Bursicon is a neurohormone that regulates cuticle sclerotization (tanning and hardening) and the wing expansion processes in insects. Although bursicon genes have been well characterized from several insect species or predicted from insect genomes, bursicon and its genes in the diamondback moth (DBM), Plutella xylostella, remain poorly understood. In this study, two bursicon genes, Pxbursα and Pxbursβ (GenBank accession numbers: KF498645 and KF498646, respectively), were cloned from P. xylostella with the fulllength cDNA of 537 bp and 360 bp, respectively. The nucleotide sequence identities of Pxbursα and Pxbursβ with the bursicon genes from other insect species range from 51% to 68%, and 37% to 57%, respectively. Based on qRT-PCR results, we found that bursicon genes were expressed at a higher level in pupae, but at lower levels in larvae and adults. When the 4th-instar larvae were fed with double-stranded RNA of Pxbursα, the expression of Pxbursα in pupae subsequently declined significantly. Thus, the development of pupae was seriously delayed, and pupae died before eclosion. We inferred that higher level expression of Pxburs genes in pupae might play a key role in the metamorphosis and wing expansion of P. xylostella.

【Aim】 To study the binding function of Acer-ASP1, a pheromone binding protein (PBP), in the Chinese honeybee (Apis cerana cerana) with pheromone and other plant volatiles. 【Methods】 In order to obtain the recombinant protein (Acer-ASP1), we successfully constructed the cloning and prokaryotic expression vector of Acer-ASP1, which was expressed in the optimized conditions. After the recombinant protein with biochemical activities was purified, the binding capability of Acer-ASP1 with pheromone and other odors was measured using competitive fluorescence assay where 1-NPN was applied as fluorescence probe. 【Results】 Seven of the 22 ligands tested showed stronger binding capability with Acer-ASP1 and were able to decrease the relative fluorescence intensity of 1-NPN by more than 50%. Among them, methyl 4-hydroxylbenzoate, a queen pheromone, showed the strongest capability to compete with 1-NPN, causing 99.31% reduction in the relative fluorescence intensity, and K_D=13.39 μmol/L; vanillyl alcohol, another queen pheromone, caused 95.5% decline in the relative fluorescence intensity, and K_D=98.44 μmol/L. Nevertheless, AcerASP1 did not bind with other kinds of pheromone at all except queen pheromone. In addition, five plant volatiles, i. e., methyl salicylate, phenylacetaldehyde, 3,4-dimethyl-benzaldehyde, 4-allylveratrole and β-ionone, showed capabilities to bind with ASP1 in various degrees. 【Conclusion】 The results indicate that Acer-ASP1 exhibits remarkable specificity with queen pheromone, and it can bind with some plant volatiles to some extent. This implies that Acer-ASP1 is probably a protein having complex physiological function, in which recognizing queen pheromone is deemed as its main function while recognizing plant volatiles its secondary function.

【Aim】 In this study, we cloned a zinc transporter 7-like (ZnT-7-like) gene in Apis mellifera carnica in order to prepare the polyclonal antibody against recombinant ZnT-7-like, and to understand ZnT-7-like gene in hypopharyngeal glands of A. m. carnica differentially expressed in different periods. 【Methods】 The ZnT-7-like gene in A. m. carnica was amplified by RT-PCR method from the total RNA of hypopharyngeal glands. To avoid the interference of transmembrane domains, the partial sequence (273 bp) was chosen as peptide sequence through bioinformatics analysis, then sub-cloned into prokaryotic expression vector pGEX-4T-1 and expressed in E. coli BL21 (DE3) host cells. Fusion protein was purified and used to immunize New Zealand white rabbits so as to prepare polyclonal antibody. We also detected the sensitivity and specificity of the polyclonal antibody prepared through indirect ELISA and Western blot methods, respectively. Finally, the expression levels of this gene in different day-old adults were detected by real-rime quantitative RT-PCR and Western blot methods. 【Results】 ZnT-7-like gene was cloned from A. m. carnica , and the obtained fragment was 1 065 bp in length. SDS-PAGE electrophoresis showed that the fusion protein was well expressed. The anti-ZnT-7-like polyclonal antibody showed high sensitivity (1∶64 000) and specificity. There was a big difference in the transcriptional level of ZnT-7-like gene in different day-old A. m. carnica adults. The expression level of 3-day-old bees was extremely significantly higher than that of other ages (P<0.01), the 12-day-old bees showed the minimum expression level, and significant (P<0.05)or extremely significant differences (P<0.01) existed among 6-day-old, 9-day-old and 12-day-old bees. The protein expression level of ZnT-7-like was generally consistent with the transcription level. 【Conclusion】 The ZnT-7-like gene was successfully cloned, the rabbit anti-A. m. carnica ZnT-7-like polyclonal antibody was prepared, and the relative expression of ZnT-7-like in hypopharyngeal glands of different day-old A. m. carnica adults were measured at the transcription and translation levels. These results lay the foundation for further studies on functions of A. m. carnica ZnT-7-like gene.

