The N-glycosylation pathway in insects differs from the mammalian pathway, which limits insect-based expression system to produce mammalian glycoproteins with biomedical value. This study aims to produce transgenic Bombyx mori capable of processing glycoproteins as mammalian glycosylation pathways. Two key genes in mammalian N-glycosylation pathway, i.e., mammalian sialic acid synthase and CMP-sialic acid synthetase genes, which were driven by B. mori actin3 promoter, were transformed into B. mori by piggyBac transgenic system. Enhanced green fluorescent protein (EGFP) which was driven by 3×P3 promoter was also introduced as a molecular selection marker. The expression of transformed genes was analyzed in G1 transgenic silkworms. The current study provides a solid evidence of simulating mammalian glycosylation pathways in the model insect B. mori.

Insects mainly rely on innate immune responses to resist foreign invasion. Activation of the serine proteinase cascades, involved in hemolymph melanization and antimicrobial peptide synthesis, plays an important role. To define the role of serine proteinases in the innate immune responses of Pieris rapae, a cDNA fragment of a serine proteinase gene, Pr-SP1 screened by RT-PCR using degenerate primer, was sorted out in this article. The full-length cDNA of Pr-SP1 was cloned using RACE strategy. The cDNA length was found to be 1 489 bp, including a 1 059 bp open reading frame, which encodes 353 amino acids with a 20-amino-acid signal peptide. The predicted protein molecular mass and pI are 36.85 kDa and 6.41, respectively. Multiple sequence alignment result revealed that Pr-SP1 shares high identities with its homologs in other insect species, which also possesses a clip and active catalytic domain at N-terminus and C-terminus, respectively. Real-time quantitative PCR and Western blotting results indicated that Pr-SP1 was primarily transcribed in granulocytes in the pupal stage and the protein product of Pr-SP1 was detected mainly in plasma. Pr-SP1 was transcribed and its protein product expressed in different developmental stages and instars. The highest and lowest transcript and expression levels appeared in 5th instar larvae and egg stages, respectively. The transcript level of Pr-SP1 and expression level of Pr-SP1’s protein product could be induced by Escherichia coli, Micrococcus luteus and Pichia pastoris. According to these results, Pr-SP1 could be considered as a candidate of Spätzle processing enzyme, which may participate in the innate immune responses in P. rapae.

Chemosensory proteins (CSPs) are a class of small soluble proteins in insects, which are supposed to recognize and transport non-volatility odour molecules and chemical stimuli. In this study, we investigated the expression pattern of BmCSP4 using semi-quantitative RT-PCR. The results showed that BmCSP4 was expressed across various developmental stages, with gradual decrease from 4th larva to pupa. The tissue expression profile in males and females at adult stage was further studied, and the results showed that the expression level of BmCSP4 was lower in female head (with antennae removed), throax and abdomen than in other female tissues. The binding specificity of BmCSP4 to 15 compounds was tested using N-phenyl-1-naphthylamine (1-NPN) as fluorescent probe, and the results showed that only benzaldehyde, p-methoxy benzaldehy, 2-pentyl-3-phenylpropenoic, 1-phenyl-1-butanol and acetophenone at the concentration of 10 μmol/L replaced 1-NPN from BmCSP4 by 50%, with the dissociation constants of 3.20, 2.24, 2.88, 2.04 and 2.52 μmol/L, respectively. These results suggest that BmCSP4 may have diverse physiological function in different developmental stages, and may be involved in the process of discriminating aromatic aldehyde and aromatic ketone compounds.

