【Aim】 Calliptamus italicus is a major plague pest in the arid and semi-arid grasslands of Xinjiang, with a lack of information of its reproduction related genes. The objective of this study is to reveal the characteristics of the transcriptome of C. italicus and to obtain the gonada development related genes in C. italicus through the transcriptome sequencing of its testicular and ovarian tissues. 【Methods】 The transcriptome of C. italicus was sequenced using the Illumina HiSeq/MiSeq platform and bioinformatically analyzed. 【Results】 A total of 718 872 unigenes were obtained, with a mean length of 631 bp and an N50 of 1 186 bp. Through a similarity search, 254 597 unigenes were annotated. Most unigenes (28.87%) were annotated to Nr database, and the unigenes of C. italicus had the highest similarity (10.80%) with those of Lasius niger annotated in Nr database. According to GO database, all unigenes were broadly annotated into three categories: 31 392 unigenes to be related to molecular function, 20 586 unigenes related to cellular component, and 57 014 unigenes related to biological processes. In KEGG database, a total of 23 666 unigenes were assigned to 251 metabolic pathways, of which many pathways are involved in the reproductive system development, including oocyte meiosis, insulin signaling pathway, Wnt signaling pathway, GnRH signaling pathway, TGF-β signaling pathway, ubiquitin mediated proteolysis, progesterone-mediated oocyte maturation, and insect hormone biosynthesis. By further screening and identification, gonadal development related genes of C. italicus, including genes of vg, vgr, sox 21b, testis-specific serine/threonine-protein kinase, spermatogenesis-associated protein and vasalike gene, were obtained. 【Conclusion】 This study acquired the transcriptome data and gonadal development related genes of C. italicus, providing a molecular foundation for further studying the mechanism of reproductive regulation of C. italicus.

【Aim】 Based on our previous work of cloning the phospholipase C gene AlPLC from Apolygus lucorum, this study aims to determine the expression profiles of AlPLC gene in different day-old nymphs of A. lucorum, to obtain the recombinant protein which has the phospholipase enzyme activity and to clarify its enzymatic characteristics. 【Methods】 Using the qRT-PCR technique, we determined the expression pattern of AlPLC in one day-old to 13 day-old nymphs of A. lucorum. The recombinant plasmid containing target gene was specifically expressed after induction by IPTG. The recombinant protein was purified by GST agarose affinity chromatography and molecular sieve chromatography. Then the enzymatic characteristics of this recombinant protein under different temperatures and pH were measured by p-nitrophenylphosphorylcholine (p-NPPC). Finally, the optimum temperature and pH for the enzyme activity of PLC were analyzed. 【Results】 AlPLC was found to be continuously expressed throughout the surveyed nymphal stage of A. lucorum, and highly expressed in 4, 8, 12 and 13 day-old nymphs. The recombinant plasmid pCzn1-AlPLC expressed the target recombinant protein of 79 kD after IPTG induction in Escherichia coli. The 12% SDS-PAGE showed that the recombinant protein was mainly present as inclusion bodies. The purified protein of AlPLC with the phosphodiesterase activity was obtained after denaturation and renaturation. This recombinant protein had higher phospholipase activity by using p-NPPC as substrates, and under the protein concentration of 0.25 mg/mL, the most suitable temperature for its reaction was 57℃ (179.54±3.96 nmol/μg·min) and the optimal pH was 8.5 (374.99±2.84 nmol/μg·min). 【Conclusion】 The expression of AlPLC shows the developmental stage-specificity. Higher enzyme activity of AlPLC can be achieved under higher temperature and alkaline environment. The results provide the basis for analyzing the AlPLC function at the protein level.

