【Aim】 The effect of low-temperature acclimation on the contents of related physiologically active substances was determined in Octodonta nipae (Coleoptera: Chrysomelidae) to reveal its cold tolerance mechanisms. 【Methods】 We compared the contents of different physiological substances such as free water, protein, amino acids, crude fat, glycerol and soluble glycogen between the non-acclimation (25℃) and acclimation (acclimated at 12.5, 15.0, 17.5 and 20.0℃ for 10 d) treated O. nipae. 【Results】 The contents of related physiologically active substances in O. nipae were significantly affected by acclimation temperatures. With the decrease of the acclimation temperature, the contents of glycerol and amino acids increased, while those of the free water, protein, crude fat and glycogen decreased. Compared with the control, the contents of free water in O. nipae subjected to acclimation at 12.5℃ had significant changes (P<0.01). The contents of protein and crude fat significantly changed when O. nipae was subjected to low-temperature acclimation at 12.5, 15.0, 17.5 and 20.0℃, so did the content of the amino acids except under acclimation at 20℃ (P<0.01). Low temperature acclimation-mediated effects on glycerol content were found in O. nipae at different developmental stages except the 2nd instar larvae. Significant changes in the contents of soluble glycogen were determined in different developmental stages except the 2nd instar larvae, pupae and adults after being acclimated at 12.5, 15.0, 17.5 and 20℃, respectively. The average glycerol content in O. nipae after being acclimated at 15.0℃ was about 9.4 times higher than that at 25℃. However, after O. nipae was acclimated at 12.5℃, its average glycerol content increased only 3.5 times as compared with the control. 【Conclusion】 These results indicated that the effect of low-temperature acclimation on the contents of related physiologically active substances in O. nipae was limited. O. nipae could make the optimal physiological adjustments based on the adverse environment conditions to adapt the future environment and maximize their fitness.

【Aim】 To study the function and binding characteristics with plant volatiles of the odorant-binding proteins in the oriental fruit moth, Grapholita molesta (Busck).【Methods】 The OBP cDNA from G. molesta was cloned by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), its tissue and developmental expression profiles were detected by RT-PCR and Real-time PCR, and the protein binding property was analyzed using fluorescence competitive binding assay with N-phenyl-1-naphthylamine (1-NPN) as the fluorescent probe. 【Results】A novel OBP cDNA from G. molesta was obtained, which was named as GmolOBP3 (GenBank accession no.: KF395363). GmolOBP3 contains a 492 bp open reading frame encoding a 163-amino-acid residue peptide. The predicted molecular weight and isoelectric point are 18.72 kDa and 4.93, respectively. The mature protein GmolOBP3 includes six typical conservative cysteine residues, which are the hallmark of insect OBPs. GmolOBP3 was expressed in the antennae and abdomen of female and male adults. In five days after eclosion, the expression level of GmolOBP3 in antennae of female moths increased with day-old after eclosion, but on the 5th day after eclosion, the expression quantity in antennae of male moths significantly reduced. In order to obtain the recombinant protein GmolOBP3, we constructed the prokaryotic expression vector of GmolOBP3, which was successfully expressed in the optimized condition. The recombinant protein was purified by anion exchange and Superose-12. The binding affinity of GmolOBP3 with 16 plant volatiles and 4 sex pheromone analogs indicated that GmolOBP3 could not bind with (E)-8-dodecenyl acetate and 1-dodecanol, while had weaker affinity with (Z)-8-dodecenyl acetate and (Z)-8-dodecenol with the association constants of 83.00 and 103.70 μmol/L, respectively. GmolOBP3 could also weakly bind with 16 plant volatiles with the strongest affinity to β-ionone and with a association constant of 49.36 μmol/L. 【Conclusion】 We so inferred that GmolOBP3 protein have characteristics of selective binding with various ligands.

