Effect of a Blood Meal on <i>Plasmodium</i> Oocyst Growth Using the Enema Injection Method

<b><i>Objective:</i></b> <i>Anopheles</i> mosquitoes transmit <i>Plasmodium</i> parasites through blood feeding. The oocyst stage in mosquitoes is crucial for <i>Plasmodium</i> transmission. Oocysts can form without a blood meal or the midgut passage and utilize nutrients from additional blood feeding. However, it remains unclear the impact of a blood meal during infection on oocysts. The present study evaluated how a blood meal during infection impacts oocyst growth to better understand oocyst development. <b><i>Methods:</i></b> We used a novel method for <i>Plasmodium</i> infection known as enema injection, which involves injecting <i>Plasmodium berghei</i> (ANKA strain) into the midgut lumen of <i>Anopheles stephensi</i> mosquitoes (STE2 strain) via the anus, traversing the midgut wall without a blood meal. We compared the size of oocysts in mosquitoes infected by enema injection alone, hemocoel injection alone, and each infection method combined with uninfected blood feeding, as well as those only with infected blood feeding. <b><i>Results:</i></b> By enema injection with <i>Plasmodium</i> ookinetes, oocysts formed solely in the mosquito's midgut. Oocysts from enema-injected mosquitoes were similar in size to those from hemocoel-injected mosquitoes. Oocysts from mosquitoes infected by enema injection combined with uninfected blood feeding were larger than oocysts from mosquitoes infected by enema injection alone. However, the size of oocysts from mosquitoes infected by hemocoel injection was not affected by the presence or absence of blood feeding. <b><i>Conclusion:</i></b> Enema injection with <i>Plasmodium</i> ookinetes is applicable to <i>Anopheles</i> mosquitoes. Using the enema injection method, we suggest that a blood meal during infection might facilitate oocyst growth within the midgut.