Nonsteroidal ecdysteroid agonists act in a similar fashion with ecdysone to affect the insect pest larval molting. The ecdysone receptor is so sensitive and specific that the new screening assay technique must be set up to detect the activity of insecticidal lead compounds. In this study, the functional domains of the ecdysone receptor and ultraspiracle protein from Spodoptera litura were cloned by RT-PCR. These two fragments were inserted into expression vectors and named as pEHISEGFPTEV-EcR_cde and pEHISEGFPTEV-USP_cde, respectively, and the fusion proteins were purified after induction. With l mg/mL fusion protein and 8 nmol/L ³H-PonA, the radioligand receptor binding assays were carried out to detect the radioactivity changes in the count per minute (CPM) in treatments with four insecticides (tebufenozide, fufenozide, dibenzoylhydrazide and chlorbenzuron) at different concentrations. The results showed that when the concentrations of tested compounds gradually increased, the CPM of the first three insecticides was reduced in different degrees. Tebufenozide had the highest degree of radioactivity reduction, followed by fufenozide and dibenzoylhydrazide, while the radioactivity of chlorbenzuron had almost no change. This study indicated that among the tested four insecticides, tebufenozide has the highest insecticidal activity under the same conditions, and the method can be used to preliminarily screen out the insecticidal activity of nonsteroidal ecdysteroid agonists or lead compounds.

【Aim】 To clone and identify the olfactory receptor co-receptor (Orco) gene in the Chinese honeybee, Apis cerana cerana, and locate its proteins in the worker’s antennae. 【Methods】 The olfactory receptor Orco gene from honeybee A. cerana cerana was cloned by RT-PCR, and the expression of this gene at different developmental stages and in different tissues of the honeybee were profiled by Real-time PCR. The putative amino acid sequence was analyzed by bioinformatics methods, and the sub-cellular localization of Orco in the worker’s antennae was detected by immunofluorescence. 【Results】 The full-length cDNA sequence of olfactory receptor Orco was obtained from A. cerana cerana, and named as AcerOrco (GenBank accession no. JF968610.1). Its open reading frame is 1 434 bp in length, encoding 477 amino acid residues. Homology analysis showed that the amino acid sequences of AcerOrco contain seven-transmembrane domains and four hydrophilic regions outside the membrane. The expression profiling at different developmental stages showed that AcerOrco was expressed in the egg, larval and pupal periods at very low levels. The expression profiling in different tissues revealed that AcerOrco was highly expressed in the antennae and legs in nurse bee, with the highest level in the antennae of the 1 day-old worker, while in forager it was highly expressed in antennae, head (without antennae), thorax, abdomen and wings. Subcellular localization displayed that AcerOrco was largely expressed in pairs in the antenna of forager, especially in the 1st flagellar segment of antenna. Furthermore AcerOrco was seemingly to be mainly expressed in the outer dendrite (OD) neurons of sensilla trihodea and the dendrites of sensilla placodea in antennae. 【Conclusion】 The full-length olfactory receptor gene AcerOrco and its expression profiles were obtained, and the encoded protein in the forager antenna of worker honeybee was successfully localized using immunofluorescence. It is inferred that AcerOrco is closely related with the olfactory development and functions of antennal sensilla in the honeybee.

