Description of potential vectors of zoonotic filarial nematodes, Brugia pahangi, Setaria digitata, and Setaria labiatopapillosa in Thai mosquitoes.

Filariasis is classified as a vector-borne zoonotic disease caused by several filarial nematodes. The disease is widely distributed in tropical and subtropical regions. Understanding the relationship between mosquito vectors, filarial parasites, and vertebrate hosts is therefore essential for determining the probability of disease transmission and, correspondingly, developing effective strategies for prevention and control of diseases. In this study, we aimed to investigate the infection of zoonotic filarial nematodes in field-caught mosquitoes, observe the potential vectors of filaria parasites in Thailand using a molecular-based survey, conduct a study of host-parasite relationship, and propose possible coevolution of the parasites and their hosts. Mosquitoes were collected around cattle farms in Bangkok, Nakhon Si Thammarat, Ratchaburi, and Lampang provinces from May to December 2021 using a CDC Backpack aspirator for 20-30 minutes in each area (intra-, peri-, and wild environment). All mosquitoes were identified and morphologically dissected to demonstrate the live larvae of the filarial nematode. Furthermore, all samples were tested for filarial infections using PCR and sequencing. A total of 1,273 adult female mosquitoes consisted of five species: 37.78% Culex quinquefasciatus, 22.47% Armigeres subalbatus, 4.71% Cx. tritaeniorhynchus, 19.72% Anopheles peditaeniatus, and 15.32% An. dirus. Larvae of Brugia pahangi and Setaria labiatopapillosa were found in Ar. subalbatus and An. dirus mosquitoes, respectively. All mosquito samples were processed by PCR of ITS1 and COXI genes for filaria nematode species identification. Both genes showed that B. pahangi was found in four mosquitoes of Ar. subalbatus from Nakhon Si Thammarat, S. digitata was detected in three samples of An. peditaeniatus from Lampang, and S. labiatopapillosa was detected in one of An. dirus from Ratchaburi. However, filarial nematodes were not found in all Culex species. This study infers that this is the first data regarding the circulation of Setaria parasites in Anopheles spp. from Thailand. The phylogenetic trees of the hosts and parasites are congruent. Moreover, the data could be used to develop more effective prevention and control strategies for zoonotic filarial nematodes before they spread in Thailand.

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 (Cytb) 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.

Culicoides Latreille (Diptera: Ceratopogonidae) as potential vectors for Leishmania martiniquensis and Trypanosoma sp. in northern Thailand.

Biting midges of genus Culicoides (Diptera: Ceratopogonidae) are the vectors of several pathogenic arboviruses and parasites of humans and animals. Several reports have suggested that biting midges might be a potential vector of Leishmania parasites. In this study, we screened for Leishmania and Trypanosoma DNA in biting midges collected from near the home of a leishmaniasis patient in Lamphun province, northern Thailand by using UV-CDC light traps. The identification of biting midge species was based on morphological characters and confirmed using the Cytochrome C oxidase subunit I (COI) gene. The detection of Leishmania and Trypanosoma DNA was performed by amplifying the internal transcribed spacer 1 (ITS1) and small subunit ribosomal RNA (SSU rRNA) genes, respectively. All the amplified PCR amplicons were cloned and sequenced. The collected 223 biting midges belonged to seven species (Culicoides mahasarakhamense, C. guttifer, C. innoxius, C. sumatrae, C. huffi, C. oxystoma, and C. palpifer). The dominant species found in this study was C. mahasarakhamense (47.53%). Leishmania martiniquensis DNA was detected in three samples of 106 specimens of C. mahasarakhamense tested indicating a field infection rate of 2.83%, which is comparable to reported rates in local phlebotomines. Moreover, we also detected Trypanosoma sp. DNA in one sample of C. huffi. To our knowledge, this is the first molecular detection of L. martiniquensis in C. mahasarakhamense as well as the first detection of avian Trypanosoma in C. huffi. Blood meal analysis of engorged specimens of C. mahasarakhamense, C. guttifer, and C. huffi revealed that all specimens had fed on avian, however, further studies of the host ranges of Culicoides are needed to gain a better insight of potential vectors of emerging leishmaniasis. Clarification of the vectors of these parasites is also important to provide tools to establish effective disease prevention and control programs in Thailand.

