the host's immunity for successful parasitism. Here we review current knowledge of the cellular and humoral immune interactions between Drosophila and its parasitoids, focusing on the defense mechanisms used by Drosophila and the strategies evolved by parasitic wasps to outwit it.

A venom protein, Kazal-type serine protease inhibitor, of ectoparasitoid Pachycrepoideus vindemiae inhibits the hemolymph melanization of host Drosophila melanogaster.

Parasitic wasps inject various virulence factors into the host insects while laying eggs, among which the venom proteins, one of the key players in host insect/parasitoid relationships, act in host cellular and humoral immune regulation to ensure successful development of wasp progeny. Although the investigations into actions of venom proteins are relatively ample in larval parasitoids, their regulatory mechanisms have not been thoroughly understood in pupal parasitoids. Here, we identified a venom protein, Kazal-type serine protease inhibitor, in the pupal ectoparasitoid Pachycrepoideus vindemiae (PvKazal). Sequence analysis revealed that PvKazal is packed by a signal peptide and a highly conserved "Kazal" domain. Quantitative polymerase chain reaction analysis recorded a higher transcript level of PvKazal in the venom apparatus relative to that in the carcass, and the PvKazal messenger RNA level appeared to reach a peak on day 5 posteclosion. Recombinant PvKazal strongly inhibited the hemolymph melanization of host Drosophila melanogaster. Additionally, the heterologous expression of PvKazal in transgenic Drosophila reduced the crystal cell numbers and blocked the melanization of host pupal hemolymph. Our present work underlying the roles of PvKazal undoubtedly increases the understanding of venom-mediated host-parasitoid crosstalk.

Identification, Characterization and Expression Analysis of TRP Channel Genes in the Vegetable Pest, Pieris rapae.

Transient receptor potential (TRP) channels are critical for insects to detect environmental stimuli and regulate homeostasis. Moreover, this superfamily has become potential molecular targets for insecticides or repellents. Pieris rapae is one of the most common and widely spread pests of Brassicaceae plants. Therefore, it is necessary to study TRP channels (TRPs) in P. rapae. In this study, we identified 14 TRPs in P. rapae, including two Water witch (Wtrw) genes. By contrast, only one Wtrw gene exists in Drosophila and functions in hygrosensation. We also found splice isoforms of Pyrexia (Pyx), TRPgamma (TRPγ) and TRP-Melastatin (TRPM). These three genes are related to temperature and gravity sensation, fine motor control, homeostasis regulation of Mg2+ and Zn2+ in Drosophila, respectively. Evolutionary analysis showed that the TRPs of P. rapae were well clustered into their own subfamilies. Real-time quantitative PCR (qPCR) showed that PrTRPs were widely distributed in the external sensory organs, including antennae, mouthparts, legs, wings and in the internal physiological organs, including brains, fat bodies, guts, Malpighian tubules, ovaries, as well as testis. Our study established a solid foundation for functional studies of TRP channels in P. rapae, and would be benefit to developing new approaches to control P. rapae targeting these important ion channels.

Molecular and pharmacological characterization of a ß-adrenergic-like octopamine receptor from the green rice leafhopper Nephotettix cincticeps.

As the counterparts of noradrenaline and adrenaline in vertebrates, octopamine (OA) regulates multiple physiological and behavioral processes in invertebrate. OA mediates its effects via binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been reported in several insects. However, little work was documented in hemipteran insects. We cloned a ß-adrenergic-like OAR (NcOA2B2) from Nephotettix cincticeps. NcOA2B2 shares high similarity with members of the OA2B2 receptor class. Transcript level of NcOA2B2 varied in various tissues and was highly expressed in the leg. After heterologous expression in CHO-K1 cells, NcOA2B2 was dose-dependently activated by OA (EC50 = 2.56 nM) and tyramine (TA) (EC50 = 149 nM). Besides putative octopaminergic agonists, dopaminergic agonists and amitraz and DPMF potently activated NcOA2B2 in a dose-dependent manner. Receptor activity was blocked by potential antagonists and was most efficiently antagonized by asenapine. Phentolamine showed both antagonist and agonist effects on NcOA2B2. Our results offer the important information about molecular and pharmacological characterization of an OAR from N. cincticeps that will provide the basis for forthcoming studies on its roles in physiological processes and behaviors, and facilitate the design of novel insecticides for pest control.

