This technique allows researchers to feed and single-handedly perform high throughput measurements to compare meal volumes consumed by hundreds of individual mosquitoes. It can be adaptive to alter the content of the meal, or to compare the volume consumed by different experimental groups. This protocol is aimed at being user-friendly and low cost.
For pharmacological and genetic manipulations, to study mosquito blood feeding and post-flood feeding behavior. Organization is critical for this protocol. Prepare the setup and reagents in advance, so that you can quickly deliver the meals to the mosquitoes.
To begin, transfer 1.98 to two milliliters of defibrinated sheep blood into a 15 milliliter conical tube. If performing fluorescence based quantification of meal size, add fluorescein solution to a final concentration of 0.002%reducing the volume of blood by the same amount as the fluorescein added. Retain one milliliter of the final meal formulation to generate the reference standard curve.
All meals and solutions containing fluorescein, should be wrapped in aluminum foil or kept in the dark. To prepare protein free saline meals, combine 600 microliters of 400 millimolar sodium bicarbonate with 1.39 milliliters of double distilled water in a 15 milliliter conical tube. If fluorescence based quantification of meal size is to be subsequently carried out, add fluorescein solution to a final concentration of 0.002%Reducing the volume of water by the same amount as the added fluorescein.
Retain at least one milliliter of the final meal formulation containing 0.002%fluorescein to generate the reference standard curve. Set aside the prepared meals until you are ready to deliver them. All meals and solutions containing fluorescein should be wrapped in aluminum foil or kept in the dark.
Place the experimental group of female mosquitoes into a container and cover them with mesh. Allow these females to acclimate in the feeding chamber. Set aside a control group of unfed mosquitoes that will not be offered a meal.
To construct a GLI tube, fill a 50 milliliter conical tube with 40 milliliters of 100%glycerol. Seal the open conical tube with two pieces of parafilm to minimize the chance of leakage. Optionally, hold the parafilm in place using rubber bands.
Invert the tube to ensure that there are no holes or gaps. To create the meal delivery device, cut a centered 2.5 centimeter hole in the screw cap of the conical tube using a sharp razor blade or a lave. Stretch, a piece of parafilm evenly so that it roughly doubles in size.
The parafilm should be thin enough that mosquitoes can easily pierce through it but there should be no leaks. Seal the outer surface of the screw cap with the parafilm to fully cover the hole, and set the cap aside. Heat both the sealed tube of glycerol and the blood and saline meals and a 42 to 45 degrees celsius water bath for at least 15 minutes.
Add ATP to the warmed meal and vortex thoroughly. Pipette two milliliters of the warmed meal into the inner chamber of the screw cap and gently placed the inverted warmed glycerol filled conical tube on it. Partially, screw the cap with the meal onto the glycerol filled tube, just enough to prevent leakage of the meal or the glycerol.
Place the assembled gly tube on top of the mosquito container, apply carbon dioxide and allow the mosquitoes access to feed for at least 15 minutes to achieve maximum feeding rates. After feeding, the GLI tube cap can be discarded as biohazard waste or reused after soaking in a low percentage bleach solution and thoroughly rinsing in water. To generate a reference standard curve, prepare a serial dilution of the same meal containing 0.002%fluorescein that was offered to the experimental group of mosquitoes.
To make the first solution of the standard curve, add 50 microliters of meal containing 0.002%fluorescein to 950 microliters of PBS and vortex thoroughly. Perform twofold dilutions for the rest of the standard curve solutions, vortexing well before each dilution. Pipette 100 microliters of each of the standard curve solutions into each of the eight wells in the first column of a 96-well PCR plate.
Add one anesthetized or frozen unfed control mosquito to each of the same eight wells. Repeat in the second column of the plate for a replicate measurement. Add 100 microliters of PBS to each remaining well for the unfed control and experimental groups.
As a negative control, add one unfed mosquito to each well in the next two columns of the plate. Add one mosquito per well to the remaining wells from the experimental groups that were offered a meal. Add three millimeter borosilicate solid glass beads to each well if disrupting tissue with bead mill homogenizer.
Seal the plate carefully and disrupt the tissue then centrifuge the plate at 2000 RPM for one to two minutes to collect the lysate. Prepare a black 96-well plate with 180 microliters of PBS in each well. Then, add 20 microliters of lysate to each well and mix.
Measure fluorescein intensity using a plate reader with the 485 excitation and 520 emission channels. Generate the reference standard curve by plotting the known volume of meal against the corresponding fluorescence intensity measurement. Using the reference standard curve generated, extrapolate the meal volume ingested by each of the experimental group mosquitoes.
Subtract the average fluorescence intensity reading of the unfed negative control group from the fluorescence intensity reading of each experimental group individual to correct for baseline tissue autofluorescence. To measure fluorescein meal size from a blood feeding experiment, a standard curve was generated by plotting fluorescence readings from the designated reference wells containing an unfed mosquito and a known volume of the meal with 0.002%fluorescein. Fluorescence Readings from the remaining wells which contain mosquitoes from either the unfed negative control group of mosquitoes, or the experimental group of mosquitoes offered a meal, are compared to this standard curve to quantify the meal volume consumed.
Although all females in the experimental group were offered the blood meal, some mosquitoes fed and some did not. This protocol can be used to deliver and quantify meals with various protein compositions. Data collected using meals with added fluoricine is shown here.
An independent experiment where mosquitoes were scored as fed or unfed by eye, after they were offered meals without fluoricine, was also performed. This protocol was used to determine the consumed volume of meals with drugs that regulate mosquito hosts seeking behavior. In these experiments, females were offered saline an ATP meals with a human NPY two receptor agonist.
This technique was also used to measure nectar feeding behavior, by exchanging the gly tube for a cotton ball saturated with 10%sucrose containing 0.002%fluorescein, demonstrating that it can be used to measure smaller or more variable meal sizes that cannot be accurately discerned from group weight measurements. Most important thing to when performing this protocol, is to organize the samples in events and to set aside the unfed mosquitoes and one ML of each meal for generating the standard curve. Following this procedure, the behavior of the animals after blood feeding can be observed and digestion can be tracked.
This method allows researchers to evaluate changes in meal consumption in genetically modified mosquitoes. It also provides a straightforward, rapid and noninvasive way to deliver drugs to mosquitoes and to test their effects on behavior.
