【Aim】 This study aims to investigate the expression patterns of the peptidoglycan recognition protein gene ApPGRP in the desert beetle Anatolica polita, its regulation of the downstream immune-related genes in vivo and the binding capability of the recombinant protein ApPGRP with bacteria in vitro. 【Methods】 The expression vector pET-28a-ApPGRP was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant protein His-ApPGRP was expressed and purified, and its binding capabilities with Staphylococcus aureus and peptidoglycan (PGN) were detected, respectively. The expression levels of immune-related genes including ApPGRP, antimicrobial peptide genes ApAttacin2 and ApAttacin1, defensin gene ApDefensin, serine protease gene ApSP and serine protease inhibitor gene ApSerpin in the 6th instar larva of A. polita infected by S. aureus were determined by qRT-PCR. The expression levels of the immune-related genes in the 6th instar larvae of A. polita after RNAi of ApPGRP and S. aureus stimulation following RNAi of ApPGRP were also detected by qRT-PCR, respectively. 【Results】 The purified recombinant protein His-ApPGRP had the binding capability with both S. aureus and PGN. Except for ApSP, the expression of the other tested immune-related genes in the 6th instar larva of A. polita was significantly up-regulated after S. aureus injection. When ApPGRP was knocked down by RNAi, the expression of all the other tested immune-related genes in the 6th instar larva of A. polita was significantly downregulated and the expression levels of these genes in response to subsequent S. aureus challenge in the RNAi-treated larva were also significantly lower than those of the control group. 【Conclusion】 These results show that ApPGRP plays an important role in recognizing exogenous microorganisms, activating signaling pathways and regulating the expression of antimicrobial peptides in the immune defense of A. polita.

【Aim】 Harmonia axyridis is an important predatory natural enemy, and trehalose plays an important role in the whole life of H. axyridis, such as metamorphosis and adult eclosion. This study aims to explore the potential functions of two trehalase genes TRE2-like and TRE2, which were obtained earlier, on trehalose utilization during adult eclosion of H. axyridis, and to provide reference for clarifying the potential mechanism of trehalose metabolism in the development from pupa to adult in H. axyridis. 【Methods】 The dsRNAs of TRE2-like and TRE2 were synthesized by designing double-stranded RNA (dsRNA) region fragments based on the cloned gene sequences of TRE2-like and TRE2, and injected into the 2-day-old pupae of H. axyridis by RNAi technology. The expression levels of carbohydrate metabolism-related genes in the 1-day-old adults of H. axyridis after RNAi were detected by real-time quantitative PCR (RT-qPCR). At the same time, the changes in the contents of main saccharides and the trehalase activity after RNAi in its 1-day-old adults were determined by fluorenone colorimetric method and enzyme labeling method, respectively, and the phenotypic changes in eclosed adults after RNAi were observed. 【Results】 The results showed that after the dsRNA of TRE2-like or TRE2 was injected into the 2-day-old pupae of H. axyridis, the expression levels of TRE2-like and TRE2 in its newly emerged adults were significantly decreased as compared with the control group (dsGFP injection group), and a few adults showed abnormal phenotypes such as difficulty in molting and wing formation. The soluble trehalase activity decreased significantly after injection of dsTRE2-like, while the membrane-bound trehalase activity decreased significantly after injection of dsTRE2. As compared with the control group, the dsTRE2 injection group had significantly decreased glycogen content, the dsTRE2-like injection group had significantly decreased glycogen and trehalose contents, and the dsTRE2-like plus dsTRE2 injection group had significantly decreased glycogen and glucose contents and extremely significantly decreased trehalose content. Meanwhile, the expression levels of soluble trehalase genes TRE1-1 and TRE1-2 in the dsTRE2-like, dsTRE2 and dsTRE2-like plus dsTRE2 injection groups decreased or significantly decreased, that of TRE1-5 increased or significantly increased, and those of trealose-6-phosphate synthase (TPS), glycogen phosphorylase (GP) and glycogen synthase (GS) genes were significantly decreased as compared to the control group. 【Conclusion】 The metabolism of trehalose and the like in H. axyridis is affected after the expression of TRE2-like and TRE2 is inhibited. The results of this study lay a foundation for exploring the potential function and regulation mechanism of membrane-bound trehalases in H. axyridis.

