The age-stage, twosex life table,called two-sex life-table for short, is an important theory and an analytical tool that are commonly used in population ecology and pest management. The user-friendly TWOSEX-MSChart program, which had been designed based on the twosex life table theory to help researchers for data analysis in insect population studies, has been more and more widely used by increasing numbers of scientists around the world. There are many statistical techniques and computer simulations embedded in the TWOSEX program, and the bootstrap technique is one of the major procedures included in the program. In this article, we describe the principles, methods, advantages/disadvantages, and the application of the bootstrap technique in the twosex life table analysis, as well as the application of the multinomial theorem in life table research. Compared with the general statistics, the bootstrap technique can be used to estimate and infer the distribution characteristics of data without the assumption of data distribution. In the twosex life table analysis, the bootstrap technique can not only be used to estimate the population parameters or the variances and standard errors of general statistics, but also to be used to assess the differences between treatments by paired bootstrap test to accurately show the population variability. The same bootstrap samples can be used to calculate the hatching rate and the contribution of different reproductive forms to population parameters, and to link the life table and predation rate analysis of natural enemies for an accurate analysis of the reproduction and predation potential of natural enemies. In addition, we also introduce the multinomial theorem, i.e., the mathematical basis of the bootstrap technique. The application of the multinomial theorem demonstrates that stable and reliable estimates can be obtained by using the bootstrap technique. We also elaborate the necessity of considering the ineffective bootstrap samples in the life table research. In recent years, although the twosex life table and the bootstrap technique had been widely adopted in research, few reports discussed the principles and methodology involved. This article will help interested researchers in entomology and ecology understand the basic theories and principles of the bootstrap technique and the multinomial theorem, and their application in twosex life table analysis, so as to better apply them in the related scientific research projects.

 Fat body is a multifunctional organ in insects, similar to the liver of vertebrates, and is distributed in the abdomen, thorax and even the head cavity of insects, with the abdominal fat body being the most developed. The fat bodies of honeybees can be divided into two types, peripheral fat body and perivisceral fat body, and are composed of trophocytes, urocytes and oenocytes. As in other insects, the fat body plays an important role in life activities in honeybees, and its morphology and function vary with the developmental stage, season, and division of labor. The structure of fat body is relatively simple, but its physiological function is very complex. The major function of fat body is the storage and metabolism of energy substances. Fat body is not only a central storage pool of nutrients (i.e., lipids, carbohydrates and proteins) for honeybees, but also an intermediate station for nutrient metabolism, with a variety of enzymatic systems for the interconversion of energy and substances, undertaking the supply of metabolic water and synthesizing purines and pyrimidines and many important proteins. At the same time, fat body is the exchange center for various hormonal and nutritional signals during insect development and behavior regulation, and fat body hormones and nutritional signals are involved in regulating fat body development, nutrient metabolism, reproduction and labor division in honeybees. Fat body has a variety of functions including energy storage and release, biosynthesis and catabolism, regulation of nutrient perception, integration of metabolic signals, endocrine regulation, immunity and detoxification, magnetic field perception, improved cold resistance, and protection of organs in the body cavity. Given the important roles of the fat body, a review of the research progresses in the morphology and function of honeybee fat body can provide references and ideas for the analysis of insect nutritional signaling pathways, highquality bee species breeding and control of honeybee diseases.

【Aim】 This study aims to determine the morphological and biological characteristics including developmental duration and adult fecundity of Trigonotylus coelestialium, so as to provide a theoretical basis for its prediction and scientific control. 【Methods】 T. coelestialium was bred with corn grains at the filling stage as the food under the indoor natural variable temperature (22.0-28.1℃) and the constant temperature of 25℃, respectively, in Zhengzhou, Henan Province, central China during September-October, 2021, and the morphological characteristics of T. coelestialium at different developmental stages were observed and recorded. The biological indexes including the duration of different developmental stages, survival rate, adult longevity and female oviposition quantity were measured. 【Results】 The egg mass of T. coelestialium is deposited in the inner glume of corn grain. The egg is cylindrical and slightly curved to one side. The antennae of the 1st instar nymphs are reddish, and the red color becomes obvious with the increase of nymphal instar. At the 5th instar nymphal stage, three clearly visible red longitudinal lines appear on the 1st segment of antennae. The 3rd instar nymphs have evident wing buds. The ovipositor of the female adult is long valvular and lies flat in the groove of genital segments. Under the indoor natural variable temperature, the egg duration of T. coelestialium was 6.27 d with the egg hatching rate of 89.90%, the 1st-5th instar nymphal duration was 2.80, 2.33, 2.70, 2.77 and 3.90 d, respectively, and the total nymphal duration was 14.50 d with the total nymplal survival rate of 85.97%. The pre-oviposition period of female adult was 4.43 d, the oviposition duration was 13.93 d, and a total of 82.55 eggs in 19.47 egg masses were produced by a single female under the indoor natural variable temperature. Under the constant temperature of 25℃, the egg duration of T. coelestialium was 7.73 d with the egg hatching rate of 81.13%, the 1st-5th instar nymphal duration was 2.17, 1.90, 1.77, 1.90 and 2.93 d, respectively, and the total nymphal duration of T. coelestialium was 10.67 d with the total nymphal survival rate of 71.84%. The pre-oviposition period of female adult was 4.17 d, the oviposition duration was 11.27 d, and a single female laid 72.22 eggs in 21.17 egg masses under the constant temperature of 25℃. 【Conclusion】 The morphological characteristics of the 1st antennal segment of the 5th instar nymph and adult of T. coelestialium can be used to distinguish it from other species of Trigonotylus. The developmental characteristics of the wing bud of T. coelestialium can be used to distinguish its nymphal stage. Variable temperature prolongs the nymphal duration and adult longevity of T. coelestialium, and increases its egg laying capacity and egg hatching rate.

 As one of the important branches of entomology, insect development and immunity, facing the national demand and scientific frontier, has made great achievements in solving major pest disasters and human health through multidimensional research. Meanwhile, the progress of new biotechnology has greatly promoted the research of insect development and immunity by deepening and widening our understanding of insect development and immune defense. Articles in this special issue of “Insect growth and development and immunity” reflect well the current status and features of research on insect development and immune in China. The growth and development part covers all developmental stages from egg to adult, mainly focusing on the signal transduction, and the immunity part focuses on biological interactions. In the context of big data, more efforts will be made to combine traditional and modern techniques, and strengthen cooperation, thus making the research branch play a greater role in pest control, insect resource utilization, and food security.

【Aim】 This study aims to analyze the function and molecular mechanism of the  ATP synthase subunit d (ATPs-d) in trehalose metabolism regulating the development and  metamorphosis of Helicoverpa armigera larva. 【Methods】 The open reading frame (ORF) of  HaATPs-d of H. armigera was cloned by PCR, and its sequence and phylogeny were analyzed by  bioinformatics methods. The qRT-PCR was employed to analyze the expression levels of  HaATPs-d in the cuticle, midgut and fat body of the 5th instar larva at the molting stage  and 6th instar larva, and in the fat body and cuticle of the 6th instar larva in response  to 20-hydroxyecdysone (20E)(0.1 mg/mL). The subcellular localization of HaATPs-d in the  Spodoptera frugiperda oocyte line Sf9 cells was studied by fluorescence photography. Yeast  two-hybrid (Y2H) was emplyed to identify the protein interacting with HaATPs-d. The  effects of the HaATPsd knockdown by RNAi on the larval development and metamorphosis, and  soluble trehalase activity and trehalose content in the midgut after injecting dsHaATPs-d  into the 6th instar larva of H. armigera were measured. 【Results】 The H. armigera HaATPs- d (GenBank accession number: LOC110375576) ORF is 525 bp in length and encodes 174 amino  acids. HaATPs-d was highly conservative, and had close relationship to ATPs-d of S.  frugiperda and S. litura. The HaATPs-d expression level peaked in the cuticle of the day- 3 6th instar larva and in the midgut and fat body of the 5th instar larva at the molting  stage. Compared with the control, 20E (0.1 mg/mL) significantly upregulated the expression  levels of HaATPs-d in the fat body and cuticle of the 6th instar larva. HaATPs-d is a  cytoplasmic protein. HaATPs-d directly bound with soluble trehalase of H. armigera.  Knockdown of HaATPs-d in the 6th instar larva of H. armigera resulted in slower larval  development, larval weight loss, increased larval mortality, decreased pupation rate and  adult emergence rate, significantly decreased soluble trehalase activity and significantly  increased trehalose content in the midgut compared with the control group (injected with  dsGFP). 【Conclusion】 HaATPs-d controls the activity of soluble trehalase and trehalose  content in larvae by binding with the soluble trehalase of H. armigera, thus affecting the  sugar source and finally the larval metamorphosis.

【Aim】 This study aims to assess the selective toxicity of six neonicotinoid insecticides and one novel insecticide triflumezopyrim to Thrips hawaiiensis and its natural enemy Orius strigicollis so as to provide a basis for the combined control of T. hawaiiensis using O. strigicollis and insecticides. 【Methods】 The acute toxicity of six neonicotinoid insecticides including imidacloprid, dinotefuran, flupyradifurone, imidaclothiz, nitenpyram and thiamethoxam, and triflumezopyrim to T. hawaiiensis adults and the 5th instar nymphs of O. strigicollis was determined using the residual film method, and their exposure risks to the 5th instar nymphs of O. strigicollis were assessed. 【Results】 The median lethal rates (LR₅₀ values) of these seven insecticides to T. hawaiiensis adults were lower than their maximum recommended field application rates. The LR₅₀ value of imidaclothiz to T. hawaiiensis adults was the lowest (0.183 g a.i/hm²), significantly lower than those of the other insecticides, whereas those of flupyradifurone and triflumezopyrim were 3.066 and 3.949 g a.i/hm², significantly higher than those of other insecticides. The LR₅₀ values of the two nitenpyram formulations 20% nitenpyram SL and 10% nitenpyram AS to T. hawaiiensis adults were 0.327 and 0.201 g a.i/hm², and those of the two thiamethoxam formulations 70% thiamethoxam WG and 25% thiamethoxam WG were 0.970 and 0.685 g a.i/hm², respectively. The toxicity of nitenpyram and thiamethoxam in different formulations and with different contents to T. hawaiiensis adults was significantly different. The LR₅₀ values of the tested six neonicotinoid insecticides to the 5th instar nymphs of O. strigicollis were lower than their maximum recommended field application rates, while that of triflumezopyrim to the 5th instar nymphs of O. strigicollis was higher than its maximum recommended field application rate. The toxicity of triflumezopyrim to the 5th instar nymphs of O. strigicollis was the lowest, with the LR₅₀ value of over 65.736 g a.i/hm², and that of imidacloprid and dinotefuran followed, with the LR₅₀ values of 21.317 and 24.486 g a.i/hm², respectively. Imidacloprid, dinotefuran, and triflumezopyrim showed high selective toxicity to T. hawaiiensis adults and the 5th instar nymphs of O. strigicollis. The risks of imidacloprid and triflumezopyrim to O. strigicollis adults were acceptable in two exposure scenarios in- and off-field. However, the risks associated with imidaclothiz and thiamethoxam to the 5th instar nymphs of O. strigicollis were unacceptable. 【Conclusion】 T. hawaiiensis adults have extremely high sensitivity to six neonicotinoid insecticides and triflumezopyrim. Imidacloprid and triflumezopyrim exhibit low risks to the 5th instar nymphs of O. strigicollis, and triflumezopyrim has high compatibility with O. strigicollis. The combination of triflumezopyrim with O. strigicollis shows a promising potential for the management of T. hawaiiensis.

