【Aim】 Odorant binding proteins (OBPs) play crucial roles, functioning as transporting vectors of odors, in host location and oviposition selection for many insects. Cloning and identification of OBP genes and clarifying their ligand-binding characteristics of the compounds may help address the molecular mechanisms in P. xylostella. 【Methods】 OBP2 was cloned from P. xylostella based on PCR approach. Signal peptide and transmembrane region were predicted and the nucleotide sequence of P. xylostella OBP2 (PxylOBP2) was aligned with that of the homologs from other insects using DNAMAN. A neighbor-joining tree was constructed using MEGA5.0. Stage- and tissue-specific expressions of PxylOBP2 were profiled by using real-time quantitative PCR (qRT-PCR). Prokaryotic expression vector was constructed to express the recombinant protein, and the purified protein was detected by SDS-PAGE. The protein binding affinity of PxylOBP2 with 39 compounds was analyzed using fluorescence competitive binding assay. 【Results】 PxylOBP2 was successfully cloned and sequenced. Its ORF is 546 bp in length (GenBank accession no. KT070562), encoding 182 amino acids, and the encoded protein has six cysteine conserved domains, with the predicted molecular mass of 22.24 kDa and isoelectric point of 5.69. SDS-PAGE analysis showed that the fusion protein was successfully expressed. The expression profiling of PxylOBP2 exhibited a higher expression level in virgin male adults than in female adults and mated male adults, and the expression levels of PxylOBP2 in legs of male and female adults were higher than those in other tissues. The binding affinity test of PxylOBP2 to three sex pheromones and 36 different plant volatiles showed that PxylOBP2 could bind with Z-11-16:Ald, with a dissociation constant of 48.951 μmol/L, and with 11 plant volatiles, exhibiting a stronger capability of binding linalool and 1-nonanol with the dissociation constants of 4.733 and 6.861 μmol/L, respectively. 【Conclusion】 The nucleotide and amino acid sequences of PxylOBP2 were characterized. Based on qRT-PCR and competitive binding test results, we infer that PxylOBP2 may play important roles in adult courtship of the moth, and host plant volatiles play a synergism role.

【Aim】 To obtain the recombinant allergen CPH30 in pupae of the silkworm, Bombyx mori, and to detect its immunogenicity. 【Methods】 The potential allergenicity of CPH30 protein was analyzed with proteomic methods. The CPH30 gene was synthesized by chemical method and introduced into pET-28a vector. The expression of the recombinant CPH30 was induced by IPTG. The allergenic activity of CPH30 protein was detected by Western blot. The potential B cell epitopes were analyzed using DNAStar. 【Results】 After induction with IPTG, CPH30 protein was largely expressed in Escherichia coli Rosetta and purified by affinity chromatography. The molecular weight of the recombinant CPH30 protein is 25 kD. The CPH30 protein show a high IgE binding activity with the serum from silkworm-allergic patients, indicating that the recombinant CPH30 protein has immunogenicity. Prediction result using DNAStar software showed that the potential B cell epitopes of CPH30 are located between the 57th-69th and 150th-158th amino acids .【Conclusion】 The purified CPH30 has high purity and immunogenicity. This study lays a foundation for the specific diagnosis, vaccine treatment and further experimental studies of allergic diseases caused by silkworm pupae.

【Aim】 Aquaporin (AQP) is one of the transmembrane proteins widely existing in mammals, plants, and microorganisms, and plays an important role in the processes of cell moisture transportation, selective ion permeability and osmotic balance. This study aims to understand the gene characteristics of AQP1 from Ectropis obliqua Prout, and its subcellular localization based on polyclonal antibody  preparation.【Methods】 The complete cDNA of AQP1 was cloned from E. obliqua by RT-PCR and RACE technologies. The bioinformatics analysis was carried out by online website and edited on the bioinformatics software. The mRNA relative expression levels in different developmental stages and different tissues of the 6th instar larvae of E. obliqua were investigated by real-time quantitative PCR (qRT-PCR). The polyclonal antibody was prepared by immunizing a male New Zealand white rabbit after prokaryotic expression and Ni-sepharose purification. Subcellular localization in Drosophila melanogaster embryonic cells (S2 cells) was observed by fluorescence microscope, and Western blot analysis was performed with the polyclonal antibody of AQP1 from E. obliqua. 【Results】 The AQP1 cDNA was cloned successfully from E. obliqua and named EoAQP1 (GenBank accession no.: KT819587). It is 1 826 bp in length, including a 780 bp of open reading frame (ORF), which encodes 259 amino acids. Phylogenetic tree analysis and alignment of amino acid sequences showed that EoAQP1 is highly conserved with AQP1 proteins of other species of Lepidoptera. Transmembrane structure and water percolating simulation showed that EoAQP1 has a classical water percolation model. The qRT-PCR result revealed that EoAQP1 was expressed in all developmental stages and various tissues of the 6th instar larvae of E. obliqua at different mRNA expression levels. Subcellular localization showed that EoAQP1 aggregated around cell membrane with circular and granular models, but was not expressed in cell membrane, cytoplasm or nuclear membrane. Western blot analysis demonstrated that the obtained polyclonal antibody had high specificity and could be used for further experiments. 【Conclusion】 The nucleotide sequence, bioinformatics and expression profiling of aquaporin 1 gene EoAQP1 in E. obliqua were clarified. With the obtained polyclonal antibody, its subcellular localization of EoAQP1 was preliminarily investigated. These results provide a foundation for further research on the mechanism of water infiltration of EoAQP1.  

