“Salivary gland cellular architecture in the Asian malaria vector mosquito Anopheles stephensi”

Wells and Andrew Parasites & Vectors (2015) 8:617 DOI 10.1186/s13071-015-1229-z RESEARCH Open Access “Salivary gland cellular architecture in the Asian malaria vector mosquito Anopheles stephensi” Michael B. Wells and Deborah J. Andrew* Abstract Background: Anopheles mosquitoes are vectors for malaria, a disease with continued grave outcomes for human health. Transmission of malaria from mosquitoes to humans occurs by parasite passage through the salivary glands (SGs). Previous studies of mosquito SG architecture have been limited in scope and detail. Methods: We developed a simple, optimized protocol for fluorescence staining using dyes and/or antibodies to interrogate cellular architecture in Anopheles stephensi adult SGs. We used common biological dyes, antibodies to well-conserved structural and organellar markers, and antibodies against Anopheles salivary proteins to visualize many individual SGs at high resolution by confocal microscopy. Results: These analyses confirmed morphological features previously described using electron microscopy and uncovered a high degree of individual variation in SG structure. Our studies provide evidence for two alternative models for the origin of the salivary duct, the structure facilitating parasite transport out of SGs. We compare SG cellular architecture in An. stephensi and Drosophila melanogaster, a fellow Dipteran whose adult SGs are nearly completely unstudied, and find many conserved features despite divergence in overall form and function. Anopheles salivary proteins previously observed at the basement membrane were localized either in SG cells, secretory cavities, or the SG lumen. Our studies also revealed a population of cells with characteristics consistent with regenerative cells, similar to muscle satellite cells or midgut regenerative cells. Conclusions: This work serves as a foundation for linking Anopheles stephensi SG cellular architecture to function and as a basis for generating and evaluating tools aimed at preventing malaria transmission at the level of mosquito SGs. Keywords: Anopheles, Salivary gland, Malaria, Drosophila, Cell architecture, Secretion Background Mosquito transmitted disease represents a major threat to human health. Hundreds of millions of infections occur each year, leading to nearly two million deaths. The majority of these deaths are caused by malaria transmitted by mosquitoes of the genus Anopheles. Thirty-nine species of Anopheles are known to contribute to malaria infection worldwide [1], and two of the major vector species are Anopheles gambiae (prevalent in Africa) and Anopheles stephensi (prevalent in India). * Correspondence: Department of Cell Biology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., G-10 Hunterian, Baltimore, MD 21205, USA These are also two of the most well-studied mosquito species. The life cycle of malaria parasites, Plasmodium species, has been characterized [2–5]. The parasite is acquired by mosquitoes that blood feed on infected humans [3]. Parasite gametes fuse inside the mosquito midgut to form zygotes that mature into motile ookinetes, which traverse the peritrophic matrix and midgut epithelium to form an oocyst in the gut wall lining [6]. Within the oocyst, the parasites multiply and mature into sporozoites, which travel via hemolymph flow to the salivary glands (SGs) after oocyst rupture. Plasmodium sporozoites acquire the ability to infect mammalian liver cells either in the hemolymph [7] or in the SGs [8]. Twenty percent of © 2015 Wells and Andrew. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wells and Andrew Parasites & Vectors (2015) 8:617 parasites that escape the midgut enter the SGs [5, 9, 10], while the rest are cleared from the mosquito. SG invasion is thought to involve receptor/ligand interactions; several parasite coat proteins (CSP, MAEBL, TRAP, UOS3, CRMP1/2), as well as SG surface sugar molecules (e.g. heparin sulfate) and proteins (SGS1, Saglin, TRAP) have been implicated in this process [4]. Once sporozoites contact the SGs, the parasite is thought to traverse the basement membrane via gliding motility and invade the SG epithelial cell by a process similar to cell engulfment, using the plasma membrane to form a second outer membrane (parasitophorous vacuole), which is subsequently lost. The parasite exits the epithelial cell into the secretory cavity, where hundreds to thousands of sporozoites collect. Only a small number of parasites can enter the salivary duct to be injected into their next host upon subsequent blood feeding. Parasites are injected along with mosquito saliva and a complement of factors that prevent clotting and host immune response [2, 3]. Despite over 100 years of discontinuous work focused on disease transmission to humans, mosquito biology at the cellular and molecular levels remains understudied. Adult An. stephensi SG morphology has been described using electron microscopy (EM) [11, 12], where a number of observations regarding cell shape, organelle localization, and secretion characteristics were made. Other accounts of Anopheles adult SG structure by light and fluorescence microscopy have illuminated additional details regarding gross morphology, but these studies are quite limited in scope [13–16]. In contrast, a number of labs have characterized the proteins produced in Anopheles SGs, either en masse through mass spectrometry [17–20], or individually through biochemistry and molecular genetics methods [21–23]. Results overlap as far as the salivary proteome at large is concerned, but studies of proteins at the cellular level, particularly of protein localization by immunofluorescence, have produced inconsistent results and are typically limited to examination of a single protein [24–30]. One group has also recently generated Anopheles stephensi RNA-seq profiles at many developmental stages, with representative time points from early embryogenesis through early adulthood in either sex [31]. The limited characterization of adult SGs is not a problem unique to Anopheles and other insect vectors of disease. Indeed, very little is known regarding adult SG architecture in Drosophila melanogaster, a major model organism in laboratory research. Aside from a study of microfilament and microtubule organization [32], almost nothing has been done to characterize Drosophila adult SGs. Several accounts exist of conservation of function between Drosophila and Anop (...truncated)