【Aim】 To explore the expression pattern and roles of dual oxidase (DUOX) in gut immunity in the silkworm, Bombyx mori. 【Methods】 The amino acid sequence of BmDUOX was analyzed by bioinformatic methods, and the outside region encoding gene, BmDUOX_OM, was cloned from B. mori and transferred into Eescherichia coli Transetta (DE3) to obtain the fusion protein. After purification by Ni affinity chromatography, the purified protein was treated as antigen to immunize mouse to prepare polyclonal antibodies, which were utilized for analyzing the expression and localization of BmDUOX in BmE cells. Semi-quantitative RT-PCR was employed to assay the expression patterns of BmDUOX in different developmental stages and different tissues and the pathogen-induced expression profiles. Additionally, the content of reactive oxygen species (ROS) in BmE cells after induction by Nosema bombycis was analyzed by using ROS detection kit. 【Results】 Bioinformatics analysis showed that BmDUOX has conserved peroxidase, ferric_reduct, EF-hand, FAD-binding and NAD-binding domains. There are also six transmembrane regions in BmDUOX, and their transmembrane form is consistent with that of hDUOX2 and DmDUOX. Multiple sequence alignment analysis showed that the peroxidase domain of BmDUOX has conserved activity sites. BmDUOX_OM gene was cloned, the recombinant protein was purified, and mouse-origin polyclonal antibody with high specificity was prepared. Indirect immunofluorescence assay (IFA) showed that BmDUOX was located on the plasma membrane of BmE cells. Expression pattern analysis showed that BmDUOX was highly expressed in the 5th instar day-3 larvae and adults of the silkworm, and in the tissues of integument, testis, ovary, head of larvae, and the ovary, testis, integument, fat body of its adults. Expression of BmDUOX in the larval midgut of the silkworm could be continuously induced by microsporidia Nosema bombycis, and the content of ROS in BmE cells was obviously increased after induction by N. bombycis, suggesting that DUOX-regulated intestinal epithelial ROS response may play important roles in the resistance against the infection of N. bombycis. 【Conclusion】 BmDUOX has the typical functional domains and active sites, showing its conservative biological function in B. mori. Expression profiles of BmDUOX in B. mori in different developmental stages, different tissues, and under induction by diverse pathogenic microorganisms suggest that BmDUOX may be involved in the immune response of host intestinal epithelium to the invasion of pathogenic microbes.

【Aim】 This study aims to screen and verify stably expressed genes under given conditions as reference genes for quantitative real-time PCR (qRT-PCR) in Conogethes punctiferalis, so as to provide the basis for quantitative studies of genes of this moth. 【Methods】 Based on transcriptomics sequencing results in C. punctiferalis and reported reference genes in other insect species, six candidate genes including β-actin gene (ACT), glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH), ribosomal protein 49 gene (RP49), alpha tubulin gene (α-tub), ribosomal protein L13 gene (RPL13) and vacuolar-type ATPase gene (V-ATPase) were cloned, and their expression levels in different developmental stages and different adult tissues were measured by qRT-PCR. Then the stabilities of these candidate genes were evaluated by a series of programs including ΔCt method, GeNorm, NormFinder, BestKeeper and an online program, RefFinder. Finally, the stabilities of selected reference genes were validated with an olfactory receptor co-receptor gene (Orco) and pheromone binding protein 1 gene (PBP1). 【Results】 Analyzed by four programs including ΔCt method, GeNorm, NormFinder and BestKeeper, similar rankings of six candidate genes were obtained, among which RP49, RPL13 and GAPDH were the most stable genes in different developmental stages and different adult tissues, and ACT was ranked as the least stable gene despite of experimental conditions by all programs. Comprehensive analysis with RefFinder further showed that in different adult tissues, RPL13 was the most stable gene, followed by RP49, and in different developmental stages, RP49 was the most stable gene, followed by GAPDH. Additionally, the optimal number of reference genes was calculated by GeNorm as 2. Finally, the stabilities of selected reference genes were validated with Orco and PBP1 as target genes. The results indicated that when two pairs of genes, RPL13 and RP49, and RP49 and GAPDH, were respectively used as reference genes, Orco and PBP1 showed reliable expression patterns, which were consistent with life habits of C. punctiferalis and the results of the previous research. The expression patterns of Orco and PBP1, however, were irregular when ACT was used as the reference gene. 【Conclusion】 In C. punctiferalis, two pairs of genes, RPL13 and RP49, and RP49 and GAPDH, are recommended as reference genes in different adult tissues and different developmental stages, respectively.

