【Aim】 The receptor of juvenile hormone (JH), methoprene-tolerant (Met), activates Krüppel-homolog 1 (Kr-h1), a key transcription factor in JH signaling pathway. Met plays a crucial role in insect metamorphosis. The aim of this study is to explore the functions of NlMet and NlKr-h1 in metamorphosis in the brown planthopper, Nilaparvata lugens. 【Methods】 An opening reading frame (ORF) of NlMet was cloned from N. lugens by PCR. The mRNA expression levels of NlMet and/or NlKr-h 1 were down-regulated by RNAi, and the effects of RNAi on metamorphosis and external genital morphology were observed. 【Results】 The open reading frame of NlMet is 1 185 bp in length, encoding 395 amino acids. The NlMet protein contains four domains, i.e., bHLH, PAS-A, PAS-B and PAC. PAS-B and PAC are highly conserved, whereas the other two show relative lower similarities. By disruption of NlMet and NlKrh-1 expression singly or both in the same time by RNAi in N. lugens, we found that down-regulating NlKr-h 1 in the 4th instar nymphs significantly increased the mortality of nymphs and the newly emerged male and female adults (P<0.05). After interference of NlKr-h 1 in the 5th instar nymphs, only the mortality of nymphs significantly increased (P<0.05), while after interference of NlMet in the 5th instar nymphs, the mortality of nymphs had no significant change (P＞0.05). After interference of both genes, the change tendency of mortality was similar to that after interference of NlKr-h 1. Furthermore, we found that NlKr-h 1 RNAi caused abnormity in female external genitalia. Although we found no visible abnormity of the external genitalia after inference with NlMet, the abnormity rate of the external genitalia increased significantly when both genes were silenced. 【Conclusion】 NlMet cooperates with the downstream transcription factor and has an important role in both metamorphosis and external genitalia development of N. lugens. Our study helps to understand the function of the NlMet and NlKr-h 1 genes in metamorphosis of N. lugens.

【Aim】 This study aims to analyze the changes in protein expression in the silkworm, Bombyx mori, under starvation stress, so as to illustrate the mechanism of starvation tolerance of the silkworm. 【Methods】 The silkworm variety 932 was used as the experimental material. The difference of protein expression was analyzed through two-dimensional electrophoresis and mass spectrometry technology between the silkworms normally feeding and subjected to 24 h starvation stress after the 5th instar newly moulting larval stage. The transcription level of BmLp-c 6 was detected by using real-time quantitative PCR. 【Results】 There were 62 specific proteins in the total proteins of the starved larvae compared with the larvae normally feeding. Their isoelectric points range from 4.22 to 6.98, and their molecular weights are distributed between 20.81 and 144.69 kDa. The specific protein No. 7111, which was expressed only in the starved larvae, was selected and identified by mass spectrometry. According to the amino acid sequence, the primers were designed and the target gene BmLp-c 6 was cloned. The BmLp-c 6 gene was induced to express successfully after being connected with the expression vector pET-21d(+). The real-time quantitative PCR analysis showed that when the 5th instar larvae suffered from starvation stress, BmLp-c 6 had a higher transcriptional level in the hemolymph, while its transcriptional level was very low in the midgut. 【Conclusion】 When the 5th instar larvae of B. mori are subjected to starvation stress, the protein expression profiles in the hemolymph change and BmLp-c 6 has a high transcriptional level.

