【Aim】 This study aims to further explore the possible role and mechanism of G-quadruplex (G4) structure in the regulation of the embryonic development of Bombyx mori by searching for the binding protein of the G4 structure of the embryonic development factor (EDF) gene BmEDF of B. mori. 【Methods】 Circular dichroism (CD) and electrophoretic mobility shift assay (EMSA) were performed to verify whether G4 sequence formed G4 structure in vitro. The effect of the G4 structure of the BmEDF promoter on the regulation of BmEDF expression was verified by the promoter activity experiment. qRT-PCR was performed to determine the expression levels of BmEDF in B. mori embryo at different developmental stages. The protein that could bind to the G4 structure of BmEDF was analyzed by EMSA combined with mass spectrometry. Then, the two candidate proteins BmeIF4H and BmADDH binding with G4 structure were cloned, expressed and purified, respectively. The binding of the candidate proteins BmeIF4H and BmADDH to the G4 structure of BmEDF was verified by EMSA. 【Results】 CD and EMSA experiments demonstrated that the G4 sequence of BmEDF could form G4 structure in vitro. Promoter activity experiments showed that the presence of the G4 structure of BmEDF had a positive regulatory effect on the transcriptional expression of BmEDF. The qRT-PCR results showed that the expression level of BmEDF was significantly increased at 120 h after oviposition. The recombinant BmeIF4H and BmADDH were obtained by prokaryotic expression and purification. EMSA experiments showed that the recombinant BmeIF4H bound to the G4 structure of BmEDF in vitro, while BmADDH did not bind to the G4 structure of BmEDF. 【Conclusion】 The BmeIF4H protein in B. mori embryo may bind to the G4 structure of BmEDF. This study provides the experimental evidence for analyzing the regulatory mechanism of DNA advanced structure in the embryonic development of B. mori.

 【Aim】 This study aims to clone the transcript variant X3 of Pangolin isoforms A/H/I/S (Pangolin X3), a key gene downstream of the Wnt signaling pathway in the silkworm, Bombyx mori, and analyze its sequence and expression characteristics. 【Methods】 Pangolin X3 of B. mori was retrieved from the NCBI database, and the primers were designed according to its coding sequence (CDS). Pangolin X3 was cloned from the midgut and hemolymph of B. mori larvae using PCR, and then verified by sequencing. The sequence characteristics of Pangolin X3 were analyzed using SilkDB 3.0, SMART, multiple sequence alignment and phylogenetic tree. The relative expression levels of Pangolin X3 in different tissues (head, hemolymph, integument, gonads, midgut, anterior silk gland, middle silk gland, posterior silk gland, fat body and Malpighian tubules) of the day-3 5th instar larvae of B. mori were analyzed using qRT-PCR. 【Results】 The CDS of Pangolin X3 (GenBank accession no.: XM_038020921) was cloned from the midgut and hemolymph of B. mori larvae. It has an open reading frame of 1 560 bp in length, encoding 519 amino acid residues with the predicted molecular weight of 55.86 kD and the isoelectric point of 7.53. Pangolin X3 contains a conserved β-catenin binding site and an HMG domain, its amino acid sequence is relatively conserved among different insects, especially the HMG domain that binds to DNA, but some amino acid residues in the N-terminal CTNNB1 domain that binds to β-catenin are variable. Tissue expression profile revealed that the relative expression levels of Pangolin X3 in the midgut, hemolymph and gonads of the day-3 5th instar larvae of B. mori were high, while those in the head, integument, anterior silk gland, middle silk gland, posterior silk gland, fat body and Malpighian tubules were low. 【Conclusion】 In this study, we cloned Pangolin X3 of B. mori and analyzed its sequence and expression characteristics, providing a basis for an in-depth study of the biological function of Pangolin in B. mori.

 【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】 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 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 of 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.

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.

Ticks are zoonotic ectoparasites transmitting pathogens to hosts via biting and feeding on hosts, which are associated with various diseases. Coagulation reaction is an important physiological process and plays critical roles in physiological hemostasis in humans and animals. During the long time of biting and feeding processes on hosts, ticks can secrete a variety of anticoagulants to inhibit coagulation reactions, which would guarantee the ticks to have blood meals for a long time. To date, the major tickderived anticoagulants which have been identified and recognized according to their functions include protease inhibitors, fibrin(ogen)olytic agents, platelet aggregation inhibitors, and vasoactive proteins. These anticoagulants can act on the intrinsic pathway, extrinsic pathway, and key steps in the common pathway of the coagulation cascade, promote fibrinolysis, and inhibit platelet activation, thereby inhibiting the coagulation response in the host blood vessels. Protease inhibitors mainly inhibit the activities of thrombin and factor Xa in the common pathway of coagulation cascade. Fibrin(ogen)olytic agents cause the hydrolysis of fibrinogen and delay the formation of fibrin clots. Platelet aggregation inhibitors can suppress platelet aggregation by degrading platelet aggregation agonists and binding thromboxane A2 (TXA2) and αIIBβ3 integrin on platelet surface. Vasoactive proteins inhibit host vasoconstriction, and wound healing and angiogenesis. In addition, there are some other protein molecules secreted by ticks that pose anticoagulation effects through different pathways. In this article, we summarized the anticoagulant proteins and small molecules found in ticks so far and their anticoagulant mechanisms, which will promote understanding of the mechanism and physiological significance of anticoagulation process in hosts triggered by ticks, and will provide important reference for developing new tickderived anticoagulant drugs and new antithrombotic therapies.