Proteomics of Anopheles gambiae

Malaria is a global disease in the world today but most common in the poorest countries of the world, with 90% of deaths occurring in sub-Saharan Africa. This book provides information on global efforts made by scientist which cuts across the continents of the world. Concerted efforts such as symbiont based malaria control; new applications in avian malaria studies; development of humanized mice to study P.falciparium (the most virulent species of malaria parasite); and current issues in laboratory diagnosis will support the prompt treatment of malaria. Research is ultimately gaining more grounds in the quest to provide vaccine for the prevention of malaria. The book features research aimed to bring a lasting solution to the malaria problem and what we should be doing now to face malaria, which is definitely useful for health policies in the twenty first century


Introduction
Mosquitoes are a family of around three and a half thousand species and belong to the order of Diptera. They are found throughout the world except in places permanently frozen. Three quarters of all mosquito species live in the humid tropics and subtropics, where the climate is warm and moist, well adapted to the development of all stages and allows adults survival. Mosquitoes are classified in three subfamilies (Toxorhynchitinae, Anophelinae, Culicinae). Anopheles mosquitoes are members of subfamily Anophelinae. Like all mosquitoes, its life cycle consists in four stages: egg, larva, pupa, and adult. Female mosquitoes lay 30-150 eggs every 2-3 days in water. Anopheles favors as breeding places, quiet water, like permanent or temporary ponds, or quite cove of rivers. Each egg is protected by an eggshell. Mosquitoes can develop from egg to adult in as little as 5 days but usually take 1014 days in tropical conditions. Anopheles can be distinguished from other mosquitoes by their palps, which are as long as the proboscis. Most Anopheles mosquitoes are not exclusively anthropophilic or zoophilic. Anopheles mosquitoes are more frequent in tropical and subtropical countries but are also found in temperate climates. They are not found at altitudes above 2000-2500 m.

Pathogens transmitted by Anopheles mosquitoes
Anopheles mosquitoes are vectors of several pathogens. They can transmit O'nyong-nyong virus as well as parasitic nematodes that cause lymphatic filariasis. Of the approximately 460 known species of Anopheles, less than 100 can transmit human malaria in nature. The primary malaria vectors in Africa, A. gambiae and A. funestus, are strongly anthropophilic and, consequently, are two of the most efficient malaria vectors in the world.
Malaria is a parasitic disease that affects 200 million people worldwide and causes 1.5 to 2.7 million deaths per year. Of the 300500 million clinical cases annually, nearly 90% are in the sub-Saharan countries of Africa where the malaria parasite, Plasmodium falciparum, is primarily transmitted by the mosquito Anopheles gambiae. The increasing resistance of the parasite to inexpensive drugs and the resistance of mosquitoes to insecticides have created an urgent need for innovative methods that block parasite transmission during its development within the insect. Strategies for the development of malaria vaccine candidates target the stages found within humans e.g. the asexual erythrocytic stages. However vaccine candidates have also been developed against both the sexual stages of malaria in the mosquito (reviewed in [1] and against the mosquito vector itself (reviewed in [2]). The

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Anopheles mosquito not only carries the parasite from infected to uninfected people, but also plays a vital role in the parasite life cycle [3; 4]. The development of Plasmodium within the mosquito is a very complex process and represents a tight coevolved system in which genetic features of both vector and parasite characterize the potential of the parasite to develop and be transmitted. Identification of promising candidate antigens for a mosquitobased transmission blocking vaccine or interference requires a good knowledge of both partners and of proteins implied in cross-talk between them.

