TITLE: Dirofilaria immitis and Brugia malayi emergence assay for individual or pools of mosquitoes
AUTHORS: Michael Povelones, Abigail McCrea

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This assay is for determining the number of infective third-stage (L3) larvae capable of emerging from individual mosquitoes. These are referred to as emerging L3 or eL3. This assay can be used to quantitatively measure how mosquito manipulations or filaria manipulations affect eL3. This assay can be performed on groups of individual mosquitoes in a 96-well plate, or en masse in a large petri dish. The latter format is useful for producing large numbers of eL3 for assays, such as motility, molting, transformation, or for infection of a naive host animal. In addition, since the assay can be performed on large number of input mosquitoes with little increase in effort or cost, it offers a favorable low-tech alternative to other mosquito infection diagnostic methods, such as PCR or dissection when live-caught mosquitoes are available.</div><div class = "text-block"><span>This protocol assumes that you are starting with a population of infected mosquitoes by a filarial nematode. We have tested the protocol with </span><a href="https://www.beiresources.org/Catalog/BEIVectors/NR-48921.aspx" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">Aedes aegypti </span><span style = ":;">(Blackeye strain, cat number NR-48921)</span></a><span> obtained from </span><a href="https://www.beiresources.org/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">BEI Resources</span></a><span> via the </span><a href="http://www.filariasiscenter.org/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Filariasis Research Reagent Resource Center</span></a><span> infected with </span><span style = "font-style:italic;">Dirofilaria immitis</span><span> and maintained at </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span> with 80% relative humidity. Emerging L3 are produced as early as day 12. The last day that we have checked is 21 days post infection, but it is very likely eL3 can be produced for the lifespan of the infected mosquitoes. We saw maximum yields at day 13 and beyond. The same </span><span style = "font-style:italic;">Ae. aegypti </span><span>strain infected with </span><span style = "font-style:italic;">Brugia malayi </span><span>was not tested with as many time points, however, we found robust and similar yields at days 12-14, but did not establish the minimum incubation time. We also tested a strain of </span><a href="https://www.beiresources.org/Catalog/BEIVectors/NR-48979.aspx" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">Ae. albopictus </span><span style = ":;">(ATM-NJ95 strain, cat number NR-48979)</span></a><span style = ":;font-style:italic;"> </span><span>following </span><span style = "font-style:italic;">D. immitis</span><span> infection and found that it also is capable of producing eL3.</span></div></div>

[STEPS]
?. As stated in the Abstract for this protocol, it is assumed that you already have groups of infected mosquitoes to examine. First, count and record the number of live and dead mosquitoes in each group and remove the dead ones. Count the total number of mosquitoes that will be assayed.
Ideal group sizes will depend on the experiment and the infection level. We typically start with mosquitoes that are infected and treated with dsRNA. The dsRNA treatment group sizes are approximately 65 mosquitoes. We house them in   paper soup cups with a mesh cover, which are stored in a secondary container (large Bugdorm mosquito cage) in our containment suite. We aim for an average uptake per mosquito of approximately 15 microfilariae and this results in about 30-40 mosquitoes per group on day 14 post infection.
?. Fill wells of 96-well plates with   of DMEM as needed. A multichannel pipette is great to have for this! If you are only performing a single emergence assay to get the eL3 number per mosquito, then you will need one well for each mosquito. If you are planning to assay heads and carcasses after the initial whole-body emergence assay, make two additional wells to accommodate those parts (three wells total per mosquito). We typically do whole mosquitoes in one plate, heads in another plate, and carcasses in another plate using the same order for each so data can be tracked back to the same mosquito. Plates can be made in advance and stored at at
200 µl
4 °C
Instead of filling wells of a 96-well plate, a 60 or 100 mm petri dish could be used to process groups of mosquitoes en masse. In this case, fill the assay plate with   for a 60 mm plate and   for a 100 mm plate.
?. Anesthetize the mosquitoes one group at a time with carbon dioxide flowing through a flypad facing down on top of the cup. Once the mosquitoes are anesthetized, remove the netting and dump them into a netted basket placed in a petri dish containing approximately   of   ethanol in water. The exact volume is not important, but it should sufficient for the mosquitoes to submerge in the basket. The ethanol will wet the cuticle and the mosquitoes will immediately submerge, but the ethanol will not penetrate into the body. This step is performed at  .
10 ml
0 Room temperature
As alternatives to carbon dioxide, ice is a commonly used  way to anesthetize mosquitoes. We have not tested this. Another alternative is to flood the cup with   ethanol and pour the contents into the netted basket. Finally, mosquitoes caught with an aspirator can be directly placed into ethanol. Expelling them against the liquid immediately causes them to submerge.
?. After  in ethanol, pick up the basket, wipe the outside of the basket with a tissue wipe or paper towel to remove excess ethanol. Dip the basket into two changes of  distilled water in a 100 mm petri dish. Leave the basket in the second water wash. The mosquitoes will be floating at the surface. Performed at  .
20 ml
0 Room temperature
?. Pick up individual mosquitoes with fine forceps and place them head down in the buffer (if possible). You can manipulate them a little, but care must be taken to not disrupt their bodies. Gently touch them with the forceps. It is fine to grab the wings, wing hinge region or and legs roughly, but not the proboscis. Alternatively, if individual mosquito data is not needed then put the mosquitoes into a 60 or 100 mm petri dish with  or   DMEM, respectively. The plate is set up at  . Mosquitoes treated this way do not recover.
5 ml
10 ml
0 Room temperature
?. Mark the lid plate with a marker so you know where each group begins and ends.
?. Put the plate or dish into a   incubator and incubate for   with the lid on. We use a standard   carbon dioxide incubator used for culture of mammalian cell lines. Carbon dioxide buffering is probably not necessary, however, plates incubated for very long times (i.e.  ) are likely to become contaminated and this will interfere with scoring of the plate.
37 °C
?. The worms can be counted with a dissecting scope or on an inverted microscope. Tip: if you use an inverted microscope, you do not need to remove the mosquito floating near the surface. If you use a dissecting scope, you will need to remove the mosquito to see the emerged worms at the bottom.
?. Following the emergence assay, mosquitoes can be dissected to assay for L3 present in the head or carcass as well as to determine the number, and developmental stage of larvae present in the Malpighian tubules (in the case of D. immitis infections).
?. Using a concavity microscope slide filled with PBS and a dissecting microscope, remove the head with fine forceps and place into a well of a new 96 well plate containing  µl DMEM.
200 µl
?. Remove the Malpighian tubules and place them in a  drop of PBS on a standard microscope slide. Cover with a 22 mm2 coverslip. Take the remaining carcass pieces and place them into a well of a new 96 well plate containing   DMEM.
15 µl
200 µl
Note: it is common to find L3 larvae moving around the drop of PBS used for dissection. We record these as coming from the carcass sample.
?. Discard PBS used for dissection and read the Malpighian tubules slide for the number and developmental stage (if desired).
Note: this works best with two people. One to dissect and the other to read the Malpighian tubule slides. Even so, It takes approximately   to process about 50 samples. Typically, we process a subset of the total used for the initial emergence assay.
?. Once the desired number of heads, carcass, and Malpighian tubule samples are processed, the head and carcass plates are placed in the  incubator for  and scored as described above for whole mosquitoes.
37 °C