【Aim】 To search conserved miRNA target sites on the 3′UTRs of insect testis stem cell self-renewal associated genes, and to provide references for studying the miRNA-gene relationship in the process of insect spermatogenesis. 【Methods】 Testis stem cell self-renewal associated genes were identified in 27 holometabolan insects by BLAST search, and the 3′ UTRs of these genes were predicted. The miRNA target sites on the 3′UTRs of these genes were predicted by using TargetScan. The 3′UTRs of Chinmo and Imp containing let-7-5p target site of Bombyx mori and Plutella xylostella were cloned, and the target-gene relationship was verified by dual-luciferase reporter system. 【Results】 Seventeen testis stem cell self-renewal associated genes were identified in 27 holometabolan insects. By using TargetScan, 203 miRNA target sites were identified. A high number of conserved miRNA target sites were found on the 3′UTRs of Zfh-1, Chinmo, Ken and Imp. The number of conserved miRNA target sites was different among various insects. The let-7-5p target sites on Chinmo and Imp were conserved in insects and could be confirmed by other miRNA target prediction programs. The 3′UTRs of these two genes were cloned from B. mori and P. xylostella. The dual-luciferase reporter system in 293T cells validated the regulation of let-7-5p on Chinmo and Imp from these two insect species. 【Conclusion】 In this study, a large number of miRNA target sites were found on the 3′ UTRs of Zfh-1, Chinmo, Ken and Imp. The let-7-5p target sites on 3′UTRs of Chinmo and Imp are conserved in holometabolan insects. The target relationships between let-7-5p and Chinmo or Imp genes of B. mori and P. xylostella were verified by dual luciferase reporter system. This study will be helpful for better understanding the functions of miRNA in insect spermatogenesis.

【Aim】 This study aims to clone and analyze the expression of a serine protease inhibitor gene from Anatolica polita borealis so as to verify its function. 【Methods】 A serine protease inhibitor gene from A. polita borealis was cloned using PCR and RACE methods. Bioinformatics approach was applied to analyze the basic properties of the gene and its coded protein, and to make the multiple sequence alignment with its homologous sequences. The recombinant expression vector pET-32a-ApSerpin-FA72 was constructed. The expression, purification and functional verification of the recombinant target protein were analyzed. 【Results】 A serine protease inhibitor gene was cloned from A. polita borealis and named ApSerpin-FA72 (GenBank accession no.: MF188125). Its opening reading frame (ORF) is 1 176 bp in length， encoding a 391 amino-acid residue with the predicted molecular weight of 43.7 kD and pI of 5.14. ApSerpin-FA72 is a hydrophilic protein with a signal peptide of 21 amino acids at the terminus. It might play roles in the stress response and immune response, and has the highest homology with Tribolium castaneum Serpin. The purified recombinant protein TrxA-ApSerpin-FA72 has the molecular weight of 63.7 kD. The functional verification results revealed that the recombinant protein TrxA-ApSerpin-FA72 had an inhibitory effect on the activities of trypsin and chymotrypsin. 【Conclusion】 The expression product of Anatolica polita serine protease inhibitor gene has an inhibitory effect on trypsin and chymotrypsin, indicating that it may inhibit the activity of digestive serine protease, and the verification of its functional activity can be helpful to further investigate the relationship between ApSerpin-FA72 and serine protease.

