The Rieske iron-sulfur protein (RISP) is a key protein subunit of mitochondrial complex Ⅲ, which plays an important role in the respiratory electron transport chain. The opening reading-frame (ORF) of SlitRISP was cloned by RT-PCR from Spodoptera litura for the construction of prokaryotic expression vector pET32a-SlitRISP. SDS-PAGE and Western blot analysis showed that the prokaryotic protein SlitRISP was mainly present as inclusion body in the bacteria precipitate, and the antibody of RISP could be applied to the immunoblot analysis of SlitRISP successfully. In order to identify the function of SlitRISP in the cultured cell line SL-1 of S. litura, RNAi was used to silence SlitRISP by transfecting siRNA into SL-1 cells. The qRT-PCR result showed that at 48 h after the SL-1 cells were treated with 50 nmol/L and 100 nmol/L siRNA, respectively, the expression levels of SlitRISP mRNA were all inhibited effectively compared with the control. The Western blotting result showed that the content of SlitRISP in SL-1 cells was obviously lower than the control. The mitochondial membrane potential (MMP), ATP content and inhibition rate of cell proliferation were detected based on the obvious silence of SlitRISP in SL-1 cells to identify the important roles of RISP in the electron-transport chain of mitochondria. The changes of MMP in SL-1 cells were monitored by flow cytometry (FCM), which decreased by 23.52% and 11.32% at 24 h after the SL-1 cells were treated with 50 nmol/L and 100 nmol/L siRNA, respectively, compared with control. However, at 48 h after the SL-1 cells were treated with 50 nmol/L and 100 nmol/L siRNA, the MMP increased by 5.58% and 27.66%, respectively. The ATP content in SL-1 cells at 48 h after treatment with 50 nmol/L and 100 nmol/L siRNA was decreased by 82.71% and 84.50%, respectively, measured by luminometer. Owning to the suppression of ATP synthesis by siRNA in SL-1 cells, the inhibition rates of cell multiplication reached to 53.64% and 67.94%, respectively, at 48 h after the SL-1 cells were treated with 50 nmol/L and 100 nmol/L siRNA. These results demonstrate that the RISP plays an important role in the MMP formation and ATP synthesis in SL-1 cells. As RISP plays important roles in the electron-transport chain of mitochondria, it could become a new target for pest control, and this may provide reference for developing new respiration inhibitors.

Serine proteases are one group of important digestive enzymes in insects. To clarify the characteristics and functions of serine proteases, we used the polyclonal antiserum of peritrophic membrane protein from Trichoplusia ni to screen cDNA expression library of the midgut of Holotrichia oblita, and obtained a full-length cDNA clone encoding serine proteases named as HoSP1 (GenBank accession no. FJ573146). The sequence analysis indicated that HoSP1 is 902 bp in length with an opening reading frame of 783 bp encoding 260 amino acid residues with the predicted molecular weight 26.7 kDa and pI 4.19. Without N-linked glycosylation site, HoSP1 has an O-linked glycosylation site at Thr₁₅₇ and six conservative cysteines forming three pairs of disulfide bonds, which play an important role in sustaining the protein tertiary structure. Amino acid sequence alignment with several kinds of serine proteases showed that HoSP1 has the catalytic active centers of histidine, aspartic acid and serine, and shares significant similarity to 14 kinds of serine proteases from Costelytra zealandica, with the highest identity (52.47%) to CzSP3. After the gene was recombined into pET21b and expressed in vitro, the activity of HoSP1 was determined with BTEE as the substrate, which was 0.0378 μmol/mg·min. The molecular cloning and expression in vitro of HoSP1 lay a foundation for further research of its expression and function in H. oblita.

