Vector virus interactions is a field of study that

Vector virus interactions is a field of study that has been focused on over time, especially with increased outbreaks of vector borne diseases that have lead to significant mortality across the world. The anatomy of the aedes aegypti mosquito is crucial in looking at virus dissemination by going directly to the root of the problem. The spread of diseases commences by a mosquito taking an infectious blood meal, which then travels through the salivary glands and into the midgut, in which it replicates itself. The midgut is surrounded by a virus impermeable basal lamina which refers to a protective layer that makes up the outer layer. Entry and exiting of viruses from the mosquito constitutes the main function of the mosquito midgut. Studies have been done to show that an additional blood meal contributes to increased efficacy of virus dissemination (Brackney, 2017). This study has been separated into two phases. For phase one, it was hypothesized that if multiple blood meals are administered, then the integrity of the basal lamina will be compromised. Other studies have shown the effect of different feeding on the basal lamina. The basal lamina of sugarfed mosquitoes was not affected, while blood meals compromised the amount of holes in the basal lamina with the chikungunya virus. (Dong, 2017). Using uninfected blood in this experiment, the independent variable is defined as the different feeding backgrounds while the dependent variable is the integrity of the basal lamina. In this study apoptosis is defined as programmed cell death of the mosquito. When looking at the role of apoptosis, if the mosquito is able to increase programmed cell death this should logically result in decreased virus emission since the infected cells are not able to multiply as fast or with as much efficiency. The problem occurs due to the fact that the virus sometimes has coding for responses to apoptosis and is able to inhibit it (Clem, 2016). Other research has demonstrated that inhibiting the AeDronc (initiator of apoptosis) gene leads to increased virus infection of dengue in aedes aegypti mosquitoes (Ocampo, 2013). Studies have shown that when apoptosis is inhibited, mortality rates increase to about 60-70% in virus infected mosquitos (Wang, 2008). The link between apoptosis and integrity of the basal lamina has currently not been researched and will constitute phase two of this experiment. It is hypothesized for phase two that if apoptosis is inhibited then the integrity of the basal lamina will be compromised. The independent variable has been defined as the induction/inhibition of apoptosis and the dependent variable is the integrity of the basal lamina. Other studies have revealed the link between increased virus prevalence and inhibiting apoptosis. Therefore, it can be assumed that the increased virus prevalence has a link between the integrity of the basal lamina as well. A connection can be proposed between the basal lamina integrity and apoptosis. This can be supported by the fact that previous studies have shown that when there are holes in the basal lamina it becomes inherently easier for viruses to enter and escape the midgut (Dong, 2017). Other research has shown that the maximum that the basal lamina can allow is about 5-8nm in diameter. Since virions are actually five times the maximum allowance and are able to pass through it gives further clues that the basal lamina is not a rigid structure but rather a flexible one that adapts to different conditions and creates these “tears” (Houk, 1981). The actual structure of the basal lamina can also hint to the proposed results. The basal lamina has been imaged to find that it is a grid-like structure, refering to the bendability (Terzakis, 1967). This grid like structure can also support how the basal lamina is “flexible” and different factors can cause stress physically or biologically, causing it to expand and create holes in the process. Materials and MethodsTo begin the experiment seven midguts of aedes aegypti mosquitoes were dissected to be imaged and held in 2.5% glutaraldehyde solution. To commence dissection of unfed mosquitos, an aspirator was employed to collect them, and they were then held in the freezer for about five minutes and then transferred to a petri dish where they were then held on ice. By utilizing water, glass slides, a light microscope and forceps the dissection can take place. As a primary step the last abdominal section is removed in one quick motion, and then with the forcep under the thorax create a tear and gently pull apart to keep the midgut in one piece. (See figure 1). Mosquitoes were transferred and held in the glutaraldehyde solution to remove any excess water. To create the next experimental group of 24 hours post blood meal a blood feed was administered to a group of aedes aegypti mosquitoes. A water circulator was used and checked to make sure that the water level was at almost full. After, an ice cream carton was utilized by removing the top and using parafilm to cover the top. It was permeable enough that the mosquitos can feed through but could not escape. A hole was then punched in the side that allows for enough room to fit the aspirator to release the held mosquitos. The hole can be plugged by using a variety of different objects but in this experiment a small plastic test tube was used. The circulator was then filled with cattle blood and two tubes are attached circulating water at 37 degrees celsius. Mosquitos were allowed to feed for 15-30 minutes to maximize the amount that will actually take a blood meal. Directly after the blood feed mosquitos were held on ice and then sorted into blood fed females which can qualitatively be determined by the “red” color of the abdomen. These mosquito midguts were then removed following the same procedure as the dissection for the unfed mosquitos and held in the same 2.5% glutaraldehyde solution. After the midguts are held in the buffer for 24 hours and degraded through an ethanol acetone series and put into a critical point dryer to again remove any excess liquid, they were then mounted onto plates and inserted into the scanning electron microscope. To utilize the scanning electron microscope,  the samples were screwed into the pocket and then the vacuum removed the air to prepare to image. Once they are ready using a connected computer, midguts were able to be imaged at different magnifications 300x, 4000x, and 7000x. Different settings were used to adjust the auto brightness and different focuses to achieve the best possible quality (See Figure 2). To begin phase two double stranded RNA was created to intrathoricacally inject 200 ng into aedes aegypti mosquitoes. This can be done through RNA interference. Reverse and forward primers were created and then added to the inhibitor/inducer gene to attach. After each specific amount is added, utilizing a PCR machine quantities can