Infectious Bloodfeeding!

I will be completely honest and start by saying that this week was long and did not go as smoothly as I might have liked! That being said, everything that needed to be completed for the experiment was successfully completed, so the week ended on a positive note.

In recent weeks, our primary goal was rearing the mosquitoes that we would need for our experiment. The eggs that we hatched just a couple weeks ago are now adults. On Monday and Tuesday, those mosquitoes needed to be sorted. The process of sorting is simply separating the female and male mosquitoes. To do this, the mosquitoes are aspirated from their cage and knocked down by chilling them on ice. Once they are knocked down, it is relatively easy to distinguish between male and female mosquitoes. Male mosquitoes have much more plumose or feathery antenna compared to females. Female mosquitoes are then placed in a paper cup in groups of 50. A fine mesh is secured on the top of the cup and the mosquitoes are given sugar water.

 

Now for the part of the week that did not go as planned… In order to feed the mosquitoes a bloodmeal infected with Zika virus, we need the virus. To do this, the virus is placed in a flask with Vero cells (see the previous post) and allowed to replicate and release from the cells. If this virus is harvested at the opportune time, it can result in a high titer (concentration) of the virus. This high titer is what we were aiming for in the experiment and we anticipated harvesting the virus on Wednesday. However, the virus had not progressed as expected and we had to delay bloodfeeding the mosquitoes by one day. Fortunately, this change was easy enough to adjust for but had me concerned for a day or so.

Before describing the infectious bloodmeal, let me apologize for the poor photo quality of the pictures below. They were on my phone that was sealed in a plastic bag (for safety reasons).

Thursday and Friday were the days we fed our mosquitoes their bloodmeal infected with Zika virus. To do this, the harvested virus was mixed with bovine blood and some ATP (to help with feeding). This mixture is loaded into small feeders that are then attached to a Hemotek system. This feeding system heats the blood so it is similar to the human body, which also improves feeding. In the pictures below, you can see the Hemotek feeders mounted on the individual cups of mosquitoes that were sorted on Monday and Tuesday. While it may be hard to make out, the 3rd picture shows a lot of very happy and bloodfed females!

 

When the mosquitoes have finished taking their bloodmeal, they have to be sorted (again). This time, we are separating the females that fed from those that didn’t. If the female doesn’t take a bloodmeal, than she can’t be infected with the virus. Therefore, females that didn’t feed were discarded and those that fed on the infectious bloodmeal were sorted into new cages.

 

The process of bloodfeeding is a pretty involved one, as you might imagine. Because we are working with disease-causing agents that can infect humans, there are a number of precautions that must be taken. The protocols in place ensure the safety of those working in the laboratory as well. Above, you can see one of the people working on this project in a full-body suit, wearing 2 pairs of gloves, and working inside of a glovebox (a sealed area that you can insert your hands in).

 

 

Mosquito Education in Elementary Schools

My most recent posts have focused on the vertical transmission of Zika virus project. However, in addition to that project, I have been working on an extension-based research project in elementary schools in Marion County.

This week, I had the pleasure of visiting with a few hundred 4th and 5th graders from multiple elementary schools, and talking to them about my favorite insect: mosquitoes! During this curriculum, the students were given a short presentation about mosquitoes, their biology, their medical importance, and how we control them. More specifically, we discussed the container mosquito species Aedes aegypti and Aedes albopictus.

Aedes aegypti and Aedes albopictus are referred to as container mosquitoes because of where the immature stages of these species are usually found. These two species tend to lay their eggs in containers (natural or artificial) and these containers can often be found close to human dwellings. Examples of where you may find container mosquitoes developing are bird baths, corrugated water pipes, the bases of flower pots, or clogged rain gutters. Because the habitats of these two species are found in such close proximity to humans, who better to control these species than the people living in those homes? This is where the elementary mosquito education program comes in.

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In Florida, 4th and 5th graders have science benchmarks that include learning about the life cycles of plants and animals, how those plants and animals impact the environment, and their adaptations. This aligns very nicely with learning about container mosquitoes, their biology, and their importance. After giving the students a short presentation with this information, they participated in an activity where they were asked to demonstrate their new knowledge about the mosquito life cycle. For this, they used M&Ms that had the different life stages of the mosquito printed on them and placed those M&Ms on the correct part of the mosquito life cycle on a worksheet. Not surprisingly, this was a huge hit amongst the 4th and 5th graders (they got to eat the candy after they had successfully completed the activity).

