Overexpression of cytochrome P450 CYP9A17v2 gene is involved in pyrethroid resistance in Helicoverpa armigera (Hübner). In order to study the mechanisms underlying regulation of CYP9A17v2, functional analysis of the promoter of CYP9A17v2 from H. armigera was performed in this study. Luciferase reporter plasmids containing serially truncated promoter fragments (-1 095-+43) of CYP9A17v2 were transiently transfected into Sf9 cells, and the promoter activity was measured with dual-luciferase reporter assay system. Functional analysis showed that all the seven deleted fragments had promoter activity, and a region between -197 and +43 exhibited the highest level of promoter activity. Transcription enhancer elements may exist in the region between -197 and -113 of CYP9A17v2 5′-regulation area, and transcription repressor elements may situate in the region between -1 095 and -197. This study would provide an important foundation for investigating transcriptional regulation mechanisms of CYP9A17v2 overexpression in H. armigera.

RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing initiated by double-stranded RNA (dsRNA) which is homologous in sequence to the silenced gene. The cadherin-like protein, similar to cadherin, lies in brush border membrane vesicles (BBMV) of insect. It had been confirmed to be a receptor of Bacillus thuringiensis (Bt) insecticidal protein in several insects. In this study, two cDNA segments (CAD1 and CAD2) encoding the cadherin-like protein gene were cloned through RT-PCR technique using gene specific primers; double-stranded RNA (dsRNA) was synthesized by using the two gene segments respectively, and microinjected into 3rd instar larvae of Plutella xylostella; and the effects of different target sites, dose and silencing time on cadherin mRNA expression level were assayed. The results showed that the expression of cadherin-like gene was reduced sharply at 48 h after the dsRNA corresponding to CAD1 segment was injected and restored at 72 h. The immunoblotting results indicated that cadherin-like protein in BBMV of P. xylostella was reduced markedly at 48 h after injection. The cadherin-like gene was silenced successfully in this experiment, and the establishment of RNAi technique can help reveal the gene function of P. xylostella including cadherin-like gene.

Chemosensory proteins (CSPs), which are assumed to be related with the process of storing and releasing the chemical odors for insects, belong to a class of small soluble polypeptides in insect chemical receptors. In this study, chemosensory protein of Microplitis mediator MmedCSP1 was expressed using pGEX-4T-1/BL21 (DE3) prokaryotic expression system, and then purified using GSTrap FF, and the binding characteristics of MmedCSP1 to 50 kinds of chemical molecules were further studied using bis-ANS (4, 4′-dianilino-1, 1′- binaphthyl- 5, 5′-sulfonate) as fluorescent probes. The results showed that MmedCSP1 could bind to methyl salicylate, pentane, ocimene, β-ionone, 3, 4-dimethylbenzaldehyde, 2-hexanone and cis-3-hexen-l-ol. However, only β-ionone replaced the bis-ANS of MmedCSP1 by 50%, and the binding constant was 16.89 μmol/L. These results suggest that MmedCSP1 is involved in the process of storing and releasing methyl salicylate, pentane, ocimene, β-ionone, 3, 4-dimethylbenzaldehyde, 2-hexanone and cis-3-hexen-l-ol for M. mediator, and has different transport capability in storing and releasing those odorants.

HSP90 gene of Tetranychus cinnabarinus was cloned and sequenced by the combination of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) approaches. A cDNA fragment of 2 595 bp was obtained, including an open reading frame (ORF) of 2 169 bp which encodes a polypeptide of 722 amino acids with a predicted molecular weight of 83.45 kDa and a theoretical isoelectric point of 4.81, 3′-untranslated region (UTR) of 249 bp, and a 5′UTR of 177 bp. By using Antheprot analysis, five signature sequences of HSP90 family and C-terminal five amino acids of Hsp90 (MEEVD) were detected. Blast analysis indicated that the nucleotide sequence of HSP90 gene coding region of Tetranychus cinnabarinus shared high similarity with other known HSP90s, especially with those from insects of arthropods. The confirmed prokaryotic recombinant expression plasmids pET43a-TcHSP90 was transformed into Escherichia coli BL21 and expressed in prokaryotic cells. The fusion proteins of recombinant TcHSP90 in E. coli BL21(origami) were separated and identified by SDS-PAGE and Western blotting, and the result showed that the constructed prokaryotic expression plasmid could stably and correctly express in the host bacteria. Cloning and prokaryotic expression of TcHSP90 from T. cinnabarinus would provide useful experimental material for the further study of the nature and function of HSP90.