【Aim】 This study aims to explore the digestive and detoxification enzyme genes highly expressed in the midgut of Grapholita molesta larvae so as to provide the theoretical basis for the future research of novel pesticides and transgenic crops targeting intestine. 【Methods】 Based on the FPKM values of high-throughput transcriptome sequencing data of the 4th instar larval midgut of G. molesta, the highly expressed genes were screened and subjected to GO function annotation and KEGG pathway enrichment analysis, and the highly expressed genes of the digestive and detoxification enzymes were screened by alignment using BLAST software. Phylogenetic analysis of these highly expressed digestive and detoxification enzymes and the homologous proteins from other lepidopteran insects was performed using MEGA software. Quantitative analysis and verification of the expression levels of the representative digestive and detoxification enzyme genes highly expressed in the midgut of G. molesta larvae at different instars were carried out by qRT-PCR. 【Results】 A total of 103 677 genes highly expressed in the migdut of the 4th instar larvae of G. molesta were annotated in GO database, including 41 branches of three function categories cellular component, molecular function and biological process. KEGG pathway analysis showed that 10 846 highly expressed genes are involved in five types of biochemical metabolism pathways. Seventeen digestive enzyme genes ［five trypsin (TRY) genes, three aminopeptidase (APN) genes and nine carboxypeptidase (CP) genes］ and 32 detoxification enzyme genes ［11 glutathione Stransferase (GST) genes, 13 cytochrome P450 (CYP450) genes and eight carboxylesterase (CarE) genes］ with complete open reading frames were obtained. The results of phylogenetic analysis indicated that the homologous clustering branches of the digestive enzymes of G. molesta were scattered, and the homologous clustering branches of the GST and CYP450 proteins of G. molesta were concentrated, but they were all clustered with the homologous proteins of at least one lepidopteran insect. qRT-PCR verification result showed that the expression levels of digestive enzyme and detoxification enzyme genes in the midgut of G. molesta larvae at different instars were significantly different, with the highest expression levels in the 4th instar larval stage. 【Conclusion】 In this study, we successfully screened and verified some digestive enzyme and detoxification enzyme genes highly expressed in the larval midgut of G. molesta, and clarified their evolutionary relationship with homologous proteins of other lepidopteran insects. These results provide references for the transcriptome analysis of other related species of Lepidoptera and the study of insect pest control targeting intestine.

【Aim】 This study aims to determine the toxicity level and field control efficacy of metaflumizone against the larval fall armyworm, Spodoptera frugiperda, and to provide a reference for scientific use of metaflumizone in the control of S. frugiperda. 【Methods】 The median lethal concentration (LC₅₀) and LC₉₀ values of metaflumizone and four commonly used insecticides including emamectin benzoate, chlorantraniliprole, lufenuron and indoxacarb against the 3rd and 6th instar larvae of S. frugiperda and the medium lethal time (LT₅₀) values of these insecticides at the concentration of LC₉₀ against the 3rd instar larvae were determined in the laboratory with diet-incorporated method. Simultaneously, the control efficacies of 22% metaflumizone suspension concentrate(6.6 g/667 m²), 22% metaflumizone suspension concentrate (17.6 g/667 m²), 5.7% emamectin benzoate water dispersant (1 g/667 m²), and 150 g/L indoxacarb suspension concentrate (2 g/667 m²) against S. frugiperda larvae were determined in corn field by artificial spray approach. 【Results】 Laboratory bioassay results revealed that among the tested five insecticides metaflumizone showed higher toxicity to the 3rd and 6th instar larvae of S. frugiperda, with the LC₅₀ values of 2.64 and 4.36 mg/kg, respectively, and its toxicity to the 6th instar larvae of S. frugiperda was only lower than that of emamectin benzoate. The LT₅₀ value of metaflumizone at the concentration of LC₉₀ against the 3rd instar larvae of S. frugiperda was 11.98 h, comparable to that of indoxacarb (11.50 h) and lower than those of chlorantraniliprole, emamectin benzoate and lufenuron (17.20, 23.40, and 39.24 h, respectively). Field efficacy test showed that 22% metaflumizone suspension concentrate at the application rate of 17.6 g/667 m² had an excellent control efficacy against S. frugiperda larvae, with the corrected control efficacies of 69.97%, 78.98% and 82.86% at 1, 3 and 7 d after application, respectively, showing no significant difference from that of the control agent 5.7% emamectin benzoate water dispersant at the application rate of 1 g/667 m². 【Conclusion】 The results of this study suggest that metaflumizone has excellent insecticidal activity and rapid action against S. frugiperda larvae, especially against the 6th instar larvae in the laboratory, and exhibits good control efficacy against S. frugiperda in the field, suggesting that it can be utilized to control S. frugiperda in China.

【Aim】 This study aims to identify the G protein-coupled receptor (GPCR) genes expressed in the fat body of the female adults of Locusta migratoria, and uncover their functions in egg development and ovarian growth of L. migratoria. 【Methods】 Based on the previously obtained transcriptome data of the fat body of the female adults of L. migratoria during the first gonadotropic cycle, identification and cluster analysis of GPCR genes were performed. qRT-PCR was employed to measure the tissue-specific expression patterns of GPCR genes in the brain, thoracic and abdominal ganglia, fat body, ovary and midgut of vitellogenic female adults of L. migratoria. RNAi experiments were carried out to analyze the functions of GPCR genes in egg development and ovarian growth. 【Results】 A total of 29 GPCR genes were newly identified from the fat body transcriptome of female adults of L. migratoria. qRT-PCR results demonstrated that PTH/PTHrPR1 and MSR were significantly highly expressed in the fat body and thoracic and abdominal ganglia of female adults of L. migratoria, respectively, while Mthl4, Mthl6 and GPR119Lwere significantly highly expressed in the midgut, and the expression levels of Octβ1R, CrzR, CCAPR, LGR2, mGluR and GABABR in the brain were significantly higher than those in the other tissues. RNAi screening revealed that knockdown of five GPCR genes, CCAPR, PTH/PTHrPR1, ADGRL3, Mthl15 and DHR, significantly inhibited the primary oocyte development and ovarian growth of L. migratoria. 【Conclusion】 Findings in the present study are helpful for unveiling the regulatory networks of powerful fecundity of insects and exploring novel targets for insect pest control.

【Aim】 The objective of this study is to unravel the regulatory role of ame-miR-14 in the developmental process of the larval guts of Apis mellifera ligustica workers.【Methods】 The expression and sequence authenticity of ame-miR-14 in the 6-day-old larval guts of A. m. ligustica workers were proved by Stem-loop RT-PCR and Sanger sequencing, respectively. Overexpression and knockdown of ame-miR-14 were conducted by feeding its corresponding mimic (mimic-ame-miR-14) and inhibitor (inhibitor-ame-miR-14), and their corresponding negative controls mimic-NC and inhibitor-NC, respectively, and then RT-qPCR was used to detect the expression levels of ame-miR-14 in the 4-6-day-old larval guts of A. m. ligustica workers. Bioinformatic software was used to predict and analyze the target genes of ame-miR-14. RT-qPCR was employed to detect the relative expression levels of the target genes FoxO and Hedgehog in the 4-6-day-old larval guts after overexpression and knockdown of ame-miR-14.【Results】 ame-miR-14 truly exists and is expressed in the 6-day-old larval guts of A. m. ligustica workers. The expression levels of ame-miR-14 in the 4-6-day-old larval guts of A. m. ligustica workers fed with mimic-ame-miR-14 were significantly up-regulated as compared to those fed with mimic-NC. The expression levels of ame-miR-14 in the 4-6-day-old larval guts of A. m. ligustica workers fed with inhibitor-ame-miR-14 were significantly down-regulated as compared to those fed with inhibitor-NC. In total, ame-miR-14 can target 309 genes, which could be annotated to 45 KEGG pathways and 36 GO terms. Further analysis showed that ame-miR-14 can target 14 genes associated with growth and development and have potential targeting relationship with target genes FoxO and Hedgehog. After overexpression of ame-miR-14, the expression level of FoxO in the 4dayold larval gut of the mimic-ame-miR-14 group was down-regulated and those in the 5- and 6-day-old larval guts significantly down-regulated as compared to those in the mimic-NC group. After knockdown of ame-miR-14, the expression level of FoxO in the 4-day-old larval gut of the inhibitor-ame-miR-14 group was down-regulated, while that in the 5-day-old larval gut was significantly up-regulated and that in the 6-day-old larval gut was up-regulated as compared to those in the inhibitor-NC group. After overexpression of ame-miR-14, the expression levels of Hedgehog in the 4-6-day-old larval guts of the mimic-ame-miR-14 group were significantly down-regulated in comparison with those in the mimic-NC group, whereas those in the 4-6-day-old larval guts of the inhibitor-ame-miR-14 group were up-regulated in comparison with those in the inhibitor-NC group. 【Conclusion】ame-miR-14 truly exists and is expressed in the larval guts of A. m. ligustica workers. Effective overexpression and knockdown of ame-miR-14 in the larval guts of A. m. ligustica workers can be achieved by feeding mimic and inhibitor, respectively. ame-miR-14 potentially participates in regulation of the larval gut development by negatively regulating the expression of FoxO and Hedgehog.

【Aim】Juvenile hormone (JH) and ecdysone (Ecd) play important roles in the growth and development of insects. The aim of this study is to clarify the regulatory mechanism of these two hormones and their metabolism-related genes on the development of Calliptamus italicus eggs.【Methods】The changes in contents of JH and Ecd during the development of C. italicus eggs were detected by ELISA assay, and the expression patterns of important genes (JHE, JHEH, DIB and EcR) in the metabolic pathways of JH and Ecd during the development of C. italicus eggs were detected by qRT-PCR.【Results】The JH content in C. italicus eggs was significantly higher at the diapause stage (stages Ⅳ-Ⅵ) than at the early developmental stage (stages Ⅰ-Ⅲ), and decreased significantly at the post-diapause developmental stage (stages Ⅶ-Ⅸ). The Ecd content in C. italicus eggs increased significantly at the early diapause stage (stage Ⅳ), then decreased significantly, and increased again at the post-diapause developmental stage. The expression level of JHE in C. italicus eggs increased firstly and then decreased at the early developmental stage and the post-diapause developmental stage, and was low at the diapause stage. The expression level of JHEH in C. italicus eggs also increased first and then decreased at the early developmental stage, and didn’t change significantly at the diapause stage, but increased significantly at the post-diapause developmental stage. The expression level of DIB in C. italicus eggs was higher at the diapause stage than at the early developmental stage and decreased at the post-diapause developmental stage. The expression level of EcR in C. italicus eggs did not change significantly at the early developmental stage and diapause stage, but increased gradually at the post-diapause developmental stage. 【Conclusion】The development of C. italicus eggs is co-regulated by JH and Ecd. JH is the important hormone to regulate diapause entry, while Ecd is the important hormone for diapause termination of C. italicus eggs. JH catabolic pathway of C. italicus eggs is mainly regulated by JHE before diapause termination and by JHEH after diapause termination. DIB and EcR can affect the development of C. italicus eggs by regulating Ecd content. These results lay a foundation for further revealing the diapause mechanism of C. italicus eggs.  

