| IntroductionThe malaria parasite is cyclically transmitted between the vector mosquito and the vertebrate animal. The gametocyte is the only stage through which the host and the vector are connected with each other. Exflagellation induction in vitro can be triggered by the combination of a decrease in temperature and an increase in pH from the temperature in the blood of the vertebrate animals to about 8.0. However, alkalization does not occur in the midgut of mosquito, where pH never exceeds 7.8 after blood feeding. Therefore, some factors other than pH must exist in the midgut to induce exflagellation. Characterization and identification of mosquito-derived factor as an inducer of exflagellation in vivo has long been studied. The gametocyte activating factor (GAP) derived from pupae and heads of Anopheles stephemi has been purified and identified as xanthurenci acid (XA). XA is a by-product in tryptophan metabolism. In insect, a branch of this metabolic pathway produces ommochromes, which are responsible for the color of wings and eyes.By using in vitro exflagellation assay of Plasmodium berghei and some other analytic methods we carried out the following studies. To clarify the effect of GAP in the salivary glands of mosquitoes on the malaria transmission, we first examined GAP activity in the extract and homogenate supernatant of An. stephensi; To make clear the role that Anopheles mosquitoes played in transmission of mammalian malaria, we compared GAP activity in the salivary glands of six mosquito species, An. stephensi, An.omorii, An. sinensis, Aedes aegypti, Ae. albopictus and Culex pipiens pallens and compared GAP activity between pre-fed and blood-fed female mosquitoes in five species among them; To further elucidate the fate of GAF after exflagellation ends in the rnidgut, the activity kinetics of GAF in the salivary glands of An stephensi, and the correlation between GAF activity in the salivary gland and oviposition in the female mosquitoes, we analyzed the varying trend of GAF activity and protein contents in the salivary glands and ovaries of An. stephensi during development.Materials and MethodsAnimals1. Parasites: a rodent malaria, P. berghei, was maintained in Department of Medical Zoology, Jichi Medical School, Japan by cyclical passage through BALB/c mice and An. stephensi mosquitoes. The freezed P. berghei parasite was intraperitoneally infected to BALB/c mice, and the tail blood of infected mice at day 10~12 after infection was used for in vitro exflagellation assay.2. Vector mosquitoes: An. stephensi, An. omorii, Ae. aegypti, Ae. albopictus and Cx. p. pallens were maintained in Department of Medical Zoology, Jichi Medical School. An. sinensis was collected around Jichi Medical School in Minamikawachi, Tochigi, Japan.3. Mice: Female BALB/c and ICR mice were purchased from SLC, Inc. (Shizuoka, Japan).Reagents and equipmentAll the reagents and equipment were provided by Department of Medical Zoology, Jichi Medical School, Japan.Experimental methods1. Blood feeding of mosquitoesBefore blood feeding, the female mosquitoes were starved for 6h. The mosquitoes fed on blood of an ICR female for 30~60min. After blood feeding, fully engorged mosquitoes were used for experiment. The salivary glands andother examined organs were collected from mosquitoes in the different phasic points according to the different experiment.2. Collection of salivary glands and preparation of extractsAfter being cold-anaesthetized, the different species of mosquitoes were dissected under microscope. The intact salivary glands were briefly rinsed, transferred into an Eppendorf tube containing 100ul of the medium mentioned above, rinsed again by centrifugation, the supernatant was removed. Pellet was subjected to three freeze-thaw cycles at -20℃and room temperature. Finally tubes were stored at -20℃ until use.3. Supernatant preparation of the salivary gland, head and ovary homogenates of An. stephensiMosquitoes were dissected and the heads, salivary glands a... |
PDF Full Text Request