The sophisticated olfactory system of insects plays a crucial role in their behaviors, such as host seeking, mating, oviposition, as well as escaping behaviors. Therefore, the mystery of arousing a series of insect behavior reactions by perceiving and distinguishing odorants in the environment will be unveiled by investigating olfactory receptive mechanism. In order to clarify the mechanism of interaction between olfactory associated proteins in Holotrichia oblita Faldermann, the HoblOBP2 bait was constructed using DUALhunter Starter Kits. Then, the normalized cDNA library from H. oblita antennae was screened. By detecting β-galactosidase activity and the Blast analysis in GenBank, six interactors were identified using HoblOBP2 bait. We inferred that the proclotting enzyme is the strongest positive interactor, which might play a role in the process of olfactory recognition.

The Asian corn borer, Ostrinia furnacalis (Guenée), is one of the most important insect pests of maize in China. Transgenic Bacillus thuringiensis (Bt) maize provide an effective mean to control this insect pest. However, evolution of resistance in the target insect will be a great threat to the continued success of Bt toxins used in insecticide formulations or expressed by transgenic maize. It is theoretically and practically important to well characterize resistant strains, which will provide the only way to empirically validate proposed management strategies. A strain of O. furnacalis was selected for resistance to Cry1Ie by exposure to the toxin incorporated into artificial diet in the laboratory. The resistance development and cross-resistance to other Bt toxins such as Cry1Ab, Cry1Ac and Cry1Fa after 14 generations of selection were determined by bioassay. In addition, the changes in biology of O. furnacalis were also observed. The results indicated that the susceptibility of the selected strain to Cry1Ie toxin declined as the selection pressure increased. The selected strain developed higher than 23-fold resistance to Cry1Ie after 14 generations of selection. However, it was as susceptible to Cry1Ab, Cry1Ac, and Cry1Fa as the unselected control strain. In addition, compared with the unselected strain, the larvae of selected strain required an average 5.7 d longer duration, the pupal weight reduced by 13.7%, and the number of eggs laid per female decreased by 40.0%. These results suggest that the widespread use of transgenic Cry1Ie maize could lead to the development of resistance in target pest O. furnacalis. The lack of cross-resistance between Cry1Ie and Cry1Ab, Cry1Ac or Cry1Fa suggests that maize hybrids expressing these two toxins (Cry1Ie and Cry1Ab, Cry1Ac or Cry1F) are likely to be compatible for resistance management of O. furnacalis.

【Aim】 To clarify the development of resistance to imidacloprid and the related enzyme mechanism of cross-resistance to other neonicotinoids, clothianidin and acetamiprid, the imidacloprid-resistant strain of the contton aphid, Aphis gossypii, was continuously selected in the laboratory after we found a mutation in nicotinic acetylcholine receptor (nAChR) β1 subunit. 【Methods】 Leaf dipping method was used to select the imidaclopridresistant strain of A. gossypii and to test the cross-resistance to acetamiprid and clothianidin. The LC₂₀ of acetamiprid and clothianidin were applied to the imidacloprid-resistant strain, and the effects on activities detoxifying enzymes and biological characteristics were determined with biochemical method and the systematic observation, respectively. 【Results】 The resistant strain of A. gossypii was continuously selected with imidacloprid for 75 generations and the resistance ratio reached to 72.6-fold as high as that of the susceptible strain. When resistance selection was suspended in the next successive 12 generations, the resistance of aphids just decreased to 72.0-fold as high as that of the susceptible strain. By generation F₇₅₊₁₂, the strain showed 11.9-fold and 20.1-fold cross-resistance to acetamiprid and clothianidin, respectively. Clothianidin could significantly inhibit the activities of CarE, GST and AChE of the imidacloprid-resistant cotton aphid, while acetamiprid showed little effect on their activities. Clothianidin had stronger inhibition on honeydew excretion and bodyweight gain of A. gossypii than other two insecticides. 【Conclusion】 The results demonstrate that the imidacloprid-resistant mutation strain not only has high-level resistance to imidacloprid, but also the resistance can be stably inherited. Therefore clothianidin has a great application value in control of the imidacloprid-resistant mutation strain of cotton aphid.

