Protocol for Mosquito Rearing (A. gambiae)

My name is Shish and I'm a postdoc In the lab of Dr.George OU at John Hopkins School of Public Health. In the Department of Molecular Microbiology and Immunology, I'll be showing how to rear mosquitoes the phis Gambi. In the laboratory, we maintain them in the insectory room, which is usually maintain at 27 degrees centigrade and at 80%humidity.

The insectory room is also maintain in a 12 hour day night circle so that we can mimic the conditions which is in the natural conditions. So this is the Insectory room that we have, which is a core facility here, maintained this insect. As you can see, these have all the different trays which have the mosquitoes at all different stages, the egg, the different stages of the larvae and the peepee.

These trays have larvae, which are like 11 or 12 day old, and this is a cage where we collect the adult mosquitoes and we maintain the amongst 10%sucrose solution. To start the mosquito wearing, We need to blood feed the mosquitoes because the mosquitoes, they need the protein of the blood to lay eggs. So the day one is after the adults are being collected in a cage and maintain on 10%sucrose.

The day one is to feed the mosquitoes with blood, and that is why the artificial membrane blood feeding. This is a glass feeder, which we use for the membrane feeding for the mosquitoes. We put the blood inside through the small hole that you see and it is contained in that.

At the bottom on the side are two nozzles, which are connected to a water birth, which is maintained at 37 degree. To maintain the blood warm at 37, to prepare the feeder for the feeding, we take a small piece of peril. We stretch from the two ends on from all the four sides, and then put that on the open side of the glass feeder so as to make a thin sheet of membrane just as to mimic the skin.

This is what is called the membrane feeding. This is a circulating water bath, which maintains the water at 37 and also circulates the water through the glass feeder so that the blood is cemented warm at 37. So we connect the glass feeder through the small capillaries and connect that to the circulating water bath and turn the water bath on so that the water circulates and the water and blood they never mix as they're in two separate chambers.

This is a cage of Phis Gambi adult mosquitoes. So this is the glass feeder, which is being connected to the circulating bath, and we put a clamp on the top of the cage and add 200 microliter of blood. The cage has both the male and the female mosquitoes.

It's only the female that comes to feed on the blood. The feeding is continued for around 30 minutes. Two days after the blood feeding, We put egg cups inside the cage of the mosquitoes for them to the eggs.

This is how it is done. We take a small, be fill up partially with some distill water, take a what and fill the paper, then wrap in a conical shape and put that in the beaker containing the distilled water and make sure that the inda pepper gets mo. Now slowly we open the mosquito cage taking care not to allow any mosquitoes to come out and put the egg cups inside.

The next day after the cups are put inside the cage, That is on the fourth day, they are taken out from the cage slowly. And these are the eggs, the black ones, which also has some dead mosquitoes. Once you have the eggs ready, you put them on a plastic tray.

The past, the plastic tray is filled up with distilled water. Take the filter pepper with the eggs and put them out. We also add a pinch of food, A, which is the ground fish food, and put the tray in the rack.

We should level the tray the day that we start the tray and also What species they are here. This is after You, this is the next day after you put the egg in the tray. That is, these are one day old, but actual protocol is fifth day of the mosquito day.

These are the black ones are the eggs, which are just going to hatch to larvae. And these are the mosquito food. For the mosquito rearing, we usually use three different kinds of foods.

Food A is actually grounded fish food, and this is, we add this food when we start the mosquito culture, when they're at the X stage, that every day we add two tablets of food, C, which is cat food bought from Purina. After eight or nine days, we change the water of the tray and give fresh water. And at that time, instead of food A, we add a pinch of food B, which is nothing but grounded food, C that is grounded cat food as well as add two tablets of food C, the eggs, the hatch to larvae.

And there are three different stages of the larvae. First in install, Second in install, and the third in install. These are trays that have the larvae, Which are five or six day old.

These Ravi, Which are at a much later stage, like nine to 10 days old. In between the water has been changed and fresh water has been added and fresh food. This tray has both the Adults And the ppe.

These are the adults, as you can see, flying under the mosquito nets. And at the edge you can see all the dark colored ppe, which is going to Hatch to the adults. Once you have the adults, they're ready to be collected in a cage, which looks like this.

It has a paper towel lining inside and a a small bottle containing 10%super solution and a cotton soap tin it, which will serve as a food source for the mosquitoes. So we covered the lid, and this is apparatus, the aspirator, which is connected to the vacuum through which will collect the adults from these trays. This part of the aspirator, it is connected here and it's on the other end.

Connected to the vacuum. We put the cage in, which we'll be collecting the adults into this small box. This is another part of the aspirator, which has a long tube, which will put inside the mosquito tray.

The other side will be connected to the cage. You have to make sure that everything is tight, and so that the vacuum works well. We close the lid and make sure everything is tight.

Slowly start the backend. Once the setup is ready, we put this long pipe slowly inside the mosquito tray, making sure that you close the net and don't allow the mosquitoes to go loose. And as you can see, the mosquitoes are getting collected in the small cage.

Take the pipe all over the place slowly to collect all the adults. Now I'm almost done. All the adults have been collected and, and they're all in this cage, which you can see.

Then slowly, we release the vacuum and dismantle the collection apparatus Out. So this is The Ana Gambi adult mosquitoes, which will be in this cage for few days. They'll be fitting on the 10%sucrose, and they'll be given blood to fit when only then we need to lay Eggs.

I've just shown you how to wear the Mosquitoes. Finis mb mosquitoes in the laboratory conditions, the important steps in words are the blood fitting. Then putting the egg cups, following the eggs, then, and then they go through different stages like larvae, ppe, and adults.

And finally, we collect the adults. The importance of this technique is that we have to mimic the conditions that are there outside in the natural conditions so that we have healthy mosquitoes, and then we infect them with the plasmodium and do the different kind of studies on the malaria, which the main aim is to eradicate The mal from the world.