【Aim】 This study aims to clone the heat shock protein Hsp70 gene in Galeruca daurica, and to analyze the gene sequence and mRNA expression profiles so as to explore the function of Hsp70 gene in the development and its responses to thermal stress in G. daurica. 【Methods】 The full-length cDNA of a Hsp70 gene was cloned and identified from the 2nd instar larvae of G. daurica using RT-PCR and RACE technique, and the putative amino acid sequence was analyzed by bioinformatics methods. The relative expression levels of the Hsp70 gene in different adult tissues (head, thorax and abdomen), different developmental stages (egg, the 1st-3rd instar larva, pupa and female and male adult), the 2nd instar larvae exposed to different temperatures (-14, -10, -5, 0, 5, 10, 15, 20, 25 and 30℃ for 1 h), and eggs exposed to 0℃ for 0 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, and 3 h, respectively, were measured using real-time quantitative PCR method. 【Results】 A heat shock protein 70 gene was cloned from G. daurica, and named GdHsp70 (GenBank accession no. KY460462), which is 2 340 bp in length and contains an open reading frame (ORF) of 1 899 bp, encoding 632 amino acid residues. The putative protein is 70.12 kD with an isoelectric point (pI) of 4.79, and has no transmembrane region and signal peptide. Subcellular localization prediction revealed that GdHsp70 is mainly located in the cytoplasm. Protein domain analysis showed that GdHsp70 has three highly conserved domains. Homologous alignment and phylogenetic analysis demonstrated that GdHsp70 has high similarity with the Hsp70 proteins from other insects which are highly close to G. daurica based on taxonomy. Tissue-and developmental stage-specific mRNA expression profiling showed that GdHsp70 had the highest expression levels in the adult thoraxes and eggs. Both heat and cold stress could induce the mRNA expression of GdHsp70 in the 2nd instar larvae with the highest expression level at -10℃ for 1 h. GdHsp70 was up-regulated in G. daurica eggs exposed to 0℃ for 15 min to 3 h, with the highest expression level at 1 h after treatment. 【Conclusion】 GdHsp70 might be associated with the development and play an important role in response to heat and cold stress in G. daurica.

【Aim】 This study aims to clone a C-type lectin gene from Plutella xylostella, to investigate its expression patterns and to elucidate its agglutination on bacteria. 【Methods】 Based on the bioinformatical analysis of genome and transcriptome database of P. xylostella, the full-length cDNA of a C-type lectin gene was cloned from P. xylostella by RT-PCR and rapid amplification of cDNA ends (RACE) techniques. Prokaryotic expression plasmid was constructed and the fusion protein was expressed in E.coli BL21. The polyclonal antibody with high serum titer was prepared using the purified fusion protein to immunize New Zealand white rabbit. Real-time quantitative PCR (RT-qPCR) was employed to analyze the expression profiles of this gene in different tissues (hemocyte, cuticle, fat body, midgut and Malpighiam tubules) of the day-1 4th instar larvae and different developmental stages （egg, 1st-4th instar larva, prepupa, pupa, and adult） of P. xylostella. RT-qPCR and Western blot were used to detect the expression difference of this gene after challenge with E. coli and B. thuringiensis at the mRNA and protein levels, respectively. The agglutination of the recombinant protein to the two bacteria was investigated by microscopic observation. 【Results】 A C-type lectin gene was cloned from P. xylostella and named PxCTL5 (GenBank accession no.: KY807084). Its full-length cDNA is 1 243 bp, with the open reading frame (ORF) of 672 bp, encoding 223 amino acid residues. RT-qPCR analysis revealed that PxCTL5 was expressed in all developmental stages, with the highest expression level in adults of P. xylostella, and mainly in the cuticle, in which the transcript level was up-regulated upon B. thuringiensis or E. coli challenge, with the highest expression level at 18 h and 24 h post infection, respectively. Western blot results also confirmed that the expression level of PxCTL5 in the hemolymph was significantly increased after challenge with B. thuringiensis and E. coli, respectively. Agglutination assay in vitro showed that in the presence of calcium ions, PxCTL5 protein had certain agglutination activity on E. coli and B. thuringiensis with a stronger agglutination effect on B. thuringiensis. 【Conclusion】 The mRNA level of PxCTL5 is significantly induced by bacteria, and PxCTL5 protein has high agglutination activity on B. thuringiensis.