【Aim】 Time interval measuring enzyme (TIME-EA4) is a diapause bioclock protein in insects. This study aims to clone TIME-EA4 cDNA and analyze its spatiotemporal expression patterns in various developmental stages and different tissues of Chilo suppressalis, including the populations collected in the fields, and diapause and non-diapause populations induced at various temperatures. 【Methods】 Full-length cDNA sequence of TIME-EA4 gene of C. suppressalis was cloned by using RACE technique. The relative expression levels of TIME-EA4 in different developmental stages, different tissues of the 6th instar larvae, larvae of field populations collected in different seasons and the 6th instar larvae from different populations of the moth treated for one hour at different temperatures were assayed by real-time PCR. 【Results】 The full-length cDNA of C. suppressalis TIME-EA4 is 821 bp, with an open readingframe of 516 bp encoding 172 aa. TIME-EA4 was ubiquitously expressed, but with higher expression levels in early pupae and adults, and in the head, fat body and midgut of the 6th instar larvae. And the expression level of TIME-EA4 in the tissues of diapause larvae was much higher than that in the non-diapause larvae. Interestingly, the expression level of TIME-EA4 was significantly increased 3-fold in diapause groups compared with that in the non-diapause individuals. The abundance of TIME-EA4 gene was elevated at ≤10℃ in the non-diapause population, and reached the peak at 10℃. However, TIME-EA4 expression was not significantly altered by temperature change in the diapause population. 【Conclusion】 These results suggest that the enhanced expression of TIME-EA4 correlates with the low temperature stress or low temperature-induced diapause in C. suppressalis. This study forms a basis for further investigation of TIME-EA4 in the diapause of agricultural pests.

 【Aim】 The sHSPs are a diverse of molecular chaperones with molecular weights ranging from 12 to 43 kDa in different species. These proteins have been shown to be crucial components of the diapause of some species, but need to be further elucidated. The study aims to clone and characterize the sHSP gene of Delia antiqua, of which orthologs were earlier reported to be diapause-related, and to investigate the expression patterns of the gene during both winter and summer diapauses of the species. 【Methods】 A sHSP gene was cloned from D. antiqua using RACE-PCR, and characterized through homology, domain and phylogenetic analyses. The existence of the intron was investigated with genome sequencing around the gene. The expression level of this gene during both winter diapause (WD) and summer diapause (SD) was detected real-time qPCR, and the expression difference was compared to reveal the association with diapause development. 【Results】 The cDNA of sHSP cloned from D. antiqua, which is named DaHSP23 (GenBank accession no. HQ392521.1), is 904 bp in length, encoding 186 amino acids with a molecular mass of 20.9 kDa and a theoretical isoelectric point of 6.42. DaHSP23 shares more than 66% amino acid identity with the known HSPs of other dipteran insects and is homologous with the known HSP23 genes of other dipteran insects. Genome sequencing around the gene revealed that it is intronless. The expression of DaHSP23 was up-regulated in both SD and WD pupae, with higher expression level detected in the late maintenance stage, and down-regulated when those diapauses were terminated. 【Conclusion】 The patterns of HSP23 regulation are substantially different between different developmental stages of both SD and WD, and its regulation in diapause might be species-specific. DaHSP23 might play an essential role in these two types of diapauses in D. antiqua.

