ABSTRACT
The Asian tiger mosquito, Aedes albopictus, is an important vector for the transmission of arboviruses such as dengue virus (DENV). Adenosine deaminase (ADA) is a well-characterized metabolic enzyme involved in facilitating blood feeding and (or) arbovirus transmission in some hematophagous insect species. We previously reported the immunologic function of ADA by investigating its effect on mast cell activation and the interaction with mast cell tryptase and chymase. The 2-D gel electrophoresis and mass spectrometry analysis in the current study revealed that ADA is present and upregulated following mosquito blood feeding, as confirmed by qRT-PCR and western blot. In addition, the recombinant ADA efficiently converted adenosine to inosine. Challenging the Raw264.7 and THP-1 cells with recombinant ADA resulted in the upregulation of IL-1ß, IL-6, TNF-α, CCL2, IFN-ß, and ISG15. The current study further identified recombinant ADA as a positive regulator in NF-κB signaling targeting TAK1. It was also found that recombinant Ae. albopictus ADA facilitates the replication of DENV-2. Compared with cells infected by DENV-2 alone, the co-incubation of recombinant ADA with DENV-2 substantially increased IL-1ß, IL-6, TNF-α, and CCL2 gene transcripts in Raw264.7 and THP-1 cells. However, the expression of IFN-ß and ISG15 were markedly downregulated in Raw264.7 cells but upregulated in THP-1 cells. These findings suggest that the immunomodulatory protein, Ae. albopictus ADA is involved in mosquito blood feeding and may modulate DENV transmission via macrophage or monocyte-driven immune response.
Subject(s)
Aedes , Dengue Virus , Dengue , Animals , Dengue Virus/physiology , Mosquito Vectors , Tumor Necrosis Factor-alpha , Adenosine Deaminase , Interleukin-6 , Virus Replication , ImmunityABSTRACT
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 melanogasterï¼GenBank No. AAF45497ï¼. 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 ï¼egg, first to fourth instar, pupa, non-blood-fed female and male mosquitoesï¼ and in different tissues ï¼salivary gland, midgut, fat body, and ovaryï¼ 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 ï¼15%-49%ï¼ homology among the proteins, but high homologyï¼60%ï¼ 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 ï¼0.018 9ï¼ and Aa-yellow-x ï¼0.023 5ï¼ in male Ae. aegypti ï¼P<0.01 or P<0.05ï¼; high expression of Aa-yellow-fc ï¼0.024 8, 0.034 9ï¼ in female Ae. aegypti and in the salivary gland ï¼P<0.01ï¼; high expression of Aa-yellow-f2 ï¼0.093 4ï¼ in the second instar stage ï¼P<0.01ï¼; high expression of Aa-yellow ï¼0.562 1ï¼, Aa-yellow-e ï¼0.004 4ï¼, and Aa-yellow-fb ï¼0.008 4ï¼ in the third instar stage ï¼P<0.05ï¼; and high expression of Aa-yellow ï¼0.569 4ï¼, Aa-yellow-e ï¼0.027 0ï¼, Aa-yellow-f2 ï¼0.006 5ï¼, Aa-yellow-fb ï¼0.001 0ï¼, Aa-yellow-h ï¼0.084 8ï¼ and Aa-yellow-x ï¼0.015 1ï¼ in the ovary. Genes other than Aa-yellow-c ï¼0.004 0ï¼ and Aa-yellow-x ï¼0.007 4ï¼ 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.
