Biology of Malaria Vectors and Parasite-Vector Relationships

1. Biology of Malaria Vectors and Parasite-Vector Relationships Dawn Wesson Tulane Department of Tropical Medicine

2. Malaria Vector Biology • Anopheline Life Cycle – habitat preferences, types of habitat, unpolluted water • Effect of human activities on habitat creation – agriculture, irrigation, etc. • Biology of Malaria Vectors – General and Specific

3. Family Culicidae > 3500 species 3 subfamilies: Anophelinae - Anopheles, Bironella and Chagasia, ~ 500 species Toxorhynchitinae - Toxorhynchites, 70+ species (allnon-bloodfeeding) Culicinae - Aedes, Culex, Haemagogus, Mansonia, and all other genera, > 3000 species Anophelinae Toxorhynchitinae time Culicinae

4. Anopheles mosquito life cycle

5. eggs

6. Anopheline Culicine Adult

7. Genus Anopheles 6 subgenera: Cellia- >230 species, most important Old World malaria vectors (Africa and Asia) Anopheles - >180 sp., were the most important malaria vectors in Europe and N. America Nyssorhynchus ->40 sp., most important New World malaria vectors Kertezia - >10 sp., NW, bromeliads Lophopodomyia – 6 sp., NW tropics Stethomyia – 5 sp., NW tropics

8. Anopheles Habitat Preferences Effects of human activities Major malaria vectors tend to be colonizing species in temporary habitats free of established predators They have evolved with humans to take advantage of these environments

9. LARVAL HABITAT - An. albimanus in Cuba WHO/TDR/Service, 1992

10. LARVAL HABITAT - An. bellator in Brazil from bromeliades WHO/TDR/Service, 1992

11. LARVAL HABITAT - An. pseudopunctipennis in Mexico WHO/TDR/Service, 1992

12. LARVAL HABITAT - An. stephensi from water tanks on rooftops in Dubai WHO/TDR/Service, 1992

13. LARVAL HABITAT - Irrigation ditches provide Anopheles breeding sites in the Gambia WHO/TDR/Lindsay, 1991 WHO/TDR/Olliaro, 1988 LARVAL HABITAT - Standing water created by road building in Benin

14. LARVAL HABITAT - Rice fields and irrigated areas provide Anopheles breeding sites in Viet Nam and the Gambia WHO/TDR/Lindsay, 1991 WHO/TDR/Martel, 1994

15. Water storage pots, breeding site of An. gambiae and other mosquitoes in Nigeria WHO/TDR/Ragavoodoo, 1992 Roof water breeding site of An. arabiensis in Mauritius WHO/TDR/Service, 1992

16. Biology of Anopheles gambiae Anopheles gambiae WHO/TDR/HOLT Studios, 1992

17. Anopheles gambiae Major malaria vector in sub-Saharan Africa Typical anopheline life cycle, but extreme preference for living around and feeding on humans Preferred oviposition sites – small temporary pools in full sunlight Seasonal abundance correlates with rainfall

18. Anopheles gambiae – life cycle Other sites – irrigated areas (rice fields); drying streams in dry season; habitats created by humans Eggs laid on water or damp soil; hatch 48 hr. – 2 weeks Larvae can crawl across damp soil from drying pool to another with water Larval development - <week with sufficient temperature and food

19. Anopheles gambiae – life cycle Larvae are filter feeders on surface film – algae and bacteria Pupation in full sunlight – can be induced in laboratory with light Pupal development in 24 hr. – 3 days; temperature dependent Adult emergence at night Both sexes need 24 hr. to reach sexual maturity – male terminalia (genitalia) rotate 180.

20. Mosquito Emerging from Pupal Exuvia

21. Anopheles gambiae – adult behavior Male mosquito swarming behavior – females fly into swarm to mate (not well documented in wild An. gambiae but does occur in lab colonies). Male activity increases at sundown. Changes in antennae (plumes folded up during day – open to detect female flight sound; Johnston's organ) Males attracted to females and mate in flight – females probably mate only once (?) – store sperm in spermathecae

22. Anopheles gambiae – host seeking Mated An. gambiae females seek blood at night (after sundown) - ~90% of bloodmeals taken from sleeping human hosts and they usually rest on the inside walls of the house to digest the meal Egg development takes about 48 hrs during warm season – longer in cooler weather Oviposition occurs at night – usually the 2nd night after a bloodmeal

