Aedes albopictus salivary adenosine deaminase is an immunomodulatory factor facilitating dengue virus replication.

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.

Genomics evolution of Jingmen viruses associated with ticks and vertebrates.

Jingmen virus (JMV) associated with ticks and vertebrates have been found to be related to human disease. We obtained the genome of a Jingmen tick virus (JMTV) strain from Rhipicephalus microplus in Guizhou province and compared the genomes of seven JMV species associated with ticks and vertebrates to understand the evolutionary relationships. The topology of the phylogenetic tree of segment 1 and segment 3 is similar, and segment 2 and segment 4 formed two different topologies, with the main differences being between Alongshan virus (ALSV), Takachi virus, Yanggou tick virus and Pteropus lylei jingmen virus (PLJV), and the possibility of genetic reassortment among these viruses. Moreover, we detected recombination within JMTV and between PLJV and ALSV. The genetic reassortment and recombination that occurs during cross-species transmission of these JMV associated with ticks and vertebrates not only complicates their evolutionary relationships, but also raises the risk of these viruses to humans.

Frequency and polymorphism of acetylcholinesterase gene involved in the organophosphate resistance of Musca domestica in Guizhou Province, China.

Organophosphate (OP) resistance has been prevalent in Musca domestica populations worldwide since 1960s. Previous studies have demonstrated that point mutations of the acetylcholinesterase gene (Ace) are one of the important molecular mechanisms underlying OP resistance. However, few studies have investigated the molecular mechanisms of OP resistance in the past 10 years in China. In this study, we investigated the status of OP resistance and genetic diversity of Ace in the field populations of houseflies in Guizhou Province of China. The bioassays showed that the houseflies had 142-304-fold resistance to dichlorvos (DDVP) and 122-364-fold resistance to temephos, compared to the susceptible houseflies. Five nonsynonymous mutations (Y226F, V260L, G342A/V, F407Y) in Ace were detected among the 7 field populations, with an average frequency of 5.4%, 55%, 68%, 32%, and 94%, respectively, of which the Y226F mutation had not been reported previously. Eleven combinations of triple mutations (at positions 260, 342, and 407) were observed, of which the combination 260L/V+342A/V+407Y was predominant. The ZY and AS populations showed greatest diversity of allelic combination and the other five populations showed different distributions among different regions. These results indicate that the resistance to OPs is prevalent among the housefly populations and target-site insensitivity is the main cause of resistance in Guizhou Province. The difference in distribution and the allelic diversity of Ace in field populations may be due to the complexity and variability of insecticide application. It is necessary to monitor resistance to insecticides and conduct management of houseflies in Guizhou Province.

A C-type lectin in saliva of Aedes albopictus (Diptera: Culicidae) bind and agglutinate microorganisms with broad spectrum.

Via complex salivary mixture, mosquitos can intervene immune response and be helpful to transmit several viruses causing deadly human diseases. Some C-type lectins (CTLs) of mosquito have been reported to be pattern recognition receptor to either resist or promote pathogen invading. Here, we investigated the expression profile and agglutination function of an Aedes albopictus CTL (Aalb_CTL2) carrying a single carbohydrate-recognition domain (CRD) and WND/KPD motifs. The results showed that Aalb_CTL2 was found to be specifically expressed in mosquito saliva gland and its expression was not induced by blood-feeding. The recombinant Aalb_CTL2 (rAalb_CTL2) could agglutinate mouse erythrocytes in the presence of calcium and the agglutinating activity could be inhibited by EDTA. rAalb_CTL2 also displayed the sugar binding ability to D-mannose, D-galactose, D-glucose, and maltose. Furthermore, it was demonstrated that rAalb_CTL2 could bind and agglutinate Gram positive bacteria Staphylococcus aureus and Bacillus subtilis, Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa, as well as fungus Candida albicans in vitro in a calcium dependent manner. However, rAalb_CTL2 could not promote type 2 dengue virus (DENV-2) replication in THP-1 and BHK-21 cell lines. These findings uncover that Aalb_CTL2 might be involved in the innate immunity of mosquito to resist microorganism multiplication in sugar and blood meals to help mosquito survive in the varied natural environment.

SARS-CoV-2 ORF3a positively regulates NF-κB activity by enhancing IKKß-NEMO interaction.