Identification and Comparative Analysis of Venom Proteins in a Pupal Ectoparasitoid, Pachycrepoideus vindemmiae.

Parasitoid wasps inject venom containing complex bioactive compounds to regulate the immune response and development of host arthropods and sometime paralyze host arthropods. Although extensive studies have been conducted on the identification of venom proteins in larval parasitoids, relatively few studies have examined the pupal parasitoids. In our current study, a combination of transcriptomic and proteomic methods was used to identify 64 putative venom proteins from Pachycrepoideus vindemmiae, an ectoparasitoid of Drosophila. Expression analysis revealed that 20 tested venom proteins have 419-fold higher mean expression in the venom apparatus than in other wasp tissues, indicating their specialization to venom. Comparisons of venom proteins from P. vindemmiae and other five species spanning three parasitoid families detected a core set of "ancient" orthologs in Pteromalidae. Thirty-five venom proteins of P. vindemmiae were assigned to the orthologous groups by reciprocal best matches with venoms of other pteromalids, while the remaining 29 were not. Of the 35 categories, twenty-seven have orthologous relationships with Nasonia vitripennis venom proteins and 25 with venoms of Pteromalus puparum. More distant relationships detected that five and two venom proteins of P. vindemmiae are orthologous with venoms of two Figitidae parasitoids and a Braconidae representative, respectively. Moreover, twenty-two venoms unique to P. vindemmiae were also detected, indicating considerable interspecific variation of venom proteins in parasitoids. Phylogenetic reconstruction based on a set of single-copy genes clustered P. vindemmiae with P. puparum, N. vitripennis, and other members of the family Pteromalidae. These findings provide strong evidence that P. vindemmiae venom proteins are well positioned for future functional and evolutionary studies.

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT7 genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.

Genome-wide characterization and transcriptomic analyses of neuropeptides and their receptors in an endoparasitoid wasp, Pteromalus puparum.

In insects, neuropeptides constitute a group of signaling molecules that act in regulation of multiple physiological and behavioral processes by binding to their corresponding receptors. On the basis of the bioinformatic approaches, we screened the genomic and transcriptomic data of the parasitoid wasp, Pteromalus puparum, and annotated 36 neuropeptide precursor genes and 33 neuropeptide receptor genes. Compared to the number of precursor genes in Bombyx mori (Lepidoptera), Chilo suppressalis (Lepidoptera), Drosophila melanogaster (Diptera), Nilaparvata lugens (Hemiptera), Apis mellifera (Hymenoptera), and Tribolium castaneum (Coleoptera), P. puparum (Hymenoptera) has the lowest number of neuropeptide precursor genes. This lower number may relate to its parasitic life cycle. Transcriptomic data of embryos, larvae, pupae, adults, venom glands, salivary glands, ovaries, and the remaining carcass revealed stage-, sex-, and tissue-specific expression patterns of the neuropeptides, and their receptors. These data provided basic information about the identity and expression profiles of neuropeptides and their receptors that are required to functionally address their biological significance in an endoparasitoid wasp.

Genomic and transcriptomic analyses of glutathione S-transferases in an endoparasitoid wasp, Pteromalus puparum.

