Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

The overall goal of this procedure is to observe the responses of gustatory neurons in insects and to measure gene expression in gustatory tissues. This is accomplished by first recording the responses of specific gustatory hairs to several tastes at different concentrations. The second step is to carefully dissect the gustatory tissues.

Then separate the samples by sex and appendage. Next, isolate the total RNA from each tissue sample and create CD NA preparations for next gen sequencing and QPCR. The final step is to analyze the gene expression data and form hypotheses, such as how the expressed chemos sensory genes mediate physiological responses to tastes in insects.

Ultimately, electrophysiological recordings are used to show the receptive range of the insect gustatory system and RNA sequencing of gustatory tissue is used to highlight the most important genetic components of the gustatory reception. Generally, individuals new to these methods will struggle because of the small size of insect chemo. Sensory appendages.

Whether dissecting or recording from small insect organs practice in patients are the keys to mastering these techniques. To begin this procedure, pull a thin tip recording electrode in a pipette puller with a glass capillary under the dissecting microscope. Carefully break the electrode tip off so that its opening is wide enough to envelop the target centum, but small enough to avoid contacting the neighboring structures.

Next, anesthetize two to four adult insects in the freezer at minus 20 degrees Celsius for 20 seconds. After that, immobilize one of the insects using a cover slip with small strips of cellophane tape. Under the microscope.

Identify the target sens by its morphology and position and orient the animal to allow free access to it from the angle of electrode entry. Then insert a tungsten wire into its dorsal thorax. To serve as the ground electrode, fill the glass electrode from the tip with a stimulating solution of interest.

Flick out the air bubbles while holding the tip down. Insert a silver wire into the electrode to serve as the recording and stimulating electrode. After that, connect the electrodes to a pre amplifier, which allows the reduction of the settling time to capture neuronal activity.

The pre amplifier is connected to a computer equipped with spike recording software to collect, store, and analyze electrical signals. Now stimulate the centum with a control solution to ensure its functionality. Count the spikes starting 200 milliseconds following the stimulus artifact.

To obtain the dose response results. Expose the centum to increasing concentrations of the experimental chemical and allow a minimum of three minute intervals between stimulations to ensure adequate recovery. In this procedure, prepare a shallow cooler with dry ice.

For the tissue collection tubes, label the 1.5 milliliter RNA free snap cap tubes for the collection of different types of tissue. Keep the tubes closed to avoid excess condensation. Next anesthetize 30 to 40 mosquitoes in the minus 20 degrees Celsius freezer for 15 to 30 seconds.

Then transfer the anesthetized mosquitoes to a cold stage and sort by sex. Turn the insects dorsal side down by grasping their wings, being careful not to damage the taste appendages. Under a dissecting microscope, dissect the paired label Bella or tarsi from either males or females by gently grasping the mosquito pro Bois just proximal to the label.

Bella with one pair of forceps to expose the inner stylus and remove the label with the other pair of forceps. Then place the tissue in the cold collection tube for tarsal tissue. Gently grasp the mosquito leg at the junction of the tibia and the first tarsal segment, and remove the tarsi.

Then place the tissue in the cold labeled tube. Spin the collection tubes for one minute in a zero degree Celsius centrifuge at 9, 000 times gravity to keep the tissue at the bottom of the tube. If moisture accumulates in the collection tubes during dissection, add 50 microliters of cold triol reagent to cover and protect the collected tissue.

Freeze the tubes in the 1.5 milliliter tube rack containing liquid nitrogen. After that, prepare the tightly fitting RNA free pestles by cooling them on dry ice before tissue disruption. Then grind the tissue into fine powder and repeat one more time.

Bring the total volume of triol to one milliliter and resus. Suspend the ground tissue by vortexing. After that, add 200 microliters of chloroform to the ground tissue and mix thoroughly.

After five minutes at room temperature, spin it down in the centrifuge at four degrees Celsius at 11, 000 times gravity for 15 minutes. Carefully add the aqueous layer directly to a genomic DNA filter column. Then spin it down at 11, 000 times gravity for five minutes.

Add an equal volume of RNAs free 70%ethanol to the flow through and mix by pipetting. Transfer the liquid to an RNA collection column and continue RNA extraction. After that, quantify and assess the purity of the total RNA on a precision spectrophotometer and proceed with any standard RNA sequencing method.

The trace recordings of action potentials from aide's E GTI gustatory illa demonstrate the effectiveness of direct stimulation with the range of chemicals. This technique can be used to quantify responses to any stimulating chemical by counting the spikes of a given amplitude and duration over a reasonable time range of less than 500 milliseconds. These three spikes correspond to three sensory neuron subtypes with different sensitivities.

The biological replicates for RNA-Seq data sets may be limited by cost or difficulty of tissue collection. Q-R-T-P-C-R offers the ability to validate small subsets of total gene expression at less cost and requires less source. RNA shown in the left panel side by side is a comparison between the relative expression determined by RN Aeq and the relative expression determined by Q-R-T-P-C-R.

These two methods rely on different chemistries for gene abundance estimation. The statistical equivalence functions displayed graphically in the right panel demonstrate the limits of certainty in each case. At after watching this video, you should have a good understanding of how to dissect insect tissue for RNA isolation and record neural signals from the gustatory hairs of live insects.