RESUMO
OBJECTIVE: Our previous transcriptome analysis of Anopheles dirus revealed upregulation of the An. dirus yellow-g gene upon ingestion of Plasmodium vivax-infected blood. This gene belongs to the yellow gene family, but its role regarding P. vivax infection is not known and remains to be validated. The aim of this study was to investigate the role of the An. dirus yellow-g gene in P. vivax infection. METHODS: The qRT-PCR was used to detect the expression of the yellow-g gene in many organs of both male and female mosquitos. The yellow-g gene silencing was performed by dsRNA membrane feeding to An. dirus. These mosquitoes were later challenged by P. vivax-infected blood. The oocyst numbers were determined. RESULTS: The yellow-g transcript was detected in several organs of both male and female An. dirus mosquitoes. Successful knockdown of yellow-g was achieved and resulted in reduced P. vivax infection in the mosquitoes. The decrease in yellow-g expression had no effect on the life span of the mosquitoes. CONCLUSIONS: These results support the yellow-g gene as having an important function in Plasmodium development in Anopheles mosquitoes.
Assuntos
Anopheles/genética , Malária Vivax/genética , Plasmodium vivax/genética , Animais , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes de Insetos , Oocistos/genética , Proteínas de ProtozoáriosRESUMO
Malaria transmission-blocking compounds have been studied to block the transmission of malaria parasites, especially the drug-resistant Plasmodium. Carboxypeptidase B (CPB) in the midgut of Anopheline mosquitoes has been demonstrated to be essential for the sexual development of Plasmodium in the mosquito. Thus, the CPB is a potential target for blocking compounds. The aim of this research was to screen compounds from the National Cancer Institute (NCI) diversity dataset and U.S. Food and Drug Administration (FDA)-approved drugs that could reduce the Anopheles CPB activity. The cDNA fragment of cpb gene from An. minimus (cpbAmi) was amplified and sequenced. The three-dimensional structure of CPB was predicted from the deduced amino acid sequence. The virtual screening of the compounds from NCI diversity set IV and FDA-approved drugs was performed against CPBAmi. The inhibition activity against CPBAmi of the top-scoring molecules was characterized in vitro. Three compounds-NSC-1014, NSC-332670, and aminopterin with IC50 at 0.99 mM, 1.55 mM, and 0.062 mM, respectively-were found to significantly reduce the CPBAmi activity.
Assuntos
Anopheles/enzimologia , Carboxipeptidase B/antagonistas & inibidores , Malária/prevenção & controle , Sequência de Aminoácidos , Animais , Anopheles/genética , Carboxipeptidase B/genética , Clonagem Molecular , Malária/transmissão , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Malaria is transmitted by Plasmodium parasites through the bite of female Anopheles mosquitoes. One of the most important mosquito vectors in the Greater Mekong Subregion is Anopheles dirus. This study reports RNA sequencing (RNA-Seq) transcriptome analysis of An. dirus at 18 hours and 7 days after a P. vivax-infected blood meal, which represent infection at the ookinete and oocyst parasite developmental stages, respectively. Following infection, 582 An. dirus transcripts were modulated. The 2,408 P. vivax transcripts could be classified into ookinete-specific, two-stage, and oocyst-specific groups. Results were validated by quantitative reverse transcription polymerase chain reaction. Gene ontology analysis of the vector and parasite revealed several biological pathways for both, providing a better understanding of Anopheles-Plasmodium interactions at the ookinete and oocyst stages.