【Aim】 To explore the roles of Hsp90 gene in development and response to heat and cold stress in the citrus red mite, Panonychus citri. 【Methods】 The full-length cDNA encoding Hsp90 from P. citri was cloned and identified using RT-PCR and RACE technique. Bioinformatics programs were used to analyze the sequence characteristics of the gene. The mRNA expression levels of the gene in this mite at different developmental stages and under cold and heat stress conditions were detected by real-time PCR. 【Results】 The complete cDNA of the cloned gene PcHsp90 with GenBank accession no. GQ495086 is 212 763 bp in length, and contains an open reading frame of 2 193 bp, which encodes a protein of 730 amino acids, with the molecular weight of 83.85 kDa and the theoretical isoelectric point of 4.99. The deduced amino acid sequence contains five highly conserved motifs of Hsp90 family and the cytoplasmic Hsp90 C-terminal region “MEEVD”. Phylogenetic analysis showed that PcHsp90 clustered firstly with Hsp90 of Tetranychus cinnabarinus, and then with that of Ixodes scapularis, suggesting the close relationship among them. PcHsp90 transcripts were all detected at different developmental stages, and the relative mRNA expression level was significantly lower at larval stage than those at nymphal and adult stages (P=0.015). No distinct change was found in relative PcHsp90 mRNA expression levels in the mites subjected to 0-10℃ cold stress (P=0.492), but a significant up-regulation was observed in the mites subjected to 35-41℃ heat stress (P=0.007); especially when the temperature was up to 41℃, the mRNA level was 6.75-fold as high as that of the control (25℃).【Conclusion】 PcHsp90 may be involved in development and plays an important role in response to heat stress in P. citri.  

【Aim】 In order to develop the molecular markers of the tea green leafhopper, Empoasca vitis, the high-throughput sequencing technologies were used in sequencing and analysis of DNA of the leafhopper. 【Methods】 Based on Illumina HiSeq sequencing technology, Illumina PE library (~400 bp) was constructed, and then the scanning of the whole genome sequences was finished using bioinformatics methods. Furthermore, MISA was used to identify the microsatellites in the genome. Ten pairs of primers were designed according to the microsatellite loci, and three-step procedure was used to screen the polymorphism of primers. 【Results】 Totally 183 194 scaffolds were examined, among which 1 545 contain microsatellites, and 1 569 microsatellites were identified in all these scaffolds. Among all 87 kinds of repeat motifs, the dominant microsatellite types were dinucleotide and trinucleotide repeats, accounting for 70.26% and 27.84% of the total SSRs, respectively, in E. vitis. CA/TG and AAT/ATTare the most frequent motifs, accounting for 33.96% and 5.86% of the total SSRs, respectively. Five primers among 10 primers designed revealed polymorphism, and 16 alleles were detected from eight individuals of E. vitis. 【Conclusion】 The results suggest that SSRs of E. vitis with polymorphism have potential for further use research. The polymorphism of SSRs may provide a molecular perspective of population divergence, dispersal mechanisms, diffusion path and influencing factors in E. vitis.

【Aim】 This study aims to select suitable internal control genes for gene expression analysis in chemosensory tissues of Monochamus alternatus. 【Methods】 Candidate reference genes were identified from our transcriptome results, and their expression patterns in chemosensory tissues were investigated by RT-qPCR in different developmental stages and both genders. Their expression stabilities were also compared with each other by using softwares geNorm, NormFinder and BestKeeper. 【Results】 Nine candidate reference genes were identified in M. alternatus, including Actin, TUB, 18S rRNA, RPS27A, RPS3, RPL10, AK and GAPDH, of which the last seven were firstly identified in this insect species. Candidate reference genes in M. alternatus have high nucleotide sequence identity with those from other insects. Designed primers of nine candidate reference genes worked well with high amplification efficiencyand specificity to M. alternatus. It was found that 18S rRNA had the highest expression level while EF1A had the lowest expression level in M. alternatus. 18S rRNA and Actin had the most variable expression levels between studied chemosensory tissues, but GAPDH and TUB had the least variable expression levels. GAPDH was the most stable reference gene, TUB was a moderately stable reference gene, and 18S rRNA and Actin were the least stable reference genes in different chemosensory tissues, which was referred from analysis with softwares geNorm and NormFinder. Analysis using BestKeeper software showed that GAPDH and TUB but not 18S rRNA and Actin were the suitable reference genes in chemosensory tissues of M. alternatus. The combination of two reference genes, GAPDH and TUB, was referred as the best internal control combination genes for normalization of RT-qPCR data conducted with chemosensory tissues of different developmental stages (larva, pupa and adult) and different genders of M. alternatus. 【Conclusion】 The results of this study provide important data for selecting internal reference genes for gene expression analysis of M. alternatus and other longhorn beetles, and also for gene expression analysis of chemosensory tissues of other insects by RT-qPCR.

