Field Trials

The lab has been a very busy place for the last couple of months! Since May, Davi (lab technician), Zach (UF IFAS Intern), and I have been busy preparing for and executing field trials in collaboration with mosquito control programs throughout the state of Florida. In May, we completed 4 field trials in Indian River County and in June were able to complete 5 more in Walton County. Thank you to Indian River Mosquito Control and both North and South Walton Mosquito Control for all of your work in helping us make those trials happen.

My primary focus (at least currently) is on insecticide resistance, particularly in mosquitoes. The bulk of our resistance work has focused on Aedes aegypti and Aedes albopictus, but we have recently been working with other species such as Culex quinquefasciatus and Aedes japonicus. There are multiple methods for evaluating the insecticide susceptibility status of an insect and one of those methods is field trials. For my research, we have been using the CDC bottle bioassay and field trials to assess insecticide susceptibility status.

CDC Bottle Bioassay
CDC Bottle Bioassay

The CDC bottle bioassay is a lab-based assay where we treat a glass bottle with pure active ingredient and evaluate mortality over time. These assays can be great indicators of resistance developing within a population. However, there are a lot of factors that can influence the success of an insecticide in the field (weather conditions, vegetation, chemical product, etc.).

Since the results in the bottle do not necessarily match the mortality you might see in a field application of an insecticide, we do field trials to assess the efficacy of a formulated product. In Florida, public health mosquito control is done by mosquito control programs located throughout the state. When adulticiding, one of two chemical types is used: a pyrethroid or organophosphate. For our field trials, we wanted to assess the efficacy of both of these chemical types.

FieldGraphic

Pictured on the left is a representation of how our field site is set up for trials. Cages are place 100, 200, and 300 ft from the line of spray. Also at each of these locations is a spinner that collects droplets from the spray truck on glass slides. Control cages are placed upwind of the line of spray to ensure that the mortality observed during the trial is due to the insecticide spray and not something pre-existing in the environment.

Prior to the trial, mosquitoes are aspirated into the field cages and those cages are hung on stakes at each of the field locations. The spinners are mounted on top of the stakes and turned on immediately prior to the trial.

 

Once the cages are up and the spinners are on, we wait for the appropriate wind speeds (2-10 mph). It is important to wait until there is sufficient wind so that 1) the insecticide will actually be carried across the field site and 2) we are using the product according to label specifications.

After the cages have been treated, they are transferred to clean holding cages. To do this, we first knock down any surviving mosquitoes with CO2. Once they have been knocked down, the field cage is cut open and mosquitoes are transferred to a clean holding cup for the remainder of the trial. The mosquitoes quickly recover from their CO2 knockdown and mortality readings can be taken for the next 24 hours.

From these trials, we will learn more about the efficacy of each mosquito control program’s product in the field, and how that relates to the results we get in the laboratory.

IMG_0667As a side note (and kind of a funny one), doing field work at night in Florida is really fun. During these trials, we try to avoid using insect repellent so it doesn’t confound our results in some way. As a result, we may become prey to hungry mosquitoes. In Indian River, the Psorophora population was out in force. Take a look at the mosquito control director’s leg covered in mosquitoes!

Next week, we will head out to Pasco County for one more round of field trials before the end of the summer. Thank you to Indian River Mosquito Control, South and North Walton Mosquito Control, Dr. Connelly, Daviela, and Zach for helping make these trials go off so smoothly! It truly was a team effort and I think the trials went as well as they could.

IMG_0674
The team for the Indian River trials
IMG_0979
Dr. Connelly, Daviela, and Casey

Culex Collections

The first few months of 2018 were primarily spent in FMEL’s Biosafety Laboratory. Now, it’s time to shift gears and do some field work! May, June, and July will be spent preparing for and executing field trials to assess the susceptibility status of field populations of Aedes aegypti (yellow fever mosquito), Aedes albopictus (Asian tiger mosquito), and Culex quinquefasciatus (southern house mosquito). These field trials will be conducted in Indian River, Walton, and Pasco County.

Culex quinquefasciatus is a species that I hadn’t worked with very much until the last year. These mosquitoes are vectors of St. Louis encephalitis virus and West Nile virus. In contrast to Aedes aegypti and Aedes albopictus, they lay egg rafts instead of laying their eggs singly. To collect eggs from ‘quinx’, we first created a bucket of ‘stink water’. This bucket of stink water was placed outside our lab in a wooded area and the next day we were able to collect egg rafts off the surface of the water.

In the above picture, Daviela and I are using a paint brush to gently remove the egg rafts from the surface of the water. The rafts are then placed on a piece of moist filter paper so we can take them into the lab. In the lab, we can hatch the egg rafts in a tray of water and rear them for our experiments. In one evening, we were able to collect 10 egg rafts from this stink water.
IMG_0392

The egg rafts may look small, each raft is composed of 100 or more eggs. The picture below shows the egg raft under a microscope and here you can see how many eggs really make up this egg raft!

IMG_0393

Our field trials in Indian River County will take place the week of May 21st, so stay tuned for more updates and pictures of field trials.

Vertical Transmission Update

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.

IMG_0336Progeny 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.

IMG_0346Many mosquitoes with capillary tubes on their proboscis!

img_0365.jpgA close-up shot of the capillary tubes on the mosquito’s proboscis.IMG_0347

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.

ESA Southeastern Branch Meeting and a Defense

Things have finally slowed down a bit and I can take the time to share some updates on what has been going on the last month and a half!

In March, I attended the Southeastern Branch meeting of the Entomological Society of America. This is a meeting that I was looking forward to for some time for a couple of reasons. 1) I was going to get to talk about some extension work we have been doing in a community in Vero Beach called Gifford and 2) I was the chair of the student affairs committee and was looking forward to seeing all of our events come together!

Overall, the meeting was excellent. The extension program that we have been working on in Gifford focuses on encouraging members of the community to participate in container-elimination. This community is one that is traditionally underserved (decreased access to information and resources). For our program, we had an established member of the community deliver the educational message and encourage container elimination. The study is ongoing, but our preliminary results tell us that this kind of program can be effective. These results could be important when it comes to times of active disease transmission (like Zika in 2016).

There were many students from the University of Florida in attendance at the meeting and they all did excellent in their respective endeavors.

To name a few accomplishments:

  • Cory Penca, DPM student, placed 2nd in the Extension, Outreach and Teaching student competition
  • Morgan Pinkerton, DPM Student, placed 1st in her student competition
  • Lindsy Iglesias, PhD student who graduates this semester, received the Friends of IPM award
  • Rachel Watson received an award for her undergraduate presentation
  • The UF Linnaean Team won 1st place in the Linnaean Games and will advance on to the national games in Vancouver
  • I was awarded the 1st place prize for my Extension presentation

UF_WinnersUniversity of Florida student award winners at the ESA SEB meeting

CaseyParker&SteveLapointe(SEB2018).jpgAccepting my student competition award from ESA SEB President, Dr. LaPointe

After returning from the SEB meeting in Orlando, a good friend of mine was getting ready to defend her MS thesis work. For the last 2 years, Kristin has worked on a project that revolves around Culicoides, otherwise known as no-see-ums. These insects are not only annoying but are vectors of a variety of diseases that can cause disease in cervids. Kristin evaluated sampling methods and created ecological niche models to predict the distribution of various Culicoides species throughout Florida.

She was successful in defending her thesis and will graduate later this semester. After graduating, Kristin will remain at the Florida Medical Entomology Laboratory to complete a PhD as a UF Fellowship student.

IMG_0180

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.

IMG_0062IMG_0065

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!