【Aim】 This study aims to clone and analyze the forkhead box protein A (FoxA)-like protein gene HarmFoxAl from Helicoverpa armigera, and to investigate its expression profiles after the larval midgut was subjected to 2-tridecanone treatment, so as to provide a theoretical basis for further studying the function and regulatory pathway of FoxA involved in the growth and developmental process of H. armigera. 【Methods】 The cDNA sequence of HarmFoxAl was cloned from the larval midgut of H. armigera, and its amino acid sequence and protein structure were analyzed. The recombinant vector pET32a-HarmFoxAl was constructed and transformed into Escherichia coli Transetta strain. The fusion protein His-HarmFoxAl was induced by IPTG in Transetta (pET32a-HarmFoxAl) strain, and was purified using the Ni-NTA affinity chromatography. The expression profiles of HarmFoxAl in different developmental stages (1st-6th instar larva and prepupa), tissues (fat body, midgut, integument and head) of the 6th instar larva, and midguts of the 10 mg/g 2-tridecanone treated 6th instar larvae of H. armigera at different time post treatment were detected by qPCR. 【Results】 The open reading frame of HarmFoxAl (GenBank accession no.: XM_021331806) is 669 bp, encoding 222 amino acids. The predicted molecular weight and isoelectric point of HarmFoxAl protein are 25.03 kD and 6.34, respectively. HarmFoxAl is a monomer protein without signal peptide, transmembrane region and disulfide bond, and its core sequence consists of four α-helixes and three β-sheets. After the Transetta (pET32a-HarmFoxAl) strain was induced by 0.5 mmol/L IPTG for 5 h at 25℃, a ~45 kD soluble fusion protein His-HarmFoxAl was expressed in the strain, which is consistent with the predicted molecular weight (42.8 kD). Developmental stage-specific expression profiles revealed that HarmFoxAl was expressed in the 1st, 2nd, 3rd and 6th instar larva and prepupa, with the highest expression level in the prepupa. Tissue expression profiles revealed that HarmFoxAl was expressed in the fat body, midgut and integument of the 6th instar larva, with the highest expression level in fat body, while not expressed in the head. When the 6th instar larvae were exposed to 10 mg/g 2-tridecanone, the expression level of HarmFoxAl in the midgut was significantly decreased first, and then gradually increased with the exposure time, being significantly higher than that of the control group at 48 h after treatment. 【Conclusion】 HarmFoxAl is highly expressed in the prepupal stage and larval fat body of H. armigera. After the H. armigera larvae are exposed to 2-tridecanone, the expression level of HarmFoxAl significantly decreases first and then gradually rises in larval midgut. Therefore, we presume that HarmFoxAl may play an important role in metamorphosis and metabolic detoxification of H. armigera.

【Aim】 To establish the transcriptome database of the spotted flesh fly, Wohlfahrtia magnifica, and to explore its genetic data, so as to provide a foundation for further studying the bactrian vaginal fly plague. 【Methods】 The transcriptomes of the larva, pupa and adult of W. magnifica were sequenced by a high-throughput sequencing platform (Illumina Hiseq^TM4000) and subjected to bioinformatics analysis. Olfaction-related genes were screened by differential gene expression analysis and GO function significant enrichment analysis based on the transcriptome data of the larva, pupa and adult of W. magnifica. The expression levels of three olfaction-related genes (OBP99b, OBP56a and OBP99a) in the larva, pupa and adult of W.magnifica were verified by qPCR. 【Results】 The results showed that each sample of W. magnifica generated the transcriptome data of above 4.96 Gb, in which the G+C content was above 35.35%, and the Q20 content was above 97%. A total of 73 303 unigenes could be annotated in NCBI_nr, GO, KEGG, Pfam, Swiss-Prot and eggNOG databases. Among them, the largest number of unigenes (35 066) were annotated in the eggNOG database, and involved in 23 protein functions, among which the unigenes involved in the translation modification and protein turnover accounted for a larger proportion. A total of 29 193 unigenes were annotated in the GO database, and involved in 50 branches of molecular function, cellular component and biological process, with a higher proportion of unigenes involved in biological process and oxidation-reduction process. There were 15 068 unigenes annotated in the KEGG database, with higher proportion of unigenes involved in the signal transduction process. The above data indicated that the transcriptome sequencing result was good, laying a foundation for the follow-up experiment. Based on the transcriptome data of the larva, pupa and adult of W. magnifica, 30 olfaction-related genes including 9 odor-binding protein (OBP) genes were screened. Further gene annotation revealed that OBP99b, OBP56a, and OBP99a showed differential expression (|log₂FC|>1, P<0.05) in different developmental stages of W. magnifica, which was verified by the results of fluorescence quantitative PCR. 【Conclusion】 In this study, the transcriptome data of larva, pupa and adult of W. magnifica were obtained, the olfaction-related genes at different developmental stages of W. magnifica were screened, and the differential expression of OBP99b, OBP56a and OBP99a in the three developmental stages of larva, pupa and adult of W. magnifica was verified. These results provide a new idea for preventing and treating the vaginosis of the bactrian alpaca.