【Aim】 The expression profile of gene encoding pyridoxine phosphate oxidase (PNPO), which is a key enzyme related to VB₆ metabolism, was analyzed in different developmental stages and tissues of the 5th instar larvae of Bombyx mori. 【Methods】 The recombinant plasmid pET-22b(+)-PNPO was transformed to Escherichia coli Rosetta for induction, expression and purification of PNPO, and then the polyclonal antibody was prepared. The expression profile of PNPO gene was analyzed by Real-time PCR and Western blot, respectively. 【Results】 The highest translation level of PNPO gene was found in the 5th instar larvae. The transcription level in tissues of 5th instar larvae was in sequence of testis＞head＞midgut＞Malpighian tubules＞ovary＞cuticle＞fat body＞silk glands. However, the highest translation level was found in testis, and then was in head, midgut, and malpighian tubules, respectively. 【Conclusion】 The expression profile PNPO gene in B. mori has been defined by this study.

 Odour chemically mediates foraging, mating and oviposition behaviour of the tobacco cutworm, Spodoptera litura (Fabricius) (Lepidoptera, Noctuidae). The olfactory receptor is a direct receptor of odours and a key component of olfactory system, which plays an important role in encoding and transmission pathway of olfactory signal. By using RT-PCR and Western blot techniques, the tissue-specific expression of S. litura olfactory receptor gene Ⅱ(SlitOR2) (GenBank accession no. DQ845292) in different developmental stages was analyzed and identified. Semi-quantitative RT-PCR analysis indicated that SlitOR2 mRNA was mainly expressed in the adult antennae. Western blot result showed SlitOR2 was expressed mainly in the adult antennae, which was consistent with the previous semi-quantitative RT-PCR results. But there were also trace proteins which are expressed in the adult legs, head and midpupa. It could be the non-specific protein bands with similar size to target protein or the trace of proteins expressed in the adult legs, head and mid-pupa, because a small number of olfactory sensilla are distributed in both tarsi and proboscis. A clear single target band indicates that the obtained peptide antibody with high specificity can be used for further experiments.

To explore the defense responses of Plutella xylostella (Lepidoptera：  Plutellidae) hemolymph against the infection of different strains of entomopathogenic fungus Isaria fumosorosea (syn. Paecilomyces fumosoroseus) (Hypocreales：  Cordycipitaceae), cellular immune responses of the infected 4th instar larvae was investigated with optics microscope and Giemsa stain. The results indicated that the invasion of P. xylosetlla by I. fumosorosea resulted in changes in the characteristics of host hemolymph, including the initial increase of the total numbers of hemocytes, and the change of the proportion of differential hemoeytes in association with fungal infection processes. During 8-45 h post inoculation, the peak response reached to 6 250/mm³ at 24 h after invasion by the highly pathogenic strain PFCF-001. The highest total hemocyte counts (3 000/mm³) were observed at 36 h when P. xylostella larvae were inoculated with the least pathogenic strain PFCF-D58, which was 12 h later than the treatment of strain PFCF-001. The major defense hemocytes were plasmatocytes and granulocytes. Different types of cellular immune responses were observed at the early stage of infection including adhesion, phagocytosis, hemocytic aggregation, encapsulation and nodule formation. However, the defense responses of the P. xyolstella against the highly pathogenic strain of I. fumosorosea just inhibited the pathogen temporarily but could not clear it completely.

Camptothecin possesses excellent inhibitive effects on insect growth and development. The apoptotic induction mode and mechanism of camptothecin are ambiguous, which restricts the application of camptothecin in plant protection. In order to investigate its inhibitive effect at the cellular level, the apoptotic induction effect of camptothecin on Spodoptera frugiperda Sf9 cells was studied. Induced with 1 μmol/L camptothecin, Sf9 cells showed typical morphological changes such as cell shrinkage, loss of microvilli and chromatin condensation under the transmission electron microscope. DNA Ladder assay indicated that camptothecin induced apoptosis in Sf9 cells. Apoptotic rate of Sf9 cells treated with 1 μmol/L camptothecin increased within 12 h, and reached the maximum of 39.67% at 12 h, which was 13.13 times as high as that of the control. In addition, the activity of Sf caspase-1, the homolog of mammalian effector caspase, increased after apoptosis induction in Sf9 cells, which reached the maximum at 12 h and 24 h, respectively. Furthermore, camptothecin inhibited Topo Ⅰof Sf9 cells to unwind supercoiled pBR322 DNA, which caused DNA damage to activate caspase. This suggests that the signal transduction pathway of caspase cascade is one of mechanisms of apoptosis induction. This study provides important clues to understanding the mechanisms of apoptosis induction.