【Aim】 The purpose of this study is to identify the autotomy sites in the German  cockroach (Blattella germanica), and to explore the relationship between autotomy and leg  regeneration of B. germanica, so as to provide a theoretical basis for the study of insect  regeneration. 【Methods】 The healthy nymphs of B. germanica at the 3rd to 6th instars were  selected and amputated separately at 11 sites of the right hindleg, including the proximal  1st segment of tarsus, proximal 2nd segment of tarsus, joint site of tarsus and tibia, one -third, one half and two-thirds from distal tibia, joint site of tibia and femur, one  half of femur, joint site of femur and trochanter, joint site of trochanter and coxa, and  base of coxa. The treated nymphs of B. germanica were observed daily to record whether the  phenomenon of autotomy occurred. And the time and sites of autotomy, and regeneration or  not after molting were also recorded. By using the length of the unamputated left hindleg  as the control, the differences in the regenerated legs between autotomy and without  autotomy in B. germanica were analyzed and compared, and the relationship between autotomy  and leg regeneration was analyzed.【Results】Two autotomy sites were recorded in all the 11  amputation sites of B. germanica nymphs. One autotomy site was at the end of trochanter  when amputation was performed at different parts of tibia, the joint site of tibia and  femur, and one half of femur, the other was at the end of tibia when amputation was  performed at the 1st and 2nd segments of proximal tarsus. There was no autotomy detected in  leg amputation treatments at the other sites. The autotomy sites were decided by amputation  sites but not affected by nymphal instars. At different amputation sites with the same site  of autotomy, there was a positive correlation between the degree of amputation and the  probability of autotomy in the same nymphal instar. While there was a negative correlation  between the nymphal instars and the probability of autotomy when leg amputation was  performed at the same site. Autotomy did not affect whether regeneration happened or not  but influenced the sites of regeneration. When autotomy occurred at the end of trochanter  or at the end of tibia, a completly new leg was regenerated or tarsus was regenerated at  the end of tibia. When autotomy did not happen, regeneration occurred at the amputation  sites. At the same time, the length of the regenerated leg of the individuals under  autotomy was significantly longer than that of the individuals without autotomy, this  phenomenon was more obvious when the leg was amputated at the joint site of tibia and femur  and at two-thirds from distal tibia. It was more coordinated for the proportion of  regenerated legs under autotomy than that without autotomy, and the length of sensilla on  regenerated legs under autotomy was much longer than that without autotomy. 【Conclusion】 B. germanica can optimize the regeneration by autotomy. There are two autotomy sites at the  end of trochanter and the end of tibia, respectively, and the regenerative ability is  strong at these two autotomy sites. B. germanica faces a choice between autotomy and limb  salvage when its leg is amputated: when limb autotomy can optimize the length and sensilla  integrity of the regenerated legs, B. germanica prefers to choose autotomy, while when the  regeneration can not be optimized by autotomy, no autotomy will happen.

 Sex determination is an outstanding question in developmental and evolutionary biology. The sex determination cascade in most of known insects is: primary signaling element→key gene of sex determination→double switch gene→sex differentiation gene. In spite of this pattern, different insects have different sex determination genes and regulatory mechanisms, especially the primary signaling element of sex determination. Since the primary signal of Drosophila melanogaster was discovered, the primary signal of mosquito, bee, wasp (Nasonia vitripennis), silkworm (Bombyx mori) and other model insects has been determined successively. There are many kinds of primary signals, including the dose of sex chromosomes, male-determining factors (M factors), heterozygosity of alleles and maternal imprinting, which make it more difficult to study non-model insects to some extent. Even so, the downstream regulatory mechanism is relatively conserved. In particular, the transformer (tra)+transformer2 (tra2)→doublesex (dsx)/fruitless (fru) pathway is fairly common in most insects. Tra produces alternative splicing by sensing primary signals, and with the help of tra2, tra regulates the splicing of itself and the downstream dsx and fru, and maintains gender development. Acting as the terminal ‘double-switch’, dsx is the most conserved gene in the insect sex determination cascade. dsx is highly conserved in regulating bisexual development, courtship behavior, genitalia and formation of sexual dimorphism. As a switch gene of Drosophila sexual behavior, fru is involved in regulating almost all male sexual behaviors of Drosophila. Its function has been verified in a variety of insects such as Bactrocera dorsalis, Aedes aegypti and B. mori, and it has become a typical gene for understanding the complex sexual behavior of insects. Understanding the sex determination cascade in insects, and clarifying the function and interaction of each sex determination gene are essential to elucidate the mechanism of sex determination. It provides a theoretical basis for revealing the general law of insect sex determination, and promoting the basic research on the upstream regulatory mechanism of insect sex determination, and realizing the genetic manipulation of insect sex determination.

Our research group previously found that destruxin A interacts with the hemocytin of the silkworm, Bombyx mori, and strongly suppresses hemolymph immunity, suggesting that hemocytin may become a novel target for pesticide. Therefore, it is necessary to further understand hemocytin. Hemocytin, as a lectin, is an important factor in insect hemolymph immunity to mediate the process of coagulation, nodulation and encapsulation and prevent hemolymph spillage and microbial invasion caused by epidermal breakage, as well as be involved in the fixation and removal of invaded pathogens. Insect hemocytin generally consists of 3 000-4 000 amino acids with multiple and repeatedly arranged domains including FA58C (coagulation factor 5 or 8 C-terminal), VWD (von Willebrand factor type D), TIL (trypsin inhibitor like cysteine rich), VWC (von Willebrand factor type C), CT (C-terminal cystine knotlike), C8 (8 conserved cysteine residues), ChtBD2 (chitin-binding domain type 2), and MUC (mucin-2 protein WxxW repeating region). The amino acid sequence similarity of hemocytin between different insects is quite low, but the domain sequence is highly conserved. Hemocytin is biosynthesized by hemocytes and secreted into the hemolymph in mature form. Hemocytin is the main component of blood clot, forming soft clot by coagulating hemocytes and clotting factors through its fibrous structure to seal the wound, and then forming hard clot and scab through crosslinking. Hemocytin plays an important role in the process of nodulation and encapsulation, agglutinating hemocytes, immune factors and pathogens, and finally combining with melanization to isolate and kill pathogens. Overall, insect hemocytin has not been studied in depth. Analysis of the molecular mechanism of hemocytin regulation of insect immunity is important to enrich the basic research of insect immunology and promote the development of new insecticides based on hemocytin as the target.

【Aim】 This study aims to uncover the function of ace-miR-3720 in the larval gut development of Apis cerana cerana workers and provide theoretical and experimental bases for further investigation of the regulation mechanism of ace-miR-3720 underlying the development of larval gut. 【Methods】 According to the nucleotide sequence of ace-miR-3720, the corresponding mimic (mimic-miR-3720) and inhibitor (inhibitor-miR-3720) were designed and synthesized, and used to feed larvae of A. cerana cerana workers to perform overexpression and knockdown of ace-miR-3720, respectively. The relative expression levels of ace-miR-3720 and its target genes CKⅠ, MAPK1 and βGBP1 in the 4-6-day-old larval guts after overexpression and knockdown of ace-miR-3720 were detected using RT-qPCR. The larval guts after overexpression and knockdown of ace-miR-3720 were weighed using analytical balance. 【Results】The expression levels of ace-miR-3720 in the 4- and 5-day-old larval guts in the mimic-miR-3720 group were extremely significantly up-regulated, while that in the 6-day-old larval gut was significantly up-regulated, as compared to those in the nonsense mimic (mimic-NC) group. The expression level of ace-miR-3720 in the 4-day-old larval gut in the inhibitor-miR-3720 group was extremely significantly down-regulated, whereas those in the 5- and 6-day-old larval guts were significantly down-regulated, as compared to those in the nonsense inhibitor (inhibitor-NC) group. There is a potential targeting relationship between ace-miR-3720 and genes CKⅠ, MAPK1 and βGBP1. In the mimic-miR-3720 group, the expression levels of CKⅠ in the 4- and 5-day-old larval guts were extremely significantly down-regulated, while that in the 6-day-old larval gut was significantly down-regulated; the expression level of MAPK1 in the 4-day-old larval gut was significantly down-regulated, whereas those in the 5- and 6-day-old larval guts were extremely significantly down-regulated; the expression level of βGBP1 in the 4-day-old larval gut was significantly down-regulated, while those in the 5- and 6-dayold larval guts were extremely significantly down-regulated; additionally, the weight of the 4- and 5-day-old larval guts in the mimic-miR-3720 group was extremely significantly decreased, whereas that of the 6-day-old larval gut was significantly decreased, as compared with those in the mimic-NC group. In the inhibitor-miR-3720 group, the expression levels of CKⅠ in the 4-6-day-old larval guts were extremely significantly up-regulated; the expression level of MAPK1 in the 4-day-old larval gut was significantly up-regulated, while those in the 5 and 6-day-old larval guts were extremely significantly up-regulated; the expression levels of βGBP1 in the 4- and 6-day-old larval guts were significantly up-regulated, whereas that in the 5-day-old larval gut was extremely significantly up-regulated; in addition, the weight of the 4- and 6-day-old larval guts was significantly increased, while that of the 5-day-old larval gut was extremely significantly increased, as compared with those in the inhibitor-NC group. 【Conclusion】 ace-miR-3720 truly exists in the larval gut of A. cerana cerana workers. Effective overexpression and knockdown of miRNAs in the larval gut of A. cerana cerana could be achieved by feeding with mimic and inhibitor, respectively. ace-miR-3720 may affect the weight of larval guts of A. cerana cerana workers and participate in larval gut development through regulation of the expression of CKⅠ, MAPK1 and βGBP1.

