In order to study the functions of chemosensory proteins (CSPs) of Helicoverpa armigera (Hübner) in olfactory recognition, we cloned and expressed the chemosensory protein HarmCSP6 of H. armigera in prokaryotic cell. This protein was expressed in the form of dipolymer, which was certified by Western blotting and fast protein liquid chromatography (FPLC). The results of expression profiles showed that the HarmCSP6 gene was highly expressed in male and female antennae, and was expressed to a certain extent in female legs and wings. The binding experiment with 22 kinds of odorant chemicals indicated that HarmCSP6 could bind obviously with aldehydes and terpenes. By studying the binding affinity, some special binding odorants can be selected. This may provide the theoretical basis for developing attractants and repellents of the cotton bollworm.

To study the regulation mechanism of silk genes in the silkworm, Bombyx mori, the upstream 1 233 bp promoter sequence of fibroin P25 gene and its regulatory elements were analyzed by using Bac-to-Bac expression system and real-time quantitative PCR technique. The results showed that there existed positive regulatory elements at position  -423 to -1 233 and -127 to -238 in P25 promoter region, and negative regulatory elements at position -238 to -423. PSGF and BMFA regulatory elements played a negative regulation role in P25 expression. PSGF element had a certain degree of enhancement to A3 promoter’s activity in posterior silk glands, which further validates the function of PSGF regulatory element. Analysis of P25 promoter activity, especially functional analysis of PSGF and BMFA regulatory elements, could be helpful in further understanding the mechanism of P25 expression and fine regulation.

Insect carboxylesterases are important enzymes involved in xenobiotic metabolism and degradation of odorants. In the present study, Bmae35 gene, which is expressed in larval olfactory tissues of the silkworm and the putative ortholog of antennal esterase Snon-EST in Sesamia nonagrioides, was cloned and exogenously expressed. Bmae35 comprises a complete coding sequence of 1 581 bp and codes 526 amino acids. The multiple sequence alignment of Bmae35 with other esterases revealed that Bmae35 has the structure characteristics of insect esterases, including the catalytic triad (Ser191, Glu313 and His429) and the conserved pentapeptide Gly-x-Ser-x-Gly in the α-esterase family. Based on the semi-quantitative RT-PCR analysis, the expression pattern of Bmae35 gene showed that it was highly expressed in head, fat body, Malpighian tubules, integument, and silk glands in day-3 5th instar larvae. In addition, the expression of Bmae35 showed a positive correlation with sex pheromone content. The results suggest that Bmae35 might play important roles in pheromone synthesis. The Bmae35 gene was sub-cloned into the expression vector pET28(a) and the recombinant protein was obtained by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Electrophoresis analysis showed that Bmae35 was expressed in Escherichia coli as inclusion body. The recombinant protein Bmae35 was purified by immobilized Ni²⁺ absorption chromatograph column. Western blotting analysis indicated that Bmae35 was correctly expressed in E. coli and purified. The results of this study provide useful data for further understanding the odorant detoxification and expression orientation.

Serine protease inhibitor 4 (serpin-4) is a member of serine protease inhibitor family. This investigation aims to research and produce high-titer polyclonal antibody against serpin-4 in Bombyx mori, which will lay a material foundation to further inquiry the physiological function of serpin-4 gene. The serpin-4 gene was first cloned from fat body of B. mori and then was cloned into pET28a prokaryotic expression vector by using genetic recombinant technology. The recombinant vector was transferred into Escherichia coli and the transferred E. coli was induced by IPTG in order to acquire prokaryotic expression recombinant fusion protein which was subsequently purified by Ni column. The molecular weight of the purified protein identified by SDS-PAGE and polyclonal antibody against His-tag was consistent with prediction, indicating that the interest protein with high purity was obtained. The acquired protein was employed as antigen to immunize KM mouse by four separate immunizations and ultimately the polyclonal antibody serum against serpin-4 was achieved. The serum titer validated by ELISA and Western blot was 1∶20 000. The successful preparation of polyclonal antibody against serpin-4 in B. mori demonstrated that the method applied in other organisms of preparing polyclonal antibody was also applicable to study serpin-4 gene of B. mori, and this study laid a material foundation to further investigate the physiological function of serpin-4 gene. 

