Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes.

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.

Developmental stages and molecular phylogeny of Hepatozoon fitzsimonsi (Dias 1953) (Adeleorina: Hepatozoidae) in tortoises Stigmochelys pardalis (Cryptodira: Testudinidae) and ticks of the genus Amblyomma (Acari: Ixodidae) from South Africa.

BACKGROUND: Hepatozoon fitzsimonsi (Dias, 1953) is a frequently found haemogregarine of southern African tortoises. At the time of this species' reassignment from the genus Haemogregarina to Hepatozoon, developmental stages such as sporocysts and sporozoites were observed in ticks associated with H. fitzsimonsi parasitised and non-parasitised tortoises. It was thus suggested that ticks may act as the potential vectors for this parasite. However, this earlier research was unable to confirm the identity of these sporogonic stages using molecular markers. In a separate study aimed at identifying tick species parasitising South African reptiles and molecularly screening these for the presence of Hepatozoon, that study identified H. fitzsimonsi in tortoise-associated ticks. Thus, the present study aimed to revisit the potential of ticks to act as vectors for H. fitzsimonsi in tortoises using a combined microscopy and molecular approach. METHODS: Specimens of Kinixys natalensis, Kinixys spekii, Kinixys zombensis and Stigmochelys pardalis were collected from Bonamanzi and Ndumo Game Reserve, South Africa. Upon capture, animals were examined for ticks, and these were collected along with blood and other tissues. Adult ticks were dissected and visceral impression slides were prepared along with thin blood and tissue smears on clean microscope slides. Smears and impression slides were stained with Giemsa, screened and micrographs of parasites were captured. Two primer sets were employed to target fragments of the 18S rRNA gene of parasites found in both tortoises and ticks and the resulting sequences were then compared with other known H. fitzsimonsi and haemogregarine sequences from the GenBank database. RESULTS: Peripheral blood gamont and liver merogonic stages were observed in S. pardalis, while the sporogonic stages were observed in the haemocoel of Amblyomma ticks. Gamont and sporocyst stages compared morphologically with previous descriptions of H. fitzsimonsi, identifying them as this species. Phylogenetic analysis revealed that the blood and tick sequences obtained in this study clustered in a monophyletic clade comprising known H. fitzsimonsi. CONCLUSIONS: The present study provides further support for ticks acting as the vectors of H. fitzsimonsi by molecularly identifying and linking observed developmental stages in tortoises (S. pardalis) with those in the invertebrate host (Amblyomma spp.).

Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes.

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.

Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes.

It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34-501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171-2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p<0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

Observation of Nitroquine Effect on Sporogonic Development of Plasmodium yoelii / 第三军医大学学报

The effects of nitroquine on the sporogonic development of Plasmo-dium yoelii were observed under electron microscopy. The female mosquitoes were fed directly with 10% sucrose solution containing 0.1%Nitroquine.It was found that the oocysts were smaller and markedly degenerated as compared with that of the control. The surface of the oocysts was rough and uneven. Under a transmission electron microscope, the cytoplasm of the affected oocysts contained vacuoles; the membane of mitochondria and uncleus was damaged; and the number of residual bodies increased.No sporoblast formation was seen in most of the affectes oocysts. The nuclear membrane of the degenerated sporozoites was thickened and the density of nuclear matrix decreased markedly as compared with that of the control. These results indicate that the nucleus and the membrane are mainly affected during the sporogonic development of P. yoelii by nitroquine.