【Aim】 To screen suitable reference genes for studying the expression levels of CYP392E subfamily genes in susceptible (SS), avermectin-resistant (Av-R) and spirotetramat-resistant (Sp-R) strains of Tetranychus urticae. 【Methods】 The mRNA expression stability of six house keeping genes, i. e.,  α-tubulin, β-actin, ELFn, GAPDH, 5.8S rRNA gene and SDHA of T. urticae was detected by quantitative real-time PCR (qRT-PCR), and the relative expression level of CYP392E subfamily reference genes of cytochrome P450 monooxygenases (P450s) was analyzed. 【Results】 ELFn was the most stable reference gene in SS, Av-R and Sp-R strains of T. urticae. When ELFn was used as the reference gene, the expression level of CYP392E7 in Av-R strain (2.18-fold as high as that in SS strain) was significantly higher than that in SS strain, while there was no significant difference between Sp-R strain and SS strain. However, the relative expression levels of other genes did not increase, and even those of CYP392E4, CYP392E9 and CYP392E10 in Av-R strain and CYP392E1 and CYP392E9 in Sp-R strain decreased significantly (P<0.05) (48%, 74%, 65%, 63% and 73% of the expression level in SS strain, respectively). 【Conclusion】 ELFn could be used as an ideal reference in SS, Av-R and Sp-R strains of T. urticae. The significant change of the expression levels of CYP392E4, CYP392E7, CYP392E9 and CYP392E10 in Av-R strain may be related with the resistance of T. urticae to avermectin; CYP392E1 and CYP392E9 in Sp-R strain may also be related with the resistance of T. urticae to spirotetramat.

To understand the effects of temperatures and photoperiods on diapause induction of Agrypon flexorium (Thunberg), the process of induction, the sensitive stage that was susceptible to photoperiod and the effect of low temperature on the storage was investigated. The results showed that when the temperature was between 17-23℃ and day length was 10-14 h, the diapause rate significantly increased with the decrease of temperature and day length. High temperature counteracted the effect of short day length. When the temperature was 26℃, photoperiod had no effect on diapause. Thus, lower temperature and shorter day length were the major factors that induced diapause of the parasitoid. The parasitoid accepted the signal for diapause at the egg and 1st instar larval stages. The pre-pupae entered diapause only if the eggs and 1st instar larvae had been reared in the temperature and the photoperiod for diapause induction till they matured. The emergence rate and the parasitic ability showed no significant difference between adults emerged from the diapause cocoons stored in the temperature of 4℃ for 80 d and those from the nondiapause cocoons without cold storage. After cold storage for 120 d, the emergence rate of the diapause cocoons still reached 71.7%. The results suggested that the photoperiod of 8L∶16D at 17℃ had diapause induction effect on parasitized host larva. The optimal storage time of diapause cocoons is 80 d and should not be more than 120 d. The study provided some reference information for indoor propagation, preventing wasp degeneration and field releasing.

【Aim】 To demonstrate the effect of transgenic Bt-crop on the bionomics of the tachinid fly Exorista civilis Rondani, the parasitism rate and life history parameters of E. civilis developed from larvae of the oriental armyworm, Mythimna separata (Walker), fed on artificial diets with Cry1Ab toxin were investigated in laboratory. 【Methods】 M. separata larvae were fed on diets containing 0, 3.125, 6.25, 12.5 and 25 μg/g of Cry1Ab toxin, respectively, and used as the host for E. civilis. The parameters related to the parasitism and the growth and development of E. civilis on the host were observed and recorded. 【Results】 The results showed that the parasitism rate and fecundity of the fly on M. separata larvae feeding on diets containing various doses of Cry1Ab protein were insignificantly different (P≥0.05), suggesting that host selective behavior of the E. civilis is not affected by the dose of Bt-toxin in the caterpillar. The developmental period (egg-larva, pupa, adult oviposition and generation), emergence rate, sex ratio, and fecundity of E. civilis developed from M. separata larvae treated by various concentrations of Cry1Ab toxin were insignificantly different (P≥0.05). However, the survival rate of the maggots in the host feeding on diet containing 3.125 μg/g of Cry1Ab toxin was significantly greater than that of the control and the rest treatments (P<0.05), while insignificantly different among the remained 4 treatments (P≥0.05). The pupal weight significantly declined as the concentration of Cry1Ab toxin fed by their hosts increased (P<0.05). The pupal weight in the control group was 49.8 mg, while that in treatment groups of 6.25 and 25 μg/g Cry1Ab toxin was only 41.0 and 36.7 mg, respectively. 【Conclusion】 These results indicate that parasitizing host M. separata larvae fed with Cry1Ab toxin has no dramatically negative effect on parasitoid E. civilis. This study provides a basis for the evaluation of the risk of transgenic Bt-crops on the tachinid parasitoids in fields.

