Effects of Plasmodium yoelii infection on the transcript abundance of prophenoloxidase from Anopheles dirus and intracellular calcium in the oocysts

Objective To explore the melanotic mechanisms in oocysts of Plasmodium yoelii in a Plasmodium refractory strain of Anopheles dirus by observing the effects of Plasmodium yoelii infection on the transcript abundance of prophenoloxidase(PPO) gene from Anopheles dirus and the changes of intracellular Ca 2+ in the oocyst during the melanization of oocysts of Plasmodium yoelii . Methods Total RNA of mosquitoes was extracted at 1, 2, 3, and 5 d before and after infection with Plasmodium yoelii by blood feeding. All samples were analyzed by RT PCR, and the PCR products were analyzed by agarose gel electrophoresis. The data were analyzed statistically. The midguts were dissected out from Plasmodium yoelii females at 5, 7, 11, and 15 d after infection with Plasmodium yoelii . The distributions and changes of Ca 2+ in the oocyst of Plasmodium yoelii at different time were observed by confocal laser scanning microscopy (CLSM) with Ca 2+ sensitive dye Fluo 3/Am and Pluronic. Results The products of PPO gene by RT PCR after infection were significantly more than those before infection with Plasmodium yoelii , especially at 1 and 2 d ( P 0.01). The mean content of Ca 2+ in normal oocysts was (137 15±7 02) nmol/L, but (18 44±1 75) nmol/L in melanotic oocysts with Ca 2+ sedimentation in the wall of the oocyst. Conclusion The transcript abundance of PPO gene from Anopheles dirus after infection increases obviously than that before infection. The content of the intracellular Ca 2+ in melanotic oocysts decreases significantly than that in the normal oocysts. The intracellular Ca 2+ in oocysts is excreted before melanizaiton.