Transmembrane emp24 domain (TMED) gene is closely related to immune response, signal transduction, growth and disease development in mammals. However, only the Drosophila TMED gene has been reported on insects. We identified the TMED family genes of silkworm, Tribolium castaneum, tobacco moth and Italian bee from their genomes, and found that the TMED family gene composition patterns of one α-class, one β-class, one δ-class and several γ-classes arose in the common ancestor of pre-divergent Hymenoptera insects, while the composition of Drosophila TMED family members has evolved in a unique pattern. Insect TMED family γ-class genes have evolved rapidly, diverging into three separate subclasses, TMED6-like, TMED5-like and TMED3-like. The TMED5-like gene was lost in Hymenoptera, duplicated in the ancestors of Lepidoptera and duplicated in Drosophila. Insect TMED protein not only has typical structural characteristics of TMED, but also has obvious signal peptide. There are seven TMED genes in silkworm, distributed in six chromosomes. One of seven is single exon and others are multi-exons. The complete open reading frame (ORF) sequences of seven TMED genes of silkworm were cloned from larval tissues and registered in GenBank database. BmTMED1, BmTMED2 and BmTMED6 were expressed in all stages and tissues of the silkworm, and all genes were expressed in the 4th and 5th instar and silk gland of the silkworm. The present study revealed the composition pattern of TMED family members, their γ class differentiation and their evolutionary history, providing a basis for further studies on TMED genes in silkworm and other insects.
Animals ; Bombyx/metabolism* ; Genes, Insect/genetics* ; Moths/metabolism* ; Insecta/metabolism* ; Drosophila ; Insect Proteins/metabolism* ; Phylogeny ; Mammals/genetics*

The observation statistics suggested that the haemolymph melanization speed of larvae became fast and the growth inhibition of Escherichia coli was strong as the quantities of feeding on mulberry leaves increased. The RT-PCR result showed that the mRNA expressions of melanin biosynthesis enzyme BmTan, BmPo-1, BmYellow-f and BmDdc were high in the haemolyph of 5 L 3 d larvae. The qPCR analysis showed Bmtan, Bmddc, Bmyellow, Bmebony and Bmblack, especially Bmddc expression were significantly higher in black disease larvae than in normal larvae. Compared with control, Ddc inhibitors drastically inhibited the lipopolysaccharide-induced haemolymph melanization. In addition, the content of Dopa and Dopamine markedly rose after E. coli injection. These indicated that haemolymph melanization was linked to immune defenses and Bmddc may play a role in melanization response of haemolymph immune in silkworm.
Animals ; Bombyx ; enzymology ; genetics ; microbiology ; Escherichia coli ; Genes, Insect ; Hemolymph ; chemistry ; Larva ; Melanins ; biosynthesis

OBJECTIVETo verify the miRNA in Aedes albopictus and characterize the expression profile of several miRNAs across all the life stages of Aedes albopictus.

METHODSBased on the published miRNA sequences of Anopheles gambiae and Aedes aegypti, 6 DIG-labeled antisense probes were synthesized. The total RNAs from Aedes albopictus in 6 developmental stages (embryo, early larvae, late larvae, pupa, male and female adults) were extracted with a mirVana miRNA isolation kit, loaded onto 15% denaturing polyacrylamide gel and hybridized with the appropriate DIG-labeled probes.

RESULTSNorthern blotting detected 5 miRNAs in Aedes albopictus, of which mir-9a was mainly expressed in embryo and larva stages, let-7 in pupa and adult stages, miR-184 in all life stages, mir-M1 only in the embryos and miR-1175 in all the life stages except for embryos. The expression profiles of these miRNAs in Aedes albopictus were similar to those in D. melanogaster and An.stepheni. miR-1174 was not detected in any of the developmental stages of Aedes albopictus.

CONCLUSIONThese results present the first direct experimental evidence of miRNA in Aedes albopictus. The expression profiles of the analyzed miRNAs in Aedes albopictus showed stage specificity and conservation with other mosquitoes. Further studies on the functions of these miRNAs may offer new insights in mosquito biology and may lead to novel approaches to the development of insecticides.