Bursicon plays a vital role in insect molting sclerosis, as it regulates cuticle sclerotization (hardening and tanning) via a G protein-coupled receptor (GPCR). To study the function of bursicon in the small brown planthopper (SBPH), Laodelphax striatellus, the full-length sequences of bursicon α and bursicon β were amplified by RT-PCR and RACE, and were designated as Lsburs-α and Lsburs-β, respectively. The products are 1 126 bp and 761 bp in length for Lsburs-α and Lsburs-β, respectively. Lsburs-α contains a 483 bp open reading frame (ORF) encoding a protein with 160 amino acid residues, which has two N-myristoylation sites, three casein kinase Ⅱ phosphorylation sites and two protein kinase C phosphorylation sites. Lsburs-β contains a 417 bp ORF encoding a protein with 138 amino acid residues, which has two N-myristoylation sites, three casein kinase Ⅱ phosphorylation sites and one tyrosine kinase phosphorylation site. Real-time quantitative PCR results showed that the transcripts of Lsburs-α and Lsburs-β were detectable in all the nymphal stages of L. striatellus, increased with the nymphal stages, reached the highest level in the emerging period, and then decreased after adult eclosion. The results suggest that bursicon closely relates to cuticle sclerotization in L. striatellus after molting. The results lay a solid foundation for the further study of bursicon’s functions, receptor regulation, signal pathway and so on.

Trehalase is a key enzyme in trehalose metabolism and plays important roles in the development and regulation of energy in insects. In order to further investigate the function of trehalase, we analyzed the molecular characteristics and mRNA expression pattern of a soluble trehalase (TRE, GenBank accession no. FJ795020) gene from Locusta migratoria. No transmembrane region was predicted for TRE of L. migratoria. Phylogenetic analysis showed that TRE of L. migratoria was first clustered with soluble trehalases of Aphis glycines, Acyrthosiphon pisum, Nilaparvata lugens and Laodelphax striatella, and its gene was so designated as LmTre-1. qPCR was used to analyze the mRNA expression pattern of LmTre-1 in different tissues and developmental tages in L. migratoria. The expression of LmTre-1 was low in early and middle stage eggs but greatly increased in late stage eggs. Tissue-specific-expression results showed that LmTre-1 was expressed in all the major tissues, with high expression level in integument and lower expression level in fat body, muscles, trachea, testis and ovaries in the 5th instar nymphs and adults. LmTre-1 was also highly expressed after molting and the expression level gradually declined during the 5th instar nymphal development. LmTre-1 was constantly expressed at a high level in the integument of adults. This expression pattern is very similar with that of chitin synthase 1 gene, and it is so inferred that LmTre-1 is associated with chitin synthesis in the cuticle. The study sheds new light on a better understanding of the functional characteristics of this gene and may pave a way for pesticide screening based on trehalase.

The female parasitoids can sense host-plant odour via sensilla on the antennae, but little information is available on agaonid antennae. To explore the host location of Ceratosolen emarginatus Mayr (Hymenoptera: Agaonidae), experiments were designed to examine the types, distribution and ultramorphology of antennal sensilla in female adults of C. emarginatus and to determine the putative functions of the identified sensilla using scanning electron microscopy and transmission electron microscopy techniques. The results indicated that the geniculate antennae of female C. emarginatus are comprised of a basal scape, pedicel, and a long threadlike flagellum which is composed of 11 flagellomeres, but the third flagellomere bears a spine-like expansion. Eleven sensillar types were found and described on the antennae of female C. emarginatus. Of the identified sensillar types, uniporous and multiporous basiconic sensilla, elongated and rounded multiporous placoid sensilla, basiconic capitate peg sensilla and coeloconic sensilla type 1 are porous and may be involved in perception of host chemical cues. However, the aporous trichoid sensilla and chaetica sensilla may have mechanosensory functions, while the coeloconic sensilla type 2 and 3 may be involved in proprioception, thermo-hygroreception or pressure reception. Here, the specific sensilla, sensilla obscura, are thick, aporous, and aligned with the longitudinal antennal axis toward the scape. Sensilla obscura should be regarded as mechanoreceptors that may help pollinators to move through fig ostiole. The results will be helpful for understanding the specific behavioral characteristics of C. emarginatus. In addition, the detailed knowledge of antennal morphology may serve as a prerequisite for future electrophysiological tests in order to draw a chemical mediation and partition in fig-wasp communities.

