**Proteomics of** *Anopheles gambiae*

V. Choumet *Institut Pasteur France* 

## **1. 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 10–14 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.

#### **1.1 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 300–500 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

Proteomics of *Anopheles gambiae* 41

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

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

*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

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

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

transmission and other mosquito-borne human illnesses.

transmission [5].

species [9].

**1.4 Genome versus proteome** 

understand the vector/pathogen interactions.

**1.3 The genome of** *Anopheles gambiae*

*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.

#### **1.2** *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.

Fig. 1. *Plasmodium* species life cycle in *Anopheles* mosquitoes

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

*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

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.

partners and of proteins implied in cross-talk between them.

Fig. 1. *Plasmodium* species life cycle in *Anopheles* mosquitoes

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

**1.2** *Plasmodium* **life cycle in mosquito** 

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.