To understand the molecular regulation of diapause in the beet webworm, Loxostege sticticalis L., we analyzed the contents and compositions of proteins and nucleic acids in non-diapause larvae, diapausing larvae with the diapause duration of 1, 2, 3, and 4 months, and post-diapause larvae of this species by using Trizol method, DNA and protein quantitative kit, and SDS-PAGE technique. The results showed that the RNA content in the diapausing larvae with the diapause duration of 1 to 4 months was significantly lower than that of the non-diapause larvae. Although RNA content in the post-diapause larvae increased significantly as compared with that of the diapausing larvae with the diapause duration of 2, 3 and 4 months, it was significantly lower than that of the non-diapause larvae. The DNA content among these treatments was not significantly different, but the RNA/DNA ratio in the diapausing larvae with the diapause duration of 1 to 4 months was lower than that in the non-diapause and post-diapause larvae. The protein content in all the diapause larvae was significantly higher than that in the non-diapause larvae, but there was no significant difference between the non-diapause and the post-diapause larvae. A protein band of 24 kDa was found in the diapausing larvae with the diapause duration of 1 to 4 months but was not detected in the non-diapause and post-diapause larvae by using the SDS-PAGE technique. These results suggest that the lower RNA content, the lower RNA/DNA ratio, and higher protein content, as well as the presence of 24 kDa-protein, are the major characteristics of the diapause larvae distinguishing from the non-diapause and post-diapause larvae of L. sticticalis.

The Chinese scholar tree looper, Semiothisa cinerearia Bremer et Grey (Lepidoptera: Geometridae), is the major defoliator of the Chinese scholar tree Sophora japonica L. in China. The elucidation of the absolute chemical structure of the sex pheromone component of S. cinerearia may provide a potential chemical for the pest control against the S. cinerearia infestations in the urban area. In this study, two sex pheromone gland compounds, i.e., (6Z, 9Z)6, 9cis-3, 4-epoxy-heptadecadiene (6Z, 9Z-cis-3, 4-epoxy-17: H) and (3Z, 6Z, 9Z)-3, 6, 9-heptadecatriene (3Z, 6Z, 9Z-17: H), were detected in a 100∶4.8±1.3 ratio (N=12) during analyses of solvent extracts from virgin female S. cinerearia (2-3 d old) by comparison of their gas chromatography (GC) retention time and mass spectra (MS) with those of synthetic standards. Furthermore, the absolute configuration of 6Z, 9Z-cis-3, 4-epoxy-17: H in the sex pheromone gland of S. cinerearia was determined as (6Z, 9Z)-3R, 4S-epoxy-heptadecadiene by using a chiral capillary column (CycloSil-B, 30 m × 0.25 mm × 0.25 μm film) GC under the optimized oven temperature program. The mixture of the two synthetic enantiomers of 6Z, 9Z-3R, 4S-epoxy-17: H and 6Z, 9Z-3S, 4R-epoxy-17: H in a ratio of 1.28∶1 was injected into the female extracts, and thus the ratio was changed to 1.55∶1. Based on this analysis, the absolute configuration of the gland component 6Z, 9Z-cis-3, 4-epoxy-17: H was further confirmed as 3R, 4S. It is anticipated that control of S. cinerearia infestation with enantiomerically pure materials will be much more effective.

In order to clarify the physiological effect of Cry1Ac toxin on the oriental armyworm, Mythimna separata (Walker), the activities of two detoxification enzymes (esterase and glutathione-S-transferase), three protective enzymes (superoxide dismutase, catalase and peroxidase) and midgut proteases (total protease, trypsin-like enzyme and chymotrypsin-like enzyme) in the 5th instar larvae of M. separata fed on Cry1Ac toxin were evaluated in the laboratory. The results showed that the activities of esterase, glutathione-S-transferase, peroxidase, trypsin-like enzyme and chymotrypsin-like enzyme in the larvae fed on an artificial diet containing Cry1Ac toxin were significantly lower than those of the control (P<0.05). However, the activity of superoxide dismutase in the larvae fed on the artificial diet containing Cry1Ac was significantly higher than that of the control (P<0.05). The activity of catalase of the treated larvae was obviously lower than that of the control at 6 h, 12 h and 24 h after treatment (P<0.05), but higher than that of the control at 36 h after treatment (P<0.05). These results suggest that the Cry1Ac toxin disturbs the regular metabolism of insects mainly through restraining the activities of detoxification enzymes and proteases and disturbing the balance among superoxide dismutase, catalase and peroxidase in the midgut of larvae, and thus kills the pests.

