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A simple device using folded Parafilm-M as an artificial blood feeder was designed for studying two important dengue vector mosquitoes, Aedes aegypti and Aedes albopictus. The efficiency of the artificial blood feeder was investigated by comparing the numbers of engorged mosquitoes that fed on the artificial blood feeder versus mice as a live blood source. Significantly more engorged females Aedes aegypti fed on the artificial blood feeder than on mice. In addition, the artificial feeder could serve as a useful apparatus for oral infection via artificial blood meals, and for saliva collection in mosquitoes. Our method enabled us to collect saliva from multiple mosquitoes at once, providing sufficient infected saliva for determination of the virus titer by plaque assay analysis. Our artificial feeder has the advantage that it is simple, inexpensive, and efficient.

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Duck Tembusu virus (DTMUV) is an important flavivirus that can be transmitted to poultry via Aedes albopictus bites. Furthermore, humans residing in the DTMUV epidemic area display activated antiviral immune responses to local DTMUV isolates during the pathogenic invasion, thereby raising the primary concern that this flavivirus may be transmitted to humans via mosquito bites. Therefore, we identified the gene AALF004421, which is a homolog of the 34-kDa salivary protein (34 kDa) of Ae. albopictus and studied the salivary protein-mediated enhancement of DTMUV infection in Ae. albopictus salivary glands. We observed that double-stranded RNA-mediated silencing of the 34 kDa in mosquito salivary glands demonstrated that the silenced 34 kDa impaired DTMUV infectivity, similar to inhibition through serine protease. This impairment occurred as a consequence of triggering the innate immune response function of a macroglobulin complement-related factor (MCR). 34-kDa in the salivary gland which had similar activity as a serine protease, results in the abrogation of antimicrobial peptides production and strong enhance DTMUV replication and transmission. Although the function of the 34 kDa in Ae. albopictus is currently unknown; in the present study, we showed that it may have a major role in DTMUV infection in mosquito salivary glands through the suppression of the antiviral immune response in the earliest stages of infection. This finding provides the first identification of a prominently expressed 34 kDa protein in Ae. albopictus saliva that could serve as a target for controlling DTMUV replication in mosquito vectors.