【Aim】 Sclomina erinacea is a common natural enemy insect in forest ecological system of China, and there are obvious morphological differences among its different geographical populations. This research aims to screen microsatellite loci based on the transcriptome data of S. erinacea, so as to develop reliable microsatellite markers for the further study of the genetic diversity and genetic differentiation of S. erinacea populations. 【Methods】 The SSR markers were searched by MISA software among 42 215 unigenes of the transcriptome data previously obtained by high-throughput sequencing platform Illumina HiSeq^TM 2000. SSR primers were designed by using Primer Premier 3 software. Fifty-four SSR primers randomly selected were tested in nine geographical populations of S. erinacea from China by PCR. 【Results】 A total of 2 395 SSR loci were found in 2 107 unigenes by MISA software. The main repeat types were trinucleotide repeats (44.43%), followed by dinucleotide repeats (40.08%) and tetranucleotide repeats (12.94%). More than 2 000 pairs of SSR primers were successfully designed by using Primer Premier 3 software. The PCR results of SSR loci of nine different geographical populations of S. erinacea with 54 pairs of primers randomly selected showed that 16 pairs of SSR primers could successfully amplify target fragments. 【Conclusion】 The results show that it is feasible to exploit microsatellite markers from the transcriptome data of S. erinacea. This study lays a foundation for the further study of population genetics of S. erinacea.

【Aim】 This study aims to investigate whether clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridizationcompatible tissue-hYdrogel (CLARITY) technique is feasible in insect tissue treatments, using guts of Apis mellifera workers as the test samples, and to test the feasibility of CLARITY in combination with fluorescence in situ hybridization (FISH) in the clarification of insect gut tissues. 【Methods】 A. mellifera worker guts were fixed and solidified according to the regular CLARITY protocol and clarified using the passive method. The in situ hybridization of the clarified gut tissues was conducted using species-specific oligonucleotide probes including Nosema ceranae-specific 16S rRNA-targeted oligonucleotide probe labeled with fluorescein isothiocyanate (FITC) and a universal eukaryotic 18S rRNAtargeted oligonucleotide probe labeled with Texas RED. The nuclei were counter-stained with DAPI (in blue), and the stained tissues were observed under a confocal laser scanning microscope. 【Results】 For the first time, A. mellifera guts were successfully processed and clarified.  The original morphology of Malpighian tubules in the abdomen was observed, and the spatial distribution of Nosema with higher density at the distal part of the midgut was clearly labeled in the 3D scanning using confocal laser scanning microscope. 【Conclusion】 CLARITY can be used in the clarity of honey bee gut tissues, and the clarified gut tissues can be stained by FISH and observed with confocal laser scanning microscopy. CLARITY in combination with FISH provides a reliable and specific labeling method for insect physiological and pathological studies without the troubles of antibody preparation and paraffin sectioning usually required for a similar study.

【Aim】 An increasing damage of the summer fruit tortrix moth, Adoxophyes orana, has been observed on jujube (Ziziphus jujube) in jujube growing regions of Northern Shaanxi in recent years. This study aims to clarify the developmental duration, fecundity and oviposition substrate selection of this jujube pest, so as to provide a theoretical reference for its prediction and prevention. 【Methods】 The growth, development and reproduction of A. orana feeding on jujube leaves were observed under the conditions of 25±1℃, 70%±5% RH and a photoperiod of 15L∶9D. The effects of supplementary nutrition on the reproduction of A. orana were assessed by measuring the fecundity per female moth when supplemented with different nutrients. The oviposition preference of female adults of A. orana to five substrates, including parchment paper, filter paper, white paper, polyethylene (PE) preservative film and plastic binding film, was investigated by counting the number of accumulated eggs on different substrates. 【Results】 The duration of egg, larval, pupal, and adult stages of A. orana was 7.38±1.22, 16.59±2.16, 7.01±0.79, and 16.65±5.15 d, respectively. The mean generation duration was 33.87±3.64 d. The intrinsic rate of increase (r_m), finite rate of increase (λ) and population doubling time (D_t) were 0.15 d^-1, 1.16 d^-1 and 4.67 d, respectively. The average fecundity of adult was 339.52±129.93 eggs laid per female. The female adults of A. orana oviposit eggs multiple times (6.26±2.09 on average), showing an obvious circadian rhythm (most in 0:00-8:00). The peak oviposition appeared at 3-5 d after emergence. The total fecundity on PE preservative film was significantly higher than that on the other substrates. There was no significant difference in the total fecundity between the female adults fed with sugar solution at different concentrations and those fed with water. 【Conclusion】 A. orana feeding on jujube leaves can accomplish its development and reproduce fertile offspring, and PE preservative film is the suitable inorganic oviposition substrate for its female moths.