23. Anopheles gambiae – host seeking The female then searches for another bloodmeal - during warm season, a female is capable of ovipositing every other night This behavior has implications for the timing of host seeking by An. gambiae females – early evening blood-seeking females are probably feeding for the first time (they have not laid eggs yet – nulliparous), while older (parous) females tend to seek blood later at night (they have to oviposit first)

24. Anopheles gambiae – host seeking Extrinsic incubation period (minimum) of Plasmodium falciparum in the mosquito is 8-10 days – so under ideal conditions, the female would take 5-6 bloodmeals in the process of acquiring parasites and living long enough to transmit them (about 2 weeks) In real life…environmental factors will usually affect time line – temperature, rainfall, wind will interfere with the ability to oviposit and blood-feed at will. Most field collected An. gambiae females with P. falciparum sporozoites in their salivary glands have taken 3-4 blood meals

25. Physiology of Gonotrophic Cycle • If, after locating host and ingesting blood, the blood meal is large, distention-induced host seeking inhibition is triggered • This tapers off as the blood is assimilated and excreted • Eggs mature producing oocyte-induced host-seeking inhibition, which gradually develops and then fades • Mature eggs induce preovipostion behavior, leading to oviposition

26. Other factors influencing host seeking… Host defensive behavior Mosquito age – older mosquitoes more likely to seek blood even when gravid Larval nutrition – if poor, blood may go to support adult metabolism Mating status – unmated less likely to host seek Nutritional status of male with which female mated – poor nutrition in male results in more host seeking Mosquito species – some, such as An. gambiae, host seek every 24 hrs. until replete (even if gravid!) All of these factors potentially contribute to multiple bloodmeals per gonotrophic cycle, increasing the potential for malaria transmission

27. Malaria Parasite-Vector Relationships • Malaria Transmission Cycle • Parasite Infection Specificity • Mosquito Immune Defenses

28. midgut infected with oocysts salivary glands gametocytes macrogametocyte microgametocyte salivary glands zygote sporozoites oocyst with sporozoites oocyst sporozoites ookinete Plasmodium Development in Anopheles cross section of oocyst

29. Alimentary Canal

30. Alimentary Canal • Within the alimentary canal, the malaria parasite encounters various structural and physiological/ biochemical characteristics that can influence its survival • The noncellular (chitinous) peritrophic membrane (PM) can be an effective physical barrier, preventing midgut infection • Vector specificity for malaria pathogens may be linked to the rate of PM formation versus the rate of ookinete production in bloodmeal • Adult mosquitoes secrete PM1, while larvae secret PM2 • PM1 secretion is triggered by dramatic extension of the midgut epithelium during ingestion of a bloodmeal

31. Alimentary Canal • After ingestion, the gametocytes go through a complete sexual cycle in the midgut lumen and develop into motile ookintes (~16-24 hrs) • Invasion of gut epithelilal cells occurs about 30 hrs after bloodmeal • In P. gallinaceum / Ae. aegypti , Plasmodium secretes a chitinase in order to penetrate the PM (inhibiting chitinase blocks transmission). Trypsin, secreted by the mosquito, activates parasite chitinase. • This system may vary in different mosquitoes – PM formation in An. stephensi variably detected

32. Bloodmeal processing - steps • Removal of excess water from the bloodmeal • Breakdown of vertebrate blood cells (hemolysis) • Hydrolytic degradation of macromolecules in the bloodmeal (digestion) • Absorption of small molecules into the midgut epithelial cells and subsequently into the hemocoel

33. Hemolysis of Bloodmeal • Hemolysis breaks down cells to release proteins and other nutrients, making them accessible to the digestive enzymes • Hemolysis may be achieved mechanically (cibarial armature) or biochemically (hemolytic factors including small peptides and free fatty acids)

35. Absorption of Bloodmeal Nutrients • Differences between insects that show continuous digestion (eg, tsetse flies -- absorption occurs through specialized cells) vs those that show batch digestion (eg, mosquitoes -- same cells that secrete enzymes also carry out absorption) • Processes range from simple diffusion (eg, absorption of sugar into the hemolymph) to active transport (amino acids); little is known about absorption of other molecules like lipids, vitamins, and minerals

36. Peritrophic Matrix (PM) • The peritrophic matrix is a layer of acellular material separating ingested food from epithelial cells • “peritrophic” comes from the Greek word peri for around; trophic is the Greek word for food. The PM surrounds the food bolus. • Peritrophic membrane was termed >100 years ago but membrane implies lipid bilayer. The PM is not -- it is a sheath of cheesy material of amorphous appearance. The word matrix is more suitable!