Plasmodium life cycle in mosquito
When a female Anopheles sucks the blood of an infected human host, it receives red blood cells (RBCs) containing different stages of erythrocytic cycle, including gametocytes. In its gut, all stages except the gametocytes are digested (Figure 1). These hatch out from RBCs into the lumen of host's midgut and become active to start the sexual cycle. After a process termed exflagellation, the male microgametes detach from the parent gametocyte. The female, macrogametocytes undergo oogenesis and becomes a haploid macro-gamete. Zygotes are formed by fusion of male and female gametes. The zygotic stage of Plasmodium species develops to form ookinete. The ookinete passes through the epithelium of the midgut and forms an oocyst on the exterior wall of the midgut. The oocyst matures to form an enlarged structure, after which several nuclear divisions take place. Oocyst ruptures and www.intechopen.com releases hundreds of sporozoites. Eventually, these sporozoites migrate to the salivary gland of the female anopheles mosquito. The interval between the acquisition of an infectious agent by a vector and the vector's ability to transmit the agent to other susceptible vertebrate hosts is called the extrinsic incubation period. This parameter is dependent on host, parasite and environmental factors. Estimates are on the order of 10-14 days in areas of high malarial transmission [5].
During the successive stages of parasite development within the mosquito vector, it is faced to several barriers that are decisive for the fate of the parasite and the potential of its transmission. The proteome analysis of these barriers constitutes a first step to better understand the vector/pathogen interactions.

The genome of Anopheles gambiae
Anopheles gambiae sensus stricto. was selected for full genome sequencing from the 60 or so anopheline mosquito species that transmit malaria, largely because of the large number of malaria fatalities attributed to bites from this mosquito [6; 7]. At 278 Mb, A. gambiae genome is considerably larger than the genome size of Drosophila melanogaster [8](180 Mb), but smaller than the size of many other mosquito disease vectors such as Aedes aegypti, which at approximately 1376 million base pairs is about 5 times the size of the genome of the malaria vector [9]. The difference in size between A. gambiae and D. melanogaster is largely due to intergenic DNA. Despite evolutionarily diverging 250 million years ago, the two insects reveal remarkable similarities in their DNA sequences. However, there are also important differences in their genomes. Almost half of the genes in both insect genomes are presently identified as orthologs and show an average sequence identity of 56% [6]. This could be explained by successive adaptation of these insects to different life strategies and/or ecological niches, or to the evolutionary adaptation of pathogens to insect vectors or vice versa. Studies have led to the identification of mosquito genes that regulate malaria parasite infection in the mosquito ( [10] and those involved in the ability to find and feed on blood of human hosts [11]. In Aedes aegypti genome, an increase in genes encoding odorant binding, cytochrome P450, and cuticle domains relative to A. gambiae suggests that members of these protein families support some of the biological differences between the two mosquito species [9].
With the genome of A. gambiae in hand, scientists possess molecular information to understand the biology of this arthropod vector better, and to possibly fight malaria transmission and other mosquito-borne human illnesses.

Genome versus proteome
Actually, DNA is a chemical instruction manual for everything a plant or animal does, while proteins are the dynamic components. DNA or mRNA sequences cannot sufficiently describe the structure, function and cellular location of proteins. Moreover, some important functional, post-translational modifications, such as glycosylation and phosphorylation, may not even be seen at the genome level. Hence, analysis of the human proteome revealed that it is much more complex than the human genome. While scientists are still deciphering the function of most of the 30,000 human genes, researchers estimate that each gene can give instructions for as many as 100 different proteins. On top of that, every organ has a different www.intechopen.com distribution of proteins, and the distribution may change frequently according to physiological modifications. The term proteome constitutes the total of proteins expressed by the genome. The technical improvements of the past decade have enhanced proteomic analyses and thereby enabled quantitative analysis of protein expression inside cells. The proteome of several A. gambiae organs were deciphered using various proteomic approaches and interesting insights in their function as well as in their putative interaction with pathogens were thereby gathered.

Proteomics methods
Thanks to the combination of developments in new instrumentation, fragmentation methods, availability of completed genome sequences and bioinformatics, there has been a shift from analysis of one protein at the time to more comprehensive proteome analyses. In the past decade, mass spectrometry (MS) has emerged as the dominant technology for indepth characterization of the protein components of biological systems {12; 13; 14] but a number of other technologies, resources, and expertise are absolutely required to perform significant experiments. These include protein separation science (and protein biochemistry in general), genomics, and bioinformatics. Proteomics has evolved from 1D and 2D gel electrophoresis (1DE and 2DE)/MALDI TOF (Matrix Assisted Laser Desorption Ionization) MS to gel-free liquid chromatography (LC)-MS/MS (tandem mass spectrometry) approaches. Present-day 1D/2D LC-MS/MS workflows exhibit much higher sensitivity, speed, quantitative dynamic range and ease of use in comparison with gel-based resolving techniques. These various approaches are summarized in Figure 2. Beyond protein identification, there are a number of well-established options available for protein quantification. Difference gel electrophoresis (DIGE) following 2DE is one option. Various mass spectrometry based methods have been employed for quantitative analysis of protein expression in a complex protein mixture upon particular modification, such as prior to and after parasite infection. These quantitative approaches utilize the stable isotopelabelling techniques combined with mass spectrometry analysis, including isotope coded affinity tag (ICAT) [15; 16]), isobaric tag for relative and absolute quantitation (iTRAQ) [17], and stable isotope labelling of amino acids in cell culture (SILAC) [18; 19].