A mark-release-recapture (MRR) experiment was conducted in June, 2009 in Inner Mongolia Autonomous Region, and the peak for the overwintering adult population of the beet webworm, Loxostege sticticalis L. (Lepidoptera: Pyralidae) was detected in order to demonstrate the migratory pathway and capacity of this species from northern region to northeast region in China. In July, about 1.8 million adults were caught by nets, marked by spraying colorant and released at 6 sites distributed in Wuchuan County (41.07°N, 111.42°E) and Chayouzhongqi (41.13°N, 112.58°E) of Inner Mongolia during June 1st-4th, 2009. At the monitoring and forecasting units of the beet webworm at various levels in the 11 main occurrence provinces or autonomous regions in northern China, methods such as trap lamps, clap nets and pheromone traps were used to recapture the adults of this moth. Nine adults, released at Chayouzhongqi, were recaptured at Keyouzhongqi, Wulanhaote, and Zhalantun County, respectively, in northeastern Inner Mongolia during June 7th-25th, and 1 adult released at Wuchuan County was recaptured at Beipiao County of Liaoning Province on June 29th. The straight distances from the released sites to the recaptured sites were from 725-1 117 km. This is the longest distance taken by the migratory flight of the beet webworm that has been recorded in the world. These results not only prove the soundness of traditional assumption that the beet webworm originated from northern China is one of the sources for the 1st generation of the moth in northeastern China, but also provide important basis to improve the forecast and management technology of the beet webworm.

Pollination structure and behavior of fig wasps, and the pollen-ovule (P/O) ratio of their host figs are all important indicators of their pollination mode. Field observation was made on Ficus annulata from August 2010 to June 2011 in Xishuangbanna, Yunnan, southwestern China and the pollination structures of Deilagaon annulatae, the pollinator of F. annulata, were examined through Scanning Electron Microscopes (SEM). The results showed that two pollen pockets disappeared and pollen grains can adhere to the bristles in peristaethium, which are all characteristics of a passive mode. No active pollen collecting behavior was observed under microscope, suggesting that the existing coxal combs have lost the function of actively collecting the pollen. Meanwhile, F. annulata is a typical passive pollinated species with a large number of male flowers in each syconium; the P/O ratio is high; and the pollen grains spread when the mature anthers split automatically. Given evidences from both sides, we deduce that the pollination mode of this system is passive. D. annulatae is the only fig wasp species reported with yellow body color in Xishuangbanna. They prefer emerging and acting at night when the temperature is low. During the receptive phase, Lipothymus sp. is the only non-pollinator that enters the fruit to lay eggs there along with the pollinator, and with much higher abundance. In a syconium, the number of wasp offspring is significantly greater than that of the seeds, showing a rare fig-wasp mutualism system in which fig wasps act as the dominator.

Encarsia Forster consists of important parasitoids of whitefly (Bemisia tabaci) pests, including E. bimaculata, E. formosa and E. sophia, the three most important aphelinid parasitoids in China. Eight populations of Encarsia from the South, Southeast, North and Southwest of China, as well as two populations from Malaysia and Egypt, respectively, were collected in the present study, and their inter-species phylogenetic relationships were analyzed based on 28S rRNA D2 and D3 expansion regions. The D2 and D3 regions were consistent with each other, confirmed a closer genetic relationship between E. sophia and E. bimaculata since they both belong to the Encarisa strenus species group, compared to those between these two species and En. formosa. Results of the genetic distance analysis using 28S rRNA D2 sequences revealed that there are certain genetic divergences within single species of the Encarsia parasitoids. The Guangzhou population of Encarsia sophia is more close to populations from Australia, Spain, Egypt and Ethiopia, but further from the population from Thailand. E. bimaculata populations from Sudan, Egypt and Guatemala as well as one population from Australia cluster together, while E. formosa Hengshui and Kunming populations cluster together with those from USA, UK and Greece, but are further from the Egypt population. The reasons for the inconsistency between the genetic and geographical distances of the Encarsia species are discussed.