Pteromalus puparum is a gregarious pupal endoparasitoid with a wide host range. It deposits eggs into pierid and papilionid butterfly pupae. Glutathione S-transferases (GSTs) are a family of multifunctional detoxification enzymes that act in xenobiotic metabolism in insects. Insect genome projects have facilitated identification and characterization of GST family members. We identified 20 putative GSTs in the P. puparum genome, including 19 cytosolic and one microsomal. Phylogenetic analysis showed that P. puparum GSTs are clustered into Hymenoptera-specific branches. Transcriptomic data of embryos, larvae, female pupae, male pupae, female adults, male adults, venom glands, carcass, salivary glands, and ovaries revealed stage-, sex-, and tissue-specific expression patterns of GSTs in P. puparum. This is the most comprehensive study of genome-wide identification, characterization, and expression profiling of GST family in hymenopterans. Our results provide valuable information for understanding the metabolic adaptation of this wasp.

The Pupal Ectoparasitoid Pachycrepoideus vindemmiae Regulates Cellular and Humoral Immunity of Host Drosophila melanogaster.

The immunological interaction between Drosophila melanogaster and its larval parasitoids has been thoroughly investigated, however, little is known about the interaction between the host and its pupal parasitoids. Pachycrepoideus vindemmiae, a pupal ectoparasitoid of D. melanogaster, injects venom into its host while laying eggs on the puparium, which regulates host immunity and interrupts host development. To resist the invasion of parasitic wasps, various immune defense strategies have been developed in their hosts as a consequence of co-evolution. In this study, we mainly focused on the host immunomodulation by P. vindemmiae and thoroughly investigated cellular and humoral immune response, including cell adherence, cell viability, hemolymph melanization and the Toll, Imd, and JAK/STAT immune pathways. Our results indicated that venom had a significant inhibitory effect on lamellocyte adherence and induced plasmatocyte cell death. Venom injection and in vitro incubation strongly inhibited hemolymph melanization. More in-depth investigation revealed that the Toll and Imd immune pathways were immediately activated upon parasitization, followed by the JAK/STAT pathway, which was activated within the first 24 h post-parasitism. These regulatory effects were further validated by qPCR. Our present study manifested that P. vindemmiae regulated the cellular and humoral immune system of host D. melanogaster in many aspects. These findings lay the groundwork for studying the immunological interaction between D. melanogaster and its pupal parasitoid.

Molecular cloning and characterization of TRPVs in two rice pests: Nilaparvata lugens (Stål) and Nephotettix cincticeps (Uhler).

BACKGROUD: Insect TRPV is a subfamily of transient receptor potential (TRP) ion channels, including two genes, nanchung and inactive. It has recently been found that two commercial insecticides, pymetrozine and pyrifluquinazon, target the heteromeric TRPV ion channel complex which is specifically expressed in the chordotonal organ neurons in Drosophila. However, information on the TRPV genes in agricultural insects is still limited. RESULTS: In this study, we cloned and characterized two TRPV genes from Nilaparvata lugens (NlNan and NLIav) and Nephotettix cincticeps (NcNan and NcIav), two serious rice pests throughout Asia. The deduced amino acid sequences share highly identity with other insect homologues (58-85%) and have the characteristic TRPV domain architecture: five ankyrin repeats and six transmembrane domains. These TRPV transcripts were expressed in all developmental stages and expression levels in male adults were significantly higher than in female adults. Moreover, expression levels in antennae were much higher than in heads and legs. CONCLUSION: NlNan, NlIav, NcNan and NcIav may have roles in male-specific behaviors, and the sequence information lays the foundation for further study on the structural and functional characterization of TRPVs in agricultural pests. © 2018 Society of Chemical Industry.

Resistance of rice to insect pests mediated by suppression of serotonin biosynthesis.

