In the last update, we had fed our mosquitoes their infectious bloodmeal containing Zika virus. After that, everything got a bit crazy so I have provided a short summary here of everything that we have done since that time.
After the infectious bloodfeeding, we allowed our mosquitoes to lay eggs. However, this first batch of eggs is not what we are interested in. We don’t expect to see infection in the progeny in the first batch because it is too soon after infection. So, we had to feed our female mosquitoes again! After this second bloodfeeding (which contained no virus), bloodfed mosquitoes were sorted into individual tubes and provided a wet paper where they could lay their eggs. THESE eggs are the ones we are interested in. If vertical transmission of Zika virus is occurring, these progeny are the ones we would expect to be infected.
Once the mosquitoes had finished laying their eggs, we needed to determine which of those mothers were infected (consuming an infectious bloodmeal doesn’t automatically result in infection). To do this, we tested all of the mosquitoes that laid eggs by doing RNA extractions and PCR.
Now, we know which females were infected with Zika virus which tells us which eggs to hatch. Those eggs were then hatched, provided all the larval diet they could need, and the progeny were allowed to emerge into a covered cup like the one you see below. The blue ball you see on top of the cup is a moist sugar ball which ensures our mosquitoes survive until the next step of the experiment.
Progeny of infected female mosquitoes emerging inside a cup.
We have finally arrived at the final step! The progeny of the infected mothers are now adults and we want to know if these mosquitoes are infected with Zika virus. As a reminder, these progeny have never bloodfed which means that if they are infected, they had to have received the virus transovarially from their mother.
For the progeny, we completed capillary assays. To do this, the adult mosquitoes were knocked down and kept on ice while their legs were removed and their wings were severed. This process does not kill the mosquito but does immobilize them so we are able to collect their saliva. To collect the saliva of these legless mosquitoes, they are secured on a piece of tape with their proboscis (mouthparts) hanging off the edge of the tape. A capillary tube with oil in it is placed on the proboscis of the mosquito and mosquitoes are allowed an hour to salivate into the capillary tube. This allows the mosquitoes saliva to be collected in the immersion oil. The oil that now (hopefully) contains the saliva is ejected into a media tube to await testing. If you are interested in what this looks like in the laboratory, check out the pictures below.
Many mosquitoes with capillary tubes on their proboscis!
A close-up shot of the capillary tubes on the mosquito’s proboscis.
At this point, we are no longer dealing with live mosquitoes. Now, we are working on completing the RNA extractions and PCR for all of these progeny. This may take a couple months to complete because of the large sample size we were able to achieve.
As an ending note, I want to emphasize how much work went into the experiment portion of this project. At times, we had 6 or 7 people in the laboratory processing samples, dissecting mosquitoes, putting on capillary tubes, etc. These kinds of projects are extremely intensive and I couldn’t have completed this project without the help of the individuals at FMEL.
Once we are able to complete all the extractions, PCR, and analyses, I will be sure to post an update on what we found.