Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae in a refractory vector, Aedes aegypti (Thailand strain)

Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae were analyzed using light and scanning electron microscopy in a refractory vector, Aedes aegypti (Thailand strain). Results showed that exsheathed microfilariae represented only approximately 1 % of the total microfilaria midguts dissected at 5-min post-infected blood meal (PIBM). The percentage of exsheathed microfilariae found in midguts progressively increased to about 20, 60, 80, 90, and 100 % at 1-, 2-5-, 6-12-, 18-36-, and 48-h PIBM, respectively. Importantly, all the microfilariae penetrating the mosquito midguts were exsheathed. Midgut invasion by the exsheathed microfilariae was observed between 2- and 48-h PIBM. SEM analysis revealed sheathed microfilariae surrounded by small particles and maceration of the microfilarial sheath in the midguts, suggesting that the midguts of the refractory mosquitoes might have protein(s) and/or enzyme(s) and/or factor(s) that induce and/or accelerate exsheathment. The microfilariae penetrated the internal face of the peritrophic matrix (PM) by their anterior part and then the midgut epithelium, before entering the hemocoel suggesting that PM was not a barrier against the microfilariae migrating towards the midgut. Melanized microfilariae were discovered in the hemocoel examined at 96-h PIBM suggesting that the refractory mosquitoes used melanization reactions against this parasite. This study provided evidence that A. aegypti (Thailand strain) has refractory mechanisms against B. malayi in both midgut and hemocoel

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

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