【Aim】 To explore the function of histone H3 Ser10 phosphorylation, an epigenetic marker, in cells of Spodoptera frugiperda cell line Sf9 during mitosis. 【Methods】 The sequence conservation of histone H3 was analyzed by sequence alignment. Antibody was prepared by synthesis of histone H3 pepetide RK(pS)TGGKAPRKQLC, in which the tenth serine is specifically phosphorylated by solid phase method. Sf9 cells were cultured, and the mitotic cells were prepared by cell climbing. The numbers of cells in different mitotic stages were counted, and then the localization characteristics of histone H3 Ser10 phosphorylation antibody in these stages were detected by cytology immunofluorescence. 【Results】 Sequence alignment analysis revealed that histone H3 1st-60th amino acids were highly conserved in most species. In Sf9 cells, histone H3 Ser10 phosphorylation began to appear near the nuclear membrane with a punctate distribution pattern in early prophase, and spread throughout the whole chromosome in early metaphase during the cell cycle. During anaphase, the fluorescent signals of Ser10-phosphorylated H3 disappeared from chromosomes, and the dephosphorylation completed in telophase. 【Conclusion】 Histone H3 Ser10 phosphorylation is associated with chromosome condensation in Sf9 cells during mitosis.

【Aim】 This study aims to screen plant volatiles attracting Cylas formicarius. 【Methods】 Plant volatiles of five sweetpotato cultivars were collected by headspace adsorption device and then identified and quantified on GC-MS. EAG response and taxis of C. formicarius adults to candidate volatiles from the sweetpotato plants were assayed. And the olfactory responses of C. formicarius adults to volatiles emanating from stems and leaves of five sweetpotato cultivars were measured by using a Y-tube olfactometer. 【Results】 The GC-MS results showed that the types and contents of volatiles collected from the five sweetpotato cultivars were different. Seven candidate volatiles (ocimene, limonene, nonanal, β-caryophyllene, humulene, farnesene, and (Z)-3-hexen-1-olacetate) elicited EAG responses in both male and female adults of C. formicarius. The EAG responses of female adults of C. formicarius to limonene, nonanal and β-caryophyllene were significantly higher than to the other chemicals tested, while those of males to ocimene, limonene and humulene were significantly higher than to other chemicals tested. In taxis assay, limonene and nonanal attracted male adults, and humulene and limonene attracted females. The response rates of C. formicarius adults to volatiles from stems and leaves of the five sweetpotato cultivars were 40.90%~65.21%, and the volatiles emitted from sweetpotato stems and leaves of the five cultivars had no significant attractivity to all adults (P>0.05). 【Conclusion】 Male and female adults of C. formicarius show different EAG and olfactory behavioral responses to the volatiles from the sweetpotato, and the volatile limonene is closely related to the preference of C. formicarius.