In order to increase the control effects, reduce the use of insecticides, enhance the safety of agricultural products, the synergism of two cationic adjuvants (1277 and C_8-10) and one silicone adjuvant (Breakthru S240) to three insecticides beta-cypermethrin, chlorpyrifos and chlorfenapyr against 3rd-instar larvae of the Spodoptera exigua (Hübner) and Helicoverpa armigera (Hübner) were studied by dipping method, and the influence of the three adjuvants on physical properties of water was determined. The results showed that: (1) all of the tested adjuvants showed significant synergism to those insecticides, and the synergism ratio was significantly correlated to the concentration of adjuvant. The synergism ability of tested adjuvants to those insecticides against S. exigua and H. armigera was C_8-10＞1227＞Breakthru S240 under the same concentration, except that Breakthru S240 showed higher synergism ability to 1227 against H. armigera. The optimum synergistic concentration of tested adjuvants to those insecticides against S. exigua was 900 mg/L, while against H. armigera it changed into 900 mg/L (1227), 300 mg/L (C_8-10) and 900 mg/L (Breakthru S240), respectively. (2) All of the tested adjuvants showed significant influence on the physical properties of water. Reducing ability of surface tension, as well as the increasing ability of expanded diameter and the maximum retention of tested adjuvants to water were Breakthru S240＞1227＞C_8-10under the same concentration. In conclusion, the synergism of tested cationic adjuvants (1277 and C_8-10) to three insecticides against both S. exigua and H. armigera was significantly higher than that of silicone adjuvant (Breakthru S240), while the latter showed better ability to enhance insecticide deposition on plant leaves than the former. These results provide theoretical proofs for the application of the tested adjuvants.

Characteristics of photosynthesis and water use of resistant (R-1) and susceptible (I-1) alfalfa clones damaged by thrips were tested to determine compensatory photosynthesis at the budding and early flowering stages in the field. The results showed that after the plants were damaged by thrips at the budding stage, the chlorophyll content and the net photosynthetic rate (Pn) of the resistant alfalfa clone R-1 increased significantly, while light compensation point (Lcp) and dark respiration rate (Rd) decreased significantly (P<0.05) compared with those of healthy plants (CK). However, the chlorophyll content of the susceptible alfalfa clone I-1 decreased (P<0.05), and the Pn had no significant difference from that of CK. Pn of I-1 plants damaged by thrips was lower than that of CK, while Lcp and Rd increased significantly (P<0.05) under lower illumination intensity. The values of intercellular CO2 concentration (Ci), transpiration rate (Tr) and stomatal conductance (Gs) of R-1 and I-1 plants damaged by thrips increased significantly, while the water use efficiency (WUE) decreased dramatically compared with those of CK. When the plants were damaged by thrips at the early flowering stage, Pn of I-1 plants increased significantly under strong illumination while decreased under weak illumination (P<0.05), while other indices showed the similar tendency as at the budding stage. In conclusion, R-1 alfalfa has significant compensatory photosynthesis to thrips damage, but I-1 alfalfa does not.

