| Malaria control will be confronted with great challenge because of the rapid spread of parasites with multiple anti-malaria drug resistance, the emergence of insecticide-resistant anopheline mosquitoes and the lack of effective vaccines fighting against malaria, but chemicals is the presently most important measures to prevent and cure malaria. Although knowledge of the mosquito immune response has recently improved, less is known about the impact of antimalarial drugs on mosquito immunity. In my previous study, we found that nitroquine│2,4 diamino-6-[(3,4- dichloro benzyl) nitros-amid] quinazoline│acetate (NA,CI-679) an antimalarial drug, is not only able to kill the erythrocytic and exoerythrocytic stages of many malaria parasites, but also can induce the melanization of Plasmodium by Anopheles stephensi., but the mechanism was unclear. In this study, we would elucidate the mechanism of melanization induced by nitroquine.The exploration of the recognition in the melanization is very important for the understanding of the mechanism of melanization. The present study showed that one recognition receptor, TEP1, could significantly influence the melanotic encapsulation by the Anopheles gambiae. Hence, we supposed that TEP1 could play a key recognition reaction in the melanization induced by antimalarial-drug.1,4 TEP cDNAs were successfully cloned from Anopheles stephensi and designated AsTEP1-4 based on the blast results.2,The transcript variation of AsTEP1-4 followed the Plasmodium infection and the antimalarial-durg induction were investigated using Real-time PCR, and the results showed that only the expression of TEP1 was rapidly upregulated at 24 h after infection. This was followed by a temporary depression in transcript abundance and then by a second peak of 2.0-fold induction at 4 and 5 day, afterward, the expression of AsTEP1 decreased. But the antimalarial-drug can induce upregulation of the AsTEP1, at the 3rd day medication of nitroquine(at 9d after infection), the transcript abundance of the AsTEP1 in the drug treated group is significantly higher than in the untreated group, and most oocysts were melanized at the same time. These data implied that the upregulation of AsTEP1 would be related to the melanotic encapsulation induced by the antimalarial-drug nitroquine.3,To explore the mechanism of AsTEP1 in the melanization induced by nitroquine, we observed the recognition reaction of AsTEP1 to the Plasmodium in the An.stephensi through the utilization of the laser confocal microscopy. Light microscopy showed that the melanized oocysts were smaller and markedly degenerated as compared with that of the control. The surface of the oocysts was rough and uneven, and was coated by some melanin. Laser confocal microscopy revealed that at the 3rd day medication, the binding of AsTEP1 with the oocyst in the drug treated group is significantly stronger than in the untreated group, and it is interesting that the TEP1 signal appeared on the lodgement of melanin, the increase of melanin, the TEP1 signal would be gradually enhancement. Based on these results, we supposed that TEP1 would directly involve in the tethering of the PPO melanization complex on the parasite surface.4,To understand the relationship between the TEP1 and the melanotic encapsulation induced by the antimalarial-drug, we observed the activity of phenoloxidase (PO) enzyme and the melanization induced by antimalarial-drug in An.stephensi that ingested anti-AsTEP1 antibody.The results showed that ingestion of this antibody would decrease the activity of PO enzyme and the melanization rate of antibody treated mosquito. Hence, we supposed that the recognition of TEP1 to the Plasmodium would be correlated with the melanotic encapsulation induced by antimalarial-drug nitroquine.In a word, in my study, we successfully cloned AsTEP1-4, and AsTEP1 was related with Plasmodium infection and the induction of nitroquine. Futher analysis of the mechanism of AsTEP1 in the melanization showed that the recognition of AsTEP1 to the Plasmodium would initiate the melanotic encapsulation. Our results would be helpful to develop effective measures of malaria control, to reasonably apply the existent drug and to supply another meaningful pathway for antimalarial drug design.
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