Carboxylesterase is a multifunctional superfamily. To determine the function of carboxylesterase in different tissues of Bombyx mori, we cloned a carboxylesterase gene named BmCarE-9 (GenBank accession no. EU523534) from B. mori by using rapid amplification of cDNA ends (RACE) and reverse transcription polymerase chain reaction (RT-PCR) methods. BmCarE-9 contains a 1 680 bp open reading frame (ORF), which encodes 559 amino acids. This cDNA-deduced protein BmCarE-9 has a predicted molecular weight (MW) of 64.2 kD, and isoelectric point (pI) of 7.13. Structure analysis showed that BmCarE-9 has a similar catalytic triad in which two residues have changed. We investigated the developmental expression patterns of BmCarE-9 in different tissues of the Day-3 5th instar larvae and in silk glands of different day-old 5th instar larvae by real-time quantitative PCR. The results showed that BmCarE-9 was expressed specifically in silk glands at high levels, and mainly expressed in the median and posterior silk glands. Furthermore, the expression of BmCarE-9 increased as silk glands developed, and decreased at the end of 5th instar stage. It is so inferred that BmCarE-9 might be involved in the development of silk glands or the synthesis of silk proteins.

【Aim】 cDNA cloning and prokaryotic expression of Plutella xylostella odorant receptor Or83b were conducted in order to provide the basis for studying the molecular mechanism of selection behaviour between P. xylostella and host plants, and developing insect behavior regulators. 【Methods】 cDNA encoding Or83b of P. xylostella was amplified by RT-PCR and cloned into pMD18-T vector for sequence analysis, the target gene was subcloned into pET-32a (+) for prokaryotic expression and then expressed under the induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. 【Results】 A gene encoding odorant receptor 83b from P. xylostella was cloned and named PlxyOr83b (GenBank accession number: GQ923610). The ORF of PlxyOr83b was 1 413 bp in length, encoding 471 amino acid residues, with the predicted pI of 7.19. Prokaryotic expression recombinant vector, pET-PlxyOr83b, was successfully constructed. SDS-PAGE assay showed that the target gene was highly expressed in BL21(DE3) and the molecular weight of fusion protein was about 32.0 kD. Western blot indicated that PlxyOr83b was expressed correctly in DE3. 【Conclusion】 PlxyOr83b was successfully cloned and expressed, and its sequence and protein structure are consistent with those of Or83b genes in Drosophila and other insects.

Myosin light chain 2 (MLC2) plays an important role in biological motility. Myosin light chain 2 gene was cloned from Bactrocera dorsalis and named as BdorMLC2. The open reading frame of BdorMLC2 is 669 bp in length encoding 222 amino acid residues. On-line SMART analysis revealed that BdorMLC2 has two EFh functional domains that can bind Ca²⁺, and belongs to one of the members of the troponin C superfamily. Homology analysis of amino acid sequence showed that BdorMLC2 has very high similarity with MLC from other insects and shares 93.2% identity with the MLC sequence from Drosophila melanogaster. Tissue expression analysis revealed that BdorMLC2 transcript was observed clearly in thorax of male B. dorsalis, and highly expressed in head (antennae removed), foreleg, middle legs, hind legs and wings. The temporal expression pattern further indicated that BdorMLC2 was expressed in different developmental stage. The highest expression level was found in newly-emerged adult compared to other developmental stages. The results suggest that BdorMLC2 might be closely related to muscle contraction of B. dorsalis. This study provides a basis for further researching the function of BdorMLC2.

Dipteran cell lines are widely used for studies of genetics, molecular biology, developmental biology, the host-parasite relationship in insect-borne pathogenic microbes and insect antimicrobial peptides. A new cell line from Sarcophaga peregrina, designated as Sp-E-HNU11, has been established. The primary culture from minced embryos of S. peregrina was initiated on November 17, 2008, grown in Shields & Sang M3 insect cell medium at 28℃, and was split into two 26 days later. Since then, it has been subcultured for 72 passages. The cells, mainly round or spindle-shaped, adhere tightly to the flask. The population doubling time was 42 h. Most cells in metaphase observed were sub-diploid and contained ten or twelve chromosomes, which were short pole-like except two micro chromosomes. β-naphthyl esterase and aspartate aminotransferase isozymes of this cell lines displayed one and three bands, respectively, in sodium dodecyl sulfate polyacrylamide gel electrophoresis. In random amplified polymorphic DNA analysis, the Sp-E-HNU11 cells had a banding pattern markedly different from the ones of Px-E-HNU12, IPLB-Sf-9 and Bm-21E-HNU5 cells. The establishment of the new cell line would provide an additional tool and vector for research in insect antimicrobial peptides and related fields.