【Aim】 This study aims to explore the characteristics of embryonic development between gamogenesis and parthenogenesis in Reticulitermes. 【Methods】 Fertilized eggs (female-male paired eggs, FM eggs) and unfertilized eggs (female-female paired eggs, FF eggs) were used for observing egg cleavage in two termite species, Reticulitermes chinensis and R. aculabialis. We used laser scanning confocal microscope for egg cleavage observation and digital microscope for observing external morphology of eggs and embryos. 【Results】 Fertilized and unfertilized eggs from queens of R. chinensis could all cleave in the nest. Comparison between the nuclei of FF and FM eggs in 24 and 48 h revealed no significant difference in the number of nuclei for FF egg, but significant difference for FM egg. On the 15th day of egg development, no significant changes occurred in the volume of FF egg, whereas the volume of FM egg significantly increased. The FF eggs died on the 15th-20th day, while the FM eggs were in normal development. There was no significant difference in the number of nuclei in the same period between the fertilized and unfertilized eggs of R. aculabialis, and the number of nuclei in 48 h was significantly more than that in 24 h. The length and width of the fertilized eggs significantly changed on the 10th day, with the volume significantly increasing at the same time. The increase of length, width and volume of unfertilized eggs, however, occurred on the 15th day. 【Conclusion】 The fertilized eggs and unfertilized eggs of facultative parthenogenesis R. aculabialis have the same cleavage rate. But the change in the shape of the fertilized eggs is earlier than that of the unfertilized eggs. Unfertilized eggs of R. chinensis can cleave, but have abnormal development, and can not hatch in the end. The length and width of eggs of these two termite species change simultaneously in the same development stage. The cleavage characteristics of FF egg of R. chinensis may be a preadaptive stage from bisexual reproduction to facultative parthenogenesis in termite reproductive evolution.

【Aim】 This study aims to investigate the anatomical organization of the central nervous system of Apolygus lucorum (Hemiptera: Miridae) and to characterize the morphology, volume, spatial relationship and fusion pattern of the composed ganglia. 【Methods】 Immunohistochemical staining with synaptic protein antibody was used to label the neuropil of the central nervous system. Digital images of the central nervous system were obtained by using a confocal laser scanning microscope, and the three-dimensional models were constructed by using an image analysis software. 【Results】 The anatomical 1observation indicated that the central nervous system of A. lucorum consists of four main ganglia. From anterior to posterior, they are the brain, suboesophageal ganglion, prothoracic ganglion and posterior ganglion. The first three ganglia are fused together, forming a brain-suboesophageal ganglion-prothroracic ganglion complex. Such a complex has been found in insects for the first time. The posterior ganglion is linked to prothoracic ganglion by a long paired connective. Viewed from the external morphology, the central nervous system of A. lucorum is composed of two large ganglionic masses, a brain-suboesophageal ganglion-prothroracic ganglion complex and a posterior ganglion. Based on the synaptic staining, three neuromeres in the brain, three in suboesophageal ganglion, three in posterior ganglion and one in prothoracic ganglion were identified. The three neuromeres of the brain are protocerebrum, deutocerebrum, and tritocerebrum. The suboesophageal ganglion is composed of mandibular, maxillary, and labial neuromeres, while the posterior ganglion is fused with mesothoracic, metathoracic, and abdominal ganglia. 【Conclusion】 The results demonstrate that the central nervous system of A. lucorum is composed of the brain, suboesophageal ganglion, prothoracic ganglion and posterior ganglion, showing highly fused pattern. The results provide the morphological data for the studies of development, plasticity and evolution of the central nervous system, as well as a framework for investigating the morphology, disposition and function of the neurons which are involved in a specific regulation for insect physiology and behavior.