Enhanced detoxification mediated by microsomal cytochrome P450s is a major mechanism responsible for pyrethroid resistance in Helicoverpa armigera. Our previous study showed that constitutive overexpression of CYP9A12 and CYP9A14 was associated with high levels of pyrethroid resistance in the pyrethroid-resistant YGF strain of H. armigera, and functional expression of CYP9A12 and CYP9A14 in yeast provided direct evidence on their involvement in pyrethroid resistance. In this study, molecular cloning, mRNA expression, and functional expression of CYP9A17v2 of H. armigera were investigated in order to verify possible involvement of CYP9A17v2 in pyrethroid resistance. The results showed that protein sequence of CYP9A17v2 has very high identity (94%) to that of CYP9A12. CYP9A17v2 in the fat body of the final-instar larva of the YGF strain had 10.9-fold overexpression compared with that of the control strain (YG), but no overexpression was detected in the midgut. Functional expression in yeast (Saccharomyces cerevisiae) showed that CYP9A17v2 had the capability of metabolizing several pyrethroids (esfenvalerate, deltamethrin, and cyhalothrin). It is so concluded that constitutive overexpression of CYP9A17v2 also contributes to pyrethroid resistance in the YGF strain of H. armigera. Together with previous findings, three P450 genes (CYP9A12, CYP9A14 and CYP9A17v2) from the CYP9A subfamily are confirmed to be involved in oxidative detoxification of pyrethroids in H. armigera.

Batocera horsfieldi is an important poplar stem-borer in south China. Studies on spatial distribution pattern and sampling technique can provide theoretical base for the damage survey and control of B. horsfieldi in poplars. In the study, the spatial distribution pattern of egg, larva, pupa or adult of B. horsfieldi in poplars was analyzed with Taylor’s power law, Iwao’s distribution function, and six aggregation indexes. The results showed that the spatial distribution pattern of egg, larva, pupa or adult of B. horsfieldi in poplars was all aggregated, and the basic component of the distribution was individual colony, and the aggregation intensity was increased with population density. By using the parameters α and β in Iwao’s m*-m regression equation, the optimal and sequential sampling numbers when the oviposition incisions, frass holes and emergence holes were selected as control indexes were determined and can be applied in the field control.

In order to achieve the massive rearing and release of the alligator weed flea beetle, Agasicles hygrophila, for controlling the damage of alligator weed, Alternanthera philoxeroides, we explored and established the method and work flow of massively rearing A. hygrophila in the laboratory. Our method and work flow involve the combination of the following steps in sequential order: hatching eggs by putting the leaves with eggs into rearing dish or the seedlings with eggs into jars containing water; rearing the 1st, 2nd, 3rd instar larvae and the adults of A. hygrophila, and having the adults lay eggs by putting the plants of A. philoxeroides into rearing box; helping A. hygrophila to complete the pupation and eclosion process by using live stems planted in a bucket filled with a layer of thin wet soil. The adults of A. hygrophila were able to feed, copulate and lay eggs around the clock. The prophase of egg-laying was about 4-5 d and the peak phase of egglaying was from the 7th to 24th day after eclosion. The average egg mass and egg number per female was 21.08 and about 570 in total in rearing box, respectively. The average egg mass on one leaf of alligator weed was 4.28 with the approximately same number on the back of leaf when reared in rearing box. The average egg mass on one leaf of A. philoxeroides was 1.46 and the eggs were mainly laid on the back of leaf when reared in cage. The ratios of A. hygrophila hatched eggs obtained from rearing box and cage were 94.02% and 92.50%, respectively. The 1st, 2nd, 3rd instar larva and the adult could be reared in high density using fresh broken seedlings of A. philoxeroides in rearing box. However, the pupation could only be completed smoothly using live stem planted in wet soil. We found that the periods from early 1st instar larva transferring to fresh plants (or leaves) and from fully developed 3rd instar larva in transition to pupation were both the critical phases for successful high-density rearing. The optimal density of fully developed 3rd instar larvae in transition to pupation was 8 individuals per plant, while the optimal density of egg-laying females was 5 individuals per plant.