Proteomic analyses of Anopheles gambiae
During the last ten years, several approaches were followed to describe the protein content of Anopheles gambiaes organs, hemolymph and saliva. The subsequent paragraphs will present protein data obtained from these various approaches.

The peritrophic matrix (PM) proteome
The peritrophic matrix (or peritrophic membrane) lines the gut of most insects at one or more stages of the life cycle [20]. This is the only region of the insect in contact with the external environment not covered by cuticle. It has important roles in the facilitation of the digestive processes in the gut and the protection of the insect from invasion by microorganisms and parasites. Since it poses a partial, natural barrier against parasite invasion of the midgut, it is speculated that modifications to the PM may lead to a complete barrier to infection. In agreement with this statement, it was showed that thickening of the PM by experimental manipulation resulted in decreased Plasmodium infectivity [21]. Within the blood mass, the Plasmodium gametes mate and develop into motile ookinetes, a process that takes 1624 h depending on the Plasmodium species. To traverse the gut epithelium, the ookinetes must first cross the PM. For this purpose, the Plasmodium parasite secretes its own chitinase, which allows it to penetrate this chitin-containing structure [22]. Detailed characterization of the structure of the PM may help to find out new strategies to block the parasite at the midgut stage. The complete PM proteome of A. gambiae was determined using 1D-electrophoresis followed by LC-MS [23]. Out of the 209 proteins identified by mass spectrometry, 123 had predicted signal peptides, of which 17 also had predicted transmembrane domains and were therefore candidate midgut surface proteins that were coextracted from the gut during sample processing (Table 1). Among them were nine new chitin-binding peritrophic matrix proteins, expanding the list from three to twelve peritrophins. As expected, the number of proteins with signal peptides is relatively more abundant than those with transmembrane domains only. Based on their annotations, the proteins fell into different classes, with diverse putative functions ranging from immunity to blood digestion. Only 5% of the detected proteins were either known PM proteins or contained clear chitin binding domains (CBDs). Based on these results, the authors proposed a structural model of A. gambiae PM to explain the putative interactions among the proteins identified in their study. During the course of blood ingestion the PM is rapidly secreted by the distended midgut epithelium. The chitin fibrils are assembled into a wide cross-hatched pattern connected by peritrophins containing multiple CBDs. Between 0 and 24 h, the crosshatch is fully stretched and then slowly contracts like an accordion between 36 and 48 h post-blood feeding as blood digestion proceeds to completion. The various classes of www.intechopen.com peritrophins that have been identified by proteomic analysis were proposed to assemble together to produce a PM as well as perform several of its functions.

Peritrophic matrix
Protein/gene ID The proteins of the PM may provide unique opportunities for the control of insect pests and vector borne diseases. Immunological control of blood and tissue-feeding insects by the effects of binding antibody to the proteins of the PM may be explored as well as other functional genetics approaches based on the understanding of the function of its components.