【Aim】 Gut bacterium Erwinia persicina is one of the dominant bacterial species in the larval gut of the diamondback moth, Plutella xylostella. The aim of the present study is to investigate the phenotypic characteristics of this bacterium. 【Methods】 The cell phenotype of E. persicina was analyzed with BIOLOG Phenotype MicroArray (PM). Totally 950 different metabolic phenotypes were tested using the PM plates 1-10. 【Results】 E. persicina was able to metabolize 39.47% of the tested carbon sources, 89.74% of nitrogen sources, 100% of sulfur sources, and 100% of phosphorus sources. Most informative utilization patterns for carbon sources of E. persicina were organic acids and carbohydrates, and for nitrogen were various amino acids. The bacterium had 261 different nitrogen metabolic pathways and 95 different biosynthetic pathways. It had a wide range of adaptabilities, and could still metabolize in osmolytes with up to 9% sodium chloride, 4% potassium chloride, 5% sodium sulfate, 20% ethylene glycol, 6% sodium formate, 2% urea, 6% sodium lactate, 200 mmol/L sodium phosphate (pH 7.0), 20 mmol/L sodium benzoate (pH 5.2), 100 mmol/L ammonium sulfate (pH 8.0), 100 mmol/L sodium nitrate, and 100 mmol/L sodium nitrite, respectively. It also exhibited active metabolism under pH values between 4.5 and 10, with optimal pH around 7.0. The gut bacterium showed both decarboxylase and deaminase activities in the presence of various amino acids. 【Conclusion】 The phenotypic characterization of E. persicina increased our knowledge of the bacterium, in particular its interactions with insect hosts and the adaptability in gut environments, and showed us some possible approaches to controlling diamondback moth through decreasing E. persicina density.

 To reveal the effect of temperature on Wolbachia titer and its function in regulating the biological characteristics of host, the relationship between the biological indices of host and Wolbachia titer at different temperatures was evaluated in this study. 【Methods】 Trichogramma embryophagum wasps infected by parthenogenesis-inducing Wolbachia were cultivated under four temperature gradients (22, 25, 28 and 31℃) for five consecutive generations, and the biological characteristics including the reproductive mode and sex ratio were observed. At the same time, the Wolbachia titer was detected by fluorescence quantitative real-time PCR, using the specific primers of the outer surface protein gene (wsp), fructose bisphosphate aldolase gene (fbpA) and glutamyl-tRNA(Gln) amidotransferase subunit B gene (gatB) in Wolbachia. 【Results】 At 22 and 25℃, the reproduction mode of the total five generations of T. embryophagum was not altered, without drones appearing in culture population, and the Wolbachia titer also was not significantly different among five generations. At 28℃, a few of drones were found in F₃ generation and the majority of drones appeared in F₅ generation significantly, while the Wolbachia titer in T. embryophagum began to decline in F₂ generation and decreased significantly till F₅ generation. At 31℃, in F₂ generation the drones appeared, and in F₅ generation all T. embryophagum had restored to the androgenetic parthenogenetic; the Wolbachia titer began to decrease in the F₂ generation, decreased significantly in F₃ generation and was not detectable by PCR in F₅ generation.【Conclusion】 The reproduction mode and Wolbachia titer in parthenogenetic thelytokous Trichogramma can be altered by high temperature, and are affected significantly with the increase of temperature and generation. In other words, the alteration degree of reproductive mode of Trichogramma due to high temperature is negatively correlated with the endoymbiont Wolbachia titer.

 The trophic relationship of herbivorous insects and host plants are complicated in agroecosystem. The common methods, such as direct field observation, morphological analysis of the gut content and stable isotope analysis, are difficult to fully analyze the trophic relationship. Recently, the rapid development of DNA-based technology, which uses a short DNA sequence to achieve the rapid identification of host plants fed by herbivorous insects, provides a new option for exploring this relationship. In this review, three DNA-based methods were comprehensively introduced, including diagnostic PCR, DNA sequencing and next generation sequencing (NGS). Diagnostic PCR, including single PCR and multiplex PCR, is applicable to analyze the trophic relationship between herbivorous insects and their known host plants. DNA sequencing can resolve the complete host data of target insects without a prior knowledge of host plant species. NGS can achieve the sequencing of mixed samples in a short time and improve the identification efficiency of host plants significantly owing to the massive expansion of the DNA database of insects and host plants. Diagnostic PCR and DNA sequencing have made significant research progress in studying the feeding behavior of soil-dwelling pests, the host breadth of herbivorous insects, the host selection and preference of herbivorous insects, etc. Taking the advantages and disadvantages together into consideration, the strategy of application of DNA-based technology in combination with stable isotope analysis or other methods is proposed to comprehensively analyze the trophic relationships between herbivorous insects and host plants in agroecosystem in future.