【Aim】 This study aims to reveal the tissue expression patterns of the odorant binding protein (OBP) gene HarmOBP16 in the cotton bollworm, Helicoverpa armigera, and the binding properties of its recombinant protein with plant volatiles. 【Methods】 According to the antenna transcriptome data, the OBP gene was cloned from the antennae of H. armigera adults and subjected to bioinformatical and phylogenetic analysis. The tissue expression patterns of the gene in adult head (without antennae and probosis), thorax, abdomen, legs, wings, antennae and proboscis were assayed by qPCR. The recombinant protein was expressed and purified with prokaryotic expression system. The binding characteristics of this recombinant protein to 85 candidate plant volatiles were measured by fluorescence competitive binding assay. 【Results】 An atypical OBP gene, HarmOBP16 (GenBank accession no.: JQ753074), was identified and cloned from the antennae of H. armigera adults by PCR. It contains a 441 bp open reading frame (ORF) encoding a protein of 146 amino acids with 10 conserved cysteine residues and the isoelectric point value of 6.87. The tissue expression patterns showed that HarmOBP16 was highly expressed in wings of the female adults. The competitive fluorescence binding assay of the expressed and purified recombinant HarmOBP16 showed that HarmOBP16 exhibited higher binding affinities to geranylacetone (Ki=14.2 μmol/L), β-ionone (Ki=15.2 μmol/L), octyl aldehyde (Ki=15.3 μmol/L), (+/-)-linalool (Ki=16.8 μmol/L), (R)-(+)-limonene (Ki=14.9 μmol/L), and (+)-β-pinene (Ki=17.3 μmol/L). 【Conclusion】 The results suggest that HarmOBP16 may play a role in the host seeking of H. armigera, which may provide a potential molecular target for the development of attractants or repellents in H. armigera.

【Aim】 This study aims to clone the coding sequence of ATP synthase B subunit gene, to perform bioinformatics analysis for the coding sequence and to characterize its association with deltamethrin resistance in Culex pipiens pallens. 【Methods】 The coding sequence of ATP synthase B subunit gene in C. pipiens pallens was amplified by PCR method. The physical-chemical properties and functional characteristics of the encoded protein were predicted by employing the web-based bioinformatics analysis. The expression of ATP synthase B subunit gene was compared between the deltamethrin-susceptible and-resistant strains selected in the laboratory by quantitative real-time PCR method, and between the deltamethrin-susceptible and -resistant individuals of the field populations for further verification. 【Results】 The coding sequence of ATP synthase B subunit gene (GenBank accession no.: KY783434) was cloned, which contains 717 nucleotides encoding 238 amino acids. Bioinformatics analysis showed that its encoded protein has a theoretical relative molecular weight of 26.96 kD and the isoelectric point value of 8.97 approximately. It was predicted that ATP synthase B subunit contains a structural domain (amino acids 70-231) of ATP-synthase_B, but lacks signal peptide and transmembrane domain. Quantitative real-time PCR results revealed that the expression level of ATP synthase B subunit gene was up-regulated in the deltamethrin-resistant strain as compared with that in the deltamethrin-susceptible strain selected in the laboratory, and also in the deltamethrin-resistant individuals of the field populations as compared with that in the deltamethrin-susceptible individuals of the field populations. 【Conclusion】 This study obtained the coding sequence of ATP synthase B subunit gene, performed bioinformatics analysis for the sequence, and confirmed its overexpression in the deltamethrin-resistant individuals, providing the basis for further studying its roles in insecticide resistance.