Genetic characterization of chikungunya virus isolates from Aedes aegypti mosquitoes collected during a recent outbreak in Bangkok, Thailand.

Chikungunya virus (CHIKV) is a mosquito-borne emerging pathogen that is transmitted to humans through the bite of female Aedes mosquitoes. CHIKV infection has become a major public health concern worldwide, as it has a significant impact on the healthcare system. Since 2004, the virus has emerged in Africa and subsequently spread to countries located near the Indian Ocean, including India, and to Europe, the Americas, and Asia. In Thailand, a large CHIKV outbreak occurred during 2008-2009 and was caused by a virus originating from the east/central/south African (ECSA) CHIKV genotype. Since then, the ECSA genotype of CHIKV has continued to circulate and has caused sporadic cases in different areas in Thailand. Approximately 20,000 reported cases have been confirmed by the Bureau of Epidemiology, Ministry of Public Health, Thailand, from January 1, 2018 to July 31, 2020. However, the causes of this CHIKV re-emergence remain unclear. To obtain a better understanding of CHIKV circulation during the recent outbreak in Bangkok, Thailand, complete genome analysis of CHIKV isolates from field-caught mosquitoes collected in outbreak areas was performed. A total of 28 Ae. aegypti samples (21 females and 7 males) were collected, and individual mosquitoes were used for CHIKV detection and isolation. Eleven of 28 (39.29%) female and three of 28 (10.71%) male mosquitoes were positive for CHIKV by E1 nested RT-PCR. Four CHIKV isolates were successfully isolated from four female Ae. aegypti mosquitoes. Based on complete genome analysis, several amino acid substitutions were identified in the protein coding region. The E1:K211E and E2:V264A mutations in the background of the E1:226A mutation were observed in all four CHIKV isolates. An important observation was the presence of one amino acid substitution, leading to an E1:K245R change. This mutation was found in all four CHIKV isolates from mosquitoes in this study and in Thai patients described previously. Additionally, phylogenetic analysis indicated that the four CHIKV isolates belonged to the Indian Ocean clade of the ECSA genotype. The results obtained in this study provide detailed information on the molecular characteristics and evolution of currently circulating CHIKV strains in Thailand, which are useful for developing prevention and control strategies.

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.

Detection of Leishmania and Trypanosoma DNA in Field-Caught Sand Flies from Endemic and Non-Endemic Areas of Leishmaniasis in Southern Thailand.

Phlebotomine sand flies are tiny, hairy, blood-sucking nematoceran insects that feed on a wide range of hosts. They are known as a principal vector of parasites, responsible for human and animal leishmaniasis worldwide. In Thailand, human autochthonous leishmaniasis and trypanosomiasis have been reported. However, information on the vectors for Leishmania and Trypanosoma in the country is still limited. Therefore, this study aims to detect Leishmania and Trypanosoma DNA in field-caught sand flies from endemic areas (Songkhla and Phatthalung Provinces) and non-endemic area (Chumphon Province) of leishmaniasis. A total of 439 sand flies (220 females and 219 males) were collected. Head and genitalia dissection of female sandflies were done for morphology identification, and the remaining parts of those sand flies were then used for the detection of Leishmania and Trypanosoma parasites. The DNA was extracted from individual female sand flies. Polymerase chain reaction (PCR) anneal, specific to the ITS1 and SSU rRNA gene regions, was used to detect Leishmania and Trypanosoma DNA, respectively. The positive PCR products were cloned and sequenced. The results showed that the female sand fly species in this study consisted of Sergentomyia khawi (35.9%); Se. anodontis (23.6%); Phlebotomus betisi (18.6%); Ph. kiangsuensis (9.5%); Ph. asperulus (6.4%); Se. barraudi (2.3%); 0.9% of each Se. indica, Ph. stantoni, and Ph. major major; and 0.5% of each Se. sylvatica and Ph. mascomai. The PCR and sequence analysis were able to detect Leishmania and Trypanosoma DNA in sand fly samples, which were identified as L. martiniquensis, 1/220 (0.45%) in Se. khawi, 3/220 (1.36%) of T. noyesi in Se. anodontis, and Ph. asperulus. Fourteen (6.36%) of the unidentified trypanosome species in Se. khawi, Se. indica, Se. anodontis, Ph. asperulus, and Ph. betisi were found in all of the areas of this study. Interestingly, we found a 1/220 (0.45%) co-infection sample of L. martiniquensis and Trypanosoma in Se. khawi from Songkhla Province. These data indicate that several species of sand flies might be potential vectors of Leishmania and Trypanosoma parasites in southern Thailand. However, more extensive study for potential vectors using a larger number of sand flies should be conducted to prove whether these sand flies can be natural vectors of leishmaniasis and trypanosomiasis in both humans and animals. In addition, our study could be useful for the future study of infection prevention, including effective vector control for leishmaniasis and trypanosomiasis in Thailand.