Aedes ; genetics ; Animals ; Female ; Gene Expression Profiling ; Genes, Insect ; Larva ; genetics ; Male ; MicroRNAs ; genetics ; isolation & purification ; Pupa ; genetics

OBJECTIVETo investigate the heterozygous genotype and molecular characteristics of Organophosphorus resistance associated with heterozygous Estbeta2 of esterase B2 gene from natural population of Culex pipiens complex.

METHODSGenomic DNA was extracted from natural populations of Culex pipiens complex in Hangzhou. The PCR-restriction fragment length polymorphism (PCR-RFLP) assay was applied to type the resistance associated esterase gene. Estbeta2 of esterase B2 gene was identified by PCR-RFLP, and the genotyping for heterozygous Estbeta2 was carried out after restriction enzyme digesting by Bfm I endonuclease.

RESULTSThe DNA was isolated from 207 Culex pipiens respectively, while 156 PCR samples showed positive and the positive rate was 75.36% (156/207). The PCR-RFLP assay of esterase B2 gene revealed that the Estbeta2 was accounted about 28.20% (44/156) in 156 positive samples. There were two genotypes identified, namely homozygous Estbeta2 (90.90%, 30/33) and heterozygous Estbeta2 (9%, 3/33), heterozygous Estbeta2 was in existence of a hybrid form as which combined with Estbeta2 and a subtype (Estbeta2/Estbeta2(1)).

CONCLUSIONHeterozygous Estbeta2 of Organophosphorus resistance associated with esterase genotype was determined in natural population of Culex pipiens, and a genotyping method was established.

Animals ; Culex ; enzymology ; genetics ; Genes, Insect ; Genotype ; Heterozygote ; Insecticide Resistance ; genetics ; Insecticides ; pharmacology ; Organophosphorus Compounds ; pharmacology ; Phenotype ; Serine Endopeptidases ; genetics

OBJECTIVE: To detect the 278 bp region of gene of the cytochrome oxidase subunit I (COI) in mitochondral DNA (mtDNA) of sarcosaphagous flies, identify the species of sarcosaphagous flies, and provide reference for forensic application. METHODS: Samples were collected in Baotou and Chifeng of Inner Mongolia, Tianjin, Nanning, Fuzhou, Linyi of Shandong, Shijiazhuang, Yinchuan, Lanzhou, Huairou of Beijing, Xinxiang and Nanyang of Henan, Datong of Shanxi, Wuhu of Anhui, Quzhou of Zhejiang, Changsha, Zhuzhou and Yongzhou of Hunan. A total of 38 flies were randomly collected from rabbits, dogs and pigs which were set outdoors, then the flies' mitochondrial DNA (mtDNA) were extracted by the improved small insects DNA homogenate method. Amplification was conducted by Perkin-Elmer 9600 thermal cycler, then vertical non-denaturing 7% polyacrylamide gelectrophoresis. PCR products were purified using the nucleic acid purification kit. Sequences of both strands were obtained by direct sequence of the double-stranded PCR product using one of the PCR primers and the ABI PRISM big dye terminator cycle sequencing dit. Sequence reactions were electrophorsed on ABI Model 3730 DNA Sequencers. A UPGMA tree was contrasted using the maximum composite likelihood method in MEGA4. RESULTS: The 38 sarcosaphagous flies belonged to 3 families(Muscidae, Calliphoridae, and Sarcophagidae), 10 genuses (Musca Linnaeus, Hydrotaea Robineau-Desvoidy, Aldrichina Townsend, Hemipyrellia Townsend, Achoetandrus Bezzi, Protophormia Townsend, Chrysomya Robineau-Desvoidy, Lucilia Robineau-Desvoidy, Helicophagella Enderlein, and Boettcherisca Rohdendorf), and 12 species [Musca domestica (Linnaeus), Hydrotaea (Ophyra) capensis (Wiedemann), Lucilia caesar (Linnaeus), Lucilia illustris (Meigen), Aldrichina graham (Aldrich), Hemipyrellia ligurriens, Achoetandrus (Chrysomya) rufifacies (Macquary), Protophormia terraenovae (Robineau-Desvoidy), Chrysomya megacephala (Fabricius), Lucilia sericata (Meigen), Helicophagella melanura (Meigen), and Boettcherisca peregrine (Robineau-Desvoidy)]. CONCLUSION The genus of the sarcosaphagous flies can be identified by 278 bp gene sequence analysis of CO I in mtDNA. This method is rapid, convenient and precise.
Animals ; China ; DNA, Mitochondrial ; genetics ; Electron Transport Complex IV ; classification ; genetics ; Forensic Medicine ; Genes, Insect ; Genes, Mitochondrial ; Larva ; genetics ; Phylogeny ; Sarcophagidae ; classification ; genetics ; Sequence Analysis, DNA ; Species Specificity