The composition and distribution of ground-dwelling spider were investigated using small plot direct searching method to assess the impact of forest type and habitat management on spider diversity. Six forest types in Xishuangbanna, Yunnan Province, southwestern China were surveyed, i.e., the tropical seasonal rain forest, limestone tropical seasonal rain forest, tropical montane evergreen broad-leaved forest, artificial forest, rubber-tea community and rubber plantation. A total of 18 plots (three repetitions for each forest type) were surveyed during three field trips in December 2006, March and July 2007. The relationships between forest type and spider diversity were explored using canonical correspondence analysis (CCA). A total of 9 849 individual spiders were collected from various habitats, of which 3 119 are identifiable adults representing 30 families. The most abundant families of ground-dwelling spider are Pholcidae, Linyphiidae, Theridiidae and Mysmenidae (62.2% of total species counts). Of the six forest types, 24 families were collected from the tropical seasonal rain forest including two endemic families; 22 families were collected from the limestone tropical seasonal rain forest; 22 families were collected from the tropical montane evergreen broad-leaved forest including two endemic families; 20 families were collected from the artificial forest; 21 families were collected from the rubber-tea community; and 19 families were collected from the rubber plantation including one endemic family. There are four families (13.3% of the total 30 families recorded) endemic to two types of rain forest, six families (20.0%) endemic to three types of natural forest, and one family (3.3%) endemic to plantations. Abundance of ground-dwelling spider in the tropical seasonal rain forest is significant higher than the remaining five types of forest. Shannon-Wiener (H′) index and Margalef index (Dmg) in the rubber plantation are significant lower than the three types of natural forest, and Pielou index (J) is also the lowest in the rubber plantation. The results of CCA and cluster analysis indicated that natural forests and plantations are well differentiated. In the group of natural forests, the tropical seasonal rain forest plots are well separated from two types of secondary forest plots. In the other group, the rubber plantation plots are well separated from the artificial forest and the rubber-tea community plots. The results suggested that: (1) human-management has an strong impact on ground-dwelling spider composition; (2) spider diversity decreases along with the increase of anthropogenic disturbance; and (3) reducing anthropogenic disturbance and increasing plant community diversity (e.g., plant tea tree in rubber plantation) is a meaningful way to conserve and restore biodiversity.

In order to investigate levels of genetic variation and differentiation of grape phylloxera, Daktulosphaira vitifoliae Fitch in China, we sequenced the mitochondrial DNA (mtDNA) gene cytochrome oxidase Ⅰ (COⅠ) of 35 samples from four populations. Of the total analyzed sites, 29 (6.13%) were polymorphic, including 17 (3.59%) parsimony informative and 12 (2.54%) singleton sites. Nucleotide frequencies of A, C, G and T were 34.8%, 15.8%, 10.2%, and 39.2%, respectively. In total, 13 haplotypes were identified in the target region. The most common haplotypes were H3 (HAP-A) shared by 5 samples from SHJ, and H13 (HAP-C) found in 19 samples from SXX, HNH and LNX populations. The SHJ population was unique and shared no haplotypes with the other three populations. Nm ranged from 0.02 to 4.03 and the genetic distance varied from 0.001 to 0.040 between populations. Nm (0.02) was smaller, and the genetic distance (0.039-0.040) larger between SHJ and other three populations. Phylogenetic analysis and haplotype network showed that all haplotypes from SHJ formed one cluster, and the other haplotypes from SXX, HNH and LNX are grouped into another, suggesting that there were at least two introductions of grape phylloxera into China.

Morphological identification of whitefly is limited by small size, the high degree of similarity and polymorphism. In this study, the method for the development of DNA markers for rapid detection of the spiny whitefly, Aleurocanthus spiniferus (Quaintance), was developed based on sequence characterized amplified region (SCAR) derived from a randomly amplified polymorphic DNA (RAPD) band. An approximately 990 bp DNA fragment of A. spiniferus, which is absent in other closely related whitefly species [e.g., Dialeurodes citri (Ashmead), Bemisia tabaci (Gennadius) (biotypes B, Q, ZHJ-1 and ZHJ-2), Trialeurodes vaporariorum (Westwood) and Aleurodicus disperses (Russell)］, was identified by RAPD-PCR analysis. After cloning and sequencing the target fragment (987 bp, GenBank accession no.: FJ613323), one pair of SCAR primers (AS-F518/AS-R938) was developed, which could amplify a single fragment of 421 bp in length. Specificity tests performed with this pair of primers showed that all A. spiniferus specimens were detectable and no cross reactions with other whitefly species were observed. The method was tested on egg (3 eggs as one sample), single 2nd and 4th instar nymph, and male and female adults, and proved to be applicable for these stages of A. spiniferus. Moreover, the 421 bp DNA fragment could be clearly identified even at the dilution as low as 1/1 920 of a whole female adult of A. spiniferus for all replicates. This technique should be useful in quarantine and detection of A. spiniferus during transportation of the seedlings of tea and orange.

The difference of genome size between different insects is due to varieties of duplication of the genome sequence in the amplification, deletion and differentiation. Fifty-nine insect species have been included in genome sequencing projects, whole-genome sequences of six insect species, i.e., Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Apis mellifera, Aedes aegypti and Tribolium castaneum, have been reported. The sizes of 725 insect genomes have been estimated, ranging from 0.09-16.93 pg (88-16 558 Mb). The estimation methods for insect genome size are introduced, and the variation of insect genome sizes and its significance are also discussed.

Cydia pomonella (L.) is an important quarantine pest in China, which is only distributed in Xinjiang Province and some areas of Western Gansu Province, but tends to spread to the east part of China. Sex pheromone-mediated monitoring and mating disruption of C. pomonella has become a commercially viable pest management technique and is used all over the world. In this article, research developments in component identification, synthesis and application of sex pheromone were reviewed. The problems and application prospects in the research of sex pheromone were also put forward and discussed.