 【Aim】 DNA methylation is one of the important ways of gene modification, mainly resulting in 5mC and 6mA. At present, there have been many studies related to 5mC, while studies on 6mA in eukaryotes are rare. Wolbachia bacteria are endosymbionts frequently found in insects. They can mediate their hosts' reproduction via several strategies. Cytoplasmic incompatibility (CI) is the most common defect induced by Wolbachia, which arrests the development of embryos from uninfected females that are mated with Wolbachia-infected males. The molecular mechanisms of CI, however, are not clear. The aim of this study is to investigate the mechanisms of Wolbachia affecting the reproduction of Drosophila from the viewpoint of 6mA methylation. 【Methods】 The model organism D. melanogaster was used as the test insect. Quantitative RT-PCR was adopted to detect the expression levels of DNA 6mA demethylase gene (DMAD) in testes, ovaries and early embryos of Wolbachia-infected and uninfected D.melanogaster flies. The embryos examined were from three different crosses: TT (the control, with both males and females uninfected), TW (viable, males were uninfected while females were Wolbachia-infected), and WT (lethal CI embryos, with males Wolbachia-infected and females uninfected). 【Results】 Wolbachia infection significantly up-regulated the expression of DMAD in testes of 1-day-old flies, but had no effects on DMAD expression in ovaries. In early CI embryos (0.5 h, pre-midblastula transition), DMAD expressionin level was significantly higher than those of the control and TW embryos. In the embryos at midblastula transition (3 h), the transcriptional levels of DMAD in TW and CI were extremely higher than that in the control embryos. In 6 h embryos (post-midblastula transition), however, DMAD expressionin level was the lowest in CI embryos as compared with the control and TW embryos. 【Conclusion】 Wolbachia infection may significantly change 6mA level in testes of their Drosophila hosts, and thus result in death of early embryos derived from Wolbachia-infected males and uninfected females.

【Aim】 Imidaclorprid is the most widely used pesticide among neonicotinoid pesticides and acts on the brain’s nicotinic acetylcholine receptor of honey bees. In addition, imidaclorprid interferes with the growth and development of honey bees. This study aims to clarify the effects of imidacloprid at field realistic doses on learning and memory of honey bees, so as to provide evidence for the prediction of the widespread death of colonies in some areas and reference for the safe use of neonicotinoid pesticide in the field. 【Methods】 Newly emerged 1-day-old bees (Apis mellifera ligustica) were marked by paint marker. They were kept in the colony for 8 d, and then were reared within boxes (50 individuals/box) in an incubator with constant temperature and humidity (30±1℃, relative humidity 40%±10%, dark) for 9 d. Meanwhile, the treatment group fed 30% (w/v) syrup containing 0.01 ng/μL of imidacloprid ad libitum, while the control group fed 30% (w/v) syrup containing 0.01 ng/μL of acetone ad libitum. Following being trained with three paired stimulations (lemon odor paired with sucrose stimulation) performed using a customized device, the 18-day-old bees were used for the olfactory associative learning and memory experiments. 【Results】 There was no significant difference in the mortality between the treatment group and the control group during the 9-day feeding period (P＞0.05). In the three olfactory associative learning experiments, the learning ability of bees of the treatment group in the 2nd and 3rd experiments was significantly reduced (P＜0.01) compared with the control group, but there was no difference in the 1st experiment ［proboscis extension response (PER)%=0］. After 24 h, the proboscis extension reflex rate was not significantly different between the treatment and control groups (P＞0.05). 【Conclusion】 The results demonstrate that 0.01 ng/μL imidacloprid does not cause acute death of honey bees. This dose of imidacloprid does not affect the 24 h long-term memory of honey bees, but impairs learning ability of honey bees significantly and may even adversely affect the foraging behavior of honey bees.

【Aim】 The objective of this study is to assess the resistance of Myzus persicae populations to conventional insecticides in tobacco production areas in Guizhou province, southwestern China. 【Methods】 Resistance of M. persicae field populations collected from 34 major tobacco production counties and cities of nine districts in Guizhou to acetamiprid, deltamethrin and pirimicarb was assayed with dipping method. 【Results】 The results of the resistance monitoring of 34 M. persicae populations to three insecticides indicated that the two populations collected from Qianxinan (Xingyi) and Bijie (Qianxi) maintained sensitivitydecreasing level to acetamiprid (RR=4.00-4.31), while other 32 populations developed moderate level resistance to acetamiprid (RR=10.09-26.35). For deltamethrin, the Bijie (Bijie) and Qiandongnan (Tianzhu) populations developed moderate level resistance (RR=32.47-38.72). The Bijie (Qianxi), Tongren (Sinan) and Anshun (Anshun) populations developed extremely high level resistance (RR=184.97-237.77). The other populations developed high level resistance to deltamethrin (RR=51.27-132.08). For pirimicarb, the populations collected from Bijie (Bijie), Qiandongnan (Shibing and Tianzhu) and Zunyi (Yuqing) developed low level resistance (RR=5.90-9.46), while the other populations developed moderate level resistance (RR=10.05-31.66). 【Conclusion】 The results suggest that most field populations of M. persicae in Guizhou have developed low to extremely high level resistance to the insecticides, which may be due to the wide use of these insecticides in tobacco production areas. The results provide important guidance for the choice and wise application of insecticides in the control of Myzus persicae in fields in tobacco production areas of Guizhou.