OBJECTIVETo clone and identify olfactory receptor odorant receptor 7 (OR7) gene of Aedes albopictus and analyze its expression profile and calcium regulation function.

METHODRT-PCR was used to amplify the olfactory receptor OR7 gene of Ae. albopictus and OR7 expression was detected in different tissues and organs. The coding sequence of OR7 gene was cloned in eukaryotic expression vector pME18s, which was then transfected into HEK293 cells. The calcium callback function in response to odor molecule stimulation was analyzed by calcium imaging technique.

RESULTSThe OR7 gene of Ae. albopictus was cloned and sequence analysis showed that its coding region was 1395 bp. RT-PCR detected OR7 expression in the larvae, pupae and adult mosquitoes, especially in female mosquitos. Preliminary analysis of calcium callback function demonstrated the specific regulation of calcium absorption by OR7 in response to odor molecule stimulation.

CONCLUSIONThe OR7 gene of Ae. albopictus has been cloned successfully. OR7 is highly expressed in female mosquitos and is capable of specific recognition of the odor molecules.

Aedes ; genetics ; Animals ; Cloning, Molecular ; Female ; Gene Expression ; Genes, Insect ; HEK293 Cells ; Humans ; Larva ; Pupa ; Receptors, Odorant ; genetics

The full length cDNA of silkworm hibadh gene was cloned by RT-PCR and RACE (Rapid amplification of cDNA ends) technique. The hibadh gene and its deduced amino acid sequences were analyzed. The tissue distribution of hibadh gene in 5th instar silkworm larvae was tested by RT-PCR. The expression patterns of hibadh gene in simulated weightless environment were analyzed by real time RT-PCR. The results showed that the full length hibadh cDNA sequence was 1074 bp in lenth, including an open read frame of 969 bp encoding the entire coding region of Hibadh (GenBank accession No. EU719652). The deduced amino acid sequence similarities of hibadh between silkworm and Burkholderia ambifaria, Drosophila melanogaster, Apis mellifera, Xenopus tropicalis, Mus musculus, Homo sapiens were 46%, 43%, 48%, 44%, 45%, 45%, respectively. Signal peptide analysis showed that Hibadh was a secretory protein. There wasn't glycosyl-phosphatidyl inositol anchor site in Hibadh amino acid sequence. Molecular weight and isoelectric point of Hibadh were 34.1 kD and 9.14 respectively. The RT-PCR tests indicated that the hibadh gene expressed in head, silk gland, midgut, cuticle, blood, fat body, tuba malpighii of the 5th instar silkworm larvae. There were different expression patterns of hibadh gene during different silkworm embryo period in simulated weightless environment. Simulated weightlessness resulted in the expression of silkworm hibadh gene up regulated 2.3-fold (P < 0.05), up regulated 4.6-fold (P<0.01), down regulated 7.6-fold (P < 0.01), down regulated 2.6-fold (P < 0.05) during apophysis formation period, inverse period, trachea formation period, and whole embryo period, respectively. There was no significant change of hibadh gene expression during other period of silkworm embryo between simulated weightless and control groups. There were different response patterns to simulated weightless environment between hibadh gene and whole body of silkworm. Gene showed much higher sensitivity compared to whole body in response to environment. This study is useful for the further research on the gravity biological mechanism of hibadh gene.
Alcohol Oxidoreductases ; genetics ; metabolism ; Amino Acid Sequence ; Animals ; Base Sequence ; Bombyx ; enzymology ; genetics ; Cloning, Molecular ; Computer Simulation ; Genes, Insect ; genetics ; Models, Biological ; Molecular Sequence Data ; Weightlessness

OBJECTIVETo identify and isolate the genes encoding the allergens of Psilgramma menephorn by screening the cDNA expression library.