Insects can specially recognize the opposite sexes of the same species. PBP1, OR1 and OR3 play an important role in the process of the male silkworm Bombyx mori perception and location of the females. For the purpose of finding out the molecular mechanism of difficult hybridization of B. mori and B. mandarina, and understanding the evolution of related genes to sexual recognition between the two species, we cloned three genes (Bmmpbp1, GenBank ID: GQ246497; Bmmor1, GenBank ID: GQ246496; Bmmor3, GenBank ID: GQ246498) of wild silkworm B. mandarina. Sequences analysis revealed that between the two species there were 4 singlenucleotide polymorphisims (SNPs) (C10A, A40T, T270C and A333G) in pbp1, two of which caused amino acid variation (Q to K, N to Y); there were 5 SNPs (T910C, A1147C, A1192T, T1276C and G1282A) in or1 and only 1 induced amino acid variation (F to L), and there were 4 SNPs (A507G, A513G, T605C and G672A) in or3 and 1 caused amino acid mutation (I to T). The genetic distances of the three genes are close and the evolution velocities are low. Secondary structure predicted by PHD program on line suggested that mutation sites had no influence on the adjoining regions, or on the functional sites. There are presumably no functional differences in these genes of the two related moths. Namely, male and female individuals could perceive and recognize mutually between the two species. Our findings were consistent with the experimental observations.

Tests were carried out to evaluate the toxicity of Chenopodium ambrosioides (Chenopodiacea) leaf against the 0-2 d old adult, 1st, 2nd, 3rd and 4th instar larvae of Anopheles gambiae (Diptera: Culicidae) as petroleum ether extract and essential oil respectively at ambient laboratory temperature. Thirty larvae at each instar stage were exposed to 50, 100, 250, 500, 750 and 1 000 mg/L of extract and essential oil in water, and the number of dead larvae recorded at 24 and 48 h post-exposure. Thirty adults were exposed to C. ambrosioides oil vapour in air tight rectangular glass cages at 0.8, 1.6 and 2.4 μL/L and mortality recorded 24 h after exposure. The results showed that the test extract and essential oil were toxic to all  larval stages and adults of An. gambiae. The median lethal concentration after 48 h exposure (48 h-LC₅₀) values show that the extract was most toxic to the 1st instar larvae (14.89 mg/L) followed by the 4th instar (18.90 mg/L) and least toxic to the 3rd instar (183.77 mg/L). The 48 h LC₅₀ values showed that the essential oil was most toxic to the 4th (36.62 mg/L) followed by the 1st instar (90.75 mg/L) larvae. The computed LC₅₀ value of C. ambrosioides oil against adult An. gambiae was 1.01 μL/L. The study highlights the potential of C. ambrosioides for the control An. gambiae.

【Objective】 To explore the extraction methods of genomic DNA of parasitic mites Demodex folliculorum (D.f.) and Demodex brevis (D.b.). 【Methods】 Mites were disrupted by repeated freezing and thawing in liquid nitrogen. Then, the improved DNA extraction method of miniinsects, alkaline lysis and the commercial extraction kit were used respectively to extract genomic DNA of D.f. and D.b., which had been kept freezing for ﹤5 or 8-10 months. 【Results】 The results of protein and nucleic acid radiometer showed the purity and quantity of genomic DNA extracted by the commercial kit were obviously better than those by other methods of mini-insects and alkaline lysis. Clear DNA fingerprints were found in the amplification results of random amplified polymorphic DNA (RAPD). There were obviously different banding patterns between D.f. and D.b. The freezing time affected the quantity of DNA extraction, but had only small effect on the purity and RAPD fingerprints of genomic DNA. The same species of mites had similar banding pattern even using different extraction methods. More and clear bands were detected by the commercial extraction kits and improved DNA extraction method of miniinsects. The bands detected by alkaline lysis method were less and obscure. 【Conclusions】 The application of repeated freezing-thawing in liquid nitrogen acts effectively to disrupt mites. The mites had better not be kept frozen more than 6 months. The commercial extraction kit is an effective approach to extract genomic DNA of mites. RAPD technique provides a simple method for detecting and classifying D.f. and D.b.

This paper reports the sciarid fly species on edible fungi of Yunnan province. Six genera and eleven species were identified, including one new record genus (Cosmosciara Frey), and two new record species (Bradysia difformis Frey and Cosmosciara perniciosa Edwards) in China. A key to the all species was given. All voucher specimens of the survey are deposited in the laboratory of Entomology Department, Yunnan Agricultural University. Analysis of the data of the sciarid fly species on different hosts indicated that Lycoriella pleuroti Yang et Zhang and B. difformis Frey were the dominant species on edible fungi in Yunnan.