Salivary gland proteome during adult development and after blood feeding of female anopheles dissidens mosquitoes (Diptera Culicidae)

Understanding changes in mosquito salivary proteins during the time that sporozoite maturation occurs and after blood feeding may give information regarding the roles of salivary proteins during the malarial transmission. Anopheles dissidens (formerly Anopheles barbirostris species A1) is a potential vector of Plasmodium vivax in Thailand. In this study, analyses of the proteomic profiles of female An. dissidens salivary glands during adult development and after blood feeding were carried out using two-dimensional gel electrophoresis coupled with nano-liquid chromatography-mass spectrometry. Results showed at least 17 major salivary gland proteins present from day one to day 21 post emergence at 8 different time points sampled. Although there was variation observed, the patterns of protein expression could be placed into one of four groups. Fifteen protein spots showed significant depletion after blood feeding with the percentages of the amount of depletion ranging from 8.5% to 68.11%. The overall results identified various proteins, including a putative mucin-like protein, an anti-platelet protein, a long form D7 salivary protein, a putative gVAG protein precursor, a D7-related 3.2 protein, gSG7 salivary proteins, and a gSG6 protein. These results allow better understanding of the changes of the salivary proteins during the adult mosquito development. They also provide candidate proteins to investigate any possible link or not between sporozoite maturation, or survival of skin stage sporozoites, and salivary proteins

Molecular detection of Indian Ocean Lineage Chikungunya virus RNA in field collected Culex quinquefasciatus Say from Bangkok, Thailand but no evidence of virus replication.

Following an outbreak of chikungunya virus (CHIKV) infections in Thailand in 2019, numerous cases of CHIKV infection have been diagnosed in Bangkok, the capital of the country. In our previous investigation of the vectors for disease transmission, we found natural infection of CHIKV in both male and female Aedes aegypti mosquitoes collected from the outbreak areas in Bangkok. Some reports mentioned the detection of CHIKV in Culex mosquitoes. In Thailand, the Culex quinquefasciatus Say mosquito is a common species found in urban and rural settings that coexists with Ae. aegypti. However, the role of Cx. quinquefasciatus mosquitoes in the spread of the Indian Ocean Lineage (IOL) of CHIKV in Thailand has never been investigated. In this study, Cx. quinquefasciatus were collected (16 males and 27 females) from an outbreak area in Bangkok. Eight of the 27 in field-caught female Cx. quinquefasciatus were positive for IOL CHIKV RNA, and 99-100% identity and full 100% coverage of sequences similar to CHIKV isolated from female Ae. aegypti in Bangkok, Thailand, whereas viral RNA was not detected in male samples using nested-RT-PCR. To determine whether CHIKV is able to replicate in Cx. quinquefasciatus, the laboratory strain of Cx. quinquefasciatus was allowed to feed on blood containing IOL CHIKV isolated from patient serum. The nested-RT-PCR, virus isolation, and immunofluorescence assay (IFA) were performed for CHIKV detection and replication. The results showed that CHIKV RNA was detected in Cx. quinquefasciatus until day 4 post infection. CHIKV did not produce any remarkable signs of infection, dissemination, or transmission in Cx. quinquefasciatus, and cytopathic effect (CPE) was not observed in C6/36 cells when infected with supernatant obtained from Cx. quinquefasciatus at days 7, 10, 14, and 21 post infection when compared to Ae. aegypti. The data from this study infer that CHIKV may be detected in Cx. quinquefasciatus but that the mosquito is not able to transmit CHIKV in Thailand

Protein expression in female salivary glands of pyrethroid-susceptible and resistant strains of Aedes aegypti mosquitoes

Abstract Background A group of insecticides called pyrethroids has been used extensively worldwide and development of pyrethroid resistance within mosquito populations, especially in Aedes aegypti, has rapidly spread through populations. In this study, SDS-PAGE, 2-DE coupled with NanoLC-MS, and bioinformatics were used to analyze the female salivary gland proteins of pyrethroid-susceptible (PMD) and pyrethroid-resistant (PMD-R and UPK-R) strains of Ae. aegypti mosquitoes for the first time. Results SDS-PAGE analysis revealed that among the three strains at least nine major proteins were detected but one protein band (20 kDa) was found only in the PMD strain. Two-dimensional gel electrophoresis analysis revealed 19 similarly expressed proteins in the salivary glands of the three strains involved in blood-feeding process, stress response, immunogenic response, and metabolic process and five additional major protein spots differentially expressed in the susceptible and resistant strains. Comparative analysis of the expression volume of each protein spot between the PMD and the PMD-R strains showed three downregulated proteins of the PMD-R mosquitoes. For UPK-R strains, six major proteins were downregulated when compared to the PMD strain. Additionally, four downregulated proteins were found in the UPK-R when compared to the PMD-R strain. These results suggest that pyrethroids might induce alteration of salivary gland proteins in resistant mosquitoes. Network analysis by STITCH database 5.0 showed that SRPN23 interacted with sodium and calcium ions, suggesting that SRPN23 might be involved in insecticide resistance. Conclusions Information obtained from this study will be useful for further studies on the roles of differentially expressed salivary gland proteins in resistance to insecticides and viral transmission