【Aim】 The aim of this study is to clarify the regulatory effects of ame-miR-bantam on the expression of target genes USP and P300 by performing overexpression and knockdown of ame-miR-bantam in the larval gut of Apis mellifera ligustica workers through feeding method. 【Methods】 Overexpression and knockdown of ame-miR-bantam in tissues of the larval gut of the 4-6-day-old workers of A. m. ligustica were conducted by feeding mimic-ame-miR-bantam and inhibitor-ame-miR-bantam, and their corresponding negative controls mimic-NC-ame-miR-bantam and inhibitor-NC-ame-miR-bantam of ame-miR-bantam, respectively. Target genes of ame-miR-bantam were predicted and analyzed using bioinformatic software. RT-qPCR was used to detect the overexpression and knockdown effects of ame-miR-bantam and the relative expression levels of target genes USP and P300 in the larval gut of the 4-6-day-old workers of A. m. ligustica. 【Results】 Compared with feeding with mimic-NC-ame-miR-bantam, feeding with mimic-ame-miR-bantam resulted in significant up-regulation of ame-miR-bantam in the larval gut of the 4-6-day-old workers of A. m. ligustica. Compared with feeding with inhibitor-NC-ame-miR-bantam, feeding with inhibitor-ame-miR-bantam resulted in significant down-regulation of ame-miR-bantam in the larval gut of the 4-day-old workers, and down-regulation of ame-miR-bantam in the larval gut of the 5- and 6-day-old workers. ame-miR-bantam can target a total of 222 genes, which can be annotated to 35 GO terms such as cellular process and 160 KEGG pathways such as Wnt signaling pathway. After overexpression of ame-miR-bantam, the expression levels of USP and P300 in the larval gut of the 4-day-old workers were up-regulated, that of USP in the larval gut of the 5-day-old workers was down-regulated, that of P300 in the larval gut of the 5-day-old workers was significantly down-regulated, those of USP and P300 in the 6-day-old larval gut were significantly down-regulated as compared to those in the negative control group fed with mimic-NC-ame-miR-bantam. After knockdown of ame-miR-bantam, the expression levels of USP and P300 in the larval gut of the 4- and 5-day-old workers were up-regulated, that of USP in the larval gut of the 6-day-old workers was significantly up-regulated whereas that of P300 in the larval gut of the 6-day-old workers was up-regulated as compared with those in the negative control group fed with inhibitor-NC-ame-miR-bantam. 【Conclusion】 Effective overexpression and knockdown of ame-miR-bantam in the larval gut of A. m. ligustica workers can be achieved by feeding with mimic and inhibitor, respectively, and ame-miR-bantam in the larval gut may negatively regulates the expression of USP and P300.

True fruit flies are important insect pests attacking fruits and vegetables. The total damage caused by them worldwide is estimated to amount to be more than 2 billion US dollars annually. The oriental fruit fly, Bactrocera dorsalis, is one of the representatives of this kind of pests, causing serious losses to China’s citrus industry every year. The techniques based on male attractant and protein bait have been used in environment friendly strategies for pest monitoring and control. However, the performances of those baits in the field are unsatisfied and need to be further improved. With the reduction of the cost of high-throughput sequencing technology and the development of modern molecular biology technology, scientists proposed to discover the key molecular targets for olfaction by resolving the molecular mechanism of pest chemosensory first and develop more stable and efficient attractants with the identified new targets. In order to promote the development of behavioral regulation technology targeting key chemosensory molecules in B. dorsalis, we reviewed the research status of important chemicals regulating the behavior of B. dorsalis and the mechanism of chemosensory perception in this article. The important volatiles regulating the behavior of B. dorsalis include sex pheromones, plant volatiles and food-derived protein odors. Among them, the specific compounds identified by the first two have a clear relationship with the behavior of B. dorsalis adults. For example, pyrazine substances obtained from sex pheromones can attract females, methyl eugenol in plant volatiles can attract males, γ-octalactone can induce females to lay eggs; while the composition of food-derived protein odor is complex, although it has a certain efficacy in the field, there is a lack of function validation of specific compounds in female and male insect behavior. In the olfactory sensory mechanism, there is only a morphological description of the peripheral nerve sensilla and the central antennal lobe, and the function of different types of olfactory neurons is not clear. A large number of chemical sensory proteins have been identified in B. dorsalis, including 49 odorant binding proteins (OBPs), 60 odorant receptors (ORs), 23 ionotropic receptors (IRs) and 17 gustatory receptors (GRs), through bioinformatics analysis at present. However, the number of olfactory genes functionally analyzed is small. In conclusion, although some compounds with behavioral activity on B. dorsalis have been identified, and a large number of olfactory proteins have been used as candidate molecular targets, the corresponding relationship between “chemical substances-olfactory molecular targets and nerve-behavior” is lacking, which greatly limits the role of olfactory molecular targets in attractant development. Therefore, on this basis, we put forward a development strategy for the behavioral regulation technology of B. dorsalis based on olfactory key molecular targets, to provide new ideas for the design and screening of effective behavioral regulators of B. dorsalis.

【Aim】 Gut microbiota might play an important role in mediating host resistance to Bacillus thuringiensis (Bt). The purpose of this study is to explore the effect of gut bacteria on the insecticidal activity of Bt in the diamondback moth, Plutella xylostella, and analyze the action mechanism of gut bacteria in host protection. 【Methods】 The survival rates of the 3rd instar larvae of P. xylostella feeding on the sterile artificial diet and on the artificial diet containing total gut bacteria, Enterobacter sp. IAE5 (EbPXG5), Bt strain Bt8010, Bt8010+total gut bacteria and Bt8010+EbPXG5, respectively, as well as the survival rates of P. xylostella after feeding on the sterile artificial diet and artificial diet containing EbPXG5 supernatant, EbPXG5 cell lysate solution, Bt8010+EbPXG5 supernatant, Bt8010+EbPXG5 cell lysate solution and Bt8010, respectively, were measured, and then the influence of gut bacteria on the Bt susceptibility of P. xylostella at different time was analyzed were analyzed. The plate culture technique was used to assay the abundance of EbPXG5 and Bt8010 in the gut and haemolymph of the 3rd instar larvae of P. xylostella fed with the artificial diets containing EbPXG5, Bt8010 and Bt8010+EbPXG5, respectively, and the inhibitory effect of EbPXG5 on Bt8010 in vitro, and the effect of gut bacteria on the proliferation of Bt8010 in the gut of P. xylostella and their invasion into the blood cavity was analyzed. The inner wall morphology of the gut tissues of the sterile 3rd instar larvae of P. xylostella and the gut tissues of P. xylostella fed with EbPXG5, Bt8010 and EbPXG5+Bt8010, respectively, were observed by scanning electron microscope to reveal the protective function of gut bacteria on the inner wall of gut. 【Results】 The survival rates of the 3rd instar larvae of P. xylostella feeding on the diet containing total gut bacteria and the diet containing EbPXG5 had no significant difference compared with that in the control feeding on the sterile artificial diet, but the survival rates of the 3rd instar larvae feeding on the diet containing Bt8010+EbPXG5 and the diet containing Bt8010+total gut bacteria were significantly higher than those feeding on the diet containing Bt8010 at 24, 36, 48 and 60 h. The survival rates of the 3rd instar larvae feeding on the diets containing EbPXG5 supernatant and EbPXG5 cell lysate solution had no difference from that of the control group, and the survival rates of the 3rd instar larvae feeding on the diets containing Bt8010+EbPXG5 supernatant and Bt8010+EbPXG5 cell lysate solution also had no difference from that of the larvae feeding on the diet containing Bt8010 alone. There was no significant difference in the abundance of EbPXG5 in the gut of the 3rd instar larvae of P. xylostella feeding on the artificial diets containing EbPXG5 alone and Bt8010+EbPXG5, respectively, but the abundance of Bt8010 in the gut of the 3rd instar larvae feeding on the diet containing Bt8010+EbPXG5 was significantly lower than that feeding on the diet containing Bt8010 alone at 24, 36 and 48 h. The abundance of EbPXG5 in the haemolymph of P. xylostella fed with the diet containing Bt8010+EbPXG5 was higher than that fed with the diet containing EbPXG5 alone at 36 and 48 h; however, the abundance of Bt8010 in the haemolymph of P. xylostella fed with the diet containing Bt8010+EbPXG5 was significantly lower than that fed with the diet contaning Bt8010 alone at 36 and 48 h. The Oxford cup bacteriostatic test showed that EbPXG5 had no inhibitory effect on Bt8010 in vitro. The results of scanning electron microscope observation showed that Bt8010 could destroy the larval gut cavity to form holes, mediate Bt8010 and other bacteria to cross the gut barrier and enter the hemolymph of P. xylostella. EbPXG5 could colonize the inner wall of the gut cavity of P. xylostella, weaken the damage of Bt8010 to the inner wall of the gut, and reduce the abundance of Bt8010 in the gut and hemolymph. 【Conclusion】 Gut bacteria EbPXG5 plays an important role in protecting P. xylostella and reducing the susceptibility of P. xylostella to Bt. It may weaken the colonization and invasion of pathogens by competing for niche and protecting the inner wall of gut, thus reducing the susceptibility of host to Bt. The results have important reference value for promoting the biological control and integrated management of P. xylostella.

【Aim】The wheat blossom midge, Sitodiplosis mosellana, an important agricultural pest, survives under temperature extremes during summer and winter by diapause. This study aims to explore the relationship between heat shock protein 60 (Hsp60) gene expression and diapause development and temperature tolerance in the diapause process of S. mosellana.【Methods】 The full-length cDNA sequence of Hsp60 of S. mosellana (SmHsp60) was amplified via RACE and RT-PCR technologies, and analyzed via bioinformatics. qPCR was used to detect the expression levels of SmHsp60 in the S. mosellana larvae at different stages from pre-diapause to post-diapause development including pre-diapause, diapause, post-diapause quiescence and post-diapause development, as well as oversummering larvae under extremely high temperature stress ［exposed to water bathes at 34, 40, 45 and 50 ℃ for 1 h, and 35 ℃ for 0 (control), 15, 30, 60 and 120 min］ and overwintering larvae under extremely low temperature stress ［exposed to 0, -5, -10 and -15 ℃ for 1 h, and -5 ℃ for 0 (control), 15, 30, 60 and 120 min］. The recombinant SmHsp60 protein was obtained by prokaryotic expression and affinity column chromatography, and its ability to suppress the thermal aggregation of mitochondrial malate dehydrogenase (MDH) at 43 ℃ was examined by colorimetry and SDS-PAGE.【Results】 The full-length cDNA sequence of SmHsp60 (GenBank accession no: KR733065) of S. mosellana obtained is 2 270 bp in length, which contains an open reading frame (ORF) of 1 722 bp in length encoding a protein of 573 amino acids with the relative molecular weight of 60.7 kD. Amino acid sequence analysis indicated that SmHsp60 contains the classical signature sequences of mitochondrial Hsp60, and displayed the highest amino acid identity and the closest relationship to Hsp60 from Contarinia nasturtii of Cecidomyiidae. qPCR detection result showed that the expression level of SmHsp60 did not change significantly in the diapause stage, but began to increase gradually in the post-diapause quiescence stage with a peak in early-to-mid phase of post-diapause quiescence (i.e. December and next January) and was significantly higher than those in the other developmental stages. Compared to the untreated control, the expression of SmHsp60 in the oversummering larvae under 35 and 40 ℃for 1 h and 35 ℃ for 30-60 min and that in the overwintering larvae under -5 ℃ for 1 h was significantly induced, while the expression level of SmHsp60 did not change significantly above 45 ℃ or below -10 ℃. The highly purified recombinant SmHsp60 was able to effectively suppress the thermal aggregation of MDH, indicating its significant molecular chaperone function.【Conclusion】 SmHsp60 is involved in diapause regulation of S. mosellana and might play a role in diapause termination, as well as heat tolerance and cold tolerance during diapause.