【Aim】 This study aims to study the function and ligand binding characteristics of pheromone binding proteins (PBPs) in the olfactory system of the yellow peach moth, Conogethes punctiferalis (Guenée). 【Methods】 A cDNA encoding pheromone binding protein of C. punctiferalis was cloned by RT-PCR and RACE. The expression pattern of the PBP gene in different sexes and developmental stages of C. punctiferalis was analyzed by real-time PCR, and the protein binding affinity was analyzed using fluorescence competitive binding assay. 【Results】 A novel PBP cDNA was obtained from C. punctiferalis and named as CpunPBP 1 (GenBank accession no. KP027486). Its open reading frame is 510 bp in length, encoding a protein of 169 amino acids with the predicted molecular weight of 19.12 kDa and theisoelectric point of 5.09. The 30 amino residues at the N-terminal hydrophobic region display the characteristics of a signal peptide. Protein signature analysis revealed that Cpun-PBP 1 shares the typical structural features of odorant binding proteins with other insects, including six conservative cysteine residues. Real-time quantitative PCR results indicated that CpunPBP1 was dominantly expressed in adult antennae, whereas scarcely expressed in egg stage and not expressed in larval and pupal stages. In addition, the recombinant protein of CpunPBP1 was prokaryotically expressed in Escherichia coli and purified. The binding affinity of CpunPBP1 to two sex pheromones and 14 plant volatiles showed that CpunPBP1 had a strong capability of binding two pheromone odorants (cis-10-hexadecenal and hexadecanoyl), with the binding constants of 7.32 and 9.39 μmol/L, respectively. Especially, CpunPBP1 could also bind with 8 plant volatiles, with the highest binding specificity to camphene (the binding constant: 3.76 μmol/L). 【Conclusion】 Based on these results, we inferred that CpunPBP1 play important roles in the process of discriminating odorants of pheromones and host plant volatiles of C. punctiferalis.

【Aim】 This study aims to identify a C-type lectin in Ostrinia furnacalis (Guenée) and to investigate its function. 【Methods】 A potential C-type lectin gene was identified from O. furnacalis transcriptome with bioinformatics analysis and designated as CTL6. Its expression profiles in different developmental stages and tissues and after different pathogen induction in this insect were analyzed with RT-PCR. The recombinant CTL6 protein was produced in the prokaryotic or baculovirus eukaryotic expression system, and its function was investigated preliminarily with bacterial agglutination assay. 【Results】 The full-length cDNA of CTL 6 is 1 034 nt, with an open reading frame of 945 nt. The deduced protein consists of 314 amino acid residues, including a predicted 22-residue secretion signal peptide. The mature CTL6 peptide contains two carbohydrate recognition domains. Database search indicated that O. furnacalis CTL6 has the highest amino acid sequence identity to IML-2 (Immulectin-2) from Manduca sexta. RT-PCR results showed that CTL 6 was transcribed at the highest level in the 5th instar larval stage, followed by the egg stage, and its expression level in the fat body was the highest among the tested tissues. CTL 6 mRNA level obviously increased after bacterial or fungal injection. Agglutination assay revealed that the purified CTL6 recombinant protein played a role in the agglutination of Escherichia coli. 【Conclusion】 The identified O. furnacalis CTL6 is a typical C-type lectin, which may be involved in the agglutination response of O. furnacalis against pathogens.

【Aim】 In order to accurately track the synthesis, transport pathway and absorption of vitellogenin (Vg) in Harmonia axyridis (Pallas) as well as the accumulation and distribution of vitellin (Vn) in the oocytes, the monoclonal antibody (McAb) against Vn of H. axyridis was prepared. 【Methods】 The hybridoma cell lines secreting McAb against Vn of H. axyridis were produced by using hybridoma techniques, based on the cells from BALB/c mouse which were immunized with the Vn. 【Results】 We produced four monoclonal antibodies to H. axyridis soluble yolk proteins, i.e., 5E2, 5E11, 1E9 and 5H8. Further identification indicated that 1E9, 5E11 and 5E2 were the isotype IgG1, and 5H8 was the isotype IgM. Western blot analysis showed that the four antibodies had high specificity and affinity to the Vn. However, they showed no immunological reaction with the male haemolymph. 5E2 and 1E9 had specific immunological reaction with the four subunits of the Vn. Finally, we selected 5E2 whose titer was the highest before ascites preparation to prepare the monoclonal antibodies. SDS-PAGE indicated that the molecular masses of 5E2 heavy chain and light chain were 50 and 27 kD, respectively, and its titer was 1:81 000. 【Conclusion】 The successful preparation and identification of H. axyridis McAb may establish an essential tool for further building the ELISA method for assaying its dynamics.

