引起疟疾的寄生虫必须在蚊媒中完成复杂的感染周期,这也涉及昆虫的先天免疫系统的攻击,尤其是在中肠感染的早期。然而,疟原虫孢子期后期的按蚊免疫,如卵囊,很少受到关注,因为它们被认为是免疫因子隐藏的,因为它们位于中肠基底层下方,并且具有复杂的细胞壁,该细胞壁包括源自基底层的外层,该外层赋予了其他外来结构的自身特性。这里,我们调查了冈比亚按蚊的伯氏疟原虫卵囊和子孢子是否容易受到基于黑色素化的免疫。黑化反应的负调节剂的沉默,CLIPA14,黑化患病率增加,而黑化卵囊的数量没有显着增加,同时共同沉默CLIPA14与CLIPA2,黑化的第二个负调节剂,导致黑化卵囊和黑化患病率显着增加。只有晚期卵囊被发现黑化,表明卵囊破裂是基于黑色素化的免疫攻击的先决条件,大概是由于它们墙壁的免疫逃避特征的丧失。我们还在卵囊和血囊中发现了黑化的子孢子,表明在不同成熟期的子孢子容易黑化。在CLIPA2/CLIPA14共同沉默的蚊子中沉默黑化促进因子TEP1和CLIPA28拯救卵囊黑化。有趣的是,沉默的CTL4,保护早期卵细胞黑化,对卵囊和子孢子没有影响,表明免疫对早期和晚期孢子期的不同调节。类似于以前关于Ookinete黑色素化阶段的研究,恶性疟原虫卵囊的黑化明显低于伯氏疟原虫。总之,我们的结果提供了确凿的证据,表明晚期孢子型疟疾寄生虫阶段容易黑化,我们揭示了促性腺激素和卵囊黑化的不同调节机制。
The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect\'s innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.