 As the most successful biological pesticide, Bacillus thuringiensis (Bt) insecticide has been used to control pests in agricultural production for about 80 years. The wide and successful use of Bt insecticide due to its specificity, safety and efficiency has greatly reduced the application rate of chemical pesticides, making a huge contribution to environmental protection. However, due to long-term application, some target insect pests have gradually developed resistance to Bt. In this article, the research advances in insect humoral immunity and the mechanisms of Bt resistance in insects were summarized. Previous studies suggested that the blockage of toxin activation and/or the mutation or reduction of toxin receptor are the main causes of the development of Bt resistance in insect pests. However, in recent years, more and more studies have shown that Bt resistance in insects is also associated to their immune system, especially to humoral immune pathways such as Toll, IMD and proPO-AS. Therefore, we reviewed and inferred the main pathways in which the humoral immune system is involved in the development of Bt resistance in insects. The IMD immune pathway may be involved in regulating Bt resistance in insects through the MAPK signaling pathway, or insects may counteract sepsis caused by the destruction of midgut tissue by Bt through multiple immune responses and promote midgut tissue healing through the JNK signaling pathway, thereby increasing their resistance to Bt. Research with the humoral immune system as the breakthrough point could be a new direction for further exploring the mechanisms of Bt resistance in insects.

【Aim】Based on the features that the P2C can be delivered into ovaries and the Gal4 protein can stably bind to the UAS sequence, to establish an efficient non-embryonic exogenous DNA delivery technical system in Anopheles sinensis.【Methods】The recombinant protein P2C-Gal4-DsRed was injected into the abdomen of female adults of A. sinensis at 20 h after sucking blood. The delivery efficiency of the recombinant protein P2C-Gal4-DsRed in the ovaries was analyzed by frozen section fluorescence observation and Western blot. The recombinant P2C-Gal4 DNA BINDING protein was prepared, transgenic plasmid and helper plasmid containing the 12×UAS repeat motif were constructed, and the in vitro binding between the recombinant protein P2C-Gal4 DNA BINDING and 12×UAS repeat motif was analyzed by electrophoretic mobility shift assay. The complexes P2C-Gal4 DNA BINDING recombinant protein+helper plasmid ITF36-12×UAS and P2C-Gal4 DNA BINDING recombinant protein+transgenic plasmid ITF2-12×UAS afm incubated in vitro were injected into the abdomen of female adults of A. sinensis at 20 h after sucking blood, and the DNA of their ovaries was extracted at 40 h after a blood meal. The delivery of exogenous DNA in vivo was analyzed by PCR amplification with specific primers and sequencing. 【Results】The ovaries of 100% female adults of A. sinensis injected with P2C-Gal4-DsRed showed obvious red fluorescence under the green filter, indicating that the P2C-Gal4-DsRed recombinant protein could be efficiently transferred into the ovaries of female adults of A. sinensis. The recombinant P2C-Gal4 DNA BINDING protein could stably bind to the 12×UAS repeat motif and the plasmid containing this repeat motif fragment. Exogenous DNA fragments were detected in the ovarian tissues of 91% and 93% of female adults of A. sinensis injected with P2C-Gal4 DNA BINDINGP2C-Gal4+ITF36-12×UAS and P2C-Gal4 DNA BINDING+ITF2-12×UAS afm, respectively. 【Conclusion】The exogenous DNA delivery technical system based on the P2C ovary-delivering peptide and the Gal4-12×UAS binding property was successfully established in A. sinensis. Through this technology platform, DNA molecules such as plasmids can be conveniently, rapidly and efficiently delivered into the ovaries of A. sinensis, laying a foundation for further simplifying genetic operations such as transgene, overexpression and gene knock-in.

Computer simulation of the dynamics of insect populations is very important for the prediction of population growth and pest management. In this article, we introduced the use of computer simulations to predict the population dynamics, fluctuation of predation, parasitism and feeding, timing of pest control, and variability of simulation based on the age-stage, two-sex life table. Using the life table program TWOSEX-MSChart and predation rate program CONSUME-MSChart to analyze the life table data and predation data, the results can then be used in the simulation program TIMING-MSChart to project the stage structure of the population dynamics, and the changes in the predation, parasitism and consumption capacities of population. Based on the population dynamics, computer simulation can be used to predict the damage caused by the pest population, predation capacity of predator and parasitism capacity of parasitoid. These data can then be used to plan the timing and frequencies of pesticide application in chemical control, and to predict the timing and number of natural enemies to be released in biological control. Furthermore, the uncertainty of population growth can be predicted by using the life table constructed based on 2.5, 97.5 and other percentiles generated through bootstrap technology. Computer simulations based on the age-stage, two-sex life table can predict the growth of pest populations and the optimal timing for chemical and biological control to achieve an economical and efficient integrated pest management, thus providing theoretical and technical support for sustainable agriculture.

 【Aim】 To evaluate the predatory capability and biological control potential of the native natural enemy Mallada basalis on the eggs and early instar larvae of the fall armyworm, Spodoptera frugiperda invading China. 【Methods】 The predatory capabilities of the 2nd and 3rd instar larvae of M. basalis on the eggs, the 1st and 2nd instar larvae of S. frugiperda were evaluated by using functional response models under laboratory conditions. 【Results】 The numbers of eggs and larvae of S. frugiperda consumed by M. basalis larvae increased and saturated as the prey density increased, while the predation rate of M. basalis larvae decreased with the increase of prey density. M. basalis larvae exhibited a type Ⅱ functional response to different developmental stages of S. frugiperda. For the 2nd instar larvae of M. basalis, their instantaneous attack rates a on the eggs, and the 1st and 2nd instar larvae of S. frugiperda were 0.150, 0.084 and 0.094, the handling time Th was 0.282, 0.333 and 0.519 h, and the theoretical daily maximum predation T/Th was 85106, 72072 and 46242, respectively. For the 3rd instar larvae of M. basalis, their instantaneous attack rates on the eggs, and the 1st and 2nd instar larvae of S. frugiperda were 2.018, 0.288 and 0.259, the handling time was 0.102, 0.311 and 0.375 h, and the theoretical daily maximum predation was 235294, 77.170 and 64.000, respectively. 【Conclusion】 Both the 2nd and 3rd instar larvae of M. basalis exhibit strong predatory capability on the eggs and early instar larvae of S. frugiperda, and the 3rd instar larvae of M. basalis show higher predation efficiency  than the 2nd instar larvae.

【Aim】To identify cytochrome P450 (CYP450) genes and their full-length transcripts in the larval guts of Apsi cerana cerana workers using nanopore sequencing technology and offer reference information and basis for further functional study.【Methods】The 4-6-day-old larval gut transcriptomes of A. cerana cerana workers were sequenced by Nanopore PromethION platform. Quality control of raw reads was performed using Guppy software to gain clean reads. Full-length transcript sequences were detected through recognizing primers at both ends. The sequences of the above-mentioned full-length transcripts were aligned to the Nr and GO databases with BLAST tool to identify CYP450 genes and their full-length transcripts. Astalavista software was used to identify alternative splicing (AS) events of genes. RT-PCR was used to validate the reliability of AS events of various types. 【Results】In the 4-6-day-old larval guts of A. cerana cerana workers, 7 338 627, 7 003 419 and 7 434 233 raw reads were obtained, respectively, and after quality control, 7 289 494, 6 959 880 and 7 387 756 clean reads were gained. The total number of identified non-redundant fulllength transcripts was 48 200. Forty-seven CYP450 genes and their 265 full-length transcripts were identified. A total of 90 AS events of CYP450 genes were identified, including 36 exon skipping events, 20 alternative 5′ splicing site events, 17 intron retention events, nine alternative 3′ splicing site events and eight mutually exclusive exon events. The RT-PCR result confirmed the authenticity of randomly selected three types of AS events. 【Conclusion】 The findings provide CYP450 genes and their full-length transcripts in A. cerana cerana, supplement the annotation of A. cerana reference genome, and reveal that CYP450 genes of A. cerana cerana can generate abundant isoforms via multiple AS types.

 【Aim】 To investigate the effects of Steinernema carpocapsae All infection on the innate immune response in larvae of the fall armyworm, Spodoptera frugiperda. 【Methods】 The hemocyte types of S. frugiperda larvae were observed and identified under an inverted microscope, and the total numbers of hemocytes in S. frugiperda larvae at different time after infection by S. carpocapsae All were counted. The encapsulation of invading S. carpocapsae All nematodes by S. frugiperda hemocytes was observed under an inverted microscope. The phagocytoic activity of fluorescent Staphylococcus aureus by hemocytes of S. frugiperda larvae was observed under an inverted fluorescence microscope. The phenoloxidase (PO) activity in the hemolymph, the relative expression levels of antibacterial peptide genes, and the antibacterial activity of plasma in S. frugiperda larvae infected with S. carpocapsae All were detected. 【Results】 Five types of hemocytes, prohemocyte, granulocyte, oenocytoide, spherulocyte and plasmatocyte, were found in S. frugiperda larvae. The total numbers of hemocytes in S. frugiperda larvae increased significantly at 9 and 12 h after injection of 1 μL S. carpocapsae All infective juveniles (IJs) at the dose of 3 IJs/μL. The hemocytes from S. frugiperda larvae failed to encapsulate the live and coldkilled S. carpocapsae All nematodes but could encapsulate heat-killed nematodes. The phagocytic activity of fluorescent S. aureus by S. frugiperda hemocytes was significantly inhibited after incubation with live S. carpocapsae All nematodes, but not with cold- and heat-killed S. carpocapsae All nematodes. The PO activity in the hemolymph of S. frugiperda larvae decreased first, then increased, and finally decreased after injection of 1 μL S. carpocapsae All at the dose of 3 IJs/μL. The relative expression levels of antimicrobial peptide genes Attacin-A2, Attacin-B1, Cecropin-B3, Cecropin-D, Gallerimycin, Gloverin-3 and Lebocin-2 in S. frugiperda larvae were significantly induced at 12 h after S. carpocapsae All infection, and then recovered to the control level or lower than the control level at 24 h after infection. The antibacterial activity of S. frugiperda plasma increased significantly at 12 h after S. carpocapsae All infection, but was not significantly different between the treatment group and the control group at 24 h after infection. 【Conclusion】 In the early stage of infection, S. carpocapsae All would inhibit the innate immune response in S. frugiperda larvae, then the immune system in S. frugiperda would be initiated for trying to defend against S. carpocapsae All, and in the late stage the immune system in S. frugiperda would be inhibited or destroyed with the successful colonization of nematodes. The results obtained in this study provide a basis for further understanding the immune mechanisms involved in the interaction between nematodes and S. frugiperda, and lay a theoretical foundation for further improving the control efficacy of entomopathogenic nematodes against S. frugiperda larvae.