In order to explore the function of glutathione S-transferases (GSTs) in olfactory recognition of insects, the full-length cDNA of a novel glutathione S-transferase gene was cloned from the antennae of Helicoverpa assulta by using RT-PCR and RACE methods (GenBank accession no. EU289223). Based on the sequence identity and the phylogenetic tree of the amino acids of GSTs in H. assulta and other species， the sequence obtained was found to belong to the Epsilon family， hence named as HaGSTe1. Genomic structure analysis of HaGSTe1 revealed that HaGSTe1 contained five introns with the length of 415, 513, 296, 333 and 269 bp, respectively. The qualitative and quantitative analyses of the expression of HaGSTe1 in male and female moths were conducted using semi-quantitative RT-PCR and real-time PCR. The results showed that HaGSTe1 transcript was clearly detected in the head (with antennae and proboscis removed), antenna, proboscis, thorax, legs, and wings of both male and female and abdomen of the female. HaGSTe1 transcript was significantly more highly expressed in male antennae than in female antennae, suggesting that it may be involved with the decomposition of pheromones and other xenobiotics in the antenna.

 Mushroom bodies (MBs) are insect brain centers involved in learning and other complex behaviors. The proliferation and programmed cell death (PCD) pattern of MBs in the honeybee, Apis cerana cerana, was comparatively studied by using 5-bromo-2-deoxyuridine (BrdU) incorporation and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) technique. The results showed that the MBs are created in their entirety by several neuroblasts per hemisphere. Asymmetric divisions produce a smaller ganglion mother cell (GMC) and regenerate the neuroblast. Kenyon cells in a proliferation cluster are formed through symmetrical division of MB neuroblasts. These neuroblasts divide continuously from early embryogenesis until the late pupal stage. Peduncular neuropil was first visible in the 3rd instar. The calycal neuropil rapidly increased in size during the pupal stage. Extensive apoptosis in the MBs could be detected only within a narrow time window from day 3 to day 6 of the pupal stage during metamorphosis. Extensive apoptosis in the MB proliferative clusters started at about the time when proliferation began to cease. This study provides a theoretical basis for behavioural research.

The internal gut of insects is a complex micro-ecosystem, in which inhabits a large and varied microbial community. This microbial community plays an important role for their hosts’ growth, development, digestion and absorption of nutrition, colonization resistance against invasion of exotic microbes. In this paper, the diversity of bacterial flora of citrus psyllids (Diaphorina citri), which is a vector of the Huanglongbing pathogen Candidatus Liberibacter asiaticus, was investigated based on analysis of 16S rDNA sequences by restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). The PCR-RFLP result showed that 31 sequences share high homology with Proteobacteria, including Pseudomonadaceae, Enterobacteriaceae, Xanthomonadaceae, Burkholderiaceae, Rickettsiaceae and Rhizobiaceae. The dominant bacterial flora in D. citri included syncytium endosymbiont (5 sequences, homology 99%, isolating frequency 31%), Mycetocyte endosymbiont and Candidatus Carsonella ruddii (5 sequences, homology 98% and isolating frequency 31%), Candidatus Liberibacter asiaticus and Wolbachia. The analysis of 16S rDNA V3 region sequencing by PCR-DGGE revealed that the inner bacteria of D. citri collected from Murraya paniculata were clustered in one branch and those from citrus were clustered in another branch, suggesting that the host plant has more obvious impact on the bacteria flora of D. citri than the geographic location. The sequencing of the isolated bands of DGGE and GenBank alignment showed that the bacteria of D. citri mainly belong to Proteobacteria and Firmicutes, including Pseudomonadaceae, Rickettsiaceae, Enterobacteriaceae, Xanthomonadaceae, Streptococcaceae and Bacillaceae. Syncytium endosymbiont (band 3-4) may be predominating population because it can be stably isolated from D. citri no matter what the host plant is and where the geographic location. The high isolation rate of Wolbachia inside of citrus psyllids revealed that D. citri being infected with Wolbachia is a common phenomenon in China. Both PCR-RFLP and DGGE analysis indicated that the syncytium endosymbiont of D. citri is a dominant bacterial flora and the two methods in combination can reveal the microbial diversity in D. citri conveniently.