【Aim】 To clone the cDNA sequence of small nuclear RNA (snRNA) U6 from Plutella xylostella (L.) and evaluate the possibility of using the U6 as a reference gene in quantification of microRNAs (miRNAs). 【Methods】 In this study, the full-length cDNA of U6 was cloned from the 4th instar larvae of P. xylostella by using the reverse-transcription PCR (RT-PCR) method. The expression stability of U6 and other eight miRNAs of P. xylostella was evaluated in different developmental stages and after different pesticide treatments with qRT-PCR.【Results】The full-length cDNA of U6 is 94 bp and shares 98.9% nucleotide sequence identity with U6 from other known insects. The qRT-PCR results analyzed by geNorm and RefFinder softwares showed that the expression of U6 was quite stable among eggs, the 1st-4th instar larvae, pupae and adults of P. xylostella, as well as in the 3rd instar larvae of P. xylostella at 48 h after treatment with nine insecticides (malathion, chlorpyrifos, phoxim, methomyl, fufenozide, beta-cypermethrin, chlorantraniliprole, chlorfenapyr and Bt CrylAc) that possess different mode of actions. 【Conclusion】U6 of P. xylostella is highly conserved among species, and its expression level is not affected by developmental stages or insecticides treated. Therefore, U6 can be taken as a qualified reference gene to evaluate the expression level of miRNA or non-coding snRNA by quantitative RT-PCR. The results pave the way for the accurate determination of snRNA expression in P. xylostella.

【Aim】 Wolbachia bacteria are maternallyinherited endosymbionts frequently found in insects. They can alter their hosts’ reproduction via several forms. Cytoplasmic incompatibility (CI) is the most commonly reported phenotype caused by Wolbachia infection and induces developmental arrest of early embryos resulting from mating between Wolbachia-infected males and uninfected females. The molecular mechanisms of CI, however, are not clear currently. Our previous studies showed that the Mst84Db gene exhibited significant down-regulation in the 3rd instar larval testes of Drosophila melanogaster when infected by Wolbachia. The objective of this research is to further assay whether Mst84Dbgene is associated with CI. 【Methods】 In this study, we first synthesized theMst84Db dsRNA in vitro and microinjected into male flies. Then we tested the fertility of these injected males by crossing with wild type females. Gene expression levels were examined by quantitative RT-PCR (qRT-PCR). The embryonic phenotypes were analyzed by DAPI staining. 【Results】 The expression level of Mst84Db was significantly down-regulated since 24 h after injecting Mst84Db dsRNA. At 72 h after injection, the expression level of Mst84Db exhibited the maximum down-regulation relative to the control. The knockdown of Mst84Db in males significantly decreased the hatch rate of embryos when crossed with wild type females. This is similar to the phenotype in CI crosses. In addition, the embryonic cuticles showed almost no segmental development, indicating that some embryos stop development at early stages. Obvious CI phenotypes with asynchronized nuclear division and chromatin bridges were also observed in part of early embryos derived from Mst84Db down-regulated males. 【Conclusion】 Wolbachia infection may inhibit the expression of Mst84Db during spermatogenesis, thus reduces sperm fertility and leads to embryonic lethality. Down-regulation of Mst84Db in male Drosophila might be causally linked to CI.