37. Other important points -- PM • The signal that activates PM secretion is the physical distention of the midgut epithelium; eg, ingestion of partial bm does not trigger PM formation • Mosquitoes, blackflies, and sandflies secrete different type of PM during larval life • PM is permeable to digestive enzymes • Possible barrier to pathogen infection

38. Structure of salivary glands • Structure varies among insect phyla • In mosquitoes, salivary glands of both sexes are paired organs located in the thorax, and each gland consists of 3 lobes connected to a main salivary gland duct (male sg’s small) • Female sg’s have two identical lateral lobes and one shorter medium lobe. Lateral lobes can be divided according to proximal and distal regions (different regions secrete different proteins)

39. Function of the salivary glands • Saliva contains enzymes that digest sugars • Salivary gland secretions play a role in the maintenance of feeding mouthparts - saliva acts as a lubricant • In ticks, water in ingested blood is cycled back through the sg’s where it is returned to the host

40. Salivary Glands and Bloodfeeding • Salivary glands produce a saliva that facilitates rapid and efficient feeding (hemagglutinin, anticoagulant, antiplatelet activity, vasodilators) • Parasites can increase the probability of their transmission by modifying arthropod salivary activities • Malaria sporozoites infect the female-specific salivary gland lobes (distal-lateral and medial)

41. Salivary Glands and Bloodfeeding -2 • Parasite invasion causes cellular damage in the glands – 4-5x reduction in apyrase activity • The salivary apyrases of blood-feeding arthropods are nucleotide hydrolysing enzymes and have been implicated in the inhibition of host platelet aggregation through the hydrolysis of extracellular ADP. • Sporozoite-infected mosquitoes take longer to probe – more sporozoites released • Also, more interrupted feedings – bite more frequently before achieving successful bloodmeal

42. Immune responses of vectors • Arthropod immune responses are not like vertebrate antigen-antibody reactions but the internal defense mechanisms are still specific and effective in destroying pathogens and parasites. • Much of what we know comes from immune studies of lepidopteran larvae.

43. Cuticular and gut barriers • The arthropods possess a rigid cuticle that functions as a barrier to potential pathogens. Microorganisms do not penetrate the exoskeleton unless there is a wound. • Many potential pathogens are ingested. Some are passed on through the feces or through regurgitation. Some are walled off by the peritrophic matrix (barrier?).

44. Possible outcomes following exposure of an arthropod to a parasite • susceptible arthropod: the parasite receives appropriate stimuli from the biochemical environment and develops successfully • resistant arthropod: some or all of the parasites are recognized as foreign by the cellular/humoral components in the hemolymph, and the arthropod immune response sequesters and destroys parasite • refractory arthropod: the parasites do not elicit an immune response but they fail to develop due to physiological or biochemical incompatability

45. Cellular immunity in insects • Phagocytosis. In mosquitoes, phagocytosis activity is a function of the numbers of hemocytes present • Encapsulation. The main defense mechanism of insects against invaders too large to be phagocytosed is encapsulation. Phenol oxidase enzymes are involved in melanotic encapsulation of parasites (worms and malaria parasites)

46. Summary • Anopheles gambiae is well adapted to take advantage of temporary aquatic habitat associated with human activities (farming, construction, etc.) • Behaviors such as preferential feeding on humans and resting in homes keep it closely associated with us. • The association between Anopheles mosquito and Plasmodium parasite is controlled by a series of physical, physiological and biochemical interactions, which may lead to a successful infection followed by transmission to a new host.

47. Additional Reading for More Detail: Biology of Anopheles mosquitoes – general Medical Entomology for Students, 4th Edition – pp. 33-51 Biology of Anopheles gambiae mosquitoes Biology of Disease Vectors, 1st Edition – pp. 75-77 Host seeking behavior in mosquitoes – general Biology of Disease Vectors, 2nd Edition (BODV) – pp. 277-287 Midgut structure and Peritrophic Matrix BODV – pp. 289-310 Bloodmeal Processing, Egg Development and Osmotic Regulation BODV – pp. 329-362 Immune Response in Vectors BODV – pp. 363-376 Salivary Glands and Saliva in Bloodfeeding Insects BODV – pp. 377-386