Genetic Characterization of Chikungunya Virus in Field-Caught Aedes aegypti Mosquitoes Collected during the Recent Outbreaks in 2019, Thailand.

Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to the genus Alphavirus. The virus is transmitted to humans by the bite of infected female Aedes mosquitoes, primarily Aedes aegypti. CHIKV infection is spreading worldwide, and it periodically sparks new outbreaks. There are no specific drugs or effective vaccines against CHIKV. The interruption of pathogen transmission by mosquito control provides the only effective approach to the control of CHIKV infection. Many studies have shown that CHIKV can be transmitted among the Ae. aegypti through vertical transmission. The previous chikungunya fever outbreaks in Thailand during 2008-2009 were caused by CHIKV, the East/Central/South African (ECSA) genotype. Recently, there have been 3794 chikungunya cases in 27 provinces reported by the Bureau of Epidemiology of Health Ministry, Thailand during 1 January-16 June 2019; however, the cause of the re-emergence of CHIKV outbreaks is uncertain. Therefore, the aims of this study were to detect and analyze the genetic diversity of CHIKV infection in field-caught mosquitoes. Both female and male Ae. aegypti were collected from endemic areas of Thailand, and CHIKV detection was done by using E1-nested RT-PCR and sequencing analysis. A total of 1646 Ae. aegypti samples (900 females and 746 males) were tested. CHIKV was detected in 54 (3.28%) and 14 samples (0.85%) in female and male mosquitoes, respectively. Seventeen samples of female Ae. aegypti collected from the Ubon Ratchathani, Chiang Rai, Chiang Mai, Nakhon Sawan, and Songkhla provinces found mutation at E1: A226V. Interestingly, E1: K211E mutation was observed in 50 samples collected from Nong Khai, Bangkok, Prachuap Khiri Khan, and Krabi. In addition, the phylogenetic tree indicated that CHIKV in Ae. aegypti samples were from the Indian Ocean Clade and East/South African Clade. Both clades belong to the ECSA genotype. The information obtained from this study could be used for prediction, epidemiological study, prevention, and effective vector control of CHIKV. For instance, a novel CHIKV strain found in new areas has the potential to lead to a new outbreak. Health authorities could plan and apply control strategies more effectively given the tools provided by this research.

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

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.

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.

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.

Identification of salivary gland proteins depleted after blood feeding in the malaria vector Anopheles campestris-like mosquitoes (Diptera: Culicidae).