【Aim】 The objective of this study is to clarify the dissimilarities of feeding behaviors of biotype E of the greenbug, Schizaphis graminum on oat (Avena sativa) lines with different phenotypic resistance so as to ascertain aphid-resistant factors. 【Methods】 The frequencies and duration of eight basic waveforms of A, B, C, Pd, E, F, G and np for probing and penetrating behaviors of apterous adults of biotype E greenbug were recorded by using direct current-electrical penetration potentiometer (DC-EPG Giga-4) on ten MN hulled oat lines with varied phenotypic resistance. 【Results】 The electrical penetration graph showed that recognition time of biotype E greenbug ranged from 1.03 to 2.62 min on ten MN hulled oat lines with varied phenotypic resistance. Non-penetrating time was longer and the time to the first penetration from initiation of electrical penetration graph was the longest on MN10253 hulled oat line with moderate-level phenotypic resistance, which were 117.77 min and 13.80 min, respectively, suggesting that interference factors adverse to host locating and recognizing of biotype E greenbug possibly exist in host surface. Total duration of penetrating into mesophyll and xylem by stylet and the duration of penetrating into xylem by stylet from initiation to the first penetration were all the shortest on MN08252 hulled oat line with moderate-level phenotypic resistance, which were 78.01, 6273 and 32.29 min, respectively, and the frequency of np waves was up to 28.57 times, suggesting that aphid-resistant factors are likely present in mesophyll and xylem. Total and mean duration of F waves were the longest on MN06203 hulled oat line with low-level phenotypic resistance, which were 4.96 min and 5.81 s, respectively, suggesting that the line has fair physical resistance. Total duration of E₁ waves was the shortest while duration of penetrating into phloem by stylet from initiation to the first was the longest on MN11213 hulled oat line with low-level phenotypic susceptibility, which were 1.94 min and 246.05 min, respectively, suggesting that aphid-resistant factors might be present in phloem. 【Conclusion】 Aphid-resistant factors are most possibly seated on the leaf surface of MN10253 hulled oat line with moderate-level phenotypic resistance, in the mesophyll and xylem of MN08252 hulled oat line with moderate-level phenotypic resistance, in the phloem of MN11213 hulled oat line with low-level phenotypic susceptibility, while the MN06203 hulled oat line with low-level phenotypic resistance has stronger physical resistance.

