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Simple Summary: Even after decades of control interventions, mosquito-borne diseases still pose a huge threat to humans. Understanding gene functions is important for discovering new targets for mosquito and mosquito-borne disease control. One of the well-established and widely used methods for understanding gene function is RNA-interference (RNAi). The use of RNAi is, however, restricted mostly to adult mosquitoes. A few studies have shown its applicability in mosquito larvae, and just one in pupae. The current double-stranded RNA (dsRNA) delivery methods for RNAi are labor-intensive and require microinjections into mosquitoes (adults, larvae, or pupae). In this study, we present a simple, fast, and less labor-intensive technique for RNAi in the pupal stage by soaking pupae in water containing dsRNA. This method will be useful in studying genes expressed in immature life stages of the mosquitoes and will hopefully open new avenues for identifying mosquito control targets in early life stages. RNA-interference (RNAi) is a standard technique for functional genomics in adult mosquitoes. However, RNAi in immature, aquatic mosquito stages has been challenging. Several studies have shown successful larval RNAi, usually in combination with a carrier molecule. Except for one study in malaria mosquito, Anopheles gambiae, none of the previous studies has explored RNAi in mosquito pupae. Even in the study that used RNAi in pupae, double stranded RNA (dsRNA) was introduced by microinjection. Here, we describe a successful method by soaking pupae in water containing dsRNA without any carrier or osmotic challenge. The knockdown persisted into adulthood. We expect that this simple procedure will be useful in the functional analysis of genes that highly express in pupae or newly emerged adults. [ABSTRACT FROM AUTHOR]

Simple Summary: Human malaria is transmitted by mosquitoes in the genus Anopheles—particularly species that prefer to feed on humans. The mosquito's sense of smell drives this preference; however, most studies have focused on species native to Sub-Saharan Africa. Malaria vectors in other parts of the world may use different odorants to choose hosts. We, therefore, focused on Anopheles stephensi, the south Asian malaria mosquito, in this study. Mosquitoes have different organs specialized for odor perception, such as the antennae; however, we focused on the mouthparts (primarily the maxillary palp and labella) in this study. We used the RNAseq technique to determine which odor receptors are present in the mouthparts and then focused on one of these receptors: Or8. Using a technique known as the Drosophila empty neuron system, we tested this receptor's ability to detect different odorants, particularly chemicals emitted by humans. This receptor in An. stephensi detected similar odors to a homologous receptor in an African species, Anopheles gambiae, with the exception of the chemical sulcatone. This chemical is an important attractant in other disease-transmitting mosquitoes and suggests that different mosquito species may be using odors differently to find hosts. Several mosquito species within the genus Anopheles are vectors for human malaria, and the spread of this disease is driven by the propensity of certain species to feed preferentially on humans. The study of olfaction in mosquitoes is important to understand dynamics of host-seeking and host-selection; however, the majority of these studies focus on Anopheles gambiae or An. coluzzii, both vectors of malaria in Sub-Saharan Africa. Other malaria vectors may recognize different chemical cues from potential hosts; therefore, in this study, we investigated An. stephensi, the south Asian malaria mosquito. We specifically focused on the mouthparts (primarily the maxillary palp and labella) that have been much less investigated compared to the antennae but are also important for host-seeking. To provide a broad view of chemoreceptor expression, RNAseq was used to examine the transcriptomes from the mouthparts of host-seeking females, blood-fed females, and males. Notably, AsOr8 had a high transcript abundance in all transcriptomes and was, therefore, cloned and expressed in the Drosophila empty neuron system. This permitted characterization with a panel of odorants that were selected, in part, for their presence in the human odor profile. The responsiveness of AsOr8 to odorants was highly similar to An. gambiae Or8 (AgOr8), except for sulcatone, which was detected by AsOr8 but not AgOr8. Subtle differences in the receptor sensitivity to specific odorants may provide clues to species- or strain-specific approaches to host-seeking and host selection. Further exploration of the profile of An. stephensi chemosensory proteins may yield a better understanding of how different malaria vectors navigate host-finding and host-choice. [ABSTRACT FROM AUTHOR]