Coronavirus disease 2019 (COVID-19) is a global pandemic caused by SARS-CoV-2 infection. Patients with severe COVID-19 exhibit robust induction of proinflammatory cytokines, which are closely associated with the development of acute respiratory distress syndrome. However, the underlying mechanisms of the NF-κB activation mediated by SARS-CoV-2 infection remain poorly understood. Here, we screened SARS-CoV-2 genes and found that ORF3a induces proinflammatory cytokines by activating the NF-κB pathway. Moreover, we found that ORF3a interacts with IKKß and NEMO and enhances the interaction of IKKß-NEMO, thereby positively regulating NF-κB activity. Together, these results suggest ORF3a may play pivotal roles in the pathogenesis of SARS-CoV-2 and provide novel insights into the interaction between host immune responses and SARS-CoV-2 infection.

Highly sensitive and rapid identification of coxsackievirus A16 based on reverse transcription multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor assay.

Introduction: One of the main pathogens responsible for human hand, foot, and mouth disease (HFMD), coxsackievirus A16, has put young children's health at danger, especially in countries in the Asia-Pacific region. Early quick identification is essential for the avoidance and control of the disorder since there are no vaccinations or antiviral medications available to prevent and manage CVA16 infection. Methods: Here, we describe the creation of an easy, speedy, and accurate CVA16 infection detection approach using lateral flow biosensors (LFB) and reverse transcriptionmultiple cross displacement amplification (RT-MCDA). A group of 10 primers was developed for the RT-MCDA system in order to amplify the genes in an isothermal amplification device while targeting the highly conserved region of the CVA16 VP1 gene. Then, without requiring any extra tools, RT-MCDA amplification reaction products might well be detected by visual detection reagent (VDR) and LFB. Results: The outcomes showed that 64°C within 40 min was the ideal reaction setting for the CVA16-MCDA test. Target sequences with <40 copies might be found using the CVA16-MCDA. There was no cross-reaction among CVA16 strains and other strains. The findings demonstrated that the CVA16-MCDA test could promptly and successfully identify all of the CVA16-positive (46/220) samples identified by the traditional real-time quantitative polymerase chain reaction (qRT-PCR) assays for 220 clinical anal swab samples. The whole process, such as the processing of the sample (15 min), the MCDA reaction (40 min), and the documenting of the results (2 min), could be finished in 1 h. Conclusion: The CVA16-MCDA-LFB assay, which targeted the VP1 gene, was an efficient, simple, and highly specific examination that might be used extensively in rural regions' basic healthcare institutions and point-of-care settings.

Molecular epidemiology and antimicrobial resistance of Haemophilus influenzae in Guiyang, Guizhou, China.

Background: The widespread use of antimicrobials and Haemophilus influenzae type b (Hib) vaccine worldwide has altered the epidemiological patterns of invasive H. influenzae. Nonetheless, little is currently known on the epidemiological characteristics of H. influenzae in Guiyang, Guizhou, China. Objective: To determine the serotype distribution, antimicrobial resistance and Multilocus Sequence Typing (MLST) of H. influenzae in hospitalized patients in Guiyang City. Methods: A total of 196 clinical isolates from hospitalized patients were collected. Serotypes were determined according to the specific capsule gene, bexA, amplified by PCR. According to the guidelines of Clinical and Laboratory Standards Institute (CLSI) 2020 drug susceptibility tested, and the results determined. The chromogenic cephalosporin nitrocefin method was used to detect ß-lactamase production, ß-lactamase negative, ampicillin-resistant (BLNAR) strains were detected by PCR amplification and sequencing of the penicillin-binding protein 3 (PBP3) locus of ftsI. Multilocus Sequence Typing was performed for molecular typing. Results: All isolates studied were non-typeable H. influenzae (NTHi). Most patients originated from the pediatrics department (78.6%, 154/196), and suffered from lung with respiratory tract infection (pneumonia and bronchitis, 68.4%, 134/196). The resistance rates of ampicillin, cefaclor and azithromycin were 71.4% (140/196), 36.7% (72/196) and 34.2% (67/196), respectively. 40.3% (79/196) of strains were ß-lactamase positive ampicillin-resistant (BLPAR). All BLPAR carried the TEM-1 gene. 9.2% (18/196) were ß-lactamase negative ampicillin-resistant strains (BLNAR). The PBP3 mutation was detected in the ampicillin-resistant strains (n = 113), of which 18 belonged to group IIa. A total of 49 sequence types (ST) and 23 clonal complexes (CC) were detected, among which CC107 (ST107, n = 27; ST1002, n = 5; ST1218, n = 5) was the most frequent clonal complexes. BLPAR isolates mostly belonged to ST107 (20/79), while BLNAR was predominantly distributed in ST12 (5/18). Conclusion: H. influenzae infections are predominately caused by genetically diverse NTHi among hospitalized patients in Guiyang. The prevalence of ß-lactamase production and PBP3 mutation may contribute to the high local ampicillin resistance rate.