【Aim】 Using repellent is one of the most important methods for mosquito control. In our previous study, we found that there is an interaction between repellent molecules and human-secreted attractant molecules during the use of repellents. To understand the true nature of this interaction, in this study the interactions between three repellents and one attractant L-lactic acid were explored through computational quantum chemistry based on the analysis of numerical changes of geometric structure, binding energy, charge population and thermodynamic parameters. This study might reveal the form, strength and law of the interactions between the repellent molecules and the human-secreted attractant molecules in silico. 【Methods】 The structures of three representative mosquito repellents 8-hydroxylcarveol formate (R1), hydroxy citronellal-1,2-propanediol acetal (R2) and N,N-diethyl-3-methyl benzoyl amide (DEET), the attractant L-lactic acid, and their complexes were constructed by using Gaussian 09 software package. Geometry optimizations and vibration frequency calculations of the repellents, the attractant and their complexes were done at the M06-2X-D3/6-311G (d,p) level. The single point energy of the repellents, the attractant and their complexes were calculated at the M06-2X-D3/6-311+G (d,p) level. 【Results】 The association distances and angles between the repellents R1, R2 and DEET and the attractant L-lactic acid were among 2.64-2.74 Å and 170-175°, respectively, and after the association, the bond length of oxygen hydrogen bond in carboxyl of L-lactic acid were stretched to 0.019, 0.029 and 0.022 Å, respectively. The binding energies (ΔE) of the association process were -50.278, -72.385 and -66.977 kJ/mol, the enthalpy change (ΔH) -43.715, -65.559 and -60.465 kJ/mol, the entropy change (ΔS) -0.138, -0.159 and -0.157 kJ/mol·K, the free energy(ΔG) -2.652, -18.090 and -13.784 kJ/mol, and the total net charge of L-lactic acid varied from 0 e to -0.0248, -0.0250 and -0.0168e, respectively. 【Conclusion】 The repellents R1, R2 and DEET and the attractant L-lactic acid are associated by moderately strong hydrogen bond. The hydrogen bond between R2 and L-lactic acid are the strongest, and the complex formed is the most stable.

【Aim】 This study aims to clarify the levels and mechanisms of indoxacarb resistance in different populations of the cotton bollworm， Helicoverpa armigera, so as to scientifically control this insect pest and to effectively avoid the rapid development of resistance to indoxacarb. 【Methods】 The resistance levels and the synergistic effects of PBO, DEF and DEM on indoxacarb in the 3rd instar larvae of different populations of H. armigera, including the susceptible population (CP), the indoxacarb-selected population (TP), Yishui population (YP) from Yishui, Shandong and Handan population (HP) from Handan, Hebei, were determined with leaf-dipping method. The activities of three detoxification enzymes ［mixed function oxidase (MFO), carboxylesterase (CarE) and glutathione S-transferase (GST)］ and acetylcholinesterase (AchE) in these populations were assayed. 【Results】 The CP population of H. armigera was still susceptible to indoxacarb, while the TP, YP and HP populations exhibited decreased sensitivity, low-level resistance and moderate-level resistance to indoxacarb, with the resistance ratios of 4.36-, 8.06- and 15.34-fold, respectively. In the TP population, the toxicity of indoxacarb to the 3rd instar larvae of H. armigera increased when they were fed on the leaves treated by three synergists for 0, 6 and 12 h. The synergistic effects of PBO and DEF to indoxacarb in the 3rd instar larvae were greater than that of DEM. After the 3rd instar larvae of H. armigera were fed on the leaves treated by PBO, DEF and DEM for 12 h, the synergistic ratios were 3.86-, 2.52-, and 4.57-fold in the HP population, while 1.11-, 0.52- and 1.09-fold in the CP population, respectively. The activities of MFO, CarE and GST in the HP population were significantly higher than those in the CP and TP populations. The activities of MFO and CarE in the YP population were significantly higher than those in the CP population, while the CarE activity in the YP population was significantly lower than that in the HP population； however, there was no significant difference in the AchE activities among different populations. 【Conclusion】 The results suggest that when a moderate-level resistance level is reached, the enhancement of MFO, CarE and GST activities confers resistance to indoxacarb in H. armigera, and the resistance to indoxacarb is significantly synergized by the metabolic inhibitors PBO, DEF and DEM.