Gynaikothrips ficorum (Marchal) is a major insect pest damaging Ficus microcarpa and has expanded to all the continents. At present, it damages F. microcarpa seriously in tropical and subtropical areas in Yunnan province. In order to reveal the genetic differentiation among geographic populations of G. ficorum, a 646 bp segment of the mitochondrial cytochrome c oxidase subunit Ⅰ (COⅠ) gene was sequenced from 145 individuals among 10 geographic populations in Yunnan province, and the sequence variability of the COⅠ gene and genetic differentiation among them were analyzed. The results showed that 38 variable sites were detected and six haplotypes were defined based on the COⅠ sequences among 10 geographic populations of G. ficorum, including one haplotype shared by eight populations. The genetic distances ranged from 0 (among populations of Ruili, Mangshi, Yuxi and Chenggong) to 0.043 (between Yiliang population and populations of Longchuan and Mojiang) among the 10 geographic populations, indicating that the genetic distances among G. ficorum populations were not associated with their geographic distances. Analysis of molecular variance showed that 10 geographic populations were clustered into three groups (group I: Longchuan, Ruili, Mangshi, Yuxi, Chenggong, Mojiang, Lincang, and Mengla; group Ⅱ: Mengzi; group Ⅲ: Yiliang). The Fst value was 0.9828 (P<0.05), and the Nm value was 0.01, indicating apparent genetic differentiation among the three groups, but there was only 0.0172 genetic variation from intragroup. A molecular phylogenic tree constructed using neighbor-joining (NJ) method showed that the six haplotypes were clustered into three clades associated with geographic regions. No shared haplotypes were found among the three groups. Molecular analysis showed that these three major groups/clades did not coincidence with the geographic distribution. Based on the information of host plants, we preliminarily presumed that the genetic differentiation between the two populations (Mengzi and Yiliang) resulted from different host variety and growth conditions. Haplotypes among all populations were distributed in different clades on the median joining network, revealing no obvious geographic distribution patterns.

In order to explore the difference in chromosome morphology and behavior at different phases in the process of meiosis of testis cells, genetic relationship among genera and species, and evolutionary law in Pentatomidae, we observed the chromosome morphology and behaviors and the formation of sperms of testis cells of 9 species belonging to 6 genera of Pentatomidae by using conventional chromosome preparation. The results showed that testis cells of Pentatomidae are "O"-type during the meiosis. The chromosomes of testis cells of Pentatomidae are similar in behavior but different in morphology. The arrangement and relative length of chromosomes, the karyotype and the sperm characteristics are different among genera and species. The average relative length of chromosome sets of the observed species in Pentatomidae is all 12.5. The amount information of chromosome set length of testis cell of Pentatomidae is constant in the evolutionary process. Eurydema, Aelia, Rubiconia and Graphosoma display a close genetic relationship, while Stollia and Carpocoris are more closely related based on the cluster analysis of chromosome relative length of the observed species.

The invasion of alien species that are morphologically indistinguishable, or hardly distinguishable, with the native species or earlier established species is regarded as cryptic invasion. This kind of invasion is often undetectable by human beings. The types of cryptic invasion include invasion of the alien sibling species, genetic lineages, geographic populations, and a hypothesized “back introduction”. These alien species are more readily hybridize or introgress with native species or earlier established species and thus result in the long-term ecological effects on the invasive species or native species. The research related to cryptic invasion should be further strengthened in view of its widespread occurrence．

Polarized light is different from common light, and it vibrates in one direction on light vectors and includes linear polarized light, circularly polarized light, elliptically polarized light, etc. It was verified that polarized light could affect behavior of many insects, such as flight navigation of Apis mellifera, mating of Heliconius cydno chioneus, selection of oviposition places by Papilio aegeus, etc. The reflection of circularly polarized light by scarab beetles could be used as a taxonomic character. Ommatidia accumulated in the dorsal rim area (DRA) of the compound eye are the main organs sensing polarized light. Electrophysiological studies recently showed that the anterior optic tubercle (AOTu) is a part of the polarization vision pathway of locusts. The polarized light traps have been developed for ground beetles in Hungary.

Chloride channel was closely related to insect resistance to pesticides. ESTs of glutamate mediated chloride channel (GluCl) gene were clustered and identified from transcriptome data of Phyllotreta striolata (Fabricius). The expression of GluCl gene in different tissues of P. striolata adults was profiled by quantitative real-time PCR. The results indicated that the cDNA of this gene is 1 430 bp in length with an open reading frame (ORF) encoding 447 amino acids. The putative protein molecule contains two functional domains: a neurotransmitter-gate ion-channel ligation binding domain and a neurotransmittergate ion-channel transmembrane domain. There are two cysteine loops in ligation binding domain regions demonstrating a typical characteristic of GluClα subunit. Phylogenetic analysis of the complete cDNA sequence showed that the GluCl of P. striolata is highly homologous to GluClα of other insects. Quantitative real-time PCR assay showed that a relatively higher expression amount of GluCl gene was found in head than in other organs including antenna, midgut, testis, ovary, fore legs, middle legs and hind legs. The expression level of GluCl gene in head of male adult was 65.7 times as high as that in testis and 227.5 times in midgut, suggesting its important mediation in center nervous system. This study provides useful information for studying molecular mechanism of pesticide resistance mediated by GluCl of P. striolata.