The aim of the study was to understand the location, surface conformation and ultrastructure of the sex pheromone glands (the release system of sex pheromone) of female moth, and type, configuration, distribution and function of male antennae sensilla (sex pheromone receiving system) of Holcocerus vicarius (Walker) adult. Sex pheromone glands of the females and the antennae of the males were observed by using scanning electron microscope (SEM) and transmission electron microscope (TEM). The results showed that the pheromone glands were formed at the mid-dorsal region of the intersegmental membrane between the 8th and 9th abdominal segments. Many plump cones were distributed on the surface of the gland. There was evident conjugation between glandular cells and there were many basal involutions in the basal membrane of cells. Microvilli were distributed on the cytoplasmic membrane and linked with endocuticle on which there were many layers of chitin. In the glandular cytoplast of 2-day-old virgin female moths, the cells contained lipid granules, a lot of empty bubbles, smooth and rough endoplasmic reticulum, and mitochondria. There were five types of sensilla on male antenna flagellum, including sensillum trichodea, sensillum chaetica, sensillum basiconica, sensillum coeloconica and sensillum trichodea curvata, among which sensillum trichodea is the most abundant and sensillum trichodea curvata is the least abundant. The sensilla were not observed on the antenna scape and pedicel for they were covered by a large number of scales. The study provides the basis for understanding the biosynthesis of sex pheromone, extraction and identification of sex pheromone, and reproductive and mating behavior of H. vicarius．

In order to well understand the pathogenesis of entomopathogenic fungus, Beauveria bassiana to pine caterpillar, Dendrolimus tabulaeformis Tsai et Liu, and to provide the scientific guidance for biological pest control in northern China, in the present study, B. bassiana strain No.1573 was used to infect the pine caterpillar, D. tabulaeformis. The fungal infection process and the host hisopathological changes were observed using light microscopy (LM) and scanning electron microscopy (SEM). The results showed that the pathogenic fungus generally infected the pine caterpillar by penetrating the integument. At 24 h after inoculation, the attached conidia were found on the vertex and the regions around ocellis, antenna and mouthpart on the head, while on the thorax and abdomen, they adhered on verrucas, tufts, the acanthae and intersegments. At 36 h after infection, the conidia germinated into hypha on the cuticle, and then the hyphal tip differentiated into appressoria and penetration pegs to penetrate the cuticle. At 48 h, hyphae penetrated the integument depending on both the mechanical pressure and degradation action from hyphal extracellular enzymes, causing cuticular rupture and melanism. At about 72 h after infection, the hyphae had entered the body cavity and infected hemolymph, fat body, muscle, alimentary canal, silk gland and nerve tissue. By utilizing the nutrition of haemolymph and internal organs and tissues, the fungus massively proliferated. The insect body showed swelling and dark. At 96 h after infection, as hyphae occupied the haemocoel, the internal tissue structure of the pine caterpillar was totally destroyed and the insects died. At last, the hyphae broke through the cuticle and released new conidia on the cadaver surface. This study revealed that the strain No.1573 of B. bassiana is an effective pathogen to the pine caterpillar, D. Tabulaeformis. It is a series of infection in the integument and inner tissues that causes the host insect to die.

The relationships between ovarian development, body size and take-off behavior as well as the indicator significance of wax on the abdomen in the spiraling whiteflies (Aleurodicus dispersus) were studied by dissecting their ovaries and measuring their body size, in order to analyze the influence of the ovarian development and body size on take-off behavior. The results showed that the ovarian development of A. dispersus could be divided into 6 stages based on the morphological characteristics of ovaries and eggs, i.e., stage 0 (No oocyte stage), stage Ⅰ (Pre-vitellogenic stage), stage Ⅱ (Vitellogenesis stage), stage Ⅲ (Egg maturation stage), stage Ⅳ (Ovipositing stage), and stage Ⅴ (Post-oviposition stage). The ovaries of most take-off female adults were at stageⅣ (Ovipositing stage). The number of ovarioles of takeoff adults with wax was less than that of non-take-off adults, while the number of eggs was more than that of non-take-off adults, and the body size of take-off adults was different from that of non-take-off adults. The number of eggs and the body length of adults with wax were both correlative with their body size, but those of the other type of individuals were not. Most female adults with wax had mature spermatothecae. Therefore, the ovarian development had an effect on take-off activity, and the wax on the abdomen of A. dispersus was an important indicator of its body growth and reproductive maturity.