【Aim】 To establish an effective method to prepare the antigen protein of the atypical insect olfactory receptor Orco, and to provide the foundation for study on the immunolocalization and function of Orco. 【Methods】 The target fragment between the 4th-5th transmembrane regions of Orco gene was amplified from the silkworm, Bombyx mori, by RT-PCR using primers containing restriction sites of BamH I and Hind III. This target fragment was digested with BamH I and Hind III directly and ligated to the pET-28a (+) expression vector digested with the same two restrictive enzymes. The ligated product was transformed into the competent Escherichia coli cell DH5α, and the positive clones were testified by PCR and sequenced. The right recombinant expression plasmids were transformed into BL21(DE3) strains, which then were induced with IPTG. SDS-PAGE was used to identify the expressed recombinant protein. A large amount of the induced protein was purified by HisTrap HP affinity chromatography. 【Results】 The target protein whose size was consistent with the predicted protein was induced in E. coli prokaryotic expression system. The target protein was expressed in the form of inclusion body detected by SDS-PAGE. A large amount of purified protein which can be used for antibody preparation was obtained by HisTrap HP affinity chromatography under denaturing conditions. 【Conclusion】 The antigen protein used for antibody preparation of silkworm olfactory receptor Orco can be obtained by prokaryotic expression system.

【Aim】 This study aims to examine the embryonic development of overwintering eggs of Calliptamus italicus (L.) 【Methods】 We examined the embryonic development progress of diapausing eggs collected from the field and reared in the laboratory in 2013 and 2014. 【Results】 C. italicus eggs went through 18 stages during the embryonic development, including three blastokinesis movement, i.e., anatrepsis, revolution, and catatrepsis. Diapause mostly occurred at the 12th stage. The overwintering eggs resumed partly developing as early as on January 21st in the field, resulting in the longest developmental time, and their number decreased with prolonged overwintering-time of diapausing eggs until all the eggs terminated the diapause on March 29th. Eggs entered overwintering diapause at the 12th stage (diapause-incident stage) when they were laid at early-to-middle embryonic stages (from July 27th to August 16th and terminated the diapause on April 6th next year when the 5-day ground temperature was 2.67-15.95℃, with the average of 7.59℃. But eggs entered diapause at the 10th stage (November 4th, the 5-day temperature was 5.18-9.00℃, with the average of 7.32℃) when they were laid later (from August 28th to September 4th), and resumed development on March 29th (the 5-day ground temperature of 0.14-10.27℃, with the average of 3.78℃). 【Conclusion】 The time of C. italicus eggs entering and terminating diapause varies depending on the time when they are oviposited.

【Aim】 The microsporidian Nosema bombycis can produce epizootic outbreaks in populations of the domestic silkworm (Bombyx mori) by the major mode of vertical transmission and horizontal transmission. In order to explore the genomic defense system against repetitive elements and potential means of gene regulation in N. bombycis, we conducted the survey of small RNAs associated with transposons and identification of the potential miRNAs in this silkworm parasite. 【Methods】 Total RNAs were extracted from silkworm midguts infected by N. bombycis spores and small RNA cDNA libraries were constructed for deep sequencing. After Solexa sequencing, we performed the functional characterization of N. bombycis small RNAs based on the reference genomic sequence through bioinformatics. 【Results】 N. bombycis has predominantly small RNAs with the length of 24 and 25 nt, most of which show a strong base bias for U at the 5′ end. Redundant small RNAs were associated with transposons in N. bombycis, and the amount of antisense small RNAs aligned with transposons was much more than that of sense small RNAs. Thirty-one candidate miRNAs were characterized by means of computational prediction, and certain miRNAs were shared by other Nosema species, suggesting that they may evolve conservatively. 【Conclusion】 There should exist small RNAs genomic defense system against transposons in N. bombycis. Tentative identification of potential thirtyone miRNAs provides a basis for further functional research of their targets.