Development and clinical application of a endonuclease restriction real-time loop-mediated isothermal amplification (ERT-LAMP) assay for rapid detection of Haemophilus influenzae.

Haemophilus influenzae is a main human pathogen that results in a series of diseases in children and adults, such as pneumonia, bacteremia, and meningitis. Although there are many detection methods, they cannot meet the requirements of an early diagnosis. For the prevention and control of H. influenzae infection, quick, sensitive, and particular diagnostics are crucial. Loop-mediated isothermal amplification (LAMP) coupled with restricted endonuclease digestion and real-time fluorescence (H. influenzae-ERT-LAMP) detection was employed to diagnose H. influenzae. H. influenzae-ERT-LAMP combines LAMP amplification, restriction endonuclease cleavage, and real-time fluorescence identification into a single-pot reaction, allowing for the rapid identification of H. influenzae in 40 min. The outer membrane protein (OMP) P6 gene of H. influenzae was employed to build a sequence of H. influenzae-ERT-LAMP primers. The limit of detection (LoD) of H. influenzae-ERT-LAMP test was 40 fg of genomic DNA per reaction, and the non-H. influenzae templates did not provide positive outcomes. To investigate the applicability of H. influenzae-ERT-LAMP method in clinical sample detection, 30 sputum specimens were obtained from individuals suspected of being infected with H. influenzae. H. influenzae-ERT-LAMP outcomes were in total agreement with LAMP-LFB and PCR. The H. influenzae-ERT-LAMP assay provides rapid, accurate, and sensitive detection making it a promising screening strategy in clinical and basic lab settings.

Aedes albopictus salivary proteins adenosine deaminase and 34k2 interact with human mast cell specific proteases tryptase and chymase.

When mosquitoes probe to feed blood, they inoculate a mixture of salivary molecules into vertebrate hosts' skin causing acute inflammatory reactions where mast cell-derived mediators are involved. Mosquito saliva contains many proteins with largely unknown biological functions. Here, two Aedes albopictus salivary proteins - adenosine deaminase (alADA) and al34k2 - were investigated for their immunological impact on mast cells and two mast cell-specific proteases, the tryptase and the chymase. Mouse bone marrow-derived mast cells were challenged with increased concentrations of recombinant alADA or al34k2 for 1, 3, and 6 h, and to measure mast cell activation, the activity levels of ß-hexosaminidase and tryptase and secretion of IL-6 were evaluated. In addition, a direct interaction between alADA or al34k2 with tryptase or chymase was investigated. Results show that bone marrow-derived mast cells challenged with 10 µg/ml of alADA secreted significant levels of ß-hexosaminidase, tryptase, and IL-6. Furthermore, both al34k2 and alADA are cut by human tryptase and chymase. Interestingly, al34k2 dose-dependently enhance enzymatic activity of both tryptase and chymase. In contrast, while alADA enhances the enzymatic activity of tryptase, chymase activity was inhibited. Our finding suggests that alADA and al34k2 via interaction with mast cell-specific proteases tryptase and chymase modulate mast cell-driven immune response in the local skin microenvironment. alADA- and al34k2-mediated modulation of tryptase and chymase may also recruit more inflammatory cells and induce vascular leakage, which may contribute to the inflammatory responses at the mosquito bite site.

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines.

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation of RNA virus transmission using Wolbachia-transfected mosquitoes. Key strategies to control viruses include the manipulation of host reproduction via cytoplasmic incompatibility and the inhibition of viral transcripts via immune priming and competition for host-derived resources. However, the underlying mechanisms of the responses of Wolbachia-transfected mosquitoes to viral infection are poorly understood. This paper presents a protocol for the in vitro identification of Wolbachia infection at the nucleic acid and protein levels in Aedes albopictus (Diptera: Culicidae) Aa23 cells to enhance the understanding of the interactions between Wolbachia and its insect vectors. Through the combined use of polymerase chain reaction (PCR), quantitative PCR, western blot, and immunological analytical methods, a standard morphologic protocol has been described for the detection of Wolbachia-infected cells that is more accurate than the use of a single method. This approach may also be applied to the detection of Wolbachia infection in other insect taxa.