The cytochrome P450 monooxygenases (P450s, CYPs) constitute a large and complex superfamily of heme-thiolate proteins, which are responsible for the oxidative metabolism of structurally diverse endogenous and exogenous compounds. In this review, recent progress in Bombyx mori P450 diversity, multiple functions, genomic distribution, intron-exon organization and the evolutionary relationships to P450s from Drosophila melanogaster is summarized, and the proposals of B. mori P450 study are also put forward. In the silkworm, the paralog count for P450s is lower than those found in other scavengers and omnivorous phytophagous insects, but substantially higher than that observed in Apis mellifera. The distribution of B. mori P450s in the genome indicates that most of them are tandem arranged on chromosomes. There is a relatively good correlation between intron-exon organization and phylogenetic relationship among these multiple P450s. Comparison of the P450s from B. mori to the P450s from D. melanogaster reveals that there are 10 pairs of recognizable orthologs and the P450s in CYP3 and CYP4 clans are present with species-specific expansion. These diverse P450s have been demonstrated to be associated with growth and development, tolerance to fluoride and resistance to insecticides. The silkworm is a good representative insect of the order Lepidoptera, and it is so expected that it might found theoretical basis and model system for developmental regulation and resistance management of other insects, especially for lepidopteran insects, with the further study of B. mori P450s.

RNA silencing is identified as a general and evolutionarily conserved antiviral defense mechanism in insects. Meanwhile, viruses adapt the strategy by encoding suppressors of RNA silencing to counter the host-defense. Based on the related achievements in Drosophila, this article reviews the mechanism of antiviral RNA silencing, the characteristics and functions of RNA silencing suppressors, and the coevolution between hosts and viruses. Studies showed that RNA interference mediated by siRNA (small interfering RNAs, siRNA) plays a vital role in antiviral defense in Drosophila. Dicer-2(Dcr-2), argonaute-2 (AGO2) and R2D2 are three key components in siRNA pathway in Drosophila. Flies with these three genes knockdowned were hypersensitive to RNA virus infection. Additionally, several core genes involved in RNA interference were identified, including vasa intronic gene, aubergine, armitage, rm62 and piwi, which also play roles in antiviral defense in Drosophila. Three viral suppressors of RNAi (VSRs) have been identified in Drosophila viruses, i.e., FHV-B2, DCV-1A and CrPV-1A. FHV-B2 and DCV-1A inhibit RNA silencing by binding dsRNA or siRNA while CrPV-1A by targeting AGO2. During the boundless process of evolution, the hosts and viruses counter each other as well as coevolve. Components of antiviral silencing against highly diverse VSRs should be continuously and rapidly evolving.

The molting hormone, 20-hydroxyecdysone (20E), is an insect steroid hormone which plays a dominant role in the regulation of insect molting, metamorphosis, reproduction and other physiological processes. The 20E nuclear receptor, EcR-USP, has been identified for 20 years, and several breakthroughs have been recently made in understanding the transcriptional regulation mechanisms of 20E-cR-USP. Recent research indicates that: (1) the 20E receptor is composed of two nuclear receptor molecules, i. e., EcR and USP. (2) The EcR-USP heterodimer obtains DNA binding activity with the assistance of a molecular chaperone-containing heterocomplex. (3) 20E transcriptionally activates the EcR-USP heterodimer by releasing corepressors and recruiting co-activators. (4) The ligand-receptor complex, 20E-EcR-USP, triggers a transcriptional cascade, including transcription of the 20E primaryresponse genes and the 20E secondaryresponse genes induced by transcription factors encoded by the 20E primary-response genes, to regulate physiological and developmental events.

 Eurygaster integriceps is a key pest of wheat and barley in Iran. Neonicotinoid insecticides, a strong group of insecticides, have good effect on sucking pests. In this study, effects of four neonicotinods (thiametoxam, imidacloprid, dinotefuran and acetamiprid) on 3rd nymphs and adults of E. integriceps via two methods (contact and chemigation) were investigated. Although tested insecticides had no strong effect on E. integriceps in contact method and acceptable mortality was seen at very high concentration (5 000 mg/L a. i.), all insecticides had acceptable toxicity to nymphs and adults through chemigation bioassay. According to current results, using chemigation can be a novel method for management of this important pest.