Significance of eggshell morphology as an additional tool to distinguish species of sand flies (Diptera: Psychodidae: Phlebotominae)

Morphological characteristics of eggshells are important in sand fly ootaxonomy. In this study, eggshells from Phlebotomus stantoni Newstead, Sergentomyia khawi (Raynal), and Grassomyia indica (Theodor) sand flies collected in Chiang Mai province, Thailand were examined and characterized using light microscopy (LM) and scanning electron microscopy (SEM). Then, eggshell morphology of these three species was described for the first time. Each gravid female was forced to lay eggs by decapitation and the eggs were collected for SEM analysis. Egg laying females were identified by morphological examination and molecular typing using cytochrome b ( Cyt b) as a molecular marker. The chorionic sculpturing of Ph . stantoni eggs combines two patterns on the same egg: unconnected parallel ridges and reticular patterns. Sergentomyia khawi and Gr . indica have similar chorionic polygonal patterns, but their exochorionic morphology and aeropylar area are different. Results indicate that eggshell morphological characteristics such as chorionic pattern, exochorionic morphology, inter-ridge/boundary area, aeropylar area (including the number of aeropyles) and basal layer, can be useful to develop morphological identification keys of eggs. These can serve as an additional tool to distinguish species of sand flies. In addition, the chorionic sculpturing of the eggs of the three species of sand flies observed by LM is useful for species identification in gravid females with spermathecae obscured by eggs

Leishmania (Mundinia) orientalis n. sp. (Trypanosomatidae), a parasite from Thailand responsible for localised cutaneous leishmaniasis

BACKGROUND: Leishmaniasis is an emerging disease in Thailand with an unknown incidence or prevalence. Although the number of properly characterized and clinically confirmed cases is about 20, it is suspected that this low number masks a potentially high prevalence, with clinical disease typically manifesting itself against an immunocompromised background, but with a substantial number of subclinical or cured cases of infection. To date leishmaniasis in Thailand has been mainly ascribed to two taxa within the recently erected subgenus Mundinia Shaw, Camargo & Teixeira, 2016, Leishmania (Mundinia) martiniquensis Desbois, Pratlong & Dedet, 2014 and a species that has not been formally described prior to this study. RESULTS: A case of simple cutaneous leishmaniasis was diagnosed in a patient from Nan Province, Thailand. Molecular analysis of parasites derived from a biopsy sample revealed this to be a new species of Leishmania Ross, 1908, which has been named as Leishmania (Mundinia) orientalis Bates & Jariyapan n. sp. A formal description is provided, and this new taxon supercedes some isolates from the invalid taxon "Leishmania siamensis". A summary of all known cases of leishmaniasis with a corrected species identification is provided. CONCLUSIONS: Three species of parasites are now known to cause leishmaniasis is Thailand, L. martiniquensis and L. orientalis n. sp. in the subgenus Mundinia, which contains the type-species Leishmania enriettii Muniz & Medina, 1948, and a single case of Leishmania infantum Nicolle, 1908. This study now enables epidemiological and other investigations into the biology of these unusual parasites to be conducted. It is recommended that the use of the taxonomically invalid name "L. siamensis" should be discontinued

Representative 2-DE gels of salivary gland proteins extracted from 80 female mosquitoes at different ages.

<p>(A) 0 day, (B) 1 days, (C) 3 days, (D) 12 days, (E) 16 days, and (F) 21 days old. Molecular mass markers are indicated on the left in kDa. Isoelectric points (pI) are indicated at the top. Arrows indicate major salivary gland proteins. Arrowhead indicates an internal control protein. Numbers are corresponding to the major salivary gland proteins in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163810#pone.0163810.t001" target="_blank">Table 1</a>. The large spot at the base of the right side gels of Fig 1A and B is an artifact of separation. It was seen on the three gels shown with these samples only.</p

Amounts of depletion of the major salivary gland proteins after blood feeding of <i>An</i>. <i>dissidens</i> mosquitoes.

<p>Amounts of depletion of the major salivary gland proteins after blood feeding of <i>An</i>. <i>dissidens</i> mosquitoes.</p