Malaria diagnosis:Giemsa-stained blood smear (traditional method)may be hard to detect low level infectionhard to identify which Plasmodium species occurs2. Immunofluorescence assay (IFA)antibody against specific malaria species, conjugated to fluorophoreobserve with fluorescence microscope (usually not available in hospital setting indeveloping countries where malaria is endemic)3. Molecular diagnostic tools to distinguish 4 malaria specieseg: PCRIngested pathogens, having already survived challenges from the vertebrate immune system,must then enter the mosquito midgut and overcome:The vertebrate immune system in ingested bloodDigestive enzymes of mosquitoHeme (i.e. oxidative stress) from the blood mealEndogenous microorganismsPeritrophic matrix of mosquitoMosquito immune responses68% of An. gambiae mosquitoes fed on blood containing P. falciparum never became infected(i.e. gametocytes in gut). Overall, 75% of infected mosquitoes never became infective (i.e.sporozoites in salivary glands).Mosquitoes have a highly effective innate immune system.Insects don’t have adaptive/acquired immunity – so no immunoglobulins.Insects have only innate immunity, the more evolutionarily ancient form.Insect innate immunity includes both cellular and humoral defenseresponses – a somewhat artificial distinction since many of thepathways and molecules are common to both systems.Cellular defenses:Hemocyte-mediatedPhagocytosis, encapsulationMosquitoes have three types of hemocytes:granulocytesoenocytoidsProhemocytesHumoral defenses:Toll, IMD, JAK-STAT, and PPO pathwaysPRRs kick off a signaling cascadeProduction of immune effectors:antimicrobial peptidesthioester-containing proteinsROS, NOSEntomological considerationsThe frequency of disease will depend on the Entomological Inoculation RateEntomological Inoculation Rate: number of infectious bites/dayEIR = Product of HBR & PIMHBR = human biting rate (# bites by vectors/person/day)PIM = proportion of mosquitoes that are infectious(e.g., for malaria, PIM = sporozoite rate)The HBR will be influenced by the behavior of vectors. Since most anopheles bite after dark(when people are inside and asleep) the tendency to follow an odor plume into houses isimportant.Endophilic: will enter houses to feed on hosts, a big problemExophilic: bites outdoors only, not as dangerous