Development and Application of a Multiple Cross Displacement Amplification Combined With Nanoparticle-Based Lateral Flow Biosensor Assay to Detect Candida tropicalis.

Candida tropicalis is an increasingly opportunistic pathogen that causes serious invasive candidiasis threatening a patient's life. Traditional methods to detect C. tropicalis infection depends on time-consuming, culture-based gold-standard methods. So, we sought to establish a new method that could detect target pathogens quickly, accurately, and straightforwardly. Herein, a combination of multiple cross displacement amplification (MCDA) and lateral flow biosensors (LFB) was employed to detect C. tropicalis. In the MCDA system, 10 primers were designed to identify the specific genes of C. tropicalis and amplify the genes in an isothermal amplification device. Then, MCDA amplification reaction products could be identified visibly by color change, and all the amplification products would be tested by LFB with no special equipment. The results demonstrated that the optimal reaction condition of C. tropicalis-MCDA assay was 64°C within 30 min, and only 10 fg DNA was required in each reaction. No cross-reaction was found between C. tropicalis strains and non-C. tropicalis strains. For 300 sputum samples, the results showed that MCDA-LFB assay could rapidly and successfully detect all of the C. tropicalis-positive (28/300) samples detected by the gold-standard method. The entire procedure, including specimen processing (40 min), isothermal reaction (30 min) and result reporting (within 2 min), could be completed within 75 min. Briefly, the study results demonstrated that the detection ability of C. tropicalis-MCDA-LFB assay was better than culture methods with more simplicity, rapidity, sensitivity and specificity. Hence, MCDA-LFB strategy is an effective tool to rapidly detect C. tropicalis in clinical samples, especially in resource-poor areas.

Identifying and Characterizing a Novel Peritrophic Matrix Protein (MdPM-17) Associated With Antibacterial Response From the Housefly, Musca domestica (Diptera: Muscidae).

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.

[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 characterization of a mannose binding C-type lectin gene from salivary gland of Aedes albopictus.

BACKGROUND: The studies on sialomes have shown that hematophagous mosquito saliva consists of a lot of pharmacologically active proteins, in which C-type lectins have been identified and regarded as an important component of saliva. The previous studies showed that C-type lectins play crucial roles not only in innate immunity but also in promoting disease transmission in mammals. However, the function and mechanism of C-type lectins from the mosquito sialome is still elusive. METHODS: A putative C-type lectin gene (Aalb_CTL1) was cloned and expressed from Aedes albopictus by RT-PCR. The deduced amino acid sequence was analyzed by bioinformatic methods. The gene expression profiles in different tissues and various blood-fed stages of Ae. albopictus were examined by Real-Time qRT-PCR and the biological functions of the recombined mature Aalb_CTL1 were tested by hemagglutination and sugar inhibitory agglutination assays. Moreover, the capabilities of rAalb_CTL1 against microorganisms were measured by microbial-agglutination assay. RESULTS: The full-length Open reading frame (ORF) of Aalb_CTL1 consisted of 462 bp, encoding 153 amino acid residues. The deduced amino acid sequence contained a putative signal peptide of 19 amino acids. It also contained a CRD domain with a WND (Trp137-Asn138-Asp-139) motif that needed calcium for the hemagglutinating activity and an imperfect EPS (Glu128-Pro129-Ser130) motif that had a predicted ligand binding specificity for mannose. The mRNA level of Aalb_CTL1 was much higher in female mosquito salivary gland than those in fat body and midgut which was down-regulated in salivary gland after blood feeding. The rAalb_CTL1 contained not only hemagglutinating activity and a high affinity with mannose but also agglutinating activity against yeast C. albicans and Gram-positive bacteria S. aureus in Ca2+ dependent manner. CONCLUSION: Aalb_CTL1 was a mannose-binding C-type lectin and constituted one of the important components in saliva of Ae. albopictus, which could be involved in the defense against yeast and Gram-positive bacteria infection.

[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.