【Aim】The 28-spotted ladybird, Henosepilachna vigintioctopunctata is an important pest of the Solanaceae plants. Chitin deacetylase 1 (CDA1) in insects catalyzes the deacetylation of N-acetylamino-D-glucosamine, facilitating the conversion of chitin to chitosan. Chitosan controls the orderly stacking of chitin fibers in insects and maintains the structural integrity of the cuticle. The silencing of CDA1 gene in insects inhibits the synthesis of chitosan which impairs the formation of cutiular structure and development, and eventually leads to death. 【Methods】The expression patterns of HvCDA1 in different developmental stages (egg, 1st－4th instar larvae and prepupa) and different tissues (cuticle, fat body, midgut and Malpighian tubules) of the 4th instar larvae of H. vigintioctopunctata were analyzed by RT-qPCR. The silencing of HvCDA1 was achieved by the oral feedings of eggplant leaves soaked in different concentrations of dsHvCDA1 solution for 1 min in the 1st instar larvae and direct oral feeding of different concentrations of dsHvCDA1 solution in the 4th instar larvae of H. vigintioctopunctata, and the RNAi-mediated silencing effects on the larval survival and development, and expression level of HvCDA1 were investigated.【Results】 Developmental expression profiling revealed that HvCDA1 was expressed across various developmental stages of H. vigintioctopunctata, with the highest expression level in the late 1st and late 2nd instar larvae. Tissue-specific expression profiling exhibited that the expression level of HvCDA1 in the cuticle of the 4th instar larvae was significantly higher than those in the other tissues. After feeding of eggplant leaves soaked in 50 ng/μL of dsHvCDA1 solution, the 1st instar larvae spines could not stand uprightly after molting, the cuticle could not form normally, and showed hindered feeding until death. The 1st instar larvae at 48 after being fed with the eggplant leaves soaked in dsHvCDA1 solution at the concentrations of 50, 200 and 400 ng/μL had the mortality rates of 84%, 94% and 100%, respectively. The oral feeding of the eggplant leaves soaked in 50 ng/μL of dsHvCDA1 solution reduced the expression levels of HvCDA1 by 38.63% and 79.00% in the 1st instar larvae at 48 and 96 h, respectively, as compared to the control (oral feeding of the eggplant leaves soaked in 50 ng/μL of dsGFP solution). On the other hand, ingestion of 50, 200 and 400 ng/individual of dsHvCDA1 solution by the 4th instar larvae resulted in 29%, 40% and 66% of larvae being deformed and unable to emerge normally after pupation, respectively. Similarly, the expression levels of HvCDA1 in the 4th instar larvae at 48 and 96 h after being fed with 200 ng/individual of dsHvCDA1 solution were significantly reduced by 52.99% and 70.09%, respectively, as compared to those in the control (fed with 200 ng/individual of dsGFP solution). 【Conclusion】 These results indicate that HvCDA1 plays an important role in the survival and development of H. vigintioctopunctata. The silencing of HvCDA1 negatively affects the ecdysis, pupation and emergence of H. vigintioctopunctata, and eventually leads to death. Collectively, the results from this study suggest that HvCDA1 can be a potential target gene for the RNAi-mediated control of H. vigintioctopunctata.

【Aim】To clarify the function of sulfakinin (SK) and sulfakinin receptor (SKR) in the feeding behavior of Nilaparvata lugens.【Methods】The full-length cDNA sequences of sulfakinin gene Nlsk and sulfakinin receptor gene Nlskr of N. lugens were cloned by PCR and subjected to bioinformatical analysis. The expression levels of Nlsk and Nlskr in different developmental stages (egg, 1st-5th instar nymphs, and male and female adults) and different tissues (head, antenna, wing, proboscis, leg, gut and Malpighian tubules) of the female adult of N. lugens were analyzed by qRT-PCR. The 3rd instar nymphs of N. lugens were injected with ds Nlskr for gene silencing and the expression level of Nlskr in the 4th instar nymph was detected by qRT-PCR. The food intake of the 4th instar nymph after the  Nlskr silencing was measured. GO and KEGG analyses of differentially expressed genes (DEGs) and qRT-PCR verification of feeding-related genes based on the previously constructed transcriptome database of the 4th instar nymphs after RNAi of Nlskr were performed. 【Results】 The full-length cDNA sequences of Nlsk (GenBank accession number: AB817281) and Nlskr (GenBank accession number: BAO01059.1) of N. lugens were cloned by PCR. Sequence alignment results showed that the NlSK mature peptide of N. lugens has a C-terminal FMRFamide polypeptide structure that is conserved with other species. NlSKR has a highly conserved transmembrane domain with homologous receptors of other insects. The results of qRT-PCR showed that Nlsk and Nlskr were highly expressed in the egg and 1st instar nymph, and mainly in the head. Nlskr was also highly expressed in the proboscis of N. lugens. Silencing Nlskr significantly increased the food intake of the 4th instar nymphs of N.  lugens. Based on transcriptome data, GO and KEGG analysis result of DEGs showed that silencing of Nlskr by RNAi significantly affected the expression of the olfactory, gustation, energy metabolism, and feeding-related neuropeptides and receptor genes. The qRT-PCR verification results of feeding-related genes showed that silencing Nlskr decreased the expression levels of Nl7tmOR, NlOAR-3R, NlUH-FAF and NlTRP-161A, and increased the expression levels of NlGr64f, NlUE-E2 and NlTHR.【Conclusion】 This study reveals that sulfakinin and its receptor are involved in regulating the feeding behavior of  N. lugens, providing a potential target for the development of pest insect feeding behavior inhibitors.

【Aim】To explore the influence of cadmium stress on the herbivore Spodoptera frugiperda and whether the parasitization capability of Trichogramma is affected by “bottom-up” cascade effect of cadmium. 【Methods】The larvae of S. frugiperda hatched within 24 h were fed with the artificial diets containing different concentrations (0, 0.2 and 51.2 mg/kg) of Cd²⁺, the growth and fecundity (number of eggs laid per female) of S. frugiperda and Cd²⁺ content in the eggs of F1 generation of S. frugiperda were investigated, and the parasitization capability and preference of T. dendrolimi adults to the eggs of S. frugiperda stressed by a low concentration (0.2 mg/kg) of Cd²⁺were also investigated. 【Results】Compared with the control (normal artificial diet), 0.2 and 51.2 mg/kg Cd²⁺in the artificial diet significantly prolonged the larval duration and significantly reduced the female pupal weight of S. frugiperda. The pupation rate, adult emergence rate, adult longevity and number of eggs laid per female of S. frugiperda in the treatment with high concentration (51.2 mg/kg) of Cd²⁺ decreased significantly. However, the number of eggs laid per female of S. frugiperda in the treatment with low concentration (0.2 mg/kg) of Cd²⁺ was slightly higher than that of the control, and the Cd²⁺ content in eggs of S. frugiperda in the low Cd²⁺concentration treatment was 1.03 mg/kg. The parasitism rate of T. dendrolimi on S. frugiperda eggs stressed by 0.2 mg/kg Cd²⁺ within 24 h was 52%, which was significantly higher than that of the control, while no significant differences were found in the adult emergence rate and proportion of females of F1 generation of T. dendrolimi between the treatment and control. When the eggs of S. frugiperda stressed by 0.2 mg/kg Cd²⁺ and the control eggs were separately and simultaneously exposed to T. dendrolimi females, the parasitism rate of T. dendrolimi adults on cadmium-stressed S. frugiperda eggs was significantly higher than that on the control eggs, while no significant differences were found in the adult emergence rate and proportion of females of F1 generation of T. dendrolimi when the two kinds of host eggs were separately and simultaneously provided. 【Conclusion】The results of this study suggest that cadmium stress affects the growth, development and fecundity of S. frugiperda, and cadmium in the artificial diet can be transferred into eggs via larval feeding, and affects the performance of T. dendrolimi.

【Aim】 The diamondback moth, Plutella xylostella, is a cosmopolitan lepidopteran pest that mainly attacks cruciferous plants. Its strong reproductive capacity is one of the main reasons why it becomes the pest most difficult to control in the field. Mating is a physiological process necessary for the bisexual reproduction of most insect species. Clarifying the mating behavior and physiological response of female and male adults is of great significance for population monitoring and control of P. xylostella. 【Methods】 The gonad and spermatophore formation of P. xylostella adults were observed anatomically. The mating and remating capacity, spermatophore formation and digestion of P. xylostella adults, and the effects of mating frequency on the spermatophore formation and female fecundity were measured and analyzed by behavioral and biological experiments. 【Results】 During the mating process, the seminal fluid of male adults of P. xylostella was transferred to female adults in the form of spermatophore, which is a white, opaque, balloonlike structure within the bursa copulatrix. The spermatophore could be sufficiently digested and absorbed after mating. The observation results of mating capacity showed that both female and male adults of P. xylostella had multiple mating behaviors. After the first mating, for the male adults there was a short delay in re-mating, and the mating success rate was 54.6% within 20 min, which was significantly lower than that of the first mating. Although the multiple mating did not affect the mating duration of male adults, the mating history significantly inhibited the spermatophore size and female fecundity. There was an obvious re-mating inhibition in mated female adults, of which the mating rate was significantly lower than that of the unmated females within 12 h, suggesting that re-mating inhibition depends on the rates of spermatophore digestion and absorption after the first mating. There was no significant difference in the oviposition amount and egg hatching rate between female adults with multiple and single mating. 【Conclusion】 Multiple mating results in delayed and inhibited re-mating of female and male adults o f P. xylostella, respectively. The spermatophore produced by male adults with multiple mating is significantly reduced, and the oviposition amount and egg hatching rate of female adults get no benefit from multiple mating. This study provides a theoretical foundation for better understanding the reproductive regulation mechanism of P. xylostella.

【Aim】 This study aims to investigate the biological characteristics of adults and the growth and development of F1 generation of Serangium japonicum at different cold-storage temperatures, so as to determine the optimal cold-storage temperature of S. japonicum adults.【Methods】 S. japonicum adults were stored at different low temperatures (7, 10, 13 and 16 ℃), their survival rates, numbers of eggs laid per female, longevity and daily predation amount against the 4th instar nymphs of Bemisia tabaci, the survival rates and developmental duration of F1 generation of S. japonicum, and the relative expression levels of the heat shock protein genes Hsp70 and Hsp90 in S. japonicum adults were determined at 10 d after storage by qRT-PCR. 【Results】At 10 d after S. japonicum adults were stored at 16 ℃, their survival rate, female and male adult longevity, number of eggs laid per female and daily predation amount, and the survival rate of F1 generation were not significantly different from those of the control stored at 26 ℃ (survival rate: 99% vs 100%; female adult longevity: 110.65 d vs 106.87 d; male adult longevity: 123.12 d vs 108.79 d; number of eggs laid per female: 399.19 grains vs 422.63 grains; daily predation amount: 34.70 individuals vs 31.95 individuals; F1 progeny survival rate: 8039% vs 94.12%); but the developmental duration of F1 generation was significantly shorter than that of the control (17.33 d vs 18.89 d). At 10 d after coldstorage, all S. japonicum adults died when stored at 7 ℃, when stored at 10 ℃, their survival rate and number of eggs laid per female and the survival rate of F1 generation were significantly affected, and when stored at 13 ℃, the number of eggs laid per female was significantly affected. Various cold-storage temperatures induced the up-regulation of the expression levels of both Hsp70 and Hsp90 in S. japonicum adults at 10 d after storage, and the lower the temperature, the higher the relative expression levels. The relative expression levels of Hsp70 and Hsp90 in the treatment group of 10 ℃ were 7.06- and 2.33-fold, respectively, as high as those in the control.【Conclusion】 Combining the above results, the optimum cold-storage temperature for S. japonicum adults is 16 ℃, and the heat shock proteins Hsp70 and Hsp90 may play a protective role in the response to cold stress in S. japonicum adults.

