An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, <em>Aedes albopictus</em>

Abstract

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes. Video LinkThe video component of this article can be found at

Transcriptome Analysis of the Breast Muscle of Xichuan Black-Bone Chickens Under Tyrosine Supplementation Revealed the Mechanism of Tyrosine-Induced Melanin Deposition

“…The number of fragments per gene was determined using StringTie (version: 1.3.0), and the values were normalized by the trimmed mean of M -values (TMM) method (Robinson and Oshlack, 2010). The fragments per kilobase of transcript per million mapped reads (FPKM) value of each gene was calculated using a Perl script (Poelchau et al, 2014).…”

Comparative performance of transcriptome assembly methods for non-model organisms

“…General procedures for generating these RNA-Seq reads from Ae. albopictus have been described previously [ 26 ]. Quality filtering of the RNA-Seq raw reads was consistent across studies and described in detail in previous publications from this laboratory [ 12 , 13 , 15 ].…”

Photoperiodic diapause in a subtropical population of Aedes albopictus in Guangzhou, China: optimized field-laboratory-based study and statistical models for comprehensive characterization

“…Five days after blood feeding, each cage was provided with a dark oviposition cup half-filled with deionized water and containing a piece of filter paper. The paper was removed three days later and was kept wet for two days post-oviposition to allow serosal cuticle formation, which increases the desiccation resistance of the eggs [ 32 ].…”

“…We compared two slurries used to stimulate eggs to hatch for the diapause determination: a yeast powder slurry and a food slurry (consisting of ground dog food and brine shrimp), as described by Poelchau et al [ 32 ]. The yeast powder slurry is less expensive and easier to obtain in China than the food slurry.…”

“…We stimulated the eggs to hatch by completely submerging individual egg paper in water in a fresh Petri dish and then re-drying them, after that the unhatched eggs were hatched again after 1 week. Then, each egg paper that had been stimulated twice was placed in a new Petri dish with Trpiš solution [ 32 , 34 ] for diapause identification. After bleaching for 30 min, the eggs were observed and counted under a stereomicroscope.…”