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Anopheles funestus is the most important malaria vector in Western and central Africa. It is also becoming the main vector of malaria in the East African region. Adult An. funestus are blood feeders and the ability to adapt to such a complex feeding strategy is believed to be linked to the expression and activity of serine proteases involved in the digestive tract during blood meal acquisition. In this study, we obtained two full-length cDNA sequences (ag4RNAN and ag4RNAN2) encoding one unique serine protease-like protein containing a signal peptide at the N-terminal region. They contain two domain architectures: a papain-like cysteine protease catalytic domain and a Kazal-type peptide inhibitor domain. A phylogenetic tree based on the deduced amino acid sequences of the ag4RNAN and ag4RNAN2, similar to the other two model Anopheles species (An. gambiae and An. quadriannulatus), revealed that they are more closely related to An. gambiae (agap) than An. quadriannulatus (alpr) serine proteases. A low level of expression of these two genes has been observed in salivary glands of the eight-day-old adult females. In contrast, a very high expression was noted in the whole body. Further immunolocalisation analyses showed that the two ag4RNAN genes were highly expressed in the midgut in all developmental stages. The ag4RNAN2 gene was not expressed in the developmental stages tested; this result differs from the expression of the agap gene which was highly expressed in the midgut of all life stages. According to these results, we suggest that the presence of the agap protease gene might be a prerequisite for the expression of the two ag4RNAN genes in the midgut.

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