METHODSThe cDNA expression library of Psilgramma menephorn was constructed in lambdaZAPIIphage, and the library was screened using the sera from the patients allergic to Psilgramma menephorn and those from the rabbits immunized with Psilgramma menephorn extracts. The positive clones were subcloned into pBluescript plas, and the cDNA in the positive clones were amplified with PCR and sequenced.

RESULTS AND CONCLUSIONFive positive clones were obtained by immunological screening of 5 x 10(4) recombinants. Sequence analysis showed that the positive clones contained the new genes of Psilgramma menephorn allergens. This success in isolating these genes may facilitate the development of specific immunotherapy against Psilgramma menephorn allergy and further research of allergic diseases.

Adult ; Allergens ; genetics ; immunology ; Animals ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Female ; Gene Library ; Genes, Insect ; Humans ; Immunization ; Lepidoptera ; genetics ; Male ; Rabbits ; Sequence Analysis, DNA

OBJECTIVETo isolate, identify and analyze the sex-determining gene Transformer 2 (Aaetra2) of the major vector mosquito Aedes aegypti.

METHODStBLASTn program, RT-PCR and RACE methods were used to obtain the full-length cDNA of Aaetra2. Multiple alignments of nucleotide and amino acid sequences were conducted, and the different domains in tra2 protein were indentified. RT-PCR of the total RNA extracted from different tissue from the mosquitoes in different developmental stages was performed using specific primers.

RESULTSTwo genes, namely Aaetra2-α and Aaetra2-β, were identified in different supercontig locations. The multi-transcripts were expressed by means of alternative promoters or terminators. The different domains in tra2 protein were defined as RS-rich N-terminal region, RNA recognition motif-RRM, linker region, and RS-rich C-terminal region. Both Aaetra2-α and Aaetra2-β showed sustained expression throughout the developmental stages of Ae.aegypti, and in all the tissues without a sex specificity.

CONCLUSIONAaetra2 gene has multiple isoforms and is mapped to multiple locations in the genome. Aaetra2 has conservative functional domains of the sex-determining gene tra2. For Ae.agypti, Aaetra2 shows the potential as a new target for release of insects carrying a dominant lethal (RIDL) technology based on transgenic mosquitoes.

Aedes ; genetics ; growth & development ; Amino Acid Sequence ; Animals ; Drosophila Proteins ; genetics ; Gene Expression Regulation, Developmental ; Genes, Insect ; Insect Proteins ; genetics ; isolation & purification ; Nerve Tissue Proteins ; genetics ; Phylogeny ; RNA-Binding Proteins ; genetics ; Ribonucleoproteins ; genetics ; Sequence Alignment ; Serine-Arginine Splicing Factors ; Sex Differentiation ; genetics

OBJECTIVE: To identify sarcosaphagous flies and their larvae, pupa. METHODS: Sarcosaphagous flies and their larvae, pupas were collected from human corpses and their surroundings in the Weifang city. A 304 bp region in COI gene was analyzed by mtDNA sequencing. RESULTS: The studied region showed no sequence divergence within same species and significant difference were found between different species in all samples. CONCLUSION It is a practical approach to identify these Sarcosaphagous flies and their larvae, pupas by sequence analysis of the 304bp region of the COI in mtDNA.
Animals ; Base Sequence ; China ; DNA Primers ; DNA, Mitochondrial/genetics* ; Diptera/genetics* ; Electron Transport Complex IV/genetics* ; Forensic Medicine ; Genes, Insect ; Humans ; Larva/genetics* ; Phylogeny ; Polymerase Chain Reaction/methods* ; Pupa/genetics* ; Sequence Analysis, DNA/methods* ; Species Specificity