【Aim】 The ADP/ATP carrier protein in microsporidian Nosema bombycis may be involved in carrying the energy from host cells to itself. In order to provide the theoretical basis for preventing and controlling the pebrine disease, the gene of N. bombycis ADP/ATP carrier protein was cloned and expressed in prokaryocytes. The antibody was prepared and indirect immunofluorescence assay was conducted. 【Methods】 The nucleotide sequences encoding three segmental peptides inside the membrane were synthesized with BglⅡ and SalⅠ sites at both ends (NbADP/ATP-△TM) and cloned into pUC57 vector for sequencing, and the target sequence was further subcloned into pQE40 vector with dihydrofolate reductase （DHFR） tag. The NbADP/ATP-△TM-DHFR was cut by BamHⅠ and SalⅠ and linked to pET30 vector for prokaryotic expression. The expression product was identified by SDS-PAGE, Ni-NTA affinity chromatography and immunoblotting. Indirect immunofluorescence assay was performed to survey the distribution of NbADP/ATP. 【Results】 The coding sequence of nbadp/atp (GenBank accession no. EOB13854.1) is 1 524 bp in length, encoding a 507-amino acid residue peptide with a calculated molecular weight of 59 kDa and a theoretical pI of 9.35. The ADP/ATP carrier protein of N. bombycis contains twelve transmembrane domains and the conserved TLC domain which has four conserved functional sites. Sequences blast analysis showed that the ADP/ATP carrier protein of N. bombycis shares 30% amino acid sequence identity with Nosema ceranae ADP/ATP carrier protein 2. Phylogenetic analysis placed the N. bombycis protein in the same subgroup as the ADP/ATP carrier. The recombinant plasmid NbADP/ATP-△TM-DHFR-pET30a was successfully constructed. SDS-PAGE analysis showed that the 37 kDa fusion protein was highly expressed and purified. Anti-NbADP/ATP serum was produced in mice with the purified protein. Immunoblotting result showed that NbADP/ATP was expressed in mature spores. Indirect immunofluorescence localization results revealed that ADP/ATP transporter protein is located in the plasma membrane. 【Conclusion】 This study provides a new clue for blocking energy source of N. bombycis to control and prevent pebrine disease of the silkworm.

【Aim】 After serial passages in some permissive insect cells, phenotype of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) has the potential to change from many polyhedra (MP) to few polyhedra (FP). This phenomenon is related to mutations occurred in a gene named fp25k which codes a 25 kDa protein. The AcMNPV fp25k mutants produce less occlusion-derived viruses (ODVs) and more budded viruses (BVs) than the wild type, usually resulting in reduction of foreign protein expression in the baculovirus expression vector system. To overcome the deficiency resulted from the mutations of fp25k in baculovirus expression vector system, we modified fp25k and constructed a transgenic insect cell line expressing FP25K protein. 【Methods】 The AcMNPV fp25k gene was modified in a site which is easy to be mutated during passage, and a modified fp25k, named mfp25k, was produced in this study. mfp25k  was combined to pIZT/V5-His vector, the recombinant vector was further used to transfect the Spodoptera frugiperda cells (Sf9), and Sf9 cells which were not successfully transformed were eliminated gradually by Zeocin selection. 【Results】 We successfully modified AcMNPV fp25k in TTAA site and got recombinant vector pIZT/V5-mfp25k. After transforming Sf9 cells and selecting with Zeocin, we finally got stable transgenic Sf9 cell line, Sf9-mfp25k, which contains mfp25k gene in the genome. Sf9-mfp25k  transgenic cells and normal Sf9 cells were both infected with AcP2, the AcMNPV fp25k mutant. The results indicated hat the transgenic Sf9-mfp25k cells expressing FP25K protein could rescue the phenotype of AcMNPV with fp25k mutations. 【Conclusion】 The transgenic Sf9-mfp25k  cells expressing FP25K protein could make up for the shortage of baculovirus caused by fp25k mutations. Our results provide a novel way to build stable baculovirus-insect cell expression system.

【Aim】To clarify the effects of methyl jasmonate (MeJA) treatments on the growth and detoxifying metabolic abilities of insects, and the effects of MeJA fumigation on the relationship between plant defense and insect detoxifying metabolism. 【Methods】 The influences of feeding MeJA and MeJA fumigation on the growth and the activities of detoxifying enzymes in the late 3rd instar larvae of H. armigera were examined, and the changes of detoxifying enzymes in H. armigera larvae, which fed the MeJA-sprayed cotton leaves after fumigation with MeJA, were also studied. 【Results】 Feeding MeJA and MeJA fumigation produced different effects on the growth and the detoxifying abilities of H. armigera larvae. Feeding the artificial diet containing 2.9 μg/g MeJA inhibited the growth of H. armigera larvae, and induced 1.92-fold increase in P450 activities. However, after fumigation with MeJA, no significant effects on the growth of H. armigera larvae were observed, while the activities of P450 and carboxylesterases (CarEs) increased to 2.94 and 1.16 times as high as the control, respectively. The P450 activities in H. armigera larvae, which experienced MeJA fumigation firstly and then fed the normal or MeJA-sprayed cotton leaves, were significantly induced, and H. armigera larvae, which fed the MeJA-sprayed cotton leaves after fumigation with MeJA, exhibited the highest P450 activity. 【Conclusion】MeJA fumigation induces the P450 related detoxifying abilities, and increases the detoxifying abilities in H. armigera against cotton defense responses induced by MeJA.