In order to investigate the molecular mechanisms of defense responses of kidney bean to feeding of the western flower thrips (WFT), Frankliniella occidentalis (Pergande), the expression of defense enzyme genes (LOX, AOS, PAL and PR-2) in kidney bean leaves induced by WFT feeding, mechanical damage, exogenous methyl salicylate (MeSA), and exogenous jasmonic acid (JA), respectively, were detected by real-time quantitative PCR (RT-qPCR). The results showed that the expression level of lipoxygenase gene (LOX) reached the peak at 24 h after JA induction and at 48 h after mechanical damage, respectively, but was not significantly different from the controls (P>0.05). LOX was hardly expressed after kidney bean leaves were treated by MeSA. The relative expression level of LOX was about 41.9 times as high as that of the control at 24 h after WFT feeding and was significantly higher than that in other three treatments (P<0.05). The allene oxide synthase gene (AOS) was expressed at a relatively lower level in different treatments and was barely expressed in the entire time of MeSA induction. The phenylalanine ammonialyase gene (PAL) was scarcely expressed during the induction of mechanical damage and JA, and reached the maximum, and its expression was 1.9 times as high as that of the control at 24 h after MeSA treatment. The expression level of PAL rose rapidly after WFT feeding, and was about 4.3 times as high as that of the control at 24 h, significantly higher than in other three treatments (P<0.05). The expression of β-1,3-glucanase gene (PR-2) was suppressed during the induction of WFT feeding and JA. The expression level of PR-2 increased at 24 h after mechanical damage and then decreased during the induction. The expression level of PR-2 dramatically increased in 24 h after MeSA induction, and was nearly 6.63 times as high as that of the control, significantly higher than that in other three treatments (P<0.05), but hardly expressed after 48 h. The findings suggest that WFT feeding could induce the SA- and JA-mediated signal transduction pathways and there are crosstalks between them.

This study aimed to explore the damage process of Apopestes spectrum (Esper) to murals in Mogao grottoes, so as to provide the effective scientific basis for the prevention of this dominant insect pest. Combined with microclimatic environment monitoring in Mogao grottoes, the spatial distribution characteristics and preferences of A. spectrum adults to murals of different pigments in a simulated cave inside cave 53 of Mogao grottoes were observed simultaneously. The environmental monitoring results indicated that in the peak active season of A. spectrum adults from August to September 2011, the temperature and relative humidity were very close in the simulated cave and cave 53, which was 17.97℃ and 18.02℃, and 37.04% and 37.13%, respectively, suggesting that the artificial cave can simulate the environmental condition of natural cave preferably. The observation results of spatial distribution showed that the numbers of A. spectrum adults differed greatly on mural surfaces in different orientation of simulated cave, distributed more on the upper southern wall and two sides of the eastern wall than on the ground, the ceiling, and the upper western wall. Tests of preferences of moths towards murals of different pigments indicated that there were distinct differences in the preference of A. spectrum adults towards different mural pigments, with the largest number of adults on briquettes with indigo, iron oxide red, and ink, and the less number of adults on briquettes with the pigments red lead and malachite. The Pearson correlation analysis showed that the number of adults on mural surfaces with different pigments had a significantly negative correlation with lightness of pigments, suggesting that lightness of pigment surface is one of the key factors that influence the tendency of A. spectrum adults towards pigments. This study can provide a significant reference for studying insect damage mechanisms to wall paintings in other cavetemple sites.