The application of nanogold in bioprobe technology is a major research field, which mainly depends on the effective binding of biomolecules with gold nanoparticles. Using the strategy of gene recombination, we studied the feasibility of combining baculoviral surface display with peptide-mediated immobilization to make bio-inorganic composites. A gene encoding an Auspecific binding peptide (Aubp) was synthesized and fused to the N-terminus of the gp64 derived from BmNPV to construct the recombinant plasmid pFB-gp64-Au, which was then transposed to shuttle vector Bacmid by Bac-to-Bac system. The recombinant AcMNPV baculovirus was harvested after transfection of the recombinant Bacmid into the Sf9 insect cells and its binding with colloid gold prepared by NaHB reduction method was primarily studied. The results showed that recombinant Bacmid was successfully constructed and envelope-modified baculovirus with gold-specific binding peptide was harvested. Au-bp mediated virus-nanoparticle complex was visualized by transmission electron microscopy (TEM) analysis. The study will be helpful to the research of fabricating bio-inorganic compound system and the application of nano materials in biological field.

To provide the theoretic basis for the scientific application of insecticides controlling the small brown planthopper, Laodelphax striatellus (Fallén), the bioactivity of chlorpyrifos to L. striatellus was determined by stem immersion method in the laboratory. Parafilm sachet and filter paper funnel were used to measure the effects of sublethal dose of chlorpyrios on such indices as honeydrew excretion, weight gain and egg production. The results obtained through parafilm sachet showed that honeydrew excretion and weight gain of single female L. striatellus treated with sublethal dose of chlorpyrios (0.21 mg/L a.i.) increased by 10.99% and 22.22% compared with the control, respectively, but with no significant difference (P>0.05）. The number of eggs laid per female L. striatellus was 79.6±26.4, with a 12.27% increase compared with the control and an extremely significant difference (P<0.01). The result of filter paper funnel method indicated that the honeydrew spot area of female L. striatellus treated with sublethal dose of chlorpyrios was 119.74±5.90 mm², with a 13.06% increase compared with the control and a significant difference (P<0.05). According to the test results, sublethal dose of chlorpyrios may cause the aggravation of the virulence of L. striatellus since its honeydew excretion, and weight gain and egg production could be promoted. The findings have scientific value for ascertaining sublethal effects of insecticides on the population of L. striatellus.

In order to determine the role of Encarsia sophia in the displacement of Bemisia tabaci B biotype by Q biotype, in this study we observed the parasitizing behavior and preference of E. sophia on the Q and B biotypes of B. tabaci and investigated the effects of B. tabaci biotype on development of E. sophia in the laboratory under 27±1℃, 16L∶8D and RH 70%-80%. The results indicated that the time for outside host examination of E. sophia was not significantly different between two whitefly biotypes, whereas the time for inside host examination and oviposition on Q biotype nymph was significantly longer than that on B biotype nymph, 190.2±14.6 s vs 140.0±7.5 s. In non-choice tests, the number of Q biotype nymphs parasitized (8.1±0.5) and the total egg number (9.3±0.6) laid by E. sophia was significantly higher than those for B biotype nymphs (6.3±0.5 and 7.0±0.6, repectively), while the number of eggs loaded per nymph parasitized was not significantly different between B and Q biotypes. In choice tests, the number of nymphs parasitized (3.1±0.4), the total number of eggs laid per wasp (3.8±0.5), and the number of eggs loaded per host (1.2±0.1) for B biotype were higher than those for Q biotype (the corresponding numbers were 1.8±0.3, 1.8±0.4 and 0.7±0.1, respectively). There were no significant differences in the number of nymphs fed by E. sophia between B and Q biotypes, but the mated female wasp fed more nymphs than the unmated female wasp did within the same biotype. The developmental duration of wasp for eggpupa (7.2±0.1 d) and pupa (5.2±0.1 d) on B biotype nymph was not significantly different from that on Q biotype (the corresponding time were 7.3±0.1 d and 5.6±0.1 d, respectively). The emergence rate of wasp pupae from B biotype nymphs (73.55%±1.42%) was not significantly different from that from Q biotype nymphs (68.42%±13.01%). The results suggest that E. sophia contributes to the displacement of B biotype by Q biotype of B. tabaci in the laboratory, but this effect left to be known in fields.