【Aim】 The objective of this study is to understand the characteristics of the complete mitochondrial genome of Parnassius nomion and to explore the higherlevel phylogenetic relationships of butterflies based on mitochondrial genome sequences.【Methods】 The PCR amplification technique and Sequencher 4.8 software were used to obtain the whole sequence of mitochondrial genome of P. nomion. Based on the known complete mitochondrial genomes of lepidopteran species, MEGA6.0 software was used to locate and annotate each gene of P. nomion mitochondrial genome. tRNA Scan-SE 1.21 was used to predict the secondary structure of the tRNA genes online. Based on the nucleotide sequences of 13 protein-coding genes of mitochondrial genome, the phylogenetic relationships among 28 butterflies of 10 families (Papilionidae, Parnassiidae, Pieridae, Satyridae, Nymphalidae, Lycaenidae, Danaidae, Acraeidae, Libytheidae and Riodinidae) of Papilionoidea were reconstructed. 【Results】 The results indicated that the complete mitochondrial genome of P. nomion is a circular molecule of 15 362 bp (GenBank accession no.: MF496134), including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and an A+T-rich region. The mitochondrial genome of P. nomion is biased toward a high A+T content (79.6%). The relative synonymous codon usage (RSCU) of UUA is the highest in the 13 protein-coding genes in the mitochondrial genome of P. nomion, the relative synonymous codon usage value is 5.08, but AGG and CCG has a relatively low RSCU of 0, which is consistent with the analysis results of Sasakia charonda coreana. All 22 tRNA genes show the classic clover-leaf structure, except that the dihydrouridine (DHU) arm of tRNASer (AGN) forms a simple loop, which is consistent with the secondary structure of tRNA genes in the mitochondrial genomes of other lepidopteran insects. The results of phylogenetic analysis showed that Riodinidae and Lycaenidae are sister groups, Pieridae is closer with the clade of (Lycaenidae+Riodinidae)+(((Nymphalidae+Acraeidae)+Satyridae+Danaidae)+Libyheidae), Parnassiidae is a sister group of Zerynthiinae, and then Parnassiidae+Zerynthiinae is clustered with Papilioninae. P. nomion is closely related with Parnassius epaphus in Parnassiidae. 【Conclusion】 Our study supports that Parnassiidae species should be included in Parnasiinae, Parnasiinae, Zerynthiinae and Papilioninae all belong to Papilionidae, and Parnasiinae and Zerynthiinae are sister groups.

【Aim】 This study aims to explore the feasibility and generalization ability of deep learning model applied to the automatic identification of butterfly images at the family level. 【Methods】 To improve the robustness and generalization performance of model, the data augmentation with images of 1 117 butterfly species of six families were performed to increase the number of images by flipping image horizontally, increasing image contrast and brightness, and adding noises for training. In Caffe framework, an ImageNet-trained convolution neural network model was obtained by 310 000 iterations. The training set of butterfly images was used to train a new CaffeNet model to automatically identify butterflies at the family level by the transfer learning method. To compare generalization ability of the CaffeNet model based on deep learning with the models based on traditional pattern recognition methods, global and local features were extracted from the same training samples, and the support vector machine (SVM) classifier was trained. All models were used to detect the two different test sample sets. 【Results】 When the test samples, same as the training samples, were from specimen images, the CaffeNet model had a mean accuracy rate of 95.8%, while the SVM classifier based on Gabor features had a mean accuracy rate of 94.8% in six butterfly families. When the test samples were from natural images of butterflies, the accuracy rates of the CaffeNet and SVM models were decreased. However, the accuracy rate of CaffeNet model still achieved 65.6% and the SVM classifier based on Gabor features only got the 38.9% accuracy rate. 【Conclusion】 The butterfly identification model based on deep learning has a high identification rate at the family level, with higher robustness and generalization ability than those traditional pattern recognition models based on global and local features by manual extraction and selection.

Bactrocera fruit flies are an enormous threat to fruit and vegetable production throughout the world, causing great economic loss. Although chemical control based on conventional insecticides and biotechnical tools including sterile insect technique (SIT) and male annihilation technique (MAT) have been the main weapons used in most control programs, they still have many restrictions, and ecofriendly control tools against Bactrocera spp. are urgently needed. Thus, current knowledge about sexual communication and related behaviours in Bactrocera spp. was reviewed in this article to help build behaviour-based control strategies. Two different polygynous mating systems in Tephritidae and behavioural sequences of males before courtship in lekking sites were summarized, and some mated female behaviours in oviposition sites, including oviposition marking behaviour and fighting behaviour for single oviposition sites, were elaborated. Future perspectives were also outlined. The knowledge about sexual communication is expected to provide new insights and references for integrated pest management programs for tephritid pests.