Heat shock proteins (HSPs) play an important role in the stress stimulation of insects. In order to study the HSP gene family of Plutella xylostella, 25 HSP genes, including 2 HSP90, 8 HSP70 and 15 sHSP （small heat shock protein， sHSP） genes, were identified from the P. xylostella genome by comparison with the HSP genes from the Bombyx mori genome using local Blast program. Phylogenetic analysis of the HSP genes from the genomes of P. xylostella, B. mori, Drosophila melanogaster and Tribolium castaneum revealed that sHSPs are species-specific, but HSP70 proteins are more conserved than sHSPs in insects. The analysis of expression patterns of HSP genes from P. xylostella revealed that the insecticide resistant-strains had different expression patterns of HSP genes from the susceptible strain. The expression patterns of HSP genes among the 1st, 2nd and 3rd instar larvae were similar, but their expression patterns were different from that in the 4th instar larva. The expression patterns of HSP genes between the 4th instar larva and pupa were similar, while those between male and female adults were significantly different. The expression of two sHSP genes ［CCG003980.1 (Px23.5) and CCG005412.2 (Px27.5)］ was up-regulated significantly in male adults compared to female adults, which were identified with a function of sperm production in D. melanogaster. The results indicate that HSPs may play an important role not only in pesticide resistance, development and metamorphosis, but in reproduction in insects. This study provides a foundation for further studies on the interactions between HSPs and the development and stress resistance of P. xylostella.

Nocturnal insects such as Noctuidae moths can precisely find mating partners, host plants and oviposition sites by olfaction system, and therefore are used as ideal models to study the molecular basis of olfaction. P450s are multifunctional monooxygenases which play dominant roles in the metabolism of a wide variety of both endogenous and xenobiotic substances. In order to investigate the roles of P450s in the olfaction, a new P450 gene from the male antennae of Helicoverpa armigera (Hübner), named HarmCYP9A33, with a full-length of 1 772 bp, was amplified by using RT-PCR and RACE methods. Sequence analysis showed that the full-length open reading frame of HarmCYP9A33 is 1 590 bp in size, encoding 529 amino acid residues, with the predicted molecular weight and isoelectric point of 61. 62 kD and 7. 97, respectively. HarmCYP9A33 shares a high amino acid sequence identity (75%) and the similar secondary protein structure with the homolog of Mamestra brassicae MbraCYP9A13, which is strongly expressed in sensilla trichodea. Moreover, six SRSs (substrate recognition sites) have 61% amino acid sequence identity, of which SRS4 responsible for the binding switch between substrate and enzyme is completely identical in both species. HarmCYP9A33 also shares certain similarities in the secondary protein structure with CYP9A subfamily proteins in H. armigera. Real-time PCR detection showed that HarmCYP9A33 could be detected in all test tissues of adult body, and the highest expression level of HarmCYP9A33 was in abdomen and then in head. The temporal expression profile analysis revealed that HarmCYP9A33 was expressed at all developmental stages with the highest expression level in the pupa. The expression level of HarmCYP9A33 in adult antennae varied with the time after emergence, which was higher than that in larvae and eggs. SDS-PAGE and Western blot results indicated the fused-protein was successfully expressed. These results provide a foundation to further investigate the cellular localization and biological functions of HarmCYP9A33 in cotton bollworm antennae.

In order to study the mechanism of stress-resistance, a heat shock protein gene hsp70 (named as Tmhsp70), was cloned from the larvae of Tenebrio molitor by PCR and RACE method, and the mRNA levels in developmental stages were detected by using semi-quantitative RT-PCR as well. The results showed the full sequence of Tmhsp70 cloned is 2 282 bp in length containing a 115 bp 5′ untranslated region (5′ UTR) rich in adenine, a 1 935 bp open reading frame and a 232 bp 3′ untranslated region (3′ UTR) rich in adenine and thymine. It also has seven repeats of the heat shock element nGAAn in its 5′ UTR and a 22 bp Poly (A) tail in the 3′ UTR. The deduced heat shock protein named as TmHSP70 contains three signature motifs of HSP70, i.e., IDLGTTYS, IFDLGGGTFDVSIL and IVLVGGSTRIPKIQQ as well as the terminal EEVD motif which is characteristic to cytoplasmic HSP70s. TmHSP70 has neither a signal peptide nor a transmembrane domain. It contains two main functional domains: a 42 kDa highly conserved N-terminal ATPase domain and a 18 kDa conserved C-terminal peptide-binding domain. The tertiary structure of ATPase domain is composed of two large globular subdomains and contains a nucleotide-binding core. Tertiary structure of the peptide-binding domain forms a sandwich of 2 four-stranded β-sheets and two α-helices, and includes a peptide-binding cavity. Furthermore, the expression of Tmhsp70 mRNA in T. molitor was characterized by heat-inducible and developmentalregulation feature. The overall increase in the levels of Tmhsp70 mRNA in different life stages when the larvae were exposed to 42℃ for 1 h, ranged from 1.4- to 26.9-fold on the basis of semi-quantitative RT-PCR analysis. At 25℃ Tmhsp70 mRNA expressed in 1-day old pupae was higher than that accumulated in other developmental stages, and after exposure to 42℃ for 1 h, Tmhsp70 mRNA expressed in 90-day old larvae became the most abundant, and was not only higher than that accumulated in 30- and 60-day old larvae but also higher than that accumulated in 15- and 30-day old adults. The results form a basis for further research on structure, function and expression regulation of HSPs from T. molitor as well as the relationship between HSPs and stress-resistance in the beetle.

