Proteomic Analysis of the Peritrophic Matrix from the Midgut of Third Instar Larvae, Musca domestica.

OBJECTIVE: To better comprehend the molecular structure and physiological function of the housefly larval peritrophic matrix (PM), a mass spectrometry approach was used to investigate the PM protein composition. METHODS: The PM was dissected from the midgut of the third instar larvae, and protein extracted from the PM was evaluated using SDS-PAGE. A 1D-PAGE lane containing all protein bands was cut from top to bottom, the proteins in-gel trypsinised and analysed via shotgun liquid chromatography- tandem mass spectrometry (LC-MS/MS). RESULTS: In total, 374 proteins, with molecular weights varying from 8.225 kD to 996.065 kD and isoelectric points ranging from 3.83 to 11.24 were successfully identified, most identified proteins were mainly related to immunity, digestion, nutrient metabolism and PM structure. Furthermore, many of these proteins were functionally associated with pattern binding, polysaccharide binding, structural constituent of peritrophic membrane and chitin binding, according to Gene Ontology annotation. CONCLUSION: The PM protein composition, which provides a basis for further functional investigations of the identified proteins, will be useful for understanding the housefly larval gut immune system and may help to identify potential targets and exploit new bioinsecticides.

[Identification and Expression of the yellow Gene Family in Aedes aegypti].

Objective: To identify the yellow family genes in Aedes aegypti and analyze the gene structure, phylogenetic evolution and their expression at various developmental stages and in different tissues. Methods: The yellow gene family was identified in Ae. aegypti by blasting the Ae. aegypti genome database with the amino acid sequence of the MRJP domain of Dm-yellow gene of Drosophila melanogaster（GenBank No. AAF45497）. The physico-chemical property and domains were analyzed with the on-line ExPaSy software. The signal peptide was predicted using SignalP4.1 software. Sequence alignment and the phylogenetic tree were made through combined use of DNAstar, MEGA6.0 and GeneDoc. Total RNA was extracted from Ae. aegypti, cDNA was generated, and expression of the yellow family genes at various developmental stages （egg, first to fourth instar, pupa, non-blood-fed female and male mosquitoes） and in different tissues （salivary gland, midgut, fat body, and ovary） was quantified using qRT-PCR. Results: Twelve yellow genes were identified from Ae. aegypti genome: Aa-yellow, Aa-yellow-b, Aa-yellow-c, Aa-yellow-d, Aa-yellow-e, Aa-yellow-f2, Aa-yellow-fb, Aa-yellow-fc, Aa-yellow-g, Aa-yellow-g2, Aa-yellow-h, and Aa-yellow-x. Bioinformatics demonstrated that all covered the MRJP domain and a signal peptide sequence. Sequence alignment revealed low （15%-49%） homology among the proteins, but high homology（60%） in the conserved domain. According to the phylogenetic tree analysis, the encoded 12 YELLOW proteins were classified into 5 subfamilies, and 11 had orthologues in D. melanogaster. qRT-PCR revealed high expression of Aa-yellow-d （0.018 9） and Aa-yellow-x （0.023 5） in male Ae. aegypti （P<0.01 or P<0.05）; high expression of Aa-yellow-fc （0.024 8, 0.034 9） in female Ae. aegypti and in the salivary gland （P<0.01）; high expression of Aa-yellow-f2 （0.093 4） in the second instar stage （P<0.01）; high expression of Aa-yellow （0.562 1）, Aa-yellow-e （0.004 4）, and Aa-yellow-fb （0.008 4） in the third instar stage （P<0.05）; and high expression of Aa-yellow （0.569 4）, Aa-yellow-e （0.027 0）, Aa-yellow-f2 （0.006 5）, Aa-yellow-fb （0.001 0）, Aa-yellow-h （0.084 8） and Aa-yellow-x （0.015 1） in the ovary. Genes other than Aa-yellow-c （0.004 0） and Aa-yellow-x （0.007 4） were hardly expressed in the midgut. Conclusion: The 12 yellow genes identified in the Ae. aegypti genome have low homology, and are differentially expressed at different developmental stages and in tissues.

[Cloning and expression of the mucin-related protein1 (Aamucin1) from salivary gland of Aedes albopictus].

OBJECTIVE: To clone the mucin-related protein (Aamucin1) gene from salivary gland of Aedes albopictus Guangzhou isolate, and analyze the expression difference due to blood-feeding. METHODS: Total RNA was extracted from the salivary gland. The coding region of Aamucin1 was amplified with a pair of specific primers by RT-PCR. The product was sequenced and analyzed by bioinformatics. Expression analysis was conducted by real-time RT-PCR. RESULTS: The product of RT-PCR was 849 bp with encoding 283 amino acids. To compare with that from Ae. albopictus Rome strain, 13 amino acids were deleted at the C end, and Aamucin1 in Guangzhou isolate shared 58% identity in amino acids with that of Rome isolate. In addition, an alternative splicing was found in Aamucin1 and located in a proline enrich area by Protscan. To compare with that of non-blood-feeding (group SG), Aamucin1 was significantly down-regulated with 0.39 fold expression at zero time after engorged (group BSG_0, mosquitoes with abdominal distention from the first 2 hours after blood-feeding, P < 0.01) and 0.61 fold expression at the 24th hour after engorged (group BSG_24,mosquitoes from the 24th hours after blood-feeding, P > 005). CONCLUSION: The full length of Aamucin1 gene of Ae. albopictus is cloned and it can be modulated by blood-feeding.

[Expression of the genes of adenosine deaminase, C-lectin and serpin in the salivary gland of Aedes albopictus].

OBJECTIVE: To express the genes of adenosine deaminase (ADA), C-lectin and serpin (serine protease inhibitor) in the salivary gland of Aedes albopictus. METHODS: Total RNA was extracted respectively from salivary glands of unfed (group SG) and engorged adult female Ae. albopictus mosquitoes (group BSG), female carcasses without head and salivary gland (group C), and male bodies without heads but with salivary glands (group M). After the primers for the genes of ADA, C-lectin and serpin were designed respectively according to the reported Ae. albopictus gene sequences in GenBank, real-time fluorescent quantitative RT-PCR was performed to detect expression level of these genes in different tissues of Ae. albopictus using beta-actin as internal reference. RESULTS: The mRNA expression level of ADA gene in the salivary glands from unfed adult female mosquitoes (group SG) was 545 and 123 times higher than those of female carcasses without head and salivary gland (group C) and male bodies without heads but with salivary glands (group M) (P < 0.01). In group SG, C-lectin was 3 929 and 4 973 times higher than that in group C and M (P < 0.01). High level of mRNA coding for serpin was detected in group SG, being 1 911 and 2 978 times higher than that in group C and M (P < 0.01). There was no significant difference in ADA, C-lectin and serpin mRNA levels between unfed and engorged salivary glands (P > 0.05). CONCLUSION: ADA gene can be expressed in various mosquito tissues, but higher in salivary glands. The genes of C-lectin and serpin have been highly expressed specifically in salivary gland of female mosquito.