**2.4** *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].

30 kDa protein, anti-platelet

Apyrase, anti-aggregation D7 precursor allergen AED A2

D7 related-5 protein precursor

GSG6, implied in blood-feeding

Hypothetical 10 kDa protein Hypothetical 10.2 kDa protein Hypothetical 8.8 kDa protein

Maltase, carbohydrate digestion

putative Salivary protein SG

Salivary gland 1-like 3 protein

Lysozyme precursor

Salivary D3 protein

Table 3. List of *Anopheles gambiae* salivary proteins possessing a signal peptide and detected

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

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

Guanine nucleotide releasing factor

Alpha-amylase, carbohydrate digestion

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

putative gVAG protein precursor, implied in defense

Putative 5' nucleotidase precursor, anti-platelet

Protein/gene ID Domain/motifs/Known/putative function/comments

GSG3

GSG7

SG3

GSG5 precursor

**Secreted salivary gland proteins** 

> GI:18389879 AGAP011971-PA AGAP008278-PA AGAP008284-PA AGAP008282-PA AGAP008283-PA AGAP008281-PA AGAP008280-PA GI:4127305 AGAP004334-PA AGAP000150-PA AGAP008216-PA AGAP008307-PA AGAP008306-PA AGAP006494-PA AGAP003168-PA AGAP006821-PA AGAP001903-PA AGAP006340-PA AGAP004038-PA AGAP000607-PA AGAP006507-PA AGAP000175-PA AGAP012335-PA

by proteomic approaches ([30; 32; 33]

behaviour of the parasitized mosquito.

**2.4** *Anopheles gambiae* **head proteome** 

rate of their vectors by a variety of mechanisms [40].

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.

#### **2.5 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

Proteomics of *Anopheles gambiae* 51

Unknown Unknown Unknown Unknown Unknown Unknown Unknown

Unknown Unknown Unknown

Genomics, transcriptomics and proteomics studies have been complemented by studies using RNAi for gene silencing. RNAi allows characterization of genes *in vivo* which can later be targeted for transmission blocking studies. Boisson et al. [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

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

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

interest of proteomic tools to complement other approaches for genome annotation.

Ribosomal protein L14 Ribosomal protein L4

Ribosomal protein S17

c-1-Tetrahydrofolate synthase

AGAP010252 AGAP002306 AGAP002830 AGAP012261 AGAP005802 AGAP005061 AGAP000547 AGAP006584 AGAP005338 AGAP003911 AGAP004887 AGAP000672 AGAP007758 AGAP006805

Table 4. Proteome of *Anopheles gambiae* eggshell

**2.6 Functional genomics approaches** 

role in blood-feeding.

**3. Conclusion** 

mechanisms.

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.


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

function/comments

chorion peroxidase

Prophenoloxidase 9

Vitelline membrane component Vitelline membrane component

Schistosoma mansoni egg protein Ionotropic glutamate receptor

Putative chorion protein Putative chorion protein Putative chorion protein Putative chorion protein Putative chorion protein Putative chorion protein Putative chorion protein

Yellow-g2-dopachrome conversion enzyme

Laccase 2

Peroxidase Thioredoxin

Unknown Unknown Unknown Unknown Unknown Unknown

OBP34/37 OBP35 OBP36 OBP11 OBP1 OBP44 OBP13

**Eggshell** Protein/gene ID Domain/motifs/Known/putative

Odorant binding proteins

AGAP000641 AGAP000642 AGAP000643 AGAP002025 AGAP011647 AGAP010648 AGAP002189 Enzymes AGAP004038 AGAP006176 AGAP005959 AGAP004978 AGAP003233 AGAP007020 Vitelline membrane

components AGAP002134 AGAP008696 Putative chorion components AGAP00655 AGAP006555 AGAP006553 AGAP006554 AGAP006549 AGAP006556 AGAP006550 Others AGAP006524 AGAP006563 AGAP003149 AGAP010147 AGAP004182 AGAP006527 AGAP003047 AGAP004969

be demonstrated.


Table 4. Proteome of *Anopheles gambiae* eggshell