Malaria sporozoites must invade the salivary glands of mosquitoes for maturation before transmission to vertebrate hosts. The duration of the sporogonic cycle within the mosquitoes ranges from 10 to 21 days depending on the parasite species and temperature. During blood feeding salivary gland proteins are injected into the vertebrate host, along with malaria sporozoites in the case of an infected mosquito. To identify salivary gland proteins depleted after blood feeding of female Anopheles campestris-like, a potential malaria vector of Plasmodium vivax in Thailand, two-dimensional gel electrophoresis and nano-liquid chromatography-mass spectrometry techniques were used. Results showed that 19 major proteins were significantly depleted in three to four day-old mosquitoes fed on a first blood meal. For the mosquitoes fed the second blood meal on day 14 after the first blood meal, 14 major proteins were significantly decreased in amount. The significantly depleted proteins in both groups included apyrase, 5'-nucleotidase/apyrase, D7, D7-related 1, short form D7r1, gSG6, anti-platelet protein, serine/threonine-protein kinase rio3, putative sil1, cyclophilin A, hypothetical protein Phum_PHUM512530, AGAP007618-PA, and two non-significant hit proteins. To our knowledge, this study presents for the first time the salivary gland proteins that are involved in the second blood feeding on the day corresponding to the transmission period of the sporozoites to new mammalian hosts. This information serves as a basis for future work concerning the possible role of these proteins in the parasite transmission and the physiological processes that occur during the blood feeding.

MIDGUT ULTRASTRUCTURE OF FOURTH INSTAR OCHLEROTATUS TOGOI (DIPTERA: CULICIDAE).

The ultrastructure of the midgut of fourth instar Ochlerotatus togoi was investigated by light, scanning and transmission electron microscopy. This study was performed to provide information to help devise future control efforts aimed at the larval stages of this vector of filariasis. The fourth instar midgut was approximately 2 mm in length and consisted of three morphologically distinct cell types: epithelial, regenerative, and endocrine cells. There was a monolayer of epithelial cells on the luminal surface of the midgut, with multiple folds of the plasma membrane where it adjoined the basement membrane. Regenerative cells were scattered throughout the basal portion of the epithelium, along with endocrine cells. No evidence of division or differentiation was seen in any of the cell types. Six layers of the peritrophic matrix were observed in the gut lumen which separated ingested food from the midgut epithelial cells. Cytoplasmic protrusions were seen in many areas of the luminal midgut surface and numerous autophagosomes were seen in the epithelial cells of both early and late fourth instar larvae, suggesting autophagy is involved in the degeneration process of the midgut in preparation for pupation. This study provides a basis for understanding normal Oc. togoi larval midgut development. Further studies are needed to determine the factors that control larval growth and the nutritional state. Such information could be used to reduce adult fecundity and develop biological control mechanisms.

DNA barcoding for the identification of eight species members of the Thai Hyrcanus Group and investigation of their stenogamous behavior.

Eight species members of the Thai Hyrcanus Group were identified based on the intact morphology and molecular analysis (COI barcoding, 658 bp) of F1-progenies. Five iso-female lines of each species were pooled in order to establish stock colonies. A stenogamous colony of each species was investigated by making 200 and 300 newly emerged adult females and males co-habit in a 30 cm cubic cage for one week. After ovipositon, the spermathecae of females were examined for sperms. The results revealed that Anopheles argyropus, Anopheles crawfordi, Anopheles nitidus, Anopheles pursati, Anopheles sinensis, Anopheles nigerrimus, Anopheles paraliae and Anopheles peditaeniatus yielded insemination rates of 0%, 0%, 0%, 31%, 33%, 42%, 50% and 77%, respectively. Continuous selection to establish stenogamous colonies indicated that An. sinensis, An. pursati, An. nigerrimus, An. paraliae and An. peditaeniatus provided insemination rates of 33-34%, 27-31%, 42-58%, 43-57% and 61-86% in 1, 2, 5, 6 and 20 generations of passages, respectively.

Peritrophic matrix formation and Brugia malayi microfilaria invasion of the midgut of a susceptible vector, Ochlerotatus togoi (Diptera: Culicidae).