Queen mating flight is an important prelude of sexual matured virgin queens mating with drones, which is usually accompanied by a series of important physiological changes in queens’ bodies. In order to explore the differences of gene expression following queen mating flight behaviour, we analyzed the gene expression differences between the flying matured virgin queens and non-flying matured virgin queens of Apis cerana cerana using a high-throughput sequencing method. Through digital gene expression (DGE) sequencing, we obtained 5.98 and 6.01 million clean tags from the flying matured virgin queen and non-flying matured virgin queen samples, respectively. A total of 250 genes were differentially expressed between both, with 133 up-regulated and 117 down-regulated in the flying matured virgin queens. These differentially expressed genes can be classified into 348 functional categories and involved in 142 biochemical pathways, indicating that there are a large number of genes whose expression levels change during mating flight process of matured virgin queens. The results provide important gene expression information for further researching the molecular mechanisms of the physiological changes during queen mating flight in A. cerana cerana.

To explore the metabolic pathway of NaF in the silkworm, Bombyx mori, the 5th instar larvae of the fluoride-resistant silkworm strain (T6) and the fluoride-susceptible strain (734) were fed on mulberry leaves soaked in 50, 100, 200 and 400 mg/kg NaF solutions, respectively, for 1-7 d, and the activities of carboxylesterases (CarE) and esterases in the midgut were examined. The results showed that the CarE activity in strain 734 was 1.21-1.98-fold as high as that in the control group, while the CarE activity in strain T6 was 0.72-1.10-fold as high as that in the control group. In both strains treated with NaF, the change trend of the esterase activity was similar with the control group within 7 d, and the esterase activity in the two strains was also similar. When the two silkworm strains were treated by NaF at the same concentration, their esterase activities on different days after treatment were significantly different （P＜0.05）. It is inferred that fluoride can increase the CarE activity in the midgut of the fluoride-susceptible strain of the silkworm, and inhibit the CarE activity in the midgut of the fluoride-resistant strain, but it has little effect on the esterase activity in the midgut of both strains of the silkworm.

The technology of microinjecting preblastodermic eggs is currently the main operation to obtain the transgenic silkworm (Bombyx mori). Microinjection will cause damage to silkworm eggs, and lead to the decrease in the hatchability of the injected eggs, which is one of the major problems of the transgenic technology of the silkworm. In this study, the eggs were microinjected at different embryonic age and on different positions of egg including dorsal side, ventral side, anterior pole, posterior pole and the center at 5 h after egg laying, to investigate the effect of these factors on egg hatchability and the occurrence of larval malformation in the silkworm. The results showed that microinjection operated at the earlier embryonic age or from the dorsal side increased the hatchability of those injected eggs. Microinjection from the dorsal side brought out normal larvae, while microinjection from the ventral side led to a lot of malformed larvae. It is so inferred that choosing the suitable injection time and correct position will eliminate the occurrence of malformation and increase egg hatchability to some extent. This study provides a useful reference for improving the transgenic operation of the silkworm.

As the frontline encountering various microbes, intestine plays critical roles in insect immune responses. In order to investigate the immune responses in the silkworm midgut, we identified 18 differentially expressed genes after oral infection by Pseudomonas aeruginosa and Staphylococcus aureus using annealing control primer (ACP) based reverse transcriptional PCR technique. The expression profile of four genes during the first 24 hours post infection was monitored by quantitative real-time PCR. The results showed that the peptidoglycan recognition protein L1 (PGRP-L1) gene and a serine protease precursor gene were up-regulated by P. aeruginosa infection exclusively, while the 30kP protease A precursor gene was up-regulated significantly by both P. aeruginosa and S. aureus infection. Our study identified genes involved in recognition of invading bacteria and immune signaling pathways in the larval midgut of the silkworm after oral infection and may give some clues for further functional investigation of these genes.

