**1.3 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.

#### **1.4 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

Proteomics of *Anopheles gambiae* 43

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],

During the last ten years, several approaches were followed to describe the protein content of *Anopheles gambiae*'s organs, hemolymph and saliva. The subsequent paragraphs will

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 16–24 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

and stable isotope labelling of amino acids in cell culture (SILAC) [18; 19].

**2. Proteomic analyses of** *Anopheles gambiae*

**2.1 The peritrophic matrix (PM) proteome** 

present protein data obtained from these various approaches.

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.

#### **1.5 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.

Fig. 2. Proteomic workflow

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

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.

pathogens were thereby gathered.

**1.5 Proteomics methods** 

Fig. 2. Proteomic workflow

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].

### **2. Proteomic analyses of** *Anopheles gambiae*

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

#### **2.1 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 16–24 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

Proteomics of *Anopheles gambiae* 45

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

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 SDS–PAGE 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

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

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 sporozoite's ability to infect vertebrate hepatocytes [27; 28]. Additionally,

components.

**2.2 Hemolymph proteome** 

following blood feeding and infection by parasites.

development of blocking vaccines against malaria.

**2.3 Proteomics of salivary glands and saliva** 


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.

Table 1. PM proteins identified by 1D and LC-MS

peritrophins that have been identified by proteomic analysis were proposed to assemble

function/comments

2 CBD 1 CBD A CBD 2 CBD 1 CBD 2 CBD 3 CBD 4-CBD 4-CBD 2 CBD; immune-responsive gene 1 CBD; mucin domain; chitinase

12.9 Conserved hypothetical protein;

33.8 Conserved hypothetical protein; putative protein binding motifs; unknown

19.7 Conserved hypothetical protein; snake toxin-like protein folds/disulfide rich;

92.2 Conserved hypothetical protein; snake toxin-like protein folds/disulfide rich;

16.3 Conserved hypothetical protein; MD2-

52.1 Conserved hypothetical protein; CD36

28.1 Conserved hypothetical protein; odorant/hormone binding domain

scavenger receptor Class B domain 31.2 Conserved hypothetical protein; galactose-like binding protein/lectin-like

35.0 Conserved hypothetical protein; Fibrillin/fibrinogen-like; globular domain;

unknown function

unknown function

unknown function

unknown function

lipid recognition domain

function

domain

Putatively secreted

> yes yes yes yes yes yes yes no yes yes yes

> yes

yes

yes

yes

yes

yes

yes

no

yes

Protein/gene ID Domain/motifs/Known/putative

together to produce a PM as well as perform several of its functions.

Peritrophic matrix

Protein with

chitin binding domains (CBD) AGAP006795 AGAP006796 AGAP009830 AGAP010364 AGAP001819 AGAP010363 AGAP011616 AGAP006433 AGAP006434 AGAP006432 AGAP006414

Proteins with no predicted CBD AGAP006442

AGAP004883

AGAP007860

AGAP007612

AGAP002851

AGAP001352

AGAP010132

AGAP006398

AGAP004916

Table 1. PM proteins identified by 1D and LC-MS

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.

#### **2.2 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 SDS–PAGE 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.

#### **2.3 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 sporozoite's ability to infect vertebrate hepatocytes [27; 28]. Additionally,

Proteomics of *Anopheles gambiae* 47

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

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

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.

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

A comparative proteomic analysis of salivary gland samples from infected or *Plasmodium* 

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

be reliably identified.

features and lack of homology to other known proteins.

Thirty percent of the proteins identified are secreted (Table 3).

immune response in the proteins from older mosquitoes*.* 

Afrotropical malaria vectors [37].

*berghei*-free mosquitoes was performed using the iTRAQ labeling.

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].


Table 2. Proteins identified in *Anopheles gambiae* hemolymph

Following SDS-PAGE of *A. gambiae* salivary proteins, under denaturing non-reducing conditions and Edman sequencing, 12 saliva proteins were identified [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.

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

> Ferritin, heavy Ferritin, light chain

Srpn2, Serpin chitinase Cystatin

aldo/keto reductase

Domain/motifs/Known/putative function/comments

OBP9, odorant binding protein apoLP-III, Apolipophorin IIII

 AgLLP1, lipocalin-like protein D7H1, salivary D-7 like protein TEP15, thioester protein

ML3, ML-domain protein

AgH-1, hemolymph glycoprotein family

CLIPA6, Clip domain serine protease CLIPB4, clip domain serine protease Srpn15, putative haplotype of Srpn9 Serpin

PPO2, prophenoloxidase subunit GST-S1-2, glutathione S transferase

GST-SI-I, glutathione S transferase

Phosphoglycerate mutase Triosephosphate isomerase

Adenylate cyclise

BR-1, chitinase-like BR-2, chitinase-like PO6, phenoloxidase

Following SDS-PAGE of *A. gambiae* salivary proteins, under denaturing non-reducing conditions and Edman sequencing, 12 saliva proteins were identified [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.

Putatively secreted

yes yes yes yes yes yes yes yes yes yes yes yes yes no yes no no no

no no no no

yes yes no

process [29].

Constitutevely expressed proteins

Proteins modulated

appearing after any injection

appearing or altered after bacterial injury

**Hemolymph** Protein/gene ID

> agCP2491 agCP1503 agCP1469 agCP1261 agCP5831 agCP10937 agCP3566 agCP9948 agCP9547 AF007166 agCP9254 agCp3768 ebiP6959 agCP1375 agCP1985 ebiP1964 AF004915 AF513639

> BM624855 agCP12756 agCP12096 BM622046

AY496420 AY496421 AJ010195

Table 2. Proteins identified in *Anopheles gambiae* hemolymph

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].

Proteomics of *Anopheles gambiae* 49

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 host–parasite 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.

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

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

adaptation to the large variety of environments exploited by these insects.

**2.5 Proteome of the eggshell** 


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.