The mitochondrial genome (mitogenome) of Ruspolia dubia (Orthoptera: Conocephalinae) contains a short control region with 70 bp in length. So, the mitogenome of Conocephalus maculates come from Conocephalinae was sequenced by using long-PCR and sub-PCR techniques. The mitogenome of C. maculates is 15 898 bp in size, its A+T content is 72.05% and the genome organization follows the ancestral insect gene arrangement. All protein-coding genes (PCGs) start with a typical ATN codon, while nine of the 13 PCGs end with TAA or TAG, and the remainder have incomplete termination codons T. Except for trnS^AGN, all 21 tRNAs have the typical clover-leaf structures. According to the unusual Type-9 of Steinberg et al. (1997), the DHU arm of trnS^AGN forms a simple 7-nt loop and the anticodon stem has nine base-pairs with a bulged nucleotide in the middle in contrast to the normal five base-pairs. The main differences between C. maculatus and other orthopteran mitogenomes was that two novel larger intergenic spacers (78 bp and 360 bp) between trnS^UCN and nad1, nad1 and trnL^CUN, respectively. The intergenic spacer between nad1 and trnL^CUN has been labeled unidentified open reading frame (UORF) because N strand comprises an open reading frame (103 amino acids) complete with start and termination codons (ATT/TAA). The usage of synonymous codon markedly is correlated with the nucleotide at the 3rd codon position, but has no relation to the anticodon of mitogenome tRNA. Generally, for all codons with their relative synonymous codon usage (RSCU) more than 1, the 3rd codon positions are A or T. Noncanonical matches of G-U base pairs accounts for the overwhelming majority of total unmatched base pairs in the orthopteran mitogenome tRNAs. The results suggest that the G-U may be one cognitive base pairing in mitogenome. The C. maculates mitogenome sequences, as well as the previously determined orthopteran mitogenomes, provide sequence source to reconstruct Orthoptera evolutionary history.

Up to now, the reports about the complete mitochondrial genome of butterflies and their corresponding molecular evolutions are still limited. In this paper, the complete mitochondrial genome (mitogenome) of Calinaga davidis was sequenced and analyzed using the long PCR and the conserved primer walking technology. The results showed that the entire mitochondrial genome sequence was 15 267 bp long (GenBank accession no. HQ658143), containing 13 proteincoding genes (ATP6, ATP8, COI-III, ND1-6, ND4L and Cytb), 22 transfer RNA genes, 2 ribosomal RNA genes (lrRNA and srRNA) and a putative control region (D-loop). All the 37 genes are arranged in the same orientations as those of other lepidopteran species determined; and there are 11 intergenic spacer sequences totalling 130 bp (1 bp to 46 bp for each sequence) and 13 overlapping sequences totalling 66 bp (1 bp to 35 bp for each sequence), interspersed throughout the genome, and the largest (46 bp) spacer region and overlapping region are located between the tRNA^Gln and ND2, and between COII and tRNA^Lys genes, respectively. The large and small rRNA genes are 1 337 bp and 773 bp in size, respectively. All the tRNA genes have typical leaf clover secondary structures, except for the tRNA^Ser(AGN), whose DHU arm forms a simple loop. The 13 protein-coding genes are 11 247 bp in length with corresponding 3 737 codons, their base compositions have a relatively higher AT bias and their codon usages are also predominantly A+T-rich. All the protein coding genes (PCGs) use standard initiation codons ATN, except for the COI gene, which uses TTG as its starting codon, and all the PCGs use common stop codon (TAA), except for the COI and ND4 genes, which terminate into a single T and TA, respectively. The A+T-rich region is 389 bp in length with the AT content up to 92.0%, and this region contains two microsatellite-like repeating sequences (TA)₆ and (AAT)₄. The results of this study provide some important molecular data to clarify the systematical status of Calinaginae, as well as its phylogenetic relationship with other nymphalid subfamilies.

