Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

The overall goal of this inoculation technique is to stimulate the mosquito immune response and to measure the melanization reaction. This method can help answer questions in the field of ecological immunology, including the genetic basis for the cost of the human response and the influence of environmental factors. Though this method can provide insight into immunization response, it can also be applied to other systems such as anti-viral or anti-bacterial response in other invertebrate systems.

Generally, individuals new to this method will struggle because of the insect damage that the injection can cause. Add one milliliter of 0.1%methyl green solution to 99 milliliters of the saline solution to color the transparent beads. This is the 0.001%methyl green injection solution.

Then, add five milliliters of 0.1%methyl green solution to 45 milliliters of the saline solution. This 0.01%methyl green dissection solution is 10 times more concentrated in methyl green to facilitate the coloration of the beads and their observation during the dissection. Autoclave the solutions at 110 degrees Celsius for 25 minutes.

To prepare the capillary, heat pull microcapillary glass tubes to obtain a very fine tip, slightly bigger than the smallest beads. Open and adjust each capillary by breaking the tip with tweezers or by sawing the tip off. To select beads with a capillary, pour 0.009 grams of negatively charged beads in a five centimeter Petri dish.

Then add five milliliters of injection solution 30 minutes in advance. This colors the beads to facilitate bead selection for injection. Using a capillary mounted in a capillary pipette bulb, visually select the smallest bead in 0.1 microliters of the saline solution under a binocular microscope.

Rear Anopheles gambiae at 26 plus or minus one degree Celsius and 70 plus or minus 5%relative humidity in a cycle of 12 hours light, 12 hours dark. Keep adult females and give them access to a 10%sugar solution. Using an insect aspirator, place each mosquito in a 50 milliliter tube.

Chill each mosquito briefly by placing the tube in crushed ice. Place a mosquito on its right side under the binocular microscope. Properly creating a mosquito is critical and demands some training.

It's very difficult to learn how to pierce through the insect cuticle and trust that the bead was injected. Inoculate the mosquito with a capillary by firmly piercing through the left side of the thoracic cavity cuticle and then injecting the liquid and the bead. Take care not to damage the flight muscles by holding the capillary as perpendicularly to the mosquito as possible.

Remove the capillary following injection. Using featherweight entomology forceps, place each female individually in a 180 milliliter plastic cup with access to a 10%sugar solution. Check if the mosquitoes are alive 24 hours after the inoculation.

If so, check their condition and keep only the mosquitoes that are able to fly. Freeze these mosquitoes at minus 20 degrees Celsius. They can be kept several days to months before dissection if needed.

Retrieving the beads from the mosquito for the first time is not an easy task. It can be difficult to trust that there were actually a bead injected into the mosquito. On a glass microscope slide and in dissection solution, use forceps to separate the thorax from the head and abdomen on a mosquito that has been frozen.

Open the thorax with forceps to retrieve the beads. Using a microscope equipped with a camera, take a digital image of each bead at a standard lighting setting and 400x magnification. Do not change the illumination between the different pictures to enable their comparison.

To begin picture analysis, open the image analysis software. Click on file and then on open to find a bead image. Then click on analyze.

Then click on set measurements. Tick the mean gray value box in the set measurements window. Next, click on the circle selection tool icon.

Select the bead perimeter, remaining inside the bead and avoiding the luminous halo around it. Then click on the analyze menu and click on measure. Obtain the mean gray value and the size of the cross-sectional area of the bead.

To obtain the melanization value of each bead, subtract the mean gray value from 256. The melanization measure is a value between zero and 256, with zero representing a totally white image. Pictured here are a non-melanized bead, a partially melanized bead, and a highly melanized bead.

Mosquitoes do not all melanize the beads in the same way, as some beads are less covered with melanin than others. In this figure, each point shows the melanization of a single bead and each column of points represents an individual mosquito. The solid points represents the highest melanization value per mosquito.

The cross points represent an intermediate melanization value. And the empty points represent the lowest melanization value. The solid lines represent the mean melanization per bead treatment.

The average bead melanization value decreased from 71 to 50 when the number of beads inoculated increased. In this figure, the black squares represent the mean bead melanization as a function of the number of beads. The red points represent the mean of the highest melanization and the blue triangles represent the mean of the lowest melanization as a function of the number of beads.

The lowest melanization decreased from 71 to 35, but the highest melanization stayed about constant. The variability of melanization increased with the number of beads. The melanization effort was not uniform amongst all beads in a given mosquito, as one bead was prioritized over others.

Once much of this technique can be done in a few hours for hundreds of mosquitoes, if it is performed properly. The main advantage of this technique is that it measures the final phenotype and not just one step of the physiological pathway. Following this procedures, other methods like genograms can be performed in order to gain a better understanding of the human pathways.

After watching this video, you should have a good understanding of how to inoculate mosquitoes and then dissect them to measure the melanization response.