In order to understand the effects of night-interruption on diapause-averting by different geographic populations of the Asian corn borer, Ostrinia furnacalis (Guenée), the photoperiodic responses and effects of night-interruption experiments on diapause-averting in two populations of O. furnacalis from Nanchang (28°41′N， 115°53′E) and Harbin (44°56′N， 127°10′E) were investigated in the laboratory, in which the scotophases of L9∶D15 and L12∶D12 were interrupted by 1 h light pulse at 25 and 28℃, respectively. The results showed that the critical daylengths in Harbin population at 25 and 28℃ were 1 h 40 min and 2 h longer than those in Nanchang population, respectively. In all night-interruption experiments, the incidences of diapause in almost all positions of light pulse in Nanchang population were significantly lower than that in Harbin population. When the light pulse was used to interrupt L9∶D15 and L12∶D12, the incidence of diapause was lower at L9∶D15 than that at L12∶D12. Furthermore, the most highly photo-sensitive position occurred in the middle of scotophase at L9∶D15, whereas the most highly photosensitive position occurred in the early scotophase at L12∶D12. The effect of the light pulse on diapause-averting at 28℃ was greater than that at 25℃. These results further suggest that effect of night-interruption on diapause-averting may be different even in the same species depending on different geographic populations and the experimental conditions.

In 2009, Southern rice black-streaked dwarf virus (SRBSDV) appeared first time in Zhejiang province, while only in Wuyi county morbidity phenomenon occurred. Since this virus is a kind of arbovirus, and the white-backed planthopper, Sogatella furcifera (Horváth) (WBPH), is its main transmitted vector, we used light trap catch data, migration trajectory simulation, synoptic meteorological background and virus infection to study the pathways of WBPH in Wuyi, Zhejiang, 2009. In this way, the possible source regions of SRBSDV transmitted by its vector WBPH were analyzed, and the biological parameters for trajectory analysis were determined based on the numerical simulation. This research aims to establish scientific basis for the macro-epidemic researching of SRBSDV. The results illustrated that: (1) through the overlapping analysis of the virus incidence area and the main WBPH source regions of Wuyi imitated through backward trajectory simulations, the possible virus source areas of Wuyi were mainly distributed in Guangdong, Guangxi, southern Fujian and southern Jiangxi provinces or regions. (2) Riding on the prevailing southwest wind or south wind, WBPH transmitted this virus from southern China to Wuyi. (3) It was the immigration of concentrated airborne WBPH with downdraft and rainfall that triggered the epidemic outbreak of SRBSDV in Wuyi in 2009.

This paper dealt with the spatial distribution pattern of Bipectilus zhejiangensis larvae and the relationship between the occurrence and environmental factors in 11 field plots of bamboo (Phyllostachys heterocycla cv. pubescens) forest sampled in Shaowu, Fujian province by positive/counter comparison frequency and discriminant analysis methods. Positive comparative frequency analysis showed that the spatial distribution pattern of B. zhejiangensis larvae fitted the negative binomial distribution in total 550 bamboo shoots sampled in the plots. Taking each plot as a unit and analyzed by counter-comparison frequency method, it was found that plots 1, 2, 4, 5 and 8 fitted the Neyman distribution, plots 7, 9, 10 and 11 fitted the negative binomial distribution, and plots 3 and 6 fitted the Neyman or the negative binomial distribution. The method of discriminant analysis could be 100% exact to discriminant function. The importance of the six environmental indexes from high to low were in the sequence of soil humus (X5), forest health (X6), slope position (X3), slope orientation (X4), bamboo structure (X2) and stand density (X1) according to the F values. It was concluded that the spatial distribution pattern of B. zhejiangensis larvae belongs to the aggregation type with nonuniform distribution whether sampled by bamboo shoot or by plot. The density of B. zhejiangensis larvae in plot with thick soil humus and bad forest sanitation were larger than in other place. The effects of the single index such as the stand density, bamboo structure, slope position and orientations on the density are not significant but their integrated effects are significant. More information can be obtained by the counter-comparison frequency method than by the aggregation index for analyzing spatial distribution pattern of insects.