【Aim】 To clarify the resistance mechanisms of Tetranychus truncatus Ehara to pyridaben by detecting the change of detoxification enzyme activities and the synergism of synergists to pyridaben. 【Methods】 The experiment was carried out in laboratory by biological assay. The specific activity, the Michaelis constant (K_m) and the maximum reaction velocity (V_max) of several detoxification enzymes in pyridabenresistant strain (Py-R) and susceptible strain (SS) were detected by microplate reader. Then the synergistic effects of piperonyl butoxide (PBO), diethyl maleate (DEM) and icresyl phosphate (TPP) to pyridaben were detected in Py-R and SS.【Results】 The resistance of Py-R at the 49th generation selected with pyridaben increased to 955.25-fold as high as that of SS. The piperonyl butoxide(PBO), triphenyl phosphate (TPP) and diethylmaleate (DEM) enhanced the toxicity of pyridaben, and the relative synergistic coefficients of 3 synergists to pyridaben were 95.97%, 85.14% and 97.37%, respectively. The activities of carboxylesterases (CarE), glutathione-Stransferase (GSTs) and mixed function oxidase (MFO) in Py-R significantly increased compared with those in SS (P<0.05), while the activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) had no significant difference between Py-R and SS (P>0.05). The Km values of CarE, GSTs and MFO in Py-R were lower than those in SS, while their V_max values in Py-R were higher than those in SS. 【Conclusion】 The increased activity of CarE, GSTs and MFO, and higher affinity with substrate might be the main resistance mechanisms to Py-R of T. truncatus. When 3 synergists (PBO, TPP and DEM) and pyridaben are mixed, a higher level of virulence to T. truncatus would be exhibited.

【Aim】 In order to evaluate the resistance risk of the cotton aphid, Aphis gossypii Glover, to imidacloprid, the selection and realized heritability analysis of resistance to imidacloprid in A. gossypii were performed in the laboratory. 【Methods】 The imidaclopridsusceptible and resistant strains were developed by individual reverse and group selection, with the LC₅₀ values of 0.176 and 14.657 mg/L，respectively. The realized heritability (h²) of A. gossypii resistance to imidacloprid was estimated by Tabashnik’s method. 【Results】 Compared with the LC₅₀ value of the original field strain of A. gossypii (0.346 mg/L), the LC₅₀ value of the imidacloprid-susceptible strain had a 2-fold decrease. The resistance index of the imidacloprid-resistant strain was 83.27-fold as high as that of the imidacloprid-susceptible strain after 40 generations of selection. The realized heritability (h²) of A. gossypii resistance to imidacloprid was 0.1478. It was predicted that A. gossypii would require 30.2 to 38.1 generations to obtain 100-fold resistance to imidacloprid under breeding pressure of imidacloprid with 80%-90% mortality for each generation of selection. 【Conclusion】 The results suggest that A. gossypii has resistance risk to imidacloprid.