Experiments were conducted to enhance the virulence of two isolates of entomopathogenic fungi, IRAN-187C of Beauveria bassiana (Balsamo) Vuillemin, and A-396 isolate of Metarhizium anisopliae (Metschnikow) Sorokin, by combination with InsectoSec formulation of diatomaceous earth (DE) against the adults of Callosobruchus maculatus F. under laboratory conditions. The results demonstrated that the combinations between diatomaceous earth and the fungal isolates at lethal and sub-lethal doses achieved high mortality rates in the adult populations; moreover, the treatments improved LT₅₀ values of the fungi. The lowest value of LT₅₀ (122.2 h) was achieved by LC₅₀ (B. bassiana, IRAN-187C) + LC₅₀ (DE) treatment which was on par with that of LC₅₀ (M. anisopliae, A-396) + LC₅₀ (DE) combination (128.2 h) based on their 95% fiducial limit values, resulting in better efficacy of each fungal isolate in combination with diatomaceous earth against C. maculatus adults. Also, effects of the three temperatures (22, 27, and 30℃) and two relative humidity levels (40% and 55%) on the activity of diatomaceous earth when applied against C. maculatus adults showed that the least value of LT₅₀ (110.11 h) was attained in 30℃ and RH of 40%, suggesting high activity of diatomaceous earth in high temperatures and low humidity conditions.

The cowpea weevil, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), is the main storage pest of cowpea seeds in Iran. Using biorational insecticides instead of conventional insecticides is an appropriate alternative for controlling this pest. In this study, the lethal and sublethal effects of diatomaceous earth and spinosad against C. maculatus adults on glass surface were evaluated under laboratory conditions. The results indicated that the LC₅₀ values at 24 and 48 h after treatment were 1.47 and 0.2 g/m² for diatomaceous earth and 102.9 and 68.8 mg ai/L for spinosad, respectively. Both compounds had high acute toxicity against cowpea weevil. Sublethal effects of LC₂₀ concentration of the diatomaceous earth and spinosad against C. maculatus adults were studied by detecting biological parameters. LC₂₀ concentration of diatomaceous earth and spinosad reduced the fecundity of the pest by 71.5% and 17.2%, the egg hatching rate by 57.5% and 27.8%, and adult longevity by 74.7% and 17.1%, respectively, compared to the control. Pupal period of the insect exposed to LC²⁰ concentration of diatomaceous earth and spinosad increased by 4.8% and 2.3%, respectively, compared to the control. The sublethal effect study showed that both diatomaceous earth and spinosad negatively affected life parameters of cowpea weevil. In general, combination of lethal and sublethal effects revealed that diatomaceous earth had high potential in controlling cowpea weevil.

 During the last decades, Zaprionus indianus, a warm adapted drosophilid species has invaded and expanded its boundaries in the Indian subcontinent. Z. indianus has successfully invaded because of its high adaptive flexibility and extreme physiological tolerance. A comparison of developmental thresholds of Z. indianus (warm specialist) and D. melanogaster (cosmopolitan) during pre-adult and adult life stages at extreme temperatures showed significant difference in mortality and threshold temperature between both species. To test for variation in stress resistance and survival in preadult/adult stages over winter, Z. indianus populations were initiated from temperate and tropical sites in India. The hatchability of eggs and adult survival of these populations were monitored in field cages at a temperate location and under constant laboratory conditions, until all adults died. Total hatchability and survival showed significant difference with latitude, due to high hatchability and survival by temperate in northern populations as compared with  southern populations. Egg to adult viability experiment indicated that eggs laid in low temperature conditions developed successfully to the adult stage when the temperature became moderate. This study is evident for climatic adaptation in immature stages and stress tolerance in adults that may provide seasonal protection to the species. When climate warming scenario was considered, i.e., temperature increase of 0.6℃, a slight increase in temperature could cause a significant increment in population viability and a decrease in developmental duration. These results explain why Z. indianus can easily cross barriers and is able to adapt to new environment.