Hemolymph proteome
Insects employ an open circulatory system for the transport of nutrients, wastes, and signalling molecules throughout the body. The insect circulatory system also functions in thermoregulation, promoting ventilation through the tracheal system, and the circulation of humoral immune molecules and immune blood cells (hemocytes) that survey tissues for foreign entities. The primary organ driving hemolymph circulation in mosquitoes is a dorsal vessel that is subdivided into an abdominal heart and a thoracic aorta. Hemolymph plays a very important role in protecting against harmful microorganisms like Plasmodium. Twodimensional SDSPAGE and microsequencing or peptide mass fingerprinting was used to identify major proteins in the hemolymph of A. gambiae [24; 25]. They found about 280 protein spots in hemolymph and identified 28 spots, representing 26 individual proteins ( Table 2). Most of these proteins have known or predicted functions in immunity. These include prophenoloxidase 2 subunit, two clip-domain serine proteases (CLIP B4 and A6), a thioester protein (TEP15), two serpins (SRPN2 and SRPN15), a cystatin and apolipophorin III (apoLPIII). Other proteins are implied, in iron transport like ferritin, or lipid biology. Many of the proteins have been found in hemolymph in other insects but one protein is novel: a new member of the MD-2-related lipid-recognition family. Fourteen spots were induced following bacterial injection but not by wounding. Three of the identified proteins increased in spot intensity or appeared de novo following bacterial injection: a phenoloxidase, and two chitinase-like proteins. A subset of proteins decreased following bacterial injections: these included the light and heavy chains of ferritin. Several proteins appeared in hemolymph following any wound or injection. These included two isoforms of phosphoglycerate mutase (PGM), triose phosphate isomerase (TPI), an actin, glutathione S transferase S1-1, and adenylate kinase. Most of these are metabolic enzymes lacking signal peptides that are likely to be released as a result of damage to muscles and other tissues by injury. The map obtained is a useful tool for examining changes in hemolymph proteins following blood feeding and infection by parasites.
Interestingly, antibodies raised against hemolymph proteins were shown to decrease mosquito infection by Plasmodium vivax [26]. These antibodies are directed against 11 different antigens, 6 of which were specific of the hemolymph and four were common to midgut and ovary. Identification of these antigens by mass spectrometry could lead to the development of blocking vaccines against malaria.

Proteomics of salivary glands and saliva
Mosquito saliva and salivary glands are central to the interaction between parasite, vector and mammalian host. Sporozoite maturation in the mosquito salivary glands before its transmission to vertebrates is a key stage for the effective transmission to humans since it increases the sporozoites ability to infect vertebrate hepatocytes [27; 28]. Additionally, www.intechopen.com sporozoites are injected into the vertebrate skin with nanoliter volumes of saliva. Saliva is a complex biologically active solution, contains a large number of biomolecules responsible for antihemostatic activity, which assist hematophagous arthropods during the feeding process [29].  [30]. Among these proteins were A. gambiae D7-related proteins 1-3, similar to Aedes aegypti D7. The D7 proteins are among the most abundant salivary proteins in adult female mosquitoes and sand flies [31]. D7 sequences were identified in Culex quinquefasciatus, An. arabiensis, An. stephensi, An. darlingi mosquitoes and Lutzomyia longipalpis and Phlebotomus papatasi sand flies.

