TITLE: Infecting and mosquitoes with Dirofilaria immitis and Brugia malayi
AUTHORS: Michael Povelones, Abigail McCrea

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for infecting </span><span style = "font-style:italic;">Aedes aegypti</span><span> and </span><span style = "font-style:italic;">Aedes albopictus</span><span> mosquitoes with the filarial nematodes </span><span style = "font-style:italic;">Dirofilaria immitis</span><span> or </span><span style = "font-style:italic;">Brugia malayi</span><span>. Although it is not necessary, it is strongly recommended to measure the number of ingested parasites, which indicates the exposure level for the mosquito and allows for better comparison between replicates. We use a post-feed treatment protocol, but if mosquito groups are treated prior to infection, then it is not just strongly recommended, but critical to measure ingested microfilariae in a group of mosquitoes immediately following the feed to confirm that treatment does not change the extent of blood feeding. Note that microfilariae are large (approximately 300 µm) and will quickly settle in blood samples. This should be considered throughout the protocol and this fact is pointed out in places in this protocol.</span></div></div>

[STEPS]
?. [Counting microfilariae]
Disperse  or  microfilaremic blood in a  spot of water on a microscope slide. This will lyse red blood cells and make the microfilariae more conspicuous and easy to count.
5 µl
10 µl
50 µl
Water type is not important. We have used deionized water or ultrapure molecular biology grade water.Before pipetting microfilaremic blood from the source container, mix evenly by gently inverting 10-15 times. Microfilariae are large and settle in the blood quickly.
?. [Counting microfilariae]
Immediately count the entire drop by scanning through the sample. Determine the concentration and express it as mf/ml by multiplying by the dilution factor. For example, if  was used, multiply by 200. If  was used, multiply by 100. Replicate counts are performed and averaged.
5 µl
10 µl
For best accuracy replicates counts are made. When counting more diluted blood, count   . If needed the water spot size could be increased and more blood can be dispersed. For example   of blood can be counted in a   spot of water.
From our infected dog, we typically count   of freshly drawn blood and count 200-300 microfilariae resulting in a concentration of between 40,000 - 60,000 mf/ml.
?. [Blood feeding mosquitoes]
We typically set up infections at  to  . We use heparinized sheep blood to dilute. The infected blood and diluent blood are aliquoted under sterile conditions in a tissue culture hood. Once the desired dilution is created, recount the diluted to sample to confirm and then keep the sample warm at  in a heat block or circulating water bath. For recounts we use  of the dilution in  drop of water. Replicate counts are performed and averaged.
37 °C
10 µl
50 µl
Because the microfilariae will settle during the course of feeding, the volume of blood and geometry and speed of blood feeding will all contribute to the number of microfilariae ingested per mosquito. Under the conditioned described here, mosquitoes typically have 10-15 microfilariae ingested.
?. [Blood feeding mosquitoes]
Once the water required for the feeder and the diluted blood are prewarmed to  then fill a plastic baby bottle with a solid cap (see picture below) with  water excluding as much air as possible without spillage. Then, invert the bottle and add  of diluted blood to the depression in the bottom of the bottle and cover with stretched Parafilm.
37 °C
37 °C
3.5 ml
Take care to not slosh water from the water bath onto the feeder as it might contaminate the sample and may also inhibit the mosquitoes from feeding. You better attract the mosquitoes for feeding, rub the side of the Parafilm that will be facing the mosquito cup/cage on a part of your body that does not have lotion or fragrance.
?. [Blood feeding mosquitoes]
Invert the bottle and place on top of a cup/cage of mosquitoes. If required for safety reasons, perform this step in a secondary container in the incubator, such as a Large Bugdorm cage. To encourage mosquito blood feeding, exhale a warm breath into the cup or cage before placing the feeder on top. Feed for . We feed at  and  relative humidity (our standard incubator conditions). If practical, turn off the light in the incubator.
27 °C
For important comparisons, dilute enough blood, so that each cup gets the same volume of the same dilution and is fed for the same time. It is ok to reuse the feeders as described below, but it is best not to compare groups that are fed serially since the conditions will not be identical.
?. [Blood feeding mosquitoes]
If required, you can refresh the water in the feeder to feed more mosquitoes. Take care not to spill water onto the Parafilm during this process. Hold the feeder, and remove the cap. Do not put it down on the Parafilm side or it will get damaged and leak. Quickly pour out the water. While the feeder is empty and inverted, give it a firm shake to disperse the blood again. Replace with fresh  water, cap, and put back on mosquito cup/cage for another round of feeding.
37 °C
?. [Blood feeding mosquitoes]
Sort blood fed mosquitoes. Mosquitoes are anesthetized using carbon dioxide and sorted on Flypad using a paint brush.
Alternatively, blood fed mosquitoes can be separated on ice.
?. [Checking uptake of microfilariae]
Remove blood meal from some of the blood fed mosquitoes and count the microfilariae uptake. The objective is to count all microfilariae in the entire blood meal. Place the mosquito in a drop of water on a depression slide. Tear the abdominal cuticle where it meets the thorax with fine forceps. Tear in several places to facilitate gut removal without damaging it. Perform the dissections in ultrapure or deionized water in a depression slide. Discard any damaged guts that are leaking blood. Transfer the gut to a new slide with a  drop of ultrapure water. After transferring, remove the gut tissue by peeling it away from the blood bolus with fine forceps and "wash" through the water to remove residual blood, then discard the gut tissue. The peritrophic matrix typically stays intact. Disrupt the blood meal with the fine forceps and make sure to remove as much residual blood from the tines as possible. Disperse the blood meal fully using a P20 pipette.
50 µl
?. [Checking uptake of microfilariae]
Count the entire drop as in step 2.  . Ideally, count 10 mosquitoes for all groups being compared.