Activity of Some Plant and Fungal Metabolites towards Aedes albopictus (Diptera, Culicidae).

Aedes albopictus (Skuse) is a widespread mosquito, a vector of important human arboviruses, including Chikungunya, Dengue and Zika. It is an extremely difficult species to control even for the onset of resistances to chemicals insecticides, therefore ecofriendly products are urgently needed. In this study, the activity of Amaryllidaceae alkaloids and some of their semisynthetic derivatives, of 2-methoxy-1,4-naphthoquinone and two analogues, of cyclopaldic acid and epi-epoformin on the survival and development of Ae. albopictus larvae was evaluated. First-instar larval exposure for 24 and 48 h to cyclopaldic acid, resulted in mortality mean per-centage of 82.4 and 96.9 respectively; 1,2-O,O-diacetyllycorine 48h post-treatment caused 84.7% mortality. Larval and pupal duration were proved to decrease significantly when larvae were exposed to cyclopaldic acid, 1,2-O,O-diacetyllycorine and N-methyllycorine iodide. The mean number of third-instar larvae surviving to 2-methyl-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone and 2-methoxy-1,4-naphthoquinone was significantly lower than the number of correspondent control larvae over the time. This study indicated that 1,2-O,O'-diacetyllycorine, N-methyllycorine iodide, cyclopaldic acid and 1,4-naphthoquinone structural derivatives have good potential for developing bioinsecticides for mosquito control programs. The obtained results are of general interest due to the global importance of the seri-ous human diseases such a vector is able to spread.

Interaction of RNA viruses of the natural virome with the African malaria vector, Anopheles coluzzii.

Mosquitoes are colonized by a little-studied natural virome. Like the bacterial microbiome, the virome also probably influences the biology and immunity of mosquito vector populations, but tractable experimental models are lacking. We recently discovered two novel viruses in the virome of wild Anopheles and in colonies of the malaria vector Anopheles coluzzii: Anopheles C virus and Anopheles cypovirus. Here, we describe biological interactions between these two viruses and An. coluzzii mosquitoes. Viral abundance varies reproducibly during mosquito development. DNA forms of these viruses were not detected, and thus viral persistence is likely based on vertical transmission of RNA genomes. At least Anopheles C virus is vertically transmitted by an intraembryonic route. Relative abundance of the two viruses is inversely correlated in individual mosquitoes. One possible mechanism for this could be interactions with host immunity, and functional genomic analysis indicated differential influence of at least the Toll and JAK/STAT immune signaling pathways upon the viruses. The nonrandom distributions and interactions with host immunity suggest that these and other members of the natural virome may constitute a source of unrecognized heterogeneity in mosquito vector populations.

Pure early zygotic genes in the Asian malaria mosquito Anopheles stephensi.

BACKGROUND: The Asian malaria mosquito, Anopheles stephensi, is a major urban malaria vector in the Middle East and on the Indian subcontinent. Early zygotic transcription, which marks the maternal-to-zygotic transition, has not been systematically studied in An. stephensi or any other Anopheles mosquitoes. Improved understanding of early embryonic gene expression in An. stephensi will facilitate genetic and evolutionary studies and help with the development of novel control strategies for this important disease vector. RESULTS: We obtained RNA-seq data in biological triplicates from four early An. stephensi embryonic time points. Using these data, we identified 70 and 153 pure early zygotic genes (pEZGs) under stringent and relaxed conditions, respectively. We show that these pEZGs are enriched in functional groups related to DNA-binding transcription regulators, cell cycle modulators, proteases, transport, and cellular metabolism. On average these pEZGs are shorter and have less introns than other An. stephensi genes. Some of the pEZGs may arise de novo while others have clear non-pEZG paralogs. There is no or very limited overlap between An. stephensi pEZGs and Drosophila melanogaster or Aedes aegypti pEZGs. Interestingly, the upstream region of An. stephensi pEZGs lack significant enrichment of a previously reported TAGteam/VBRGGTA motif found in the regulatory region of pEZGs in D. melanogaster and Ae. aegypti. However, a GT-rich motif was found in An. stephensi pEZGs instead. CONCLUSIONS: We have identified a number of pEZGs whose predicted functions and structures are consistent with their collective roles in the degradation of maternally deposited components, activation of the zygotic genome, cell division, and metabolism. The pEZGs appear to rapidly turn over within the Dipteran order and even within the Culicidae family. These pEZGs, and the shared regulatory motif, could provide the promoter or regulatory sequences to drive gene expression in the syncytial or early cellular blastoderm, a period when the developing embryo is accessible to genetic manipulation. In addition, these molecular resources may be used to achieve sex separation of mosquitoes for sterile insect technique.

Functional analysis of the promoter of an early zygotic gene KLC2 in Aedes aegypti.

BACKGROUND: Aedes aegypti is an important mosquito vector that transmits arboviruses that cause devastating diseases including Zika, dengue fever, yellow fever and chikungunya. Improved understanding of gene regulation in the early development of Ae. aegypti will facilitate genetic studies and help the development of novel control strategies of this important disease vector. RESULTS: In this study, we demonstrated through transgenic assays that the promoter of an endogenous early zygotic gene KLC2 could drive gene expression in the syncytial blastoderm and early cellular blastoderm, which is a stage that the developing germline and the rest of embryo are accessible to genetic manipulation. An unexpected expression of the reporter gene in transgenic male testes was also observed. Further analysis confirmed the expression of the endogenous KLC2 in the testes, which was not detected in the previous RNA sequencing data. CONCLUSIONS: Our finding provided a new promoter element that can be used in future genetic studies and applications in Ae. aegypti. Moreover, our transgenic reporter assays showed that cautions are needed when interpreting RNA sequencing data as transient or tissue-specific transcription may go undetected by RNAseq.

Assessment of Aedes albopictus reference genes for quantitative PCR at different stages of development.

Members of the Aedes genus of mosquitoes are widely recognized as vectors of viral diseases. Ae.albopictus is its most invasive species, and are known to carry viruses such as Dengue, Chikugunya and Zika. Its emerging importance puts Ae.albopictus on the forefront of genetic interaction and evolution studies. However, a panel of suitable reference genes specific for this insect is as of now undescribed. Nine reference genes, namely ACT, eEF1-γ, eIF2α, PP2A, RPL32, RPS17, PGK1, ILK and STK were evaluated. Expression patterns of the candidate reference genes were observed in a total of seventeen sample types, separated by stage of development and age. Gene stability was inferred from obtained quantification data through three widely cited evaluation algorithms i.e. BestKeeper, geNorm, and NormFinder. No single gene showed a satisfactory degree of stability throughout all developmental stages. Therefore, we propose combinations of PGK and ILK for early embryos; RPL32 and RPS17 for late embryos, all four larval instars, and pupae samples; eEF1-γ with STK for adult males; eEF1-γ with RPS17 for non-blood fed females; and eEF1-γ with eIF2α for both blood-fed females and cell culture. The results from this study should be able to provide a more informed selection of normalizing genes during qPCR in Ae.albopictus.