【Aim】 To investigate the genetic differentiation of different geographic populations of Loxostege sticticalis in China. 【Methods】 The mitochondrial COI, Cytb and COII sequences from 11 geographic populations of L. sticticalis in Northwest and North China were amplified by PCR, and the genetic distance, molecular phylogeny and degree of genetic differentiation were analyzed based on their sequence variation, Bayesian inference (BI) phylogenetic tree and haplotype network. 【Results】 The sequence fragments of mitochondrial COI, Cytb and COII from 11 geographic populations of L. sticticalis had 24, 12 and 69 variable sites, respectively (accounting for 3.6%, 2.7% and 8.8% of the total length, respectively), 22, 14 and 16 haplotypes with the haplotype diversity (Hd) of 0.7600, 0.5842 and 0.7341, respectively, and the average nucleotide difference (K) of 1.704, 0.752 and 3.997, respectively. The average genetic distance based on mitochondrial COI, Cytb and COII sequences between different haplotypes was 0.004, 0.005 and 0.013, respectively. Both the Tajima’s D and Fu’s Fs of the total population were not significant, suggesting that the genetic differentiation among different geographic populations of L. sticticalis is not significant and the population size remains stable. The phylogenetic tree and haplotype network based on the haplotypes showed that the haplotypes were highly mixed in different geographic populations, without clear geographical distribution pattern. 【Conclusion】 The genetic distance between geographic populations of L. sticticalis is not significantly correlated with their geographical distance, and there is no obvious genetic differentiation between these populations.

 【Aim】 Chromosomal characteristics play significant roles in phylogenetic analyses of insects. However, chromosomes in Bittacidae (Mecoptera) have rarely been studied to date. 【Methods】 Larvae, pupae and adults of the hangingfly Bittacus flavidus Huang & Hua were obtained by rearing in the laboratory. Chromosome preparations for the testes of the last (4th) instar larvae, pupae and newly-emerged adults of the hangingfly were stained with 4′,6-diamidino-2-phenylindole (DAPI) to investigate the karyotype, meiotic behavior, and sex determination. 【Results】 The results showed that the chromosome number of B. flavidus is 2n=26+X0, and all the chromosomes are metacentric, showing a symmetric karyotype. The absolute length of bivalents ranged from 3.20±0.07 to 1.53±0.19 μm, and the relative length from 5.31±0.29 to 2.73±0.24, with their size decreasing gradually from pair to pair. The meiosis of B. flavidus is chiasmate with a mean chiasma frequency of 11.5 per nucleus and a mean chiasma frequency of 0.88 per autosomal bivalent. The sex determination mechanism is of the XX/X0 type. Fluorescent bands with DAPI staining revealed a large AT-rich block covering one terminal region on all pachytene bivalents. 【Conclusion】 The chromosomes exhibit marked variations with respect to the diploid number, fundamental number and karyotype formula, suggesting that chromosome rearrangements (especially fusion, fission and inversion) play an important part in the lineage differentiation and chromosome evolution in Bittacidae.