 In order to study the distribution and morphometric characteristics of populations of the Chinese honeybee, Apis cerana cerana, and the diversity and genetic characteristics of A. c. cerana in different ecological regions in Fujian, Southeast China, a total of 780 worker bees of A. c. cerana collected from 11 samples throughout Fujian province were studied using morphometric methods. The 30 morphometric characters according to Ruttner et al. (1988) were measured. The data were statistically analyzed by analysis of significance of difference, discriminant analysis and cluster analysis. The results showed that a high degree of morphometric difference existed among the honeybees in Fujian. There were at least three populations of A. c. cerana, i.e., northern Fujian population, central Fujian population and southern Fujian population. The honeybees of northern Fujian had significantly larger size of body and wax mirror, longer fore wing and proboscis, and special forewing angles (P<0.01). The honeybees of Wuyi had the biggest wing angles G18, J10 and L13, and the smallest wing angle E9 (P<0.01). The honeybees of Guangze and Zhenghe had the smaller wing angles K19, O26 and L13, and the larger wing angles B4 and N23 (P<0.01). The honeybees of central Fujian population from Fuzhou, Youxi, Jiangle and Ningde differed significantly from those of other samples with larger body size, organs and the larger wing angles G18 and K19, the median N23 (P<0.01). The honeybee population from southern Fujian, Longyan, Yongding, Wuping and Zhangzhou differed significantly from those of other samples with smaller size of body and organs (P<0.01). Morphometric analysis of angles of forewing veins might be a useful tool for biodiversity studies of honeybees and other bees. This study provides more theoretical support for effectively preserving Fujian indigenous A. c. cerana as a genetic resource for future utilization in Asian honeybee breeding programs.

Rhus gall aphids include six genera and 12 species, and are mainly distributed in East-Asia with only one species Melaphis rhois in North America. Based on morphological characteristics and molecular data, we studied the taxonomic status of the North America species M. rhois. The numerical taxonomy on eight species from six genera of Rhus gall aphids was studied using hierarchical clustering analysis. A total of 108 morphological characters from 179 individuals of the alate adults were evaluated from all parts of aphid body, among which 48 characters from head, thorax and abdomen were directly measured, 31 characters were the ratio from two measured characters, and the rest were numerical values converted from morphological characters. The cladistic analysis based on the Euclidean distance showed that the eight Rhus gall aphid species are clustered into three clades, among which the North America species M. rhois is closely clustered with the two species from the genus Schlechtendalia; the two species from Nurudea and Floraphis meitanensis, Meitanaphis elongallis and two subspecies from the genus Kaburagia are clustered as a group, respectively. In addition, the 1.2 kb DNA sequences of mtDNA COI gene from 16 individuals of eight Rhus gall aphid species were obtained. Both of the MP and ML phylogenetic trees showed that the eight Rhus gall aphid species are clustered into three clades, and the North America species M. rhois clustered closely with the genus Schlechtendalia, which are consistent with the results of the morphological analysis. However, the relationship between the three clusters are inconsistent with that based on the morphological data. In molecular trees, North America species M. rhois and two species from the genus Schlechtendalia, F. meitanensis and M. elongallis, and two species from Nurudea and two subspecies from the genus Kaburagia are clustered as three groups I, II and III, respectively. The group I and II have relatively close relationship, but with very low bootstrap value (<50%). It is necessary to collect more species and use more molecular data to resolve the phylogeny of Rhus gall aphids.

Aphids are a group of phloem-feeding hemipteran insects. Due to their some unique biological features, aphids are good model organisms for understanding many significant theoretical issues in adaptive evolution. Aphids maintain an obligate endosymbiotic association with Buchnera, their primary endosymbionts, which are nutritionally and developmentally indispensable to aphids. Studies on phylogenetic relationships between aphids and Buchnera help us understand the evolution of obligate symbiosis. In this article we reviewed the advances in phylogenetic relationships between aphids and Buchnera at different taxonomic levels (from higher to lower). Available evidences indicate that parallel diversifications of aphids and Buchnera occur at lower taxonomic level. However, such relationships may not exist at higher taxonomic level, which contradicts with the traditionally accepted parallelism hypothesis. On the basis of the detailed review of previous studies, it is proposed that more taxonomic groups should be sampled and more genomic data as well as robust phylogenetic concordance tests should be used. Furthermore, Buchnera horizontal transfer and consistency of evolutionary rates of Buchnera genes among different aphid lineages should be investigated to clarify the evolutionary relationships between aphids and Buchnera.