【Aim】 Bombyx mori nucleopolyhedrovirus nucleocapsid protein VP39 is necessary for virus assembly. This study aims to explore the functions and characteristics of VP39 in the infection process of virus in silkworm cells and so to make a contribution to the antivirus research of the silkworm. 【Methods】 The prokaryotic expression vector was constructed to induce and produce the VP39 polyclonal antibody and to test the time course of protein expression by Western blot. The location of VP39 and the effect of VP39 on BmNPV proliferation in silkworm cells were observed by transfection of eukaryotic expression vector pIZ-vp39-V5/His and immunofluorescence. 【Results】 We obtained the VP39 polyclonal antibody, and verified that VP39 was mostly localized in cells nucleus areas during BmNPV infection, but just localized in the cytoplasm when overexpressed. The overexpressed VP39 inhibited the proliferation of BmNPV in BmN-SWU1. 【Conclusion】 The results suggest that the overexpression of VP39 in BmNSWU1 cells may inhibit the proliferation of BmNPV, thus offering a new way for exploring the regulation of VP39 in the interaction between host cell and virus.

【Aim】 This study aims to select suitable reference genes in the multi-pesticide (abamectin, fenpropathrin and spirodiclofen) resistant strain (Mp-R) of T. urticae by quantitative real-time PCR (RT-qPCR). 【Methods】 Eight candidate reference genes including 5.8S rRNA, α-tubulin, TBP,β-actin , ELFn, RPL13a, GAPDH and SDHA were chosen. The stability of these candidate reference genes was investigated by using three softwares (GeNorm, NormFinder and BestKeeper). Then the expression of CYP392A subfamily gene in P450 enzyme system of T. urticae was analyzed by using different reference genes. 【Results】 EFLn was confirmed the most suitable reference gene in the sensitive strain (SS) and the multi-pesticide resistant strain (Mp-R) of T. urticae by geNorm, NormFinder and BestKeeper softwares. The expression level of CYP392A1 was up-regulated significantly in the eggs of the Mp-R strain, and the expression levels of CYP392A16 gene in various developmental stages of the Mp-R strain was significantly higher than the same stage of the sensitive strain of T. urticae after 40-generation selection with the multi-pesticides. The expression levels of CYP392A16 gene in various developmental stages of the Mp-R strain was significantly higher than those in the same stage of the sensitive strain of T. urticae. The expression levels of other genes were not significantly different between the Mp-R strain and the sensitive strain. 【Conclusion】 EFLn gene is confirmed the most suitable reference gene in the multi-pesticide resistant strain and the susceptible strain of T. urticae. This work is contributable to the foundation for future gene expression studies in the resistant strain of T. urticae.

【Aim】 In order to understand the interaction between the parasitoid Haplogonatopus apicalis and its host white-backed planthopper (WBPH), Sogatella furcifera, the effects of parasitism on the developmental performance of both H. apicalis and S. furcifera were studied. 【Methods】 Developmental time of S. furcifera nymphs parasitized at different instars and developmental duration from egg to adult of H. apicalis in different instars of S. furcifera nymphs were observed at 25℃ in the laboratory. 【Results】 The developmental durations of the 2nd, the 3rd, the 4th, or the 5th instar nymphs of the host S. furcifera were significant prolonged when they were parasitized by H. apicalis wasps. When the hosts were parasitized at the 2nd and the 3rd instars, the eclosion rates of S. furcifera adults were 54.29% and 60.95%, respectively, which were much lower than those parasitized at the 4th and the 5th instars (96.20% and 100%, respectively). The generation time of H. apicalis parasitizing on the 5th instar nymphs of the host (23.77 d) was obviously shorter than that of the parasitoids parasitizing on the 2ndinstar nymphs (27.77 d). The parasitoid showed the highest eclosion rate (56.19%) when the parasitism was at the 3rd instar nymphal stage of the host, and the highest male adult proportion (77.12%) when the parasitism was at the 5th instar nymphal stage of the host. 【Conclusion】 The developmental duration of S. furcifera nymphs is obviously delayed by parasitization of H. apicalis. The 2nd and the 3rd instar nymphs of S. furcifera are the optimum host stages for H. apicalis.