The Proxenus lepigone, a new pest insect of maize production in China, suddenly outbroke in the Huang-Huai-Hai plain in 2011, which threatened the production of summer maize. In order to know the population dynamics of P. lepigone to ascertain whether this species could migrate or not, monitoring on P. lepigone moths was respectively carried out in Luancheng, Hebei province, and the urban and Yanqing county of Beijing with searchlight traps and vertical-looking insect monitoring radar. The properties of their source were determined by combining the population sizes with the meteorological data. From this study, we clarified that the mature larvae with cocoon could overwinter in Yanqing, Beijing. During the monitoring season in 2012, the trapped moths summed up to 33 951, which included 3 generations. Population size of the 1st generation did not meet the normal distribution, and differences of catch sizes in sister-light traps suggested that P. lepigone is a migratory species. The environment was not suitable for P. lepigone in Yanqing, Beijing, but the average trapped catch size was higher than that of Luancheng, Hebei. Furthermore, the catch sizes were considered to be related to the direction of airflow and P. lepigone moths could be the targets of echoes of radar. These evidences approve that the trapped moths of the 1st generation must include the individuals migrating from surrounding regions and this species is supposed to be a facultative migratory species. This study provides some migration events for the future study of this species, which would benefit the related forecasting and integrated control.

Three species of the genus Liaopodisma Zheng, 1990 are described and illustrated from China, including two new species, i. e., Liaopodisma taichungensis sp. nov. and Liaopodisma taiwanensis sp. nov. The new species L. taichungensis sp. nov. is allied to L. qianshanensis Zheng, 1990, but differs from the latter by: black postocular band indistinct, midian keel of pronotum almost invisible, length of tegmen 1.8 times width in male, interspace of mesosternum of male backward broadening gradually and hind tibia with 10 spines on outer side. The new species L. taiwanensis sp. nov. differs from two known species of the genus Liaopodisma Zheng by the hind femur red on lower side and tegmen not widened in the middle, anterior and posterior margin almost parallel. A key to species of the genus Liaopodisma is also given.

Insect olfactory receptors are important proteins which are required for the insect olfactory identification process. As a receptor strikingly different from all other insect olfactory receptors, Orco is highly conserved in insect species and widely expressed. Orco plays a critical role in insect olfaction. Insects show severe olfaction defects when Orco is silenced by gene targeting or RNA interference. Orco can form heteromeric complexes by binding the conventional olfactory receptors rather than binding odorant ligands to facilitate the localization and stability of the conventional olfactory receptors in the olfactory sensory neurons (OSNs) dendrites and improve efficient odor response of conventional olfactory receptors. The structure of insect olfactory receptors is similar to the G protein-coupled receptors (GPCRs) of vertebrates with seven transmembrane regions. However, they adopt a membrane topology distinct from that of GPCRs, with their N-termini located intracellularly and C-termini located extracellularly. Orco interacts with the conventional olfactory receptors forming the Or-Orco complexes by the conserved C-terminal region wihch is a new ligandgated ion channel. Clarifying the functional mechanism of Orco in insect olfaction recognition may provide the basis for developing a new pest control measures based on insect olfactory behavior interference.

With the continuous development of biotechnoglogy and progresses in phylogenetics, researchers now are facing more and more challenges and difficulties in reconstructing phylogenetic trees: 1) species number (or individual number) of the specific taxon of research is always increasing; 2) the number of taxonomical characters (for example molecular information) of each species (or individual) is also enlarging. Especially with the efforts of genomesequencing technology, phylogenetic reconstruction based on molecular information requires massive computation. Mathematical methods, computer technologies and other auxiliary means play key roles in enhancing the efficiency and accuracy of phylogenetic reconstruction. Maximum parsimony (MP) is a very important method for phylogenetic reconstruction, and it needs efforts of both biologists and computer scientists to enhance its computational efficiency. In this article, we elaborated the calculation procedure of the MP method in details and analyzed the influences of parameter selection on computational efficiency, in order to help more computer researchers without detailed knowledge of phylogenetics to present better, quicker and more precise solutions to phylogenetic reconstruction in practice. In the meantime, we tried to explain the basic principles and computational logic of the MP method for phylogenetic researchers to push forward continuous improvement and optimization of using maximum parsimony in biology.

 Three main parameters of virus propagation, viz., larval stage, inoculation dose and incubation temperature were studied in three nucleopolyhedrovirus isolates of Helicoverpa armigera, i. e., Maragheh (MRG), Nebrin (NBN), and Marand (MRD), collected from tomato fields of East-Azarbaijan, Iran, to select the highly promising isolate under laboratory conditions. Among the larval stages evaluated, the early 5th instar larvae recorded the maximum virus yield. The optimum inoculation dose as well as incubation temperature have been determined as 1 965.87 OB/mm² and 25℃, respectively. However, the NBN isolate collected from Nebrin recorded the optimum mass production behavior in all the assays.