【Aim】 This study aims to observe the types, morphology, quantity and distribution of sensilla on adult antenna and larval head of the tea geometrid Ectropis grisescens, so as to explore the behavioral mechanism of E. grisescens. 【Methods】 The ultrastructure of sensilla on adult antenna and larval head of E. grisescens was observed with scanning electron microscopy. 【Results】 The results showed that there are eight kinds of sensilla on the adult antennae, including sensilla styloconica, sensilla auricillica, sensilla trichodae (type Ⅰ-Ⅳ), Böhm bristles, sensilla coeloconica (type Ⅰ and Ⅱ), sensilla squamiformia, sensilla basiconica (type Ⅰ and Ⅱ), and sensilla chaetica. Sensilla styloconica were found only on female adults, while sensilla auricillica and sensilla trichodae (ST Ⅰ-Ⅲ) only on male adults. One sensillum styloconicum, one sensillum basiconicum and two sensilla chaetica were found on the antenna of the 5th instar larvae, six pairs of sensilla chaetica on the labrum, three pairs of sensilla chaetica and one pair of sensilla digitiformia on the epipharynx, two sensilla chaetica on the outer face of mandible, five sensilla chaetica, nine sensilla basiconica and two sensilla styloconica on the mandible and maxillary palp, one sensillum basiconicum and one sensillum chaeticum on the labial palp, and one pair of sensilla chaetica on the front of spinneret. 【Conclusion】 The antennal sensilla show sexual dimorphism in E. grisescens, with more kinds and quantity of sensilla on male antenna. It is so inferred that males have stronger ability to identify host plants and sex pheromones. The sensilla on larval head are associated with gustation and olfaction, and play a vital role in judging the varieties and suitability of foods.

 Inwardly-rectifying potassium channels (Kir) play important roles in various tissues of animals. Although the research on insect Kir channels is limited, some important findings about insect Kir had been achieved in the recent five years, which were reviewed in this article. Until now the studies on insect Kirs have been mainly focused on the insect orders Diptera and Hemiptera. The analysis on genomic data and gene cloning demonstrated that insects have much fewer Kir genes than mammals. The dipteran insects Aedes aegypti and Anopheles gambiae harbor five to six Kir genes, Drosophila melanogaster has only three Kir genes, and the hemipteran insects Nilaparvata lugens and Cimex lectularius also have only three Kir genes. The number of Kir genes in soybean aphid (Aphis glycines) reduced to only two, and the third one was lost, which conjecturally is related with the degeneration of Malpighian tubules during the evolution of aphids. Phylogenetic analysis reveals that insect Kirs belong to three subfamilies; however, they are not orthologous to the seven subfamilies of mammal Kirs. Even so, insect Kirs have similar structural characteristics with mammal Kirs: insect Kirs are tetramer channels composed by four Kir subunits, each subunit has two transmembrane domains (TM1 and TM2), and K⁺ selective filter sequence is present between TM1 and TM2. The Kir genes are highly expressed in salivary gland and Malpighian tubules of insects. Kir inhibitors, which block the inward potassium current of Kir in insects, suppress the secretion activities of salivary gland and Malpighian tubules, and therefore disrupt the processes of insect feeding and excretion, and finally lead to the death of insects. These results suggest that the secretion activities of salivary gland and Malpighian tubules are regulated by Kir, and the transmembrane transport of K⁺ mediated by Kir drives the secretion activities in these epithelial cells. The most important discovery is that flonicamid, an insecticide with long-sought-after mode of action, has high blocking activity on Kir in N. lugens, and disrupts the physiological function of salivary gland and renal tubes. These results demonstrate that Kir is the molecular target of flonicamid, suggesting that Kir is the excellent insecticidal target. Finally, the author analyzed the emerging scientific issues in insect Kir research remaining to be solved and highlighted the research prospects on the development of new insecticides targeting at Kir channels.

Non-coding RNAs (ncRNAs) are a family of important regulatory molecules in organisms, and the regulation of circadian rhythms by ncRNAs has received increasing attention from researchers. In this article, we reviewed the regulation of circadian rhythms by microRNAs (miRNAs) and long-chain non-coding RNAs (lncRNAs) in the context of the related studies of Drosophila melanogaster and mammals. miRNA-mediated regulation of circadian rhythms includes: there are miRNAs with rhythmic expression in organisms, especially in clock neurons; the interaction between input systems and miRNAs mainly via the zeitgeber of light; miRNAs can directly regulate the core oscillator or indirectly affect the core oscillator by regulating other genes; and the regulation of miRNAs on the output system mainly focuses on metabolic feeding rhythms, locomotion rhythms, and sleep rhythms. The circadian rhythm and lncRNAs can regulate each other, and lncRNAs have a wide range of effects and complex mechanisms, which has broad research prospects. This article will be useful for further studies on the regulation of circadian rhythms by non-coding RNAs.