The Curculionoidea is one of the richest groups in Coleptera, but the research about its complete mitochondrial genome has not been reported. In this study, the complete mitochondrial genome (mitogenome) of Hylobitelus xiaoi Zhang was determined using long PCR (L-PCR) and primer walking technology. The entire mitochondrial DNA (mtDNA) molecule is 16 123 bp in length and its overall nucleotide composition is heavily biased towards As and Ts, which account for 75.66% of the whole genome. The H. xiaoi genes are arranged in the same order and orientation as the other hexapod species, except for the occurrence of one 69 bp trnV-like region with anticodon GAC that overlaps 65 bp with the reversely encoded trnD and the presence of 232 bp long intergenic spacer between trnS^UCN and nad1. All protein-coding genes (PCGs) start with a typical ATN codon. Nine of 13 PCGs have a complete termination codon TAA, while the remaining nad1 and cox2 are terminated with TAG, nad4 and nad5 with the incomplete T. All tRNAs show the typical clover-leaf structure, except for trnS^AGN. It habors anticodon TCT instead of GCT, a lengthened 9 bp anticodon stem with a bulged nuleotide in the middle, an unusual 6 bp TΨC-stem, and a mini 1 bp DHU-stem and no connector nucleotides. The control region (CR) contains one 14 bp Poly-T sequence (ten Poly-T sequences more than 5 bp and two microsatellite-like repeating sequences (TA)₆ and (TA)₉. The results of this study will provide some important molecular data for discovering phylogenetic relationships among the superfamilies of Coleptera.

Trehalase (Treh) plays key roles in energy metabolism and is the first enzyme in chitin biosynthesis pathway of insect. Expression profile and enzyme activity of Treh are related to many important physiological processes of insects, including development, molting, metamorphosis and reproduction. To date, two kinds of Treh genes have been successfully cloned in different insect species, and it has been found that the gene family of insect Trehs is composed of multiple members. Most proteins encoded by Treh genes contain signal peptides in their leader regions, and partial proteins possess 1 or 2 transmembrane domains, based on which, Trehs are divided into two types, named the soluble (Treh1) and membrane-bound (Treh2) Trehs, respectively. In addition, there are two specific motifs (“PGGRFREFYYWDSY” and “QWDYPNAWPP”) in Treh2. The core function of Treh is to degrade the extra and intercellular trehalose into glucose in order to provide energy for insects, by participating in insect chitin biosynthesis to regulate chitin synthesis in the cuticle and midgut or cooperating with hormone to control the concentrations of trehalose and glucose dynamically in insects, so in this way insect cells would be effectively protected in adverse environment and their capacities of stress resistance be significantly improved. In view of the important roles it plays in energy metabolism and chitin biosynthesis, trehalase has been a potential novel target for insect pest control. The functional research of trehalase and development of its inhibitors may contribute to the biological control of insect pests in the future.

To study the developmental process of the reproductive system in the male adult of bumblebee Bombus patagiatus Nylander following emergence, semen volume, sperm count, gonad and spermatheca size of the reproductive system in virgin male adults at different day-old (1-15 day-old) were measured. The results showed that the volume of seminal fluid of drones at 1 day-old (5.95±0.39 μL) was maximal, while the sperm count of drones at 2 dayold reaches the peak under microscope. The number of motile sperms in the semen presented repeated wave trends. The motile sperm count (25.93±1.06) and the total number of sperm in the semen (160.67±17.11 ten thousand) reached the maximum at 9 day-old. The highest percentages of motile sperms were 86.38%±2.02% and 86.45%±2.50%, respectively, at 8 and 9 day-old. The length of gonad (1 522.01±37.93 μm) was minimum at 1 day-old. The middle width of spermatheca (86.38±2.96 μm) reached the maximum at 7 day-old, but had no significant difference with that at 9 day-old. The results indicate that the indexes of the reproductive system are relatively stable in drones at 8-9 day-old, and drones reach sexual maturity and could mate during this stage. The study provides a theoretical basis for artificial breeding of B. patagiatus.