As a model organism, Drosophila has the advantages of short growth cycle, high reproductive capacity, and low research cost. Moreover, 65% of genes in Drosophila are homologous with those in human, especially the simple genetic background of Drosophila makes Drosophila play an important role in the study of biological growth and development, pathological mechanism and gene expression regulation. So far, eight insulins, Drosophila insulin-like peptides 1-8 (Dilp1-8), have been identified in Drosophila, and studies of the Drosophila insulin signaling pathway have focused on its regulation of growth and development and energy metabolism, which is mainly mediated through Dilp1-7. Little is known about the function of Dilp8 and the molecular mechanism of its action. In this article, we summarized the results of studies on Dilp8 since its discovery. Dilp8 was mainly expressed in imaginal discs and ovaries of adult female Drosophila. Dilp8 allows Drosophila to grow into individuals with a normal-sized and symmetrical body by regulating tissue growth and developmental timing. Dilp8 in damaged larvae mitigates abnormal growth by delaying developmental time. After being activated, Dilp8 enters the central nervous system and specifically binds to its receptor leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), thereby inhibiting the synthesis of ecdysone to control the growth and development of Drosophila. It has been suggested that Lgr3 is associated with sex regulation in Drosophila. Dilp8 regulates the ovulatory capacity of adult female Drosophila. In addition, tumour-derived Dilp8 is associated with anorexia of Drosophila. Dilp8/Lgr3 is highly homologous with human INSL3/RXFP2. The molecular mechanism of action of Dilp8 needs further exploration.

【Aim】The grain aphid, Sitobion miscanthi, is one of the most important pest insects in agricultural production in China. Its saliva contains many effector proteins with various functions, which are involved in aphid-plant interactions during the feeding process. The aim of this study is to clarify the functions of the salivary protein SmHMp1 in the processes of feeding and reproduction of S. miscanthi, and to explore the feasibility of using this gene as a silencing target to control S. miscanthi.【Methods】Based on salivary gland transcriptome data of S. miscanthi, the full-length cDNA sequence of SmHMp1 of S. miscanthi was cloned, and bioinformatically analyzed. The RT-qPCR was used to determine the expression levels of SmHMp1 in different development stages (1st-4th instar nymphs and wingless adult), tissues (salivary gland, head, thorax, abdomen, embryo and whole body) of wingless adults, adults of different wing morphs (winged and wingless adults) and wingless adults of S. miscanthi fed with different diets (artificial diet and wheat seedlings). Subcellular localization of the SmHMp1 protein was analyzed by Agrobacterium tumefaciens-mediated transient expression in tobacco. The recombinant virus vector of barley stripe mosaic virus (BSMV) was constructed and the host-induced gene silencing (HIGS) technology was used to silence SmHMp1 of S. miscanthi, and the silencing effects on the growth and development, reproduction and feeding of S. miscanthi were analyzed by anatomy, life table and electrical penetration graph (EPG) techniques. 【Results】 The full-length cDNA sequence of SmHMp1(GenBank accession no.: OP021692) of S. miscanthi was cloned. SmHMp1 has an open reading frame (ORF) of 426 bp in length, encoding 141 amino acid residues with the predicted molecular weight of 16.2 kD, theoretical isoelectric point of 8.74, and an N-terminal signal peptide with 19 amino acid residues. The phylogenetic analysis showed that SmHMp1 is most closely to the uncharacterized protein LOC100165393 precursor (GenBank accession no.: NP_001155659.1) of the pea aphid, Acyrthosiphon pisum. The RT-qPCR results showed that SmHMp1 of S. miscanthi was expressed at all developmental stages, and had the highest expression level at the 1st instar nymphal stage, showing an initial downward and later upward overall trend with the developmental time. The expression level of SmHMp1 in salivary glands of wingless adult was significantly higher than those in other tissues. There were no significant difference in the expression level of SmHMp1 in adults between both wing forms or between fed with both diets (artificial diet and wheat seedlings). Subcellular localization results showed that SmHMp1 protein is localized in the membrane and nucleus of tobacco cells. The expression level of SmHMp1 in S. miscanthi wingless adults fed with the wheat seedlings inoculated with the recombinant virus BSMV-SmHMp1 decreased extremely significantly to 43.64% of that of the control group (inoculated with the recombinant virus BSMV-GFP). When SmHMp1 was silenced, the number of aphids produced in 8 d and the number of embryos of S. miscanthi wingless adults decreased significantly (54.17% and 46.25% of the control, respectively), and the phloem-feeding time was significantly shortened to 64.95% of the control group.【Conclusion】The salivary protein SmHMp1 may play an important role in the feeding and reproduction processes of S. miscanthi, and has the potential to be used as a HIGS target to control S. miscanthi. This study is conducive to the deep understanding of aphid-host interactions at the molecular level and development of green control measures for S. miscanthi.

 【Aim】 The aim of the study is to test the validity of DNA barcoding for the identification of broad-headed bugs (Hemiptera: Coreidae) from China. 【Methods】 The DNA barcode sequences of mitochondrial COI gene of 207 samples from 23 species of 13 genera of Alydidae from China were amplified, and 31 internal transcribed spacer 1 (ITS-1) sequences of three Leptocorisa species were amplified as auxiliary markers. The interspecific and intraspecific genetic distances (Kimura 2-parameter model, K2P) were calculated by MEGA 11 software. The species cluster analysis was performed using neighborjoining (NJ) method. The holotype networks were constructed using median joining network algorithm. 【Results】 Based on the DNA barcode sequences of mitochondrial COI, the mean intraspecific K2P distances of all the tested 23 species of Alydidae from China were below 2%, and the interspecific K2P distances ranged from 0.98% to 23.98%, with an average of 17.50%. Most species were separated from each other with a high bootstrap value. This COI barcode section could not distinguish between Leptocorisa chinensis and L. oratoria because of the partial COI haplotypes they shared. The ITS-1 sequences did distinguish the two species in the haplotype network analysis. 【Conclusion】 The DNA barcoding results from our data are congruent with most of the taxonomic units of the family Alydidae from China based on morphological characteristics. However, for extremely closely related species, mitochondrial data alone, especially COI barcode sequences, sometimes are insufficient for accurate species delimitation, and other DNA sequences or other types of data need to be introduced.

【Aim】 This study aims to clarify the expression pattern of odorant binding protein 1 (OBP1) in Aethina tumida and analyze the role of AtOBP1 in olfactory recognition of A. tumida.【Methods】 The cDNA full-length sequence of AtOBP1 was cloned based on the transcriptome and genome database of A. tumida and analyzed by bioinformatics. RT-qPCR was used to detect the expression levels of AtOBP1 in different developmental stages (egg, larva, pupa, female adult and male adult), different tissues (head, cuticle, wing, leg, fat body, gut, Malpighian tubules, testis and ovary) of the 7-day-old adult and in the head of different day-old adults of A. tumida after eclosion. The biological function of AtOBP1 in olfactory recognition of A. tumida was studied by RNA interference (RNAi) and Y-tube behavior choice experiment. 【Results】The cDNA full-length sequence of AtOBP1 gene (GenBank accession no.: MT211982.1) has six exons and an open reading frame (ORF) of 447 bp in length. AtOBP1 encodes 148 amino acid residues with PBP_GOBP superfamily conserved domain, and the predicted molecular mass and isoelectric point are 15.9 kD and 4.73, respectively. AtOBP1 protein is a dimer composed of six α-helics with six conserved cysteines that form three disulfide bonds. The phylogenetic tree also showed that AtOBP1 was closely clustered into one branch with TmOBP8 from Tenebrio molitor of Coleoptera. The RT-qPCR results showed that AtOBP1 was highly expressed in the pupal stage and the male adult stage, and was highly specifically expressed in the head and testis of adults. In addition, the expression level of AtOBP1 in adult head increased gradually with the day-old age, reached two peaks in the 5- and 7-day-old adult stages, respectively, and decreased in the 8-day-old adult stage. RNAi in combination with Y-tube behavior choice experiment results revealed that silencing of AtOBP1 resulted in significantly reduced preference of A. tumida adults to the pollen volatile compounds ethyl palmitate and ethyl linolenate.【Conclusion】AtOBP1 belongs to Classical OBPs family. AtOBP1 is mainly expressed in the head and testis of A. tumida adults, and may participate in the recognition process of the pollen volatile compounds ethyl palmitate and ethyl linolenate in A. tumida.
Key words: Aethina tumida; odorant binding protein; AtOBP1; RNAi; olfactory behavior

【Aim】This study aims to lay the foundation for the functional study of cuticular protein (CP) 17 of Apolygus lucorum (AlCP17) and to provide a preliminary basis for further analyzing the signal pathway map of cuticular development of A. lucorum by cloning its gene AlCP17, preparing its polyclonal antibody and analyzing its expression profiles.【Methods】Based on the previous transcriptome data, the full-length cDNA sequence of AlCP17 of A. lucorum was cloned and subjected to bioinformatics analysis. The prokaryotic expression vector pCZN1-AlCP17 was constructed and transformed to Escherichia coli to conduct in vitro expression. The purified recombinant protein was used to prepare polyclonal antibody of AlCP17. qRT-PCR was used to analyze the expression profiles of AlCP17 in different developmental stages (1st-5th instar nymphs and adult) and different tissues (head, thorax, leg, midgut, fat body and cuticle) of the early 3rd instar nymphs of A. lucorum.【Results】The full-length cDNA sequence of AlCP17 (GenBank accession no.: OM302231) was cloned from A. lucorum with an open reading frame of 594 bp in length, encoding a putative protein of 197 amino acids with the predicted molecular weight of 21.69 kD and the theoretical isoelectric point of 6.11. AlCP17 has the typical structure characteristics of cuticular protein, containing the conserved chitin binding domain (non-cysCBD) of arthropod cuticular protein, namely Chitin_bind_4 domain. Amino acid sequence alignment result showed that AlCP17 shows the highest amino acid sequence identity (76.29%) with the CPA2B-like of Cimex lectularius of Cimicidae of Hemiptera. Phylogenetic tree showed that CP is a highly conserved protein with high similarity to CPA2B-like of C. lectularius and CP7-like of Halyomorpha halys. The prokaryotically expressed recombinant AlCP17 was obtained by IPTG induction. After purification, the polyclonal antibody of AlCP17 was obtained and had good purity for subsequent experiments. AlCP17 was expressed in different developmental stages and different tissues of the early 3rd instar nymphs of A. lucorum, and its expression level reached the peak in the newly-hatched 1st instar nymphs and increased significantly at the end of each instar. In addition, the expression level of AlCP17 in the midgut of the early 3rd instar nymphs was the highest.【Conclusion】The full-length cDNA sequence of AlCP17 gene of A. lucorum was cloned. AlCP17 has the typical characteristics of insect cuticular protein. The expression of AlCP17 in A. lucorum shows developmental stage specificity and tissue specificity. These results lay the foundation for the future research on the physiological function of this protein in A. lucorum development.