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The other identified proteins were GSG6, hypothetical protein 8 (CB1), similar to hypothetical protein 9 (bB2) and herein called HP 9-like, SG1-like 2, putative 5 nucleotidase and SG1. Edman degradation for other bands was reported to be unsuccessful, either because the protein's amino terminus was blocked, or because PTH-amino acids could not be reliably identified.
A proteomic analysis of salivary glands from female A. gambiae mosquitoes was carried out [32]. Salivary gland extracts were hydrolyzed with trypsin using a 1-DE in-gel and an in solution digestion and analyzed by LC-MS/MS. This led to identification of 69 unique proteins, 57 of which were novel. A large proportion of the identified proteins were involved in protein, carbohydrate and nucleic acid metabolism, transport or energy pathways. Almost 25% of the proteins could be not be ascribed any biological function. A subcellular localization was assigned to each protein either based on the literature or the presence of particular domains/motifs. The majority of the proteins were classified as extracellular proteins. D7 family proteins, apyrases and proteins of the salivary gland-like (SGlike) family were the commonest extracellular proteins. Proteins involved in translation and protein folding were the predominant cytoplasmic proteins with a small number of proteins classified as nuclear, vesicular or lysosomal proteins. Approximately 40% of proteins could not be assigned any specific localization because of lack of any distinctive features and lack of homology to other known proteins.
To further describe A. gambiae salivary gland and saliva contents, several techniques: 1-DE, 2-DE and LC-MS/ MS were combined to characterize the protein content of 8 year-old and 21 year-old mosquito salivary glands [33]. Overall, this study has identified five saliva proteins and 122 more proteins from the salivary glands, including the first proteomic description for 89 of these salivary gland proteins. Proteomic analysis of 8 year-old bloodfed mosquitoes allowed the identification of 55 proteins. LC-MS/ MS and 2-DE-MS identified a similar number of proteins and both appear more effective than 1-DE-MS. Thirty percent of the proteins identified are secreted (Table 3).
2D-E profiles suggested that several secreted proteins may present sequence divergence or be extensively processed and/or post-translationally modified in A. gambiae salivary glands.
Since the invasion and sporozoite maturation take place during the process of salivary glands ageing, the effect of salivary gland age on salivary component composition was examined. LC MS/MS profiling of young versus old salivary gland proteomes suggests that there is an over-representation of proteins involved in signalling and proteins related to the immune response in the proteins from older mosquitoes.
A comparative proteomic analysis of salivary gland samples from infected or Plasmodium berghei-free mosquitoes was performed using the iTRAQ labeling.
The expression levels of five secreted proteins were altered when the parasite was present. The levels of GSG6, apyrase, D7 related-1 protein precursor and D7 precursor allergen AED A2 are decreased whereas the level of gVAG is increased in infected salivary glands. Apyrase, GSG6 and D7 precursor allergen AED A2 were shown to be implied in blood feeding [34; 35; 36]. In addition, GSG6 was found to be a serological indicator of exposure to Afrotropical malaria vectors [37].
www.intechopen.com 30 kDa protein, anti-platelet Alpha-amylase, carbohydrate digestion Apyrase, anti-aggregation D7 precursor allergen AED A2 D7 related-1 protein precursor, anti-inflammatory D7 related-2 protein precursor, anti-inflammatory D7 related-3 protein precursor, anti-inflammatory D7 related-4 protein precursor, anti-inflammatory D7 related-5 protein precursor GSG3 GSG5 precursor GSG6, implied in blood-feeding GSG7 Guanine nucleotide releasing factor putative gVAG protein precursor, implied in defense Hypothetical 10 kDa protein Hypothetical 10.2 kDa protein Hypothetical 8.8 kDa protein Lysozyme precursor Maltase, carbohydrate digestion Putative 5 nucleotidase precursor, anti-platelet putative Salivary protein SG Salivary D3 protein Salivary gland 1-like 3 protein SG3 Table 3. List of Anopheles gambiae salivary proteins possessing a signal peptide and detected by proteomic approaches ([30; 32; 33] The D7 short proteins bind serotonin with high affinity, as well as histamine and norepinephrine, thus antagonizing the vasoconstrictor, platelet-aggregating, and paininducing level of these factors [38]. gVAG is a defense-related protein [39]. These observations suggest an important role of these proteins in the interaction between A. gambiae, Plasmodium and the mammalian host and a putative modulation of the feeding behaviour of the parasitized mosquito.

Anopheles gambiae head proteome
Many of the most dangerous pathogens have been shown to manipulate the behaviour of their vectors, such as feeding behaviour, in ways that enhance the contact with the vertebrate host and consequently favour pathogen transmission {40]. Several studies with different systems support the idea that parasites indeed increase the probing and feeding rate of their vectors by a variety of mechanisms [40].