The mosquito midgut is the first site that vector-borne pathogens contact during their multiplication, differentiation, or migration from blood meal to other tissues before transmission. After blood feeding, the mosquitoes synthesize a chitinous structure called peritrophic matrix (PM) that envelops the blood meal and separates the food bolus from the midgut epithelium. In this study, a systematic investigation of the PM formation and the interaction of Brugia malayi within the midgut of a susceptible vector, Ochlerotatus togoi, were performed using scanning electron microscopy (SEM). SEM analysis of the midguts dissected at different time points post feeding on a B. malayi-infected blood meal (PIBM) revealed that the PM was formed from 45 min PIBM and gradually thickened and matured during 8-18 h PIBM. The PM degraded from 24 to 72 h PIBM, when digestion was completed. The invasion process of the microfilariae was observed between 3 and 4 h PIBM. In the beginning of the process, only sheathed microfilariae interacted with the internal face of the PM by its anterior part, and then the midgut epithelium before entering the hemocoel, after that they exsheathed. Microfilarial sheaths lying within the hemocoel were observed suggesting that they may serve as a decoy to induce the immune systems of the mosquitoes to respond to the antigens on the sheaths, thereby protecting the exsheathed microfilariae. These initial findings would lead to further study on the proteins, chemicals, and factors in the midgut that are involved in the susceptibility of O. togoi as a vector of filariasis.

Salivary gland proteome of the human malaria vector, Anopheles campestris-like (Diptera: Culicidae).

Anopheles campestris-like is proven to be a high-potential vector of Plasmodium vivax in Thailand. In this study, A. campestris-like salivary gland proteins were determined and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis, and nano-liquid chromatography-mass spectrometry. The total amount of salivary gland proteins in the mosquitoes aged 3-5 days was approximately 0.1 ± 0.05 µg/male and 1.38 ± 0.01 µg/female. SDS-PAGE analysis revealed at least 12 major proteins found in the female salivary glands and each morphological region of the female glands contained different major proteins. Two-dimensional gel electrophoresis showed approximately 20 major and several minor protein spots displaying relative molecular masses from 10 to 72 kDa with electric points ranging from 3.9 to 10. At least 15 glycoproteins were detected in the female glands. Similar electrophoretic protein profiles were detected comparing the male and proximal-lateral lobes of the female glands, suggesting that these lobes are responsible for sugar feeding. Blood-feeding proteins, i.e., putative 5'-nucleotidase/apyrase, anti-platelet protein, long-form D7 salivary protein, D7-related 1 protein, and gSG6, were detected in the distal-lateral lobes (DL) and/or medial lobes (ML) of the female glands. The major spots related to housekeeping proteins from other arthropod species including Culex quinquefasciatus serine/threonine-protein kinase rio3 expressed in both male and female glands, Ixodes scapularis putative sil1 expressed in DL and ML, and I. scapularis putative cyclophilin A expressed in DL. These results provide information for further study on the salivary gland proteins of A. campestris-like that are involved in hematophagy and disease transmission.

Proteomic analysis of salivary glands of female Anopheles barbirostris species A2 (Diptera: Culicidae) by two-dimensional gel electrophoresis and mass spectrometry.

Salivary gland proteins of adult female Anopheles barbirostris species A2, a potential vector of Plasmodium vivax in Thailand, were analyzed using a proteomic approach (two-dimensional gel electrophoresis followed by nanoLC-MS). Two-dimensional gel electrophoresis revealed approximately 75 well-resolved spots on the reference gel. Most of the protein spots displayed relative molecular masses from 14 to 85 kDa and isoelectric points ranging from 3.9 to 10. The proteome profiles of A. barbirostris species A2 female salivary glands were affected by aging. The typical electrophoretic pattern of the female salivary glands was reached in 48 h post emergence, suggesting the maturation of salivary glands and saliva contents for blood feeding. Proteins involved in blood feeding, i.e., putative 5' nucleotidase/apyrase, anti-platelet protein, long form D7 salivary protein, D7-related 1 protein, and gSG6 salivary protein, start to accumulate from emergence and gradually increase becoming predominant within 48 h. There are different salivary components expressed within each region of the female glands. The blood-feeding proteins were detected in the distal-lateral lobes and/or medial lobes. Proteins detected and/or identified by this approach could be tested in strategies developed to control pathogen and disease transmission. Moreover, the information of a 2D map of the female salivary gland could be used for comparison with other related species in the A. barbirostris complex to distinguish species members in the complex.