Genetics of mosquito vector competence

Abstract

Mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. Efforts to control mosquito-borne diseases have been impeded, in part, by the development of drug-resistant parasites, insecticide-resistant mosquitoes, and environmental concerns over the application of insecticides. Therefore, there is a need to develop novel disease control strategies that can complement or replace existing control methods. One such strategy is to generate pathogen-resistant mosquitoes from those that are susceptible. To this end, efforts have focused on isolating and characterizing genes that influence mosquito vector competence. It has been known for over 70 years that there is a genetic basis for the susceptibility of mosquitoes to parasites, but until the advent of powerful molecular biological tools and protocols, it was difficult to assess the interactions of pathogens with their host tissues within the mosquito at a molecular level. Moreover, it has been only recently that the molecular mechanisms responsible for pathogen destruction, such as melanotic encapsulation and immune peptide production, have been investigated. The molecular characterization of genes that influence vector competence is becoming routine, and with the development of the Sindbis virus transducing system, potential antipathogen genes now can be introduced into the mosquito and their effect on parasite development can be assessed in vivo. With the recent successes in the field of mosquito germ line transformation, it seems likely that the generation of a pathogen-resistant mosquito population from a susceptible population soon will become a reality.

FIG. 1

Migratory routes and developmental sites within the mosquito for viruses, malaria parasites, and filarial worms. Developmental sites within the mosquito are defined by the letters A to H, and migratory routes are represented by lines. Following ingestion in a blood meal (A), all the pathogens enter the midgut (B). Viruses (represented by ——) then enter the midgut epithelial cells (D), replicate, exit the cells, and travel through the hemolymph-filled hemocoel (E) to the salivary glands (H), where they again replicate and reside until injected into a vertebrate host. Malaria parasites (designated by · · · · · ·) remain in the midgut for several hours, where they undergo syngamy and ookinete formation before they migrate through the formed peritrophic matrix (C). They then pass through the midgut epithelium and lodge between this epithelial layer and the basal membrane of the midgut, where they undergo sporogony in oocysts to produce sporozoites. At maturity, the sporozoites rupture the oocyst, travel through the hemocoel, and penetrate the salivary glands, where they reside until injected into a host. When the filarial worms responsible for human disease (––––) and dog heartworm (–·–·–) are ingested in a blood meal, the former penetrate the midgut epithelium and migrate to their developmental site in the thoracic musculature (G) and the latter travel through the midgut lumen, migrate up the lumen of the Malpighian tubules, and enter the distal cells of the tubules, where they develop intracellularly (F). Following a period of development, infective third-stage filarial larvae break out of the thoracic musculature or the Malpighian tubules and enter the hemocoel, where they migrate thorough the open circulatory system to the head region. Unlike malaria sporozoites and viruses, which are directly injected into a host when a blood meal is ingested, infective-stage filarial worms actively emerge from the head region of the mosquito and are deposited on the surface of the vertebrate skin, which they enter through the wound made by the mosquito bite.

FIG. 2

Mosquito immune responses to pathogens include melanotic encapsulation, phagocytosis, and production of antibacterial compounds and immune peptides. The hemocyte is a multifaceted cell that is probably involved in pathogen recognition, cell signaling, production of enzymes and immune system-associated molecules (e.g., transferrin), and phagocytosis. Invasion by bacteria results in phagocytosis by hemocytes and in the production of antibacterial compounds. If the pathogen is too large to be phagocytosed (e.g., filarial worms), mosquito hemocytes may recognize the pathogen as foreign and recruit other hemocytes to participate in the melanization response. Activated hemocytes also produce transferrin, which is a melanization-associated molecule, and a prophenoloxidase (ProPO), which is activated by a serine protease to become phenoloxidase (PO). After a phenoloxidase-catalyzed hydroxylation of tyrosine, other enzymes like dopa decarboxylase (DDC) and dopachrome conversion enzyme (DCE) catalyze other critical steps, ultimately resulting in melanin production. Also depicted is a proposed pathway for immune system peptide production. This pathway is probably induced when hemocytes recognize a foreign pathogen and relay a signal to the fat body, where signaling pathways, like Toll, IMD, and IRD, are activated by a serine protease. Following transcriptional activation, the fat body then produces defensin, cecropin, and proline-rich and glycine-rich peptides that have antimicrobial activity. Although the humoral immune response of mosquitoes does depend upon the fat body for immune system peptide production, other tissues, such as the midgut and salivary glands, do transcribe immune system peptides following activation by a pathogen (61).

FIG. 3

In the proposed melanotic encapsulation pathway of mosquitoes, a serine protease (SP) proteolytically cleaves an inactive prophenoloxidase (ProPO) to form an active phenoloxidase (PO). Tyrosine, the initial substrate, then is hydroxylated by the activated PO to form dopa, a key branchpoint substrate. Next, dopa is oxidized by PO to form dopaquinone, which then forms a dopachrome intermediate. A dopachrome conversion enzyme (DCE) converts this intermediate to 5,6-dihydroxyindole, which subsequently is oxidized by PO to form indole-5,6-quinone. This latter compound forms eumelanin or cross-links with proteins to eventually produce a melanized capsule. Dopa also can be decarboxylated by dopa decarboxylase (DDC) to form dopamine, which forms another branch point. A PO-based oxidation of dopamine produces dopaminequinone, which can cross-link with proteins or form melanin via the indole pathway. Dopamine also may be acetylated by N-acetyltransferase (NAT) to form N-acetyldopamine (NADA). PO oxidation of NADA then produces NADA-quinone, which cross-links with other proteins to form a melanotic capsule. Solid arrows designate likely major pathways, and dashed arrows denote probable minor pathways. Adapted from reference .

FIG. 4

Eye color phenotypes in Hermes-transformed adult A. aegypti. Transformation of the kh^w (white-eye) strain of Aedes aegypti with a Hermes transposon carrying a wild-type copy of the D. melanogaster cinnabar gene (encoding kynurenine hydroxylase) restores eye color. Counterclockwise from the top left: head of a wild-type mosquito showing deep purple eyes; head of a kh^w/kh^w mosquito showing white eyes; three heads of transformed mosquitoes from independent Hermes insertions showing different eye colors. The variability in the eye color among transformed lines presumably results from insertion site effects that modulate the expression of the transgene.