In addition to the activity, all of the students went home with a goodie bag that contained a comic book titled “Fight the Bite“, bracelets and writing utensils with the Fight the Bite slogan, a magnet with a reminder about eliminating water-holding containers once a week, and a folder with a take-home activity to reinforce the concepts learned during the lesson.

To evaluate what the students learned, we used surveys to assess their pre- and post-knowledge of the topics covered. In about a month and a half, they will take the same survey one more time to assess their retention of the information.

The ultimate goal of this program is to equip students with the knowledge necessary to combat container mosquitoes around their home and incorporate container-elimination, and therefore mosquito prevention, into their weekly routine. Additionally, if the parents talk to their kids about what they learned in school, they may also become aware of the importance of container-elimination and make it a priority in their household. Engaging the community in controlling container mosquitoes is something that I am a huge advocate for (see my TEDx talk). In times of active virus transmission by container mosquitoes, the community is one of the most effective tools we have if we can properly engage them.

 

Larvae, Pupae, and Vero Cells

Earlier this week, we hatched our eggs, separated them into groups of 250 larvae per tray, fed them, and let them develop. After sorting all the larvae, we ended up with 24 trays of each mosquito population resulting in a grand total of 12,000 mosquitoes! These pictures are all of the larval rearing trays that we currently have in the rearing room.

Under ideal temperature conditions and with adequate food, this is about the time we should start seeing pupae. Sure enough, when I came to the lab this morning, I was greeted by hundreds and hundreds of pupae! A good portion of the morning was spent transferring the pupae from the larval rearing trays into small cups. These cups were then placed in a cage with sugar water. Within the next 1-2 days, these pupae will be adult mosquitoes.

This weekend, in preparation for the experiment, we also had to seed our flasks with Vero cells. Vero cells are a lineage of cells that are used in cell culture and these are cells that we will eventually infect with our virus. However, before we can infect the cells, we must first know how many cells are in each flask. This is where the process of seeding comes in. Seeding is spreading a defined amount of cells into the flask. The cells that we distribute in the new flask are pulled from an existing cell culture. After a few steps, the cells are dislodged and present in a cell suspension (video below – ignore the mosquito stuck somewhere in the microscope). From here, we calculate how much cell suspension is needed to get 21.6×10^6 cells per flask, add some media, and allow the cells to incubate for a few more days.

The major task this week will be picking pupae, but on Thursday, Friday, and Saturday, we will also be inoculating our cells with Zika virus. One step closer to our infectious bloodfeeding!

And so it begins…

Today marked the beginning of an experiment that I have been preparing for months. This experiment will consume a majority of my thoughts, time, and energy for the next 2-3 months, and I could not be more excited!

First off, if you are wondering what you are looking at in the picture below, that is THOUSANDS of mosquito larvae that I hatched this morning. Believe it or not, this is only half of the mosquitoes that will be hatched for this project! They will be used in an experiment evaluating the potential for two populations of Aedes aegypti (the yellow fever mosquito) to vertically transmit Zika virus to their offspring. Vertical transmission is the transmission from the mother to her offspring.

In the coming weeks, we will infect these mosquitoes with Zika virus in the laboratory, allow them to lay eggs, hatch those eggs, and test the progeny for the presence of Zika virus… That’s the simplified version, anyways! Over the next few months, I will share the progress of the experiment and the methods we use to investigate something like the vertical transmission of an arbovirus.

From this point forward, everything is on a very strict and planned schedule. Many people will spend many hours on various aspects of the study from bloodfeeding, to sorting mosquitoes, to RNA extractions and PCR. For this week, we are focusing on getting these immature mosquitoes to their adult form to be used in the study. Tomorrow, we will hatch more eggs, separate the larvae into more rearing trays (so they aren’t crowded and have plenty of room to develop), and make final preparations for the study. Stay tuned for more updates!

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Welcome!

My name is Casey and this is my new website! I am an early-career scientist currently located at the Florida Medical Entomology Laboratory in Vero Beach, FL. My current research is on mosquitoes, insecticide resistance, and vector competency. I also have been conducting extension research in elementary schools and underserved communities in Florida.

Please explore my page and stay tuned for updates! I am getting ready to start some experiments that look at the vertical transmission potential of Zika virus in Aedes aegypti and the extrinsic incubation period of Zika virus in Aedes aegypti. I will be sure to post updates!

 

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