Long noncoding RNAs (lncRNAs) play diverse roles in biological processes. Aedes aegypti ( Ae . aegypti ), a blood-sucking mosquito, is the principal vector responsible for replication and transmission of arboviruses including dengue, Zika, and Chikungunya virus. Systematic identification and developmental characterisation of Ae . aegypti lncRNAs are still limited. We performed genome-wide identification of lncRNAs, followed by developmental profiling of lncRNA in Ae . aegypti . We identified a total of 4,689 novel lncRNA transcripts, of which 2,064, 2,076, and 549 were intergenic, intronic, and antisense respectively. Ae . aegypti lncRNAs share many characteristics with other species including low expression, low GC content, short in length, and low conservation. Besides, the expression of Ae . aegypti lncRNAs tend to be correlated with neighbouring and antisense protein-coding genes. A subset of lncRNAs shows evidence of maternal inheritance; hence, suggesting potential role of lncRNAs in early-stage embryos. Additionally, lncRNAs show higher tendency to be expressed in developmental and temporal specific manner. The results from this study provide foundation for future investigation on the function of Ae . aegypti lncRNAs.

The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. Although numerous genetic expression studies utilizing quantitative real-time PCR (qPCR) have been conducted with regards to Ae. aegypti, a panel of genes to be used suitably as references for the purpose of expression-level normalization within this epidemiologically important insect is presently lacking. Here, the usability of seven widely-utilized reference genes i.e. actin (ACT), eukaryotic elongation factor 1 alpha (eEF1α), alpha tubulin (α-tubulin), ribosomal proteins L8, L32 and S17 (RPL8, RPL32 and RPS17), and glyceraldeyde 3-phosphate dehydrogenase (GAPDH) were investigated. Expression patterns of the reference genes were observed in sixteen pre-determined developmental stages and in cell culture. Gene stability was inferred from qPCR data through three freely available algorithms i.e. BestKeeper, geNorm, and NormFinder. The consensus rankings generated from stability values provided by these programs suggest a combination of at least two genes for normalization. ACT and RPS17 are the most dependably expressed reference genes and therefore, we propose an ACT/RPS17 combination for normalization in all Ae. aegypti derived samples. GAPDH performed least desirably, and is thus not a recommended reference gene. This study emphasizes the importance of validating reference genes in Ae. aegypti for qPCR based research.

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