To investigate the effect of cantharidin (CTD) on insect cell membrane and its mechanism, the cell membrane integrity and potential in Spodoptera frugiperda Sf9 cells treated with CTD were tested by transmission electron microscope (TEM) and laser scanning confocal microscope (LSCM) combined with fluorescent dyes FDA/PI and DiBAC4(3). The results showed that after the Sf9 cells were treated with 32 μmol/L CTD, their membrane structure was not disrupted at 6 h and 12 h after treatment, respectively. The FDA fluorescence intensities of the treated cells evidently declined compared with the control (P<0.05) while the proportion of the PI-staining cells was not significantly different from that of the control (P≥0.05) at 0.5 h after treatment under TEM observations. The membrane potential (MP) in Sf9 cells was depolarized significantly both at 140 s after treatment with 32 μmol/L CTD and in the instantaneous treatment with 64 μmol/L CTD (P<0.05). The MP in Sf9 cells depolarized obviously within 3 h after treatment with 32 μmol/L CTD or 2 h with 64 μmol/L CTD (P<0.05), and then the degree of deplorization declined. At 6 h after treatment with 32 μmol/L CTD or at 3 h with 64 μmol/L CTD, MP deplorization in treated cells was not significantly different from that in the control (P≥0.05). The results suggest that CTD causes the depolarization of cell membrane potential in Sf9 cells, which will maintain for a period of time, and induces an irreversible decline in cell activity, but not destroys the integrity of the cell membrane structure.

【Aim】In this study, the sublethal effects of indoxacarb and beta-cypermethrin on Plutella xylostella (L.) were investigated so as to further understand the mechanism of insecticides and provide a theoretical basis for proper application of pesticides and mitigating the damage to environment and negative effects on agriculture. 【Methods】 Leaf dipping method was used to test the acute toxicity of indoxacarb and beta-cypermethrin to the 2nd instar larvae of P. xylostella, and the LC₁₅, LC₃₀ and LC₅₀ values of indoxacarb (designated as TI-LC₁₅, TI-LC₃₀ and TI-LC₅₀, respectively) and beta-cypermethirn (designated as TB-LC₁₅, TB-LC₃₀ and TB-LC₅₀, respectively) were determined. After the 2nd instar larvae of P. xylostella were exposed to the six lethal concentrations above for 48 h, the biological characteristics including the survival rate, developmental duration, and larval and pupal weight were recorded, the fecundity and adult longevity were also investigated, and the age-stage and two-sex life tables was established to analyze the subthal effects of indoxacarb and beta-cypermethrin on biological characteristics of the offsprings of P. xylostella. 【Results】 The developmental duration in TI-LC₃₀, TI-LC₅₀, TB-LC₁₅, TB-LC₃₀ and TB-LC₅₀ groups were significantly longer than that of the control group （P<0.05）, and the larval and pupal weight and fecundity tended to decrease (P<0.05). But the developmental duration of the 3rd and 4th instar larvae in TI-LC₁₅ group was significantly shorter than that of the control group （P<0.05）. The total oviposition periods of offsprings in TI-LC₃₀, TI-LC₅₀, TB-LC₃₀ and TB-LC₅₀ groups were significantly shortened (P<0.05). Meanwhile, the fecundity of these groups was significantly decreased compared to that of the control (P<0.05). The mean values of the intrinsic rate of increase (r_m), finite rate of increase (λ), gross reproductive rate (GRR) and net reproductive rate (R₀) were significantly lower in all the treatment groups than in the control group (P<0.05). 【Conclusion】 The low concentrations of indoxacarb and beta-cypermethrin could restrain the growth and development of P. xylostella, decrease the fecundity of its parents and offsprings and reduce the population growth of its offsprings.