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In the Plasmodium-Anopheles interaction, facts showing that Plasmodium spp. influence the behaviour of their mosquito vectors, Anopheles spp. have accumulated [41; 42; 43; 44; 45; 46]. In this system, an altered behaviour of the vector has been shown when sporozoites have invaded the salivary glands [43; 45; 46]. For instance, malaria parasites were shown to manipulate their mosquito vectors in two different ways and in a stage-dependent mode: when the sporozoites are ready to be transmitted to the vertebrate hosts, the parasite increases the biting rate of its vector [45; 46]. In contrast, at the oocyst stage and yet not transmissible to the vertebrate host, the parasite decreases the contact between vector and vertebrate host by decreasing the natural host-seeking behaviour of the insect [46]. Moreover, it was recently shown that the presence of the parasite extends the oviposition cycle of the mosquito, thereby enhancing malaria transmission [47]. Interestingly, few studies have been undertaken to demonstrate the existence of a general manipulative mechanism by which the parasite manipulate the host central nervous system. A 2-D DIGE coupled with MS was used to analyse and compare the head proteome of A. gambiae infected with Plasmodium berghei with that of uninfected mosquitoes [48]. This approach allowed detecting modulations of 12 protein spots in the head of mosquitoes infected with sporozoites. After their identification by MS, these proteins were functionally classified as belonging to metabolic, synaptic, molecular chaperone, signalling and cytoskeletal groups.
These results indicate an altered energy metabolism in the head of sporozoite-infected mosquitoes. Some of the up-/down-regulated proteins identified, such as synapseassociated protein, 14-3-3 protein and calmodulin, have previously been shown to play critical roles in the CNS of both invertebrates and vertebrates. Interestingly, two proteins revealed in the study have been demonstrated to be involved in behavioural modifications in other hostparasite systems. Tropomyosin has been shown to be involved in the behavioural manipulation of crustacean gammarids by acanthocephalans [49], while phosphoglycerate mutase was involved in cricket behavioural manipulation induced by hairworms [50]. Furthermore, a heat shock response (HSP 20) and a variation of cytoarchitecture (tropomyosins) have been shown. Discovery of these proteins demonstrates potential molecular mechanisms that trigger behavioural modifications and offers new insights into the study of close interactions between Plasmodium and its Anopheles vector.

Proteome of the eggshell
Insect eggshells offer the embryo protection from physical and biological damages and assure their survival. Most current knowledge of insect eggshell morphology and composition are based on studies of Drosophila melanogaster [51; 52]. Mosquito eggshells show notable diversity in physical properties and structure, presumably resulting from adaptation to the large variety of environments exploited by these insects.
In contrast to Aedes mosquitoes, which are highly resistant to desiccation allowing embryos to survive for months in dry conditions [53], eggshells of the human malaria vector, A. gambiae are more permeable, restricting their survival and development to humid environments [54]. Greater knowledge of the proteins that comprise eggshells is required to understand these differences and how they contribute to successful mosquito reproductive strategies. A mass spectrometry/proteomics approach was used to identify 44 proteins as putative components of the eggshell (Table 4) [55]. Among the identified molecules are two vitelline membrane proteins and a group of seven putative chorion proteins. Enzymes with www.intechopen.com peroxidase, laccase and phenoloxidase activities, expected to be involved in cross-linking reactions that stabilize the eggshell structure, were also identified. Seven odorant binding proteins were found in association with the mosquito eggshell, although their role has yet to be demonstrated.  [56] demonstrated the role of the gene AgApy, which encodes an apyrase, in the probing behaviour of A. gambiae. An RNAimediated gene silencing approach has also been used to assess the potential involvement of 10 selected A. gambiae salivary gland genes in regulating mosquito blood-feeding capacity [36]. Silencing of several salivary gland transcripts; D7L2, anophelin, peroxidase, 5'nucleotidase and SG2 precursor, produced a significantly lowered blood-feeding phenotype and increased probing time, confirming that these genes could play an important role in blood-feeding.

Conclusion
In a recent publication, the results of an extensive qualitative proteomic analysis of Anopheles gambiae to better understand gene structures and their functions were presented [57]. In their manuscript, the authors reported validation of existing genes, correction of existing gene models, identification of novel genes, identification of novel splice variants, confirmation of splice sites and assignment of translational start sites based on highresolution mass spectrometry-derived data. A total of 2,682 peptides were identified that could not be mapped onto existing VectorBase annotations. This study emphasizes on the interest of proteomic tools to complement other approaches for genome annotation.
Dissecting the molecular basis of the interplay between vector and pathogen is essential for vector-borne disease transmission. Deciphering the proteome of the main mosquito barriers for parasite development and transmission may pave the way to novel disease control mechanisms. Malaria is a global disease in the world today but most common in the poorest countries of the world, with 90% of deaths occurring in sub-Saharan Africa. This book provides information on global efforts made by scientist which cuts across the continents of the world. Concerted efforts such as symbiont based malaria control; new applications in avian malaria studies; development of humanized mice to study P.falciparium (the most virulent species of malaria parasite); and current issues in laboratory diagnosis will support the prompt treatment of malaria. Research is ultimately gaining more grounds in the quest to provide vaccine for the prevention of malaria. The book features research aimed to bring a lasting solution to the malaria problem and what we should be doing now to face malaria, which is definitely useful for health policies in the twenty first century.