To investigate the molecular basis and regulatory mechanism of antennal development, structure and function of Bombyx mori, high resolution and twodimensional polyacrylamide gel electrophoresis followed by matrixassisted laser desorption ionization and time of flight mass spectrometry (MALDI-TOF/MS) was applied to analyze the total proteins in antennae and their expression difference between male and female moths. The ImageMaster 6.0 software was used to analyze the two-dimensional gel electrophoresis profiles. Approximately a total of 550 protein spots were detected in the antenna of silk moth, most of which were arranged from 14 to 70 kD with pI value 4-8. In the two-dimensional gel electrophoresis profiles of male and female antenna, 419 and 489 protein spots were detected, respectively, and the number of matched protein spots between male and female was 326. The matching rate was 71.81%. There were 34 protein spots which have different gray value but were distributed in both gel electrophoresis profiles of male and female antenna. Meanwhile, 9 and 20 specific protein spots were detected in gel electrophoresis profiles of female and male, respectively. Sixty-three protein spots were identified by MALDI-TOF/MS and five proteins were obtained, including imaginal disk growth factor, cuticular protein RR-1 motif 15, thiol peroxiredoxin, vacuolar ATPase B subunit and gasp precursor. These proteins are involved in cell proliferation, epidermis formation, the removal of H₂O₂ in vivo, building up of a H⁺ gradient, etc. These five proteins were expressed differentially between female and male antenna, and all had higher expression levels in male antenna than in female antenna. The results provide some basic data for the further research of antenna proteins of the silk moth.

We developed a method of RNA interference based on bacterially expressed dsRNA in the silkworm, Bombyx mori. By inserting the target, FTZ-F1 gene fragment, between the two convergent T7 polymerase promoters in opposite orientation in L4440 dsRNA expression vector, the recombinant plasmid was formed. Then the recombinant plasmid was transformed into Escherichia coli HT115, an RNase-III deficient strain. dsRNA was extracted from the E. coli HT115 after being treated with isopropyl-β-D-thio-galactopyranoside (IPTG). RNAi treatment was performed by injecting the extracted dsRNA (25 μg) into body cavity of silkworm at the 7th day of 5th instar. The RNAi of FTZ-F1 gene resulted in 85% of the insects with delay of pupal metamorphosis and disablement in pupa formation. Real-time quantitative PCR analysis revealed that the expression of FTZF1 gene was specially inhibited after the insects were treated with dsRNA of FTZ-F1 gene. The results suggest that the bacterially expressed dsRNA has potential to be used in silkworm functional genome analysis in an economical and efficient way.

In order to explore the mechanism of interference competition of the red imported fire ant, Solenopsis invicta Buren, against native ants and the success of invasive species, important invasive species S. invicta and two native ants Tapinoma melanocephalum (Fabricius) and Pheidole fervida Smith were used as test insects in this study. Interference competition between S. invicta and two native ants at one-on-one and community levels was studied in the laboratory. The aggressiveness test at the one-on-one level between S. invicta and T. melanocephalum revealed that the mutual aggressiveness was weak because workers of both species did not perform stronger tussle, but only displayed threatening posture although S. invicta had more competitive advantage. The aggressive level was concentrated on level Ⅲ. The aggressive indices between one of minor, medium workers of S. invicta and soldiers of P. fervida were significantly higher than those between major workers of S. invicta and soldiers of P. fervida, suggesting that the aggression between those two ants are more intensive. In tests at the community level, no dead S. invicta ants were found in competitive combination of S. invicta against T. melanocephalum with the average mortality of 31.80% for T. melanocephalum ants, although the mortality for S. invicta was 0.20%-12.00% in competitive combination of S. invicta against P. fervida with the average mortality  of 49.91% for P. fervida ants. The results indicated that the aggressive intensity of S. invicta was stronger than the two native ants, and the aggressiveness between S. invicta and P. fervida was stronger than that between S. invicta and T. melanocephalum. The lower mortality of T. melanocephalum may owe to its chemical defensive ability. This work laid a theoretical foundation for further study on protection and utilization of the dominant native species T. melanocephalum against S. invicta.

Automatic identification of insects is an important and emerging area of research. The screening features and transforming multi-class classification into two-class classification properly are two key procedures in the process. In this article, a novel method for automatic identification of multi-class insects was developed based on support vector classification (SVC). Firstly, the initial multi-class samples were transformed into two-class samples with interaction transformation. Secondly, a symmetrical kernel function was inducted to solve the rank problem of the two initial samples in interaction sampling pair. Thirdly, irrelevant and redundant features were eliminated nonlinearly with SVC and the relative importances of kept features were listed. Lastly, the prediction results were further corrected by simple-vote decision. The new method was applied to identify the butterflies of seven species at species level and family level, and the accuracies at both levels are 100%. The results show that the new method can be widely used in the prediction area of multi-class classification, such as automatic identification of insects.