Rice is one of the world's most important foods, but its production suffers from insect pests, causing losses of billions of dollars, and extensive use of environmentally damaging pesticides for their control1,2. However, the molecular mechanisms of insect resistance remain elusive. Although a few resistance genes for planthopper have been cloned, no rice germplasm is resistant to stem borers. Here, we report that biosynthesis of serotonin, a neurotransmitter in mammals3, is induced by insect infestation in rice, and its suppression confers resistance to planthoppers and stem borers, the two most destructive pests of rice2. Serotonin and salicylic acid derive from chorismate4. In rice, the cytochrome P450 gene CYP71A1 encodes tryptamine 5-hydroxylase, which catalyses conversion of tryptamine to serotonin5. In susceptible wild-type rice, planthopper feeding induces biosynthesis of serotonin and salicylic acid, whereas in mutants with an inactivated CYP71A1 gene, no serotonin is produced, salicylic acid levels are higher and plants are more insect resistant. The addition of serotonin to the resistant rice mutant and other brown planthopper-resistant genotypes results in a loss of insect resistance. Similarly, serotonin supplementation in artificial diet enhances the performance of both insects. These insights demonstrate that regulation of serotonin biosynthesis plays an important role in defence, and may prove valuable for breeding insect-resistant cultivars of rice and other cereal crops.

The rice planthopper parasitoid Anagrus nilaparvatae is not at risk when feeding on honeydew derived from Bacillus thuringiensis (Bt) rice.

BACKGROUND: Honeydew is a sugar-rich excretion produced by sap-feeding Sternorrhyncha and is an important source of carbohydrates for natural enemies, especially for parasitoids. Honeydew derived from genetically modified (GM) crops can contain amounts of the transgene product. Thus, it is a possible route of exposure for natural enemies feeding on honeydew. In the present study, the potential effects of Nilaparvata lugens honeydew derived from Cry1C and Cry2A rice on different life-table parameters and parasitism dynamics of the egg parasitoid Anagrus nilaparvatae were evaluated under laboratory and field conditions. Furthermore, the Bacillus thuringiensis (Bt) levels and the sugar and amino acid composition of honeydew were analyzed. RESULTS: Results indicated that A. nilaparvatae was exposed to Bt proteins by feeding on N. lugens honeydew produced from Bt rice. However, honeydew derived from the tested Cry1C and Cry2A rice lines did not affect the development, longevity, emergence rate and fecundity of A. nilaparvatae. Also, the parasitism dynamics in the field remained unaffected. In addition, the sugar and amino acid composition of N. lugens honeydew was not significantly altered for the tested Bt rice lines compared with the parental non-Bt plant. CONCLUSION: The quality of honeydew derived from the tested Bt rice lines as a food resource for natural enemies was maintained. © 2018 Society of Chemical Industry.

A new Drosophila octopamine receptor responds to serotonin.

As the counterparts of the vertebrate adrenergic transmitters, octopamine and tyramine are important physiological regulators in invertebrates. They control and modulate many physiological and behavioral functions in insects. In this study, we reported the pharmacological properties of a new α2-adrenergic-like octopamine receptor (CG18208) from Drosophila melanogaster, named DmOctα2R. This new receptor gene encodes two transcripts by alternative splicing. The long isoform DmOctα2R-L differs from the short isoform DmOctα2R-S by the presence of an additional 29 amino acids within the third intracellular loop. When heterologously expressed in mammalian cell lines, both receptors were activated by octopamine, tyramine, epinephrine and norepinephrine, resulting in the inhibition of cAMP production in a dose-dependent manner. The long form is more sensitive to the above ligands than the short form. The adrenergic agonists naphazoline, tolazoline and clonidine can stimulate DmOctα2R as full agonists. Surprisingly, serotonin and serotoninergic agonists can also activate DmOctα2R. Several tested adrenergic antagonists and serotonin antagonists blocked the action of octopamine or serotonin on DmOctα2R. The data presented here reported an adrenergic-like G protein-coupled receptor activated by serotonin, suggesting that the neurotransmission and neuromodulation in the nervous system could be more complex than previously thought.

Characterization of three serotonin receptors from the small white butterfly, Pieris rapae.