【Aim】 This study aims to reveal the impacts of the key ecological factors on the larvae of the large black chafer Holotrichia parallela so as to provide the theoretical basis for forecasting its occurrence. 【Methods】 By automatically controlling the temperature and manually adjusting the moisture, the effects of temperature, soil water content and food on the survival rates and developmental duration of H. parallela larvae were observed in the laboratory. 【Results】 The results showed that the optimum temperature range for the survival of H. parallela larvae was between 20℃ and 28℃, and the highest survival rate (74.9%±3.1%) was obtained at 24℃. The developmental duration was the longest at 12℃,the shortest at 32℃, and moderate at 20℃ and 24℃. Both the 1st and 2nd instar larvae could survive when the soil water content was between 7% and 19%. The highest survival rate (76.4%±2.5%) was observed when the soil water content was 16%. However, the survival rates were less than 50% when the soil water contents were 7% and 19%, but none survived when the soil water content was 25%. When H. parallela larvae were fed on the six tested foods including wheat root, maize root, cotton root, peanut root, soybean root and potato slices, respectively, the survival rate of the larvae fed on peanut roots was the highest (68.7%±3.2%). The larvae fed on different diets showed significant differences in the developmental duration, the developmental duration of the larvae fed on peanut roots was the shortest (only 259 d, including 227 d of the 3rd instar larval duration), while that of the larvae fed on cotton roots was the longest (346 d, including about 303 d of the 3rd instar larval duration). 【Conclusion】 Temperature, soil water content and food all have significant influences on the survival and development of H. parallela larvae. Controlling temperature and humidity and changing host plant structure during its larval stage can effectively control the occurrence and harmfulness of this pest.

【Aim】 The increasing popularization of the application of sex pheromone trapping in the monitoring and control of the rice leaf roller, Cnaphalocrocis medinalis, has also increased the necessity to distinguish the developmental status of the internal reproductive system of the trapped male adults, which, in turn, has been essential to obtaining the key forecasting indexes and evaluating the control effects by sex pheromone trapping. This study aims to obtain the morphological indicators of the developmental status of the male internal reproductive system of C. medinalis adults, and furthermore to distinguish the population developmental status on the basis of the age composition of the trapped males for sex pheromone forecasting. 【Methods】 At different days post eclosion from the laboratory-cultured pupae, different organs of the male internal reproductive system of C. medinalis adults were dissected and measured under the microscope. A standard based on the half length of testis was developed and used to distinguish the age of male adults daily collected in sex pheromone traps. The speculated age composition of the trapped males was then used to postulate the population dynamics of the insect. 【Results】 After comparing the morphology of testes, seminal vesicle, vas deferens, accessory gland and ejaculatory ducts, we found that the half length of testis reduced stably and was negatively correlated with the age in days of the cultured male adults. The semi-major axis length of testes of the male adults at the 8th day post eclosion was averagely reduced by 41.55% as compared with that in males at the 1st day post eclosion. The field data in 2015 and 2016 both showed two peaks, representing respectively the occurrence of the 1 and 4 day-old male adults, in the age composition curve of the males trapped by sex pheromones. According to the speculated age composition of the daily collected male adults in sex pheromone traps in Meishan in 2015 and Zigong in 2016 in Sichuan, respectively, the emergence dynamics of the 1 day-old male adults could be postulated, which were found to be in coincidence to the data of field investigation. 【Conclusion】 The changes in the testis size of C. medinalis could reflect the age of its male adults. The investigation of the testis size and the consequently obtained age composition of the males could help the forecasting of the population dynamics of this pest.

【Aim】 Analyzing the mitochondrial genomes of insects can well indicate the genetic relationship among insect species. This study aims to explore the mitochondrial genome of Antheraea assama and to understand the intergeneric and interspecific molecular and evolutionary relationships of Saturniidae based on the mitochondrial genome sequences. 【Methods】 The complete mitochondrial genome sequence of Antheraea assama, a rare silk-spinning insect, was determined by using PCR and primer walking, and its general features and base composition were analyzed. The phylogenetic tree of mitochondrial proteincoding genes of fourteen species of Saturniidae and the outgroup was constructed by using the neighbor-joining method (NJ), and the phylogenetic relationships of A. assama within Saturniidae were analyzed. 【Results】 The mitochondrial genome of A. assama (GenBank accession no.: KU301792) is 15 312 bp in length, and contains 13 proteincoding genes (PCGs), 22 tRNA genes, 2 ribosomal RNA genes and a 332 bp A+T-rich region. The sequenced genome displays the genomic components and gene order of the typical lepidopteran mitochondrial genome. The sequence analysis revealed that the mitochondrial genome of A.assama is biased toward a high A+T content (80.18%). All protein-coding genes start with a typical ATN initiation codon, except that COX1 starts with the CGA codon. Majority of the 13 PCGs have a complete termination codon (TAA or TAG), except that COX1, COX2 and ND5 genes terminates with the incomplete stop codon T. The secondary structure of 22 tRNA genes by predicting showed the classic clover-leaf structure, except that the dihydrouridine (DHU) arm of tRNA^Ser(AGN) forms a simple loop. The NJ tree based on the mtDNA protein-coding gene sequences showed that A. assama formed the clade of Antheraea (Lepidoptera: Saturniidae) with Antheraea pernyi, Antheraea yamamai and Antheraea frithi. In nine species of Saturniidae studied, A. assama has the closest relationship with A. yamamai of Antheraea, and has a farther relationship with Attacus atlas of Attacus. 【Conclusion】 The gene arrangement pattern of the mitochondrial genome of A. assama is identical to those of other lepidopteran insects that have been assayed. The phylogenetic relationships of Saturniidae constructed based on the mitochondrial genomes are consistent with those in traditional morphological taxonomy, i.e., A. assama belongs to.