【Aim】 To elucidate the resistance mechanisms of different rice varieties (i.e., Fengyuanyou 272, R9810-T, Huahuiyihao, Minghui 63, Manuogu and Rathu Heenati) against the white-backed planthopper, Sogatella furcifera. 【Methods】 Electronic penetration graph (EPG) was used to record the feeding behavior of S. furcifera on the seedlings of these six rice varieties at the 3rd leaf stage. Nine non-phloem variables and 22 phloem variables based on typical EPG waveforms were considered in the analysis. 【Results】 During 8 h recordings, the total time of non-penetration waveform (np) on Rathu Heenati was the longest , followed by that on Manuogu, their total time of np had significant difference, and both of them were significantly longer than that on the other four varieties (P<0.05). The total time of pathway waveform (Nc) on Rathu Heenati (8 523.41 s) was 1.24 times longer than that on susceptible variety Minghui 63. S. furcifera spent significantly less time on Rathu Heenati for phloem sap ingestion (N4-b) than other varieties. All the EPG variables on Huahuiyihao or R9810-T had no significant difference from those of susceptible variety Minghui 63 (P≥0.05). Longer average duration of watery salivation (N4-a) followed by long time of sap ingestion was recorded on Fengyuanyou 272. 【Conclusion】 It was inferred that antixenosis components and resistance factors out of or in the phloem region might prevent this insect pest from feeding on Rathu Heenati. Only antixenosis components might restrict S. furcifera to feed on Manuogu. However, Huahuiyihao and R9810-T might not possess obvious resistance to S. furcifera, and Fengyuanyou 272 might be a more susceptible variety than Minghui 63. Combined with the correlation between feeding behavior of insect vector and transmission of persistent plant viruses, these results also provide some information for control of Southern rice black-streaked dwarf virus (SRBSDV) by using S. furcifera-resistant rice varieties.

【Aim】 Bactrocera is the most economically significant genus of tephritid flies. The present study aimed to make molecular identification and phylogeny analysis of common species of the genus Bactrocera. 【Methods】 Twenty-one species belonging to eight subgenera of the genus Bactrocera that were frequently intercepted from customs were identified using DNA barcoding technology based on mitochondrial cytochrome c coxidase subunit I (COI) gene. The partial fragments (about 650 bp) of the mtDNA COI gene were amplified using DNA barcoding universal primers. The obtained COI gene fragments were sequenced and aligned. The phylogenetic tree was established by a neighbor-joining (NJ) method. The intra-and inter-species genetic distances were calculated with MEGA version 5.0 software using the Kimura 2-parameter model. 【Results】 The identification results based on the phylogenetic tree were consistent with those based on morphological analyses for the 21 fruit fly species. In these species, 11 species of Bactrocera formed monophylies of themselves, the other 10 species shared one monophyly， and all bootstrap values of the original divergence among different haplotypes within the same species were over 99%. The average inter-species genetic distance between the 21 species was 35.8 times higher than the average intraspecies genetic distance of the 10 species (0.1540 vs. 0.0043). There was no overlap between intra- and inter-species genetic distances. 【Conclusion】 The results indicated that the DNA barcoding based on the partial sequence of mtDNA COI gene cauld provide rapid and accurate identification of Bactrocera species. The technology could be used in identifying and monitoring tephritid fruit fly species.

 Cis-regulatory sequences are parts of the non-coding DNA regions that are associated with regulation of gene expression, and they can be recognized and bound specifically by their regulatory factors. Cis-regulatory sequences can precisely regulate gene expression patterns spatially and temporally by increase or decrease of transcriptional binding sites and reorganization of gene regulatory networks, and then cause animals to develop different physical and morphological consequences. Cis-regulatory hypothesis considered that evolution of cis-regulatory sequences is the main genetic mechanism responsible for evolution of a variety of animal phenotypes in nature. Firstly this review briefly describes the structure and function of cis-regulatory sequences, and then focuses on the recent advances in evolution of cis-regulatory sequences regulating Drosophila phenotypes, such as bristles, pigmentations and embryo development, which demonstrate that evolution of cis-regulatory sequences is one of the major mechanisms responsible for evolution of animal phenotypes. Finally, the review suggests the future research directions for the cis-regulatory sequences, such as functional genomics and encyclopedia of DNA elements, which provide a good reference for the cis-regulatory sequence research in evolutionary developmental biology.