【Aim】 In order to solve the test sample shortage of Bradysia odoriphaga Yang et Zhang in the laboratory, we researched the artificial massive rearing technology. 【Methods】 By the controlled variable method, we screened the formulae of artificial diet. By the two-sex life table method, we compared the rearing effect between the artificial diet and the natural diet, Chinese chives (Allium tuberosum), under the conditions of temperature 25℃, a photoperiod of 16L∶8D and the relative humidity of 75%. 【Results】 We screened eight formulae and obtained the optimum ingredients and their proportions of artificial diets to B. odoriphaga: mushroom powder 6 g, Chinese chive powder 5 g, agar powder 1.25 g, sorbic acid 0.04 g, benzoate 0.05 g, yeast powder 0.5 g, vitamin C 2.5 g, and water 50 mL. This formula had similar rearing effects to Chinese chive (P>0.05). Furthermore, we compared the feeding effects of the artificial diet with the natural diet (Chinese chive) in the laboratory. The results showed that the mean generation time (T) of B. odoriphaga fed on the artificial diet was 23.6800 d, while that fed on Chinese chive was 25.9000 d, with significant difference between both (P<0.05). The intrinsic rates of increase (r_m) of B. odoriphaga fed on Chinese chive and the artificial diet were 16.3200 and 41.1800/d, and the net reproduction rates (R₀) of B. odoriphaga fed on Chinese chive and the artificial diet were 0.1078 and 0.1570/d, respectively. 【Conclusion】 Both artificial diet and Chinese chive can be used to rear B. odoriphaga, and the artificial diet is slightly better than Chinese chive. Moreover, the artificial diet is more economical and with low pesticide residue.

【Aim】 The moth Orvasca subnotata Walker (Lepidopetra: Lymantriidae) is a severe defoliator of Dalbergia odorifera T. Chen found in manmade forests, and its biological characteristics and the determination of its larval instars are the important basis for the pest forecast and control in the forest. 【Methods】 The morphological characteristics, the habit of each developmental stage and the life history of O. subnotata were investigated though regular field sampling. Through regular forest sampling and morphological characterization, three indicators of larval instar features including the head capsule width, prothoracic verruca width and body length were recorded and analyzed. 【Results】 We described the biological characteristics of O. subnotata with an emphasis on its developmental features. Our results showed that as the larvae grow, the prothorax gradually darkens, and the abdominal back line and poison gland color gradually deepen. Statistical analysis of the body measurements showed that both the head capsule width and prothoracic verruca width could be used as indicators of developmental stages with head capsule width being superior to prothoracic verruca width, and the prothoracic verruca width and head capsule width were significantly correlated (y=0.994 x-0.114, R=0.999). The body length distribution fits the Dyar’s law. However, due to the overlap distribution among developmental stages, body length is not a reliable indicator of larval instar determination. 【Conclusion】 The results will help the identification of O. subnotata in the field and provide some crucial information for developing control and prevention strategies.

【Aim】 The aphid species in Pterocommatinae are important forestry pests. However, the aphids are in small size, their morphological characteristics tend to be simplified, and effective features for species identification are very limited, so the fast and accurate identification is hard based on external morphological characteristics. DNA barcode is an effective method for species identification. This study aims to compare the efficiency of rapid identification of Pterocommatinae based on three different molecular markers, to get the standard sequences of DNA barcode of this subfamily aphids and to solve the taxonomical problem of some species. 【Methods】 Based on COI, Cytb and Buchnera gnd genes, 197 samples of ten species in two genera were selected for the neighborjoining tree analysis, distance analysis and similarity-based DNA identification analysis under K-2P model and p-distance model. 【Results】 Compared with the K-2P model, p-distance model got the smaller genetic distance and the overlap region of intra- and interspecific divergences. COI sequences had the highest success rate of species identification. The nearly 200 standard sequences of DNA barcode for aphids of Pterocommatinae had been gotten, and the DNA barcode sequence library of the subfamily species had been built based on the three markers. 【Conclusion】 The p-distance model is superior to K-2P model, and COI sequences have the highest analysis efficiency. Plocamaphis assetacea may be a synonym of Plocamaphis flocculosa.