 Temperature is one of important environmental factors affecting on insects in the Tibetan Plateau, a region sensitive and vulnerable to climate warming, for the cold plateau climate. As an endemic species, Locusta migratoria tibetensis Chen may respond to warming climate on the plateau by increase of the accumulated degree-days (ADD) from habitat and expansion of its distribution. In order to depict the relationship between temperature changes and the ADD of the locust and to predict the distribution and occurrence of the locust in this region under the conditions of climate warming, we used surface temperature data from 90 weather stations on the Tibetan Plateau during the period of 1961-2005 to calculate the ADD of the locust and the annual area of potential distribution (APD) in ArcGIS according to the linear regression models of the ADD to geographical position. The results suggested that climate warming could increase the locust achievable ADD and APD to a great extent. The raster ADD maps indicated that in most cases the locust was distributed along the valley of main rivers over the plateau, and the potential distribution area of the locust was about 91 081 km²(3% of the Tibetan Plateau area). The largest APD occurred in 1998, the second hottest year, was 142 988 km², which was 1.9 times the APD in 1968, the coldest year. There was a significant correlation between the APD and the annual surface temperature. The annual surface temperature increased at the speed of 0.0301℃ per year, while the APD increased at the speed of 504.38 km² per year. The ratio of the increasing speed of APD to that of temperature was 16 756.8, indicating that a little increment in temperature would bring a great extension in distribution of L. migratoria tibetensis. This study presents an example of influence of global warming on plateau ecology.

 The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is a serious pest of rice in South China including Fujian Province. The earlier huge immigration peaks of WBPH had been seen from April to May 2007. In this study, HYSPLIT (Hybrid Single-particle Lagrangian Integrated Trajectory), a trajectory analysis platform developed by the National Oceanic and Atmospheric Administration (NOAA) of US and Australian Bureau of Meteorology, was used to simulate the early migratory pathways of WBPH from their source areas in April and May from 2007 to 2010. GrADS (Grid Analysis and Display System), a meteorological data analysis and displayed software, was used to analyze and display the synoptic meteorological background during the early immigration periods in May 2007. The results showed that: (1) the source areas of the earlier mass immigration population of WBPH occurred from 2007 to 2010 were mostly in Guangdong and Hainan Provinces, and a few sources were from Taiwan Province and Philippines. (2) The co-occurrence of low-level jet and continuous rainfall directly resulted in the concentration of the airborne migrants and the mass immigration of WBPH in 2007. During the appearance of major immigrants in May, 2007, there were strong southwest lowlevel jets at 850 hPa simultaneously. Meanwhile, the West Pacific subtropical high was stronger than normal and extended towards west, and the northern jump of its ridge postponed, that caused the formation of a stationary front in South China and brought the sustained rainfall. (3) The high early-immigration of the 1st generation of WBPH from late March to early April in Guangdong and Hainan, and their high offspring population of the 2nd generation bred during March to May in 2007 was the key reason for mass immigration in Fujian. It is assistant to the forecasting and management work for rice planthopper in Fujian Province to investigate the rice planthopper situation in Hainan, Guangdong and Guangxi, in more rain and stronger West Pacific subtropical high years.

The gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae), is one of the most important agricultural and forest insect pests. It is composed of several subspecies, among which the Asian gypsy moth female adults with stronger flight ability have become serious international quarantine pests. Unfortunately, it can be very difficult to distinguish these subspecies morphologically. Herein, the random amplified polymorphic DNA (RAPD) markers were used to analyze the genetic polymorphism of six gypsy moth populations collected from different provinces in China. The results showed that the genetic differentiation coefficient (G_st) among the gypsy moth populations was 0.7571 and the deduced effective number of migrants (N_em, a gene flow index) was 0.1604, suggesting that these populations are highly differentiated and the gene flow between them is very low. Based on genetic analyses, four populationspecific loci were located, followed by cloning and sequencing, sequence analyses and locusspecific primer designing. Finally, the sequence characterized amplified region (SCAR) markers for two gypsy moth populations were developed. The results of primer testing showed that these SCAR markers could be used for rapid and accurate identification of the two gypsy moth populations, which is helpful for monitoring the distribution and dispersal of gypsy moth populations.

Insect-baculovirus expression system has been widely used to produce recombinant proteins. However, due to its deficiency in the post-translation modification, complex terminal sialylated N-linked glycans can not be obtained, thereby largely limiting the application of this system in biopharmaceutical industry. By comparing the glycosylation pathways in mammalian and insect cells, it is known that the initial steps are the same and then diverge. The differences mainly include that insect cells lack the mammalian glycosyltransferases like N-acetylglucosaminyltransferase II, galaetosyltransferase/N-acetylgalactosyltransferase, α-2,3-sialyltransferase and α-2,6-sialyltransferase. On the other hand, insect cells possess specific α-1,3-fucosyltransferase and N-acetylglucosaminidase which can specifically remove the terminal GlcNAc from GlcNAcMan3GlcNAc(±α3/6-Fuc)GlcNAc. Based on the comparison, this article summarized the research efforts to produce humanized proteins in insect cells through the inhibition of GlcNAcase and knock-in of the mammalian glycosyltransferases. The results showed that engineered insect cells could produce humanized glycoproteins, which would greatly expand the application of insect-baculovirus expression system. In addition, the feasibility of production of humanized proteins by selection of novel insect cell lines and/or culture condition was discussed.

【Aim】 This study aims to reveal the mechanism of epoxyxanthatin Ⅰ (sesquiterpe lactone from Xanthium sibiricum) on Pieris rapae larvae. 【Methods】 The activity of midgut protease, amylase and carboxylesterase in P. rapae larvae were investigated by feeding to 4th larvae of P. rapae. 【Results】 Epoxyxanthatin Ⅰ showed the most obvious inhibition on midgut protease, whose activities were inhibited 20.95%, 29.38% and 50.06% at 12 h, 24 h and 48 h after treatment respectively, while it showed the less obvious inhibition on midgut amylase, whose activities were inhibited 11.89%, 39.01% and 31.92%, respectively. Meanwhile, no significant differences in carboxylesterase activity were detected between the controls and the epoxyxanthatin Ⅰ treated samples at 12 h after treatment, while the activity was obviously inhibited at 24 h after treatment and significantly increased at 48 h after treatment compared with that of the control. 【Conclusion】 The results suggest that the inhibition effect of epoxyxanthatin Ⅰ on midgut digestive enzymes of insects may be one of the causes to inhibit feeding and growth of insects.

In India, the shot-hole borer, Euwallacea fornicatus (Eichhoff, 1868), feeds inside the galleries of tea plant twigs in natural condition and also known as tea borer and a key pest of tea. The natural presence of this beetle on som plantations, Persea bombycina Kost. (Lauraceae) at Farm No. 3 Central Muga Eri Research and Training Institute, Lahdoigarh (Jorhat-Assam) has been documented with appreciable damage during May to August, 2010. The host range, distribution, biology and bionomics of E. fornicatus (Eichhoff) have been discussed in this manuscript along with the new record on P. bombycina Kost for the first time.