Autoparasitoids (heteronomous hyperparasitoids) are parasitoids whose males and females are parasitic in different species of hosts. Female eggs develop as obligate primary parasitoids, while male eggs develop as hyperparasitoids. The sex ratio of offsprings produced by females depending on the type of hosts is affected by host density, as well as the relative abundance of the primary and secondary hosts. Autoparasitoids can suppress the population of pests by parasitizing and feeding on the primary hosts. Also they can parasitize and feed on the hosts that have been parasitized by conspecific or heterospecific females, leading to the lethal interference competition. The window of secondary hosts for hyperparasitizing is at late instar larval to prepupal stage. Autoparasitoids prefer to hyperparasitize and feed on heterospecific hosts than conspecific hosts or have no obvious parasitization tendency. As a result, the specific reproductive pattern has led the application of autoparasitoids in biological control to become the focus of controversy. We should evaluate both the positive and negative influences caused by autoparasitoids before using it in biological control. In this article, we reviewed the recent advances on the reproductive traits of autoparaitoids, lethal interference competition effects on secondary hosts, and their influences on biological control. This will provide a theoretical foundation for the optimum use of autoparasitoids in integrated pest management.

【Aim】 Calliptamus italicus is a serious plague in arid and semiarid grasslands of Xinjiang. Previous studies claimed that its outbreak is closely related to climate warming in Xinjiang since 1980s. This research aimed to explore the effect of temperature increasing on the respiratory metabolism of C. italicus.【Methods】 We detected the respiratory rate, metabolic rate, CO₂ release rate, Q₁₀ (rangeability of the respiratory rate per 10℃ rising) and the respiratory quotient of C. italicus adults by using Sable Systems at different temperatures (15, 20, 25, 30 and 35℃). 【Results】 The respiratory rate, metabolic rate and CO₂ release rate of the locust all increased as the temperature went up, with the values at 15℃ significantly lower than at the other temperatures (P<0.01), but significantly higher at 35℃ (P<0.01), suggesting that both the conditions below 15℃ and over 35℃ have significant impacts on C. italicus. However, the respiratory rate, metabolic rate and CO₂ release rate measured between 20℃ and 30℃ were not significantly different (P>0.01), revealing that this temperature range is suitable for C. italicus development. The values of respiratory rate (Q₁₀) at different temperatures showed that C. italicus is sensitive to temperature change. The Q₁₀ value reached the maximum (2.42) when the temperature ranged from 25℃ to 35℃, and the minimum (1.03) from 20℃ to 30℃, suggesting that the temperature ranging from 20℃ to 30℃ is the optimal condition for C. italicus development. With an average of 0.9450, the respiratory quotient has no significant difference among different temperature treatments (P>0.01), suggesting that carbohydrates are consumed during respiration. 【Conclusion】 Temperature between 20℃ and 30℃ is suitable for growth and development of C. italicus, and the continuing warming would still make C. italicus as one of the important pests in Xinjiang grasslands.

 【Aim】 Athetis lepigone (Moschler) is a new insect pest of summer corn at seedling stage in North China. With the transformation of cultivation, the damage area of A. lepigone has been gradually expanding. In this study, the cold hardness of A. lepigone was measured to reveal its cold resistance mechanisms. 【Methods】 We measured body weight, the supercooling point (SCP), the freezing point (FP) and the contents of water, fat and glycogen in mature larvae at different overwintering stages (the early overwintering stage, November 7th, 2012; the overwintering stage, January, 20th 2012; while the late overwintering stage, March 5th, 2013). 【Results】 The SCP values on different dates during overwintering were significantly different, the lowest SCP value (-23.16±0.38℃) appeared in January， while the highest (-16.24±1.24℃) appeared at the beginning of winter. The variation tendency of FP value was consistent with that of SCP. According to the quantitative detection, we found that the fresh body weight had no correlation with SCP (r=0.17, P=0.12). The highest fat content appeared at the beginning of winter, while the lowest appeared at the late overwintering stage. The lowest glycogen content appeared at the overwintering stage. Supercooling capability enhanced as the free water content decreased, while reduced as the free water content increased. However, the change of bound water content was opposite to that of the free water content. 【Conclusion】 The cold hardiness of overwintering larvae of A. lepigone changes significantly at different overwintering stages, which gradually strengthens as temperature decreases in winter and weakens as temperature increases after overwintering.