Serotonin (5-hydroxytryptamine, 5-HT) plays a key role in modulating diverse physiological processes and behaviors in both protostomes and deuterostomes. These functions are mediated through the binding of serotonin to its receptors, which are recognized as potential insecticide targets. We investigated the sequence, pharmacology and tissue distribution of three 5-HT receptors (Piera5-HT1A, Piera5-HT1B, Piera5-HT7) from the small white butterfly Pieris rapae, an important pest of cultivated cabbages and other mustard family crops. Activation of Piera5-HT1A or Piera5-HT1B by 5-HT inhibited the production of cAMP in a dose-dependent manner. Stimulation of Piera5-HT7 with 5-HT increased cAMP level significantly. Surprisingly, with the exception of 5-methoxytryptamine, agonists including α-methylserotonin, 8-Hydroxy-DPAT and 5-carboxamidotryptamine activated these receptors poorly. The results are consistent with previous findings in Manduca sexta. All three receptors were blocked by methiothepin, but ketanserin and yohimbine were not effective. The selective mammalian 5-HT receptor antagonists SB 216641 and SB 269970 displayed potent inhibition effects on Piera5-HT1B and Piera5-HT7 respectively. The results we achieved here indicate that the pharmacological properties of Lepidoptera 5-HT receptors are quite different from those in other insects and vertebrates and may contribute to development of new selective pesticides. This study offers important information on three 5-HT receptors from P. rapae that will facilitate further analysis of the functions of 5-HT receptors in insects.

Protein Discovery: Combined Transcriptomic and Proteomic Analyses of Venom from the Endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae).

Many species of endoparasitoid wasps provide biological control services in agroecosystems. Although there is a great deal of information on the ecology and physiology of host/parasitoid interactions, relatively little is known about the protein composition of venom and how specific venom proteins influence physiological systems within host insects. This is a crucial gap in our knowledge because venom proteins act in modulating host physiology in ways that favor parasitoid development. Here, we identified 37 possible venom proteins from the polydnavirus-carrying endoparasitoid Cotesia chilonis by combining transcriptomic and proteomic analyses. The most abundant proteins were hydrolases, such as proteases, peptidases, esterases, glycosyl hydrolase, and endonucleases. Some components are classical parasitoid venom proteins with known functions, including extracellular superoxide dismutase 3, serine protease inhibitor and calreticulin. The venom contains novel proteins, not recorded from any other parasitoid species, including tolloid-like proteins, chitooligosaccharidolytic ß-N-acetylglucosaminidase, FK506-binding protein 14, corticotropin-releasing factor-binding protein and vascular endothelial growth factor receptor 2. These new data generate hypotheses and provide a platform for functional analysis of venom components.

Pharmacological characterization of dopamine receptors in the rice striped stem borer, Chilo suppressalis.

Dopamine is an important neurotransmitter and neuromodulator in both vertebrates and invertebrates and is the most abundant monoamine present in the central nervous system of insects. A complement of functionally distinct dopamine receptors mediate the signal transduction of dopamine by modifying intracellular Ca2+ and cAMP levels. In the present study, we pharmacologically characterized three types of dopamine receptors, CsDOP1, CsDOP2 and CsDOP3, from the rice striped stem borer, Chilo suppressalis. All three receptors show considerable sequence identity with orthologous dopamine receptors. The phylogenetic analysis also clusters the receptors within their respective groups. Transcript levels of CsDOP1, CsDOP2 and CsDOP3 were all expressed at high levels in the central nervous system, indicating their important roles in neural processes. After heterologous expression in HEK 293 cells, CsDOP1, CsDOP2 and CsDOP3 were dose-dependently activated by dopamine and synthetic dopamine receptor agonists. They can also be blocked by different series of antagonists. This study offers important information on three dopamine receptors from C. suppressalis that will provide the basis for forthcoming studies investigating their roles in behaviors and physiology, and facilitate the development of new insecticides for pest control.

Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae).

Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop ligand-gated ion channel (cysLGIC) superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressalis, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs (cDNAs) via reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine α-type and three ß-type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species.