【Aim】This study aims to understand the monophyletic and phylogenetic positions of the Scoliidae by phylogenetic analysis of the mitochondrial genome of Aculeata insects.【Methods】The mitochondrial genomes of five species of three genera of Scoliidae were sequenced by using Illumina Hiseq2500 next-generation sequencing technology, annotated and analyzed. Maximum likelihood (ML) and Bayesian inference (BI) methods were used to construct the phylogenetic tree of Aculeata insects based on the sequences of 13 protein-coding genes (PCGs) and two rRNA genes of 36 mitochondrial genomes. 【Results】The five newly sequenced mitochondrial genomes of the Scoliidae, including the mitochondrial genomes of Colpa quinquecincta (GenBank accession number: OM103696), Colpa tartara (GenBank accession number: OM103697), Megacampsomeris grossa (GenBank accession number: OM103796), Megacampsomeris formosensis (GenBank accession number: OM142776) and Scolia schrenkii (GenBank accession number: OM103797), are 15 367-20 649 bp in length, containing 37-39 genes including 13 PCGs, 2 rRNA genes ( rrnL and rrnS) and 22-24 tRNA genes, with the AT content of over 75%. The start codons of all the 13 PCGs are ATN, and the stop codons are TAA or T--. In contrast to the deduced ancestral sequence, complex gene rearrangement events have occurred in the five newly sequenced mitochondrial genomes of the Scoliidae, which are mainly the changes of some gene positions in the gene region trnS1-trnE-trnF-nad5-trnH, a remote inversion phenomenon of the gene region trnI-trnQ-trnM-nad2-trnW-trnC-trnY-cox1-trnL2-cox2-trnK and the position changes of the genes trnF, trnS1, trnE, trnH and nad5. Both Ka/Ks and nucleotide diversity π of 13 PCGs indicated that cox1 is the most conserved gene. The results of the ML tree and BI tree both showed that the Scoliidae is a monophyletic group, and the Scoliidae and the Pompilidae are sister groups to each other. In the Scoliidae, Colpa and Scolia, and Megacampsomeris and Camposmeris all show close relationship.【Conclusion】This study preliminarily clarified the characteristics of the mitochondrial genomes of the family Scoliidae, laying the foundation for further research. The phylogenetic analysis based on the mitochondrial genome shows that the family Scoliidae is a single lineage.

【Aim】The purpose of this study is to explore whether glutamate-gated chloride channels (GluCls) of the beet armyworm, Spodoptera exigua (SeGluCls) encoded by different transcription variants of SeGluCl have different susceptibilities to insecticides using CRISPR/Cas9 gene editing technology. 【Methods】 The full-length cDNA sequence of SeGluCl from S. exigua was obtained using RT-PCR and RACE, and analyzed by bioinformatics. Two splicing variants SeGluCl 3a and SeGluCl 3b of SeGluCl were knocked out by CRISPR/Cas9 gene editing technique, and two knockout strains (3a-KO and 3b-KO) of S. exigua were established. The difference in the susceptibilies of the 3rd instar larvae of the susceptible strain WH-S (control strain) and 3a-KO and 3b-KO strains to three insecticides abamectin, emamectin benzoate and fipronil was determined by bioassay. 【Results】 The full-length cDNA sequence of SeGluCl of S. exigua was obtained, and its two splicing variants (SeGluCl 3a and SeGluCl 3b) were found. The open reading frame (ORF) of SeGluCl 3a (GenBank accession no.: OM304353) is 1 362 bp in length encoding 454 amino acids. The ORF of SeGluCl 3b (GenBank accession no.: OM304354) is 1 365 bp in length encoding 455 amino acids. The two SeGluCls encoded by the splicing variants SeGluCl 3a and SeGluCl 3b contain four transmembrane regions and one cysteine loop. Phylogenetic analysis indicated that SeGluCl was most closely related to GluCls of S. litura and S. frugiperda. Compared to the control strain WH-S, the knockout strains (3a-KO and 3b-KO) showed no significant difference in the susceptibilities to abamectin, emamectin benzoate and fipronil. 【Conclusion】 There is no difference in the susceptibility of SeGluCls encoded by the splicing variants SeGluCl 3a and SeGluCl 3b of SeGluCl to the three insecticides abamectin, emamectin benzoate and fipronil in S. exigua.

【Aim】 This study aims to clone four antennal odorant receptors (OR) genes and analyze their expression profiles in tissues of different dayold female and male adults of Callosobruchus chinensis, so as to lay the foundation for further investigation of gene function. 【Methods】 The candidate OR genes of C. chinensis were predicted based on the antennal transcriptome data of C. chinensis adults. The full-length cDNA sequences of four OR genes of C. chinensis were cloned by RT-PCR, and subjected to bioinformatic analysis. qPCR was used to detect the expression levels of the four OR genes in female and male adult tissues (antenna, head without antenna, abdomen, leg and wing) and antennae of 1-, 3- and 5-day-old female and male adults of C. chinensis. 【Results】 Based on the predicted gene sequences, the full-length cDNA sequences of four OR genes of C. chinensis were cloned, and named CchiOR8, CchiOR10, CchiOR16 and CchiOR39 with the GenBank accession numbers of MW732665, MN832705, MW732664 and MN832706, respectively, encoding 369, 352, 400 and 367 amino acids, respectively. CchiOR8, CchiOR10 and CchiOR16 are highly related to the homologous proteins of Colaphellus bowringi, while CchiOR39 clusters into a clade with the homologous proteins of Drosophila biarmipes and D. simulans. The qPCR result showed that the four OR genes were expressed in various tissues of female and male adults and highly expressed in antennae of C. chinensis. The expression levels of CchiOR10 and CchiOR39 in antennae of female adults were significantly higher than those in antennae of male adults. The expression level of CchiOR16 in antennae of male adults was significantly higher than that in antennae of female adults. The expression levels of CchiOR8 in antennae showed no significant difference between female and male adults. In addition, the expression levels of the four OR genes in antennae of different day-old adults were different to some degree. The expression levels of CchiOR8, CchiOR16 and CchiOR39 in antennae of male adults increased first and then decreased, all reaching the peak at the 3-day-old. The expression level of CchiOR10 in antennae of male adults was low and had no significant difference in different day-old adults. The expression levels of both CchiOR8 and CchiOR16 in antennae of the 5-day-old female adults reached the highest level, while the expression levels of CchiOR39 and CchiOR10 in antennae of the 1-day-old female adults were significantly higher than those in antennae of the other day-old female adults. 【Conclusion】 Four CchiOR genes of C. chinensis are highly expressed in the adult antennae, and are speculated to play an important role in the olfactory recognition of C. chinensis.

【Aim】 The aim of this study is to ascertain whether the sweetpotato flea beetle, Chaetocnema confinis has invaded the Chinese mainland based on morphological identification and molecular biology technique, determine its mitochondrial genome sequence, and analyze its genome structure and phylogenetic relationship. 【Methods】 The morphological characteristics of C. confinis adults collected from different localities in Guangdong were observed using microscope, and the DNA sequence of cox1 gene was amplified for molecular identification. The mitochondrial genome of C. confinis was sequenced using the Illumina MiSeq sequencing platform, and its sequence was subsequently assembled, annotated and characterized. Collinear analysis was conducted and phylogenetic tree was constructed based on the mitochondrial genome sequences of closely related species to analyze the gene rearrangement and phylogenetic relationship. 【Results】 The morphological and molecular identification results indicated that the flea beetle on sweetpota found in the Chinese mainland is the sweetpotato flea beetle, C. confinis. The mitochondrial genome of C. confinis is 15 685 bp in size, with 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one non-coding control region. All the 37 genes are in compact arrangements with a total interval of 101 bp, the order of gene arrangement is consistent with that in the model insect Drosophila yakuba. The A+T content of the mitochondrial genome of C. confinis is 773%, showing obvious AT bias. The start codons of the 13 protein-coding genes are all ATNs. Except for trnS1 without DHU arm and trnD, trnG, trnN and trnT without TψC rings, a typical cloverleaf-shaped secondary structure can be formed in the remaining 17 tRNA genes, and the mutated anticodons of trnK and trnS1 are UUU and UCU, respectively. The control region fragment of C. confinis is only 60 bp in length, and is the shortest control region in the reported insect mitochondrial genomes. Based on the phylogenetic analysis, C. confinis is closely related to Phyllotreta striolata of Alticinae. 【Conclusion】 C. confinis has invaded the Chinese mainland. The mitochondrial genome sequence of C. confinis has been obtained in this study, which provides a basis for management of C. confinis and phylogenetic analysis of divergent genera in Chrysomelidae.

【Aim】 This study aims to analyze and compare the differences in the transmission of Tomato chlorosis virus (ToCV) on Capsicum annuum plants between Bemisia tabaci MEAM1 and MED adults, and to investigate the insect-virus-host plant interaction so as to provide reference for the control of ToCV on C. annuum in the field. 【Methods】 The ToCV acquisition and transmission of B. tabaci MEAM1 and MED adults on C. annuum plants were compared by using B. tabaci MEAM1 and MED adults and cDNA infectious clones of ToCV, respectively. The feeding preference of B. tabaci MEAM1 and MED adults to healthy and ToCV-infected C. annuum plants was compared. 【Results】 When inoculated on ToCV-infected C. annuum plants for 96 h, the virus accumulation in B. tabaci MEAM1 and MED adults gradually increased, faster acquisition and higher accumulation of ToCV in MED adults than in MEAM1 adults were found, and the level of virus acquisition of MED adults was 1.74 times as high as that of MEAM1 adults. The level of virus transmission of MED adults on C. annuum plants was 10 times as high as that of MEAM1 adults, and the transmission rate of MED adults was 29% higher than that of MEAM1 adults. MEAM1 and MED adults showed similar feeding preference to healthy and ToCV-infected C. annuum plants, with an obvious feeding preference to ToCV-infected C. annuum plants. 【Conclusion】 The ability of B. tabaci MED adults to acquire and transmit ToCV on C. annuum plants is higher than that of B. tabaci MEAM1 adults, which is an important reason for the rapid spread of ToCV in China.