【Aim】 To explore the complete mitochondrial genome structure and molecular phylogenetics of Limenitis helmanni. 【Methods】 The complete mitochondrial genome of L.  helmanni was sequenced and analyzed by using PCR and primer walking. Based on the sequences of 13 protein-coding genes and two rRNA genes of the mitochondrial genome, the phylogenetic tree of 66 lepidopteran species were constructed with Bayesian inference method. 【Results】 The complete mitochondrial genome of L. helmanni is a circular molecule of 15 178 bp （GenBank accession no.: KY290566）, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a 346 bp A+T-rich region. The mitogenomic gene arrangement is consistent with those of other closely related species. The mitochondrial genome of L. helmanni is biased toward a high A+T content (81.1％). All protein-coding genes start with a typical ATN initiation codon, except that COI starts with the CGA codon and ND5 with the GTT codon. Most of the 13 PCGs have a complete termination codon (TAA), except COII and ND4 genes which have incomplete stop codons (T). All tRNA genes show the classic clover-leaf structure, except that the dihydrouridine (DHU) arm of tRNA^Ser(AGN) forms a simple loop. The A+T-rich region of L. helmanni contains some conserved structures such as the motif ATAGA, 20 bp poly(T) stretch and some tandem repeat units, which is similar to those of other related lepidopteran species. Bayesian phylogenetic analyses supported that the relationship of Nymphalidae subfamilies is (Calinaginae＋Satyrinae)＋((Nymphalinae＋Apaturinae)＋(Heliconiinae＋Limenitidinae)). 【Conclusion】 Limenitini is closely related with the Euthaliini, and Parthenini may be the earliest diverged lineage in Limenitidinae. The phylogenetic relationship of Limenitidinae species based on the mitochondrial genome is not consistent with the results of the traditional morphology-based taxonomy

 Insect immunity has gradually attracted broad attention in recent years because of its important physiological functions in the process of insect resistance to harmful exogenous stimuli. Although both internal and external immunity are parts of the insect immune defense system, it is generally believed that the internal immunity is an immediate and primary immune response of insects while the external immune defense is only considered as a kind of secondary immune response. Nevertheless, the external immunity using defensive secretions as the core component constitutes the first barrier to foreign substances which invade insects. Accordingly, external immunity and internal immunity are two kinds of immune defense strategies for insects. When insects suffer exotic organism infection or adverse environmental factors, the external immunity is often the priority to be employed to fight against the invasion of natural enemies or adversity. However, the internal immunity and external immunity are both synchronized in the whole process of defense, which will cause a dilemma on energy allocation between the two defense strategies. Moreover, it is essential for insects to employ a series of immune reactions to prevent potential threats, which will seriously impair the effectiveness of pest biological control. This review focuses on insect external immunity and summarizes the current understandings on defensive secretions in insects, as well as the two immune defense strategies and their trade-offs, aiming to provide a synthesis of important updates, potential applications, and novel ideas and technologies for further research in this area. Also, the related research will not only contribute to further analysis about the immune interaction and diversified immunization strategies between insects and exogenous factors to improve the control effect in the field, but also pave the way for the development of novel insecticides targeting the insect immune system.