In order to promote the effective utilization of pesticides in cotton fields, field tests were conducted during 2011-2012 to determine the appropriate spray method and volume used to control the cotton aphid, Aphis gossypii (Glover). Deposition of insecticides applied with the lever-operated knapsack sprayer and the knapsack mist blower at different spray volumes in cotton fields were studied by adding tracer allura red in insecticide solution. The results indicated that at the seedling stage of cotton, when 3% acetamiprid EC was applied with the mist blower at the dose of 450 mL/ha, the run-off rates were 24.4%, 28.9% and 26.7% at the spray volumes of 75, 150 and 225 L/ha, respectively. The runoff rates of 3% acetamiprid EC from the lever-operated sprayer operated at the the spray volumes of 300, 450 and 600 L/ha were 35.6%, 37.8% and 46.7%, respectively. Efficacy of acetamiprid applied with two sprayers at different water volumes against cotton aphids had no significant difference (P>0.05). At the adult-plant stage of contton, when 25% pymetrozine WP was applied at the dose of 300 g/ha, the run-off rates of pymetrozine for the lever-operated sprayer at the the spray volume of 600 L/ha and the mist blower at the the spray volume of 150 L/ha were 13.3% and 3.3%, respectively. The efficacy of pymetrozine applied at the dose of 225 g/ha using the mist blower at the the spray volumes of 150 and 300 L/ha had no significant difference compared with the treatment of 300 g/ha pymetrozine applied with the lever-operated sprayer at the spray volume of 600 L/ha (P>0.05). It is so concluded that low volume spray with the mist blower could facilitate not only to reduce the insecticide dosage used in cotton fields for aphid management, but also to decrease the run-off rate of insecticide onto soil and so reduce environmental pollution.

 The n-alkanes in plant cuticular wax have been used as markers to estimate the diet composition and intake of grazing herbivores, but limited information is available about diet pattern of grasshoppers in natural grasslands based on the n-alkane technique. The objective of this research was to estimate the diet composition and trophic niche of Oedaleus asiaticus, a dominant grasshopper species, using the n-alkane technique in combination with quadrats. Experiments were conducted in three typical plant communities (i.e., Stipa klemenzii, Leymus chinensis, and Stipa grandis communities) along precipitation gradients in the Inner Mongolian steppe from July to August of 2003. Twenty quadrats were selected randomly and clipped to ground level in each community to measure plant species diversity and aboveground biomass. Main plant species in each community and the feces of O. asiaticus were analyzed for concentration patterns of n-alkanes. Our results indicate that the diet composition of O. asiaticus in natural grasslands can be accurately estimated using the n-alkane technique, and it is significantly different under different grazing pressures and in different plant communities. The grasshopper shifted its diet pattern from a specialist (graminivorous) in L. chinensis and S. grandis communities to a generalist (mixed graminivorous) in S. klemenzii community. The overlapping indexes of trophic niche between O. asiaticus and sheep were 0.0619, 0.0172 and 0.1815 in L. chinensis, S. grandis and S. klemenzii community, respectively. The community structures of the vegetation (plant species diversity, biomass proportion, and frequency distribution) had significant influences on the diet composition of O. asiaticus. Grazing altered the plant community and indirectly affected the grasshoppers’ food selection. The results suggest that certain competition may exist for food resources between grasshoppers and livestock.

 Nuclear mitochondrial pseudogenes (NUMTs) are DNA fragments transferred from mitochondria to the nucleus. They are a double-edged sword for phylogenetic analyses because of their independent evolutionary histories. Herein, we employed a targeted, PCR-based procedure to study NUMTs originated from a Nad1-12S fragment in two sibling fig wasps associated with Ficus hispida: Philotrypesis pilosa and Philotrypesis sp. These sibling fig wasps arising from sympatric speciation live in the same syconium, which makes them good models to investigate subtle behavioral and genetic divergences in similar ecological niches. Further investigation relies on a correct estimate of the separation time of both species. Through the analysis based on the acquired NUMTs, we found that the origin of the NUMTs in both species appears to be a very recent event; however they may arise in the common ancestor of both species. Since the NUMTs integration is a recent event, we assumed that these newly produced NUMTs evolve similarly to genuine mtDNA. Then we calculated the genetic divergence timing based on mitochondrial genes, an average substitution rate of about 2.3×10^-8 substitutions/site/year. These typical characters make them good ‘fossil’ markers to trace back the speciation time of both species back to 0.40-0.48 mya. The results suggest that some NUMTs are good ‘fossil’ markers for tracing back important evolutionary events including speciation.

To assess the genetic diversity and genetic structure of Apis cerana cerana populations in Hainan Island, southern China and their relationship with the mainland populations, 627 individual workers from 627 colonies in 11 localities of Hainan Island and 102 individuals from 102 colonies in two localities of mainland China were genotyped using 10 microsatellite DNA loci. The results showed a high level of allelic diversity with 5-17 alleles across all the loci and an average of 4.5-7.0 alleles per locus across Hainan Island populations. The mean expected heterozygosity (He) values across all samples ranged from 0.59 to 0.65. The populations from Hainan Island showed similar allelic structure pattern at ten loci, but Wenchang and Tunchang populations shared a unique allelic pattern at AT101. The F_ST between Hainan Island populations and adjacent mainland ones ranged from 0.06 to 0.13. The F_ST of Wenchang and Tunchang compared to other nine island populations ranged from 0.06 to 0.12, which was larger than that between the nine island populations (F_ST=0-0.05). The results showed Hainan Island A. c. cerana populations diverged from the neighboring mainland ones. Both Wenchang and Tunchang populations showed a higher level of differentiation from other nine island populations among which slight differentiation occurred. This study provides a theoretical support for efficiently preserving Hainan indigenous A. c. cerana as a genetic resource for future utilization.

