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

The overall goal of the following experiment is to identify transcriptional components of the photo periodic diapause response. In a non-model mosquito eighties elbow pictus, this is achieved by first producing biologically replicated samples of diapause and non diapause eggs via different photo periodic treatments. As a second step, RNA is extracted from each replicate for high throughput RNA sequencing.

The RNA sequences are then processed computationally to assemble them into congs and to annotate them. Ultimately variations in the gene expression in response to diapause inducing versus non diapause inducing photo periods can be inferred from Reeds MAP to the RNA assembly. The main advantage of this technique over existing methods, such as a microray analysis, is that a prior genetic resources such as the genome sequence or a microarray, are not required to obtain global estimates of gene expression in response to diapause conditions.

This method can help answer key questions in the field of ecological genomics, such as what is the transcriptional basis of DPOs or other key ecological adaptations in non-model organisms. Generally, individuals new to this method will struggle as it can be difficult to obtain sufficient quantities of diapause and non DPOs eggs and the downstream bioinformatics analysis can be challenging. We first had the idea for this method when we noticed that high throughput DNA sequencing technologies were becoming more affordable and offered de tractable method for obtaining whole genome expression profiles in non-model organisms Before starting the procedure.

Set two photo period cabinets with programmable lighting at 21 degrees Celsius for optimal diapause expression and approximately 80%relative humidity Program one cabinet for a non diapause inducing photo period with a 16 eight light dark cycle and the other for a diapause inducing eight 16 light dark cycle with the lights on at the same time for both cabinets to synchronize the circadian time between the photo periods. Next submerge egg papers for the appropriate number of eggs in 500 milliliters of deionized water and add one milliliter of ground dog food and brine shrimp food slurry. Cover the container with mesh placing a rubber band around the mesh to keep it in place.

Then incubate the eggs in the 16 eight light dark long day photo period cabinet for about 24 hours. The next day, transfer about 30 hatched larvae per group to individual 10 by 10 by two centimeter Petri dishes filled with 90 milliliters of deionized water. Every 48 to 72 hours, transfer the larvae to a clean dish, feeding them with fresh food slurry at each transfer.

After about six days, line the bottom of three to four adult cages for each photo period treatment with wet filter paper and dampen the filter paper with enough deionized water to increase the local humidity in the cage while avoiding standing water. Note the photo period, replicate number cage start date, and other information relevant to the experiment with permanent marker on the side of the cage. Then every other day for 10 days, transfer the puy into small cups of clean water at no more than 50 puy per 25 milliliters of water into the appropriate adult cages for the experiment and place organic raisins on the top mesh of the cage to provide sugar for the emerged adults To stimulate ova position four to five days after the first blood meal, fill dark colored 50 milliliter cups lined with a textured non bleached paper towel halfway with deionized water and place one cup into each cage to collect and store the eggs.

Replace the egg papers in each cage with fresh paper, placing the recently removed papers in Petri dishes and storing them in the eight 16 light dark short day photo period cabinet to avoid confounding effects on the egg storage. Approximately 48 hours after the collection, dry the egg papers in the open air such that they are limp and slightly damp to the touch, but not so wet that the papers are dark from water or stimulate hatching. To measure the diapause incidents, record the number of eggs on each egg paper.

Then completely submerge the individual papers in a 90 milliliter Petri dish with approximately 80 milliliters of deionized water. To stimulate their hatching, add approximately 0.25 milliliters of food slurry, and then after 24 hours, transfer the Petri dish onto a black surface. Now place a light source on one side of the, the larvae will move away from the light, allowing for a clear tally of the individual first in star larvae.

Use a pipette to remove each larvae after counting to prevent recounting individuals. Then place the egg papers into a new Petri dish to red dry. After counting a second batch of hatchlings a week later, place the egg papers with the remaining unhatched eggs in new 90 milliliter Petri dishes with approximately 80 milliliters of fresh bleaching solution taking care that the papers are completely submerged in the bleaching solution.

Leave the papers under a fume hood overnight and then the next day use a light microscope to tally the number of unhatched eggs. If an egg is embryonated, it will exhibit an off-white color with the eyes appearing as two small black dots opposite each other on the dorsal side. To begin, carefully clean the bench with RNA decontamination solution to remove any residual nucleases and to avoid RNA degradation.

Next, use a camel hair brush to brush at least 400 mosquito eggs containing developing embryos at the developmental time. Point of interest into glass grinders. In a fume hood, add one milliliter of triol and grind the eggs until they are completely pulverized, and then perform the RNA extraction in triol, followed by isopropanol precipitation according to the manufacturer's instructions.

Now use up to one microliter of D ns per up to 10 micrograms of RNA in 50 microliters of reaction for 30 minutes at 37 degrees Celsius according to the manufacturer's instructions. Next, incubate the reaction in five microliters of suspended DNA inactivation reagent for five minutes at room temperature. Gently vortexing the suspension three times during the incubation period.

Then centrifuge the RNA extracts for 1.5 minutes at 10, 000 times G and Eloqua, the RNA containing supernatants into fresh tubes for sequencing. Finally see the accompanying text for details on how to process the resulting next generation sequencing data from read, preparation, assembly, and annotation to differential expression analysis. In this first figure, the fluorometry of two representative RNA samples shows two bands at approximately 2000 nucleotides.

The insect 28 s ribosomal RNA is comprised of two polynucleotide chains held together by hydrogen bonds that are easily disrupted by brief heating or agents that break hydrogen bonds. The resulting two components are approximately the same size as an 18 s ribosomal RNA in the second RNA sample high levels of degradation can be observed in this representative experiment. The photo periodic treatment of a representative group of a albopictus mosquitoes resulted in a high diapause incidents in short day reared mosquitoes and a low diapause incidents in long day reared mosquitoes.

Although there was some variation among the replicates, for example, the short day two group exhibited a lower diapause incidents than the other replicates. A Trinity assembly of digitally normalized reads resulted in 76, 377 contigs with a mean conig length of 1020 3.1 and a maximum Conti length of 20, 892. Note that the distribution of the Conti lengths is heavily skewed towards shorter contigs.

This is typical of Denovo transcriptome assembly. In this final graph, the differential expression analyses from a similar workflow of embryos reared under diapause inducing conditions at 11 and 21 days post ova position reveal 3, 128 differentially expressed genes between these two time periods. Once mastered, this technique can be completed in six to eight months from tissue generation divine informatics analysis if it is performed properly.

While attempting this procedure, it is important to remember that success at each step should be verified before moving to the next step. For example, confirm that the experimental conditions have produced DPAs and non diapause eggs before testing the RNA integrity Following this procedure. Other methods such as quantitative R-T-P-C-R or comparative genomics analysis can be performed to answer additional questions concerning tissue or development specific expression of the genes of interest and or processes of molecular evolution.

Don't forget that working with triol can be extremely hazardous and that precautions such as using gloves, a lab coat, and a fume hood should always be taken while performing this procedure.