To understand the ecological significance of wing dimorphism in a cricket species, Velarifictorus asperses (Walker), the development of flight muscles and ovary, fecundity and longevity in long-winged (LW) and short-winged (SW) female adults of this cricket bred in the laboratory were compared. On the day of emergence, LW females had better-developed flight muscles than SW females, and the weight of their flight muscles was 38.68±9.15 mg and 17.53±4.44 mg, respectively. No significant difference was observed in ovary mass between LW and SW females (P>0.05), and the weight of them was 4.69±1.04 mg and 4.88±0.97 mg, respectively. Within 8 days after emergence, the weight of the LW females’ flight muscles increased 48.9%, while the weight of the SW females’ ovaries increased up to 93.5±11.7 mg, which was about 4.5 times higher than that of the LW females. The SW females were observed to reproduce earlier and had higher total number of eggs laid than the LW females, but the significant difference in number of eggs laid was only found in early adulthood (P<0.05). No significant difference was observed in longevity between the two wing forms (P>0.05). In addition, some LW females had their flight muscles histolyzed from day 12 after emergence, and the ovaries developed faster than the LW females with fully-developed flight muscles. These results suggest that a physiological trade-off between the development of flight muscles and reproductive organs existed in female V. asperses, which may cause a differentiation of life history strategy among LW and SW females, that is to say, the LW females are able to fly and the SW females benefit more in reproduction, and the histolysis of flight muscles might be a physiological signal for LW females to switch from fly to reproductive development.

To understand the effects of Wolbachia in Andricus mairei Kieffer, an oak gall wasp (Cynipini, Cynipidae) that forms clusters of up to 30 monothalamous galls in the catkins of Quercus glandulifera var. brevipetiolata Nakai, we examined the presence and infection frequency of Wolbachia in three widely separated geographic populations of A. mairei in Hunan Province, South China, using polymerase chain reaction and sequence determination of the wsp gene of Wolbachia from the three populations. Our survey results showed that Wolbachia infection rates were unusually high in the three test populations: 100% for the male wasps from all populations, while 100%, 100% and 80% for the Yueyang, Changsha and Shaoyang populations of the female wasps, respectively. The sequencing results showed that the gene from all the three populations is 561 bp in length. Comparison with the wsp gene sequences in A. mairei and Neuroterus macropterus, Biorhiza pallida, Andricus solitarius (strain 1) and Synergus crassicornis revealed a 95% identity. The phylogenic relationship of Wolbachia strains in Cynipidae with the known wsp sequences indicated that the Wolbachia strain of A. mairei is closely related to that of N. macropterus, B. pallida, A. solitarius (strain 1), and S. crassicornis, and belongs to the A group. We also discovered strikingly male-biased sex ratios in all the test populations: females only accounted for 15.3%, 12.1%, and 19.8% of the Yueyang, Changsha, and Shaoyang populations, respectively, indicating the extremely male-biased sex ratios. These results suggest that the symbiosis of Wolbachia with A. mairei does not induce parthenogenesis in A. mairei, which can not be properly explained and is worth further investigation.

Polistes mandarinus paper wasps construct multiform nests with plant materials in different environments. We collected 62 paper wasp nests of P. mandarinus from Bomi, Tibet and studied the nesting habits of the wasp from May to August in 2009. P. mandarinus paper wasps construct their nests under the logs of wooden houses using plant fibers and resin in Bomi, Tibet. A total of 42 nests in use were collected. The average number of cells of nests in use is 79.64±65.28 at the end of August, and the average number of pupae in used nests is 13.1±10.68. The average number of cells in 20 empty nests or older nests from the past year is 12.95±3.99. Successful haplometrosis had more than 80 individuals at the end of August. Three nest construction modes (mode 1-3) are used by P. mandarinus paper wasps, of which mode 1, however, is the most common. Paper wasps choose mode 1 most frequently to construct their nests because the nests constructed in mode 1 are most stable. Other two nesting modes are used rarely only when the wasps are short of nesting materials.