As eusocial insects, the honeybees Apis mellifera, are characterized by the extreme reproductive division of labor. The queen monopolizes reproduction in a colony, and the workers refrain from, are coerced not to, or have lost the ability to reproduce, undertaking all the functions inside and outside of the hive except for egg laying and mating. However, in occasional “anarchistic” queenright colonies, many workers have activated ovaries and lay eggs, so the majority of drones are the offsprings of workers but not the queen. These anarchistic bees provide a superb model for investigating the mechanisms underlying the sterility in honeybee workers. In this article the characteristics, causes and genetic basis of anarchistic colonies are reviewed. Many workers lay eggs in anarchistic colonies and these eggs could escape the worker policing. The anarchistic behavior is affected by several factors, including environmental conditions, genetic constitution and gene expression. Moreover, the genetic structure system of anarchistic behavior is quite complex and many genes may be involved in the regulation. Researches on the behavioral mechanisms of anarchistic colonies will shed light on the studies of the regulation of honeybee worker sterility and the identification and characterization of the genes that control worker sterility in social insects.

Malathion is an efficient but low toxic organophosphate insecticide with a large molecular weight and a special structure. It is widely used in the prevention and control of various agricultural pests. Mutation in carboxylesterases is one of important metabolic resistance mechanisms to organophosphate insecticides in insects. In a previous study, seven non-specific carboxylesterase genes from Aphis gossypii, Nilaparvata lugens, Spodoptera litura, Bombyx mori, Harmonia axyridis, Tribolium castaneum and Apis mellifera, respectively, were cloned, mutated at position 151 or 271 and expressed in Escherichia coli. In this experiment, the hydrolysis of the purified recombinant proteins of the seven insects was further examined towards malathion. The results showed that the wild-type carboxylesterases from A. gossypii, A. mellifera, S. litura and T. castaneum were capable of degrading malathion and the two single mutations did not improve their hydrolysis activity. The wild-type carboxylesterases from B. mori, H. axyridis and N. lugens could not degrade malathion while the G/A151D and/or W271L mutation made these esterases acquire malathion carboxylesterase activity, implying that these two single mutations could confer resistance to malathion in the three insects. Malathion carboxylesterase activity in different species had large difference. S. litura had the highest malathion carboxylesterase activity, and its K_cat/K_m was 1.8-1.9 L/μmol·min, followed by T. castaneum, with the K_cat/K_m of 0.87-0.95 L/μmol·min. Malathion carboxylesterase activity in other insects was relatively low, with ten-fold difference.

In order to study the effects of green leaf volatiles (GLVs) emitted by broad-leaved plants to the Yunnan pine shoot beetle, Tomicus yunnanesis, three most abundant GLVs ［(E)-2-hexenal, (E)-2-hexen-1-ol and (Z)-3-hexen-1-ol］ were chosen in the experiments. Single components and blends of two components were used to study the effects on the host-location behavior of T. yunnanesis by laboratory pine twig feeding test. At 12, 24 and 48 h after testing, the beetles which got into the pine twigs were counted. The results showed that the three GLVs and their blends can interrupt the host-location behavior of T. yunnanesis at different degree. At 12 h after testing, three single components ［A: (E)-2-hexenal, P＜0.01; B: (E)-2-hexen-1-ol, P＜0.01; C: (Z)-3-hexen-1-ol; P＜0.01］ and two blends ［D: (E)-2-hexenal+(E)-2-hexen-1-ol, P＜0.01; E: (E)-2-hexenal+(Z)-3-hexen-1-ol, P＜0.01］ had significant differences in the number of beetles stayed outside the pine twigs when compared to the control. These GLVs significantly inhibited the infestation probability of P. yunnanesis by T. yunnanesis. At 24 h after testing, group D (P＜0.01) and E (P＜0.01) exhibited significant differences in the number of beetles stayed outside the pine twigs from the control group. However, only group D (P＜0.01) had significant difference in the number of beetles stayed outside the pine twigs from the control group at 48 h after testing. This study provides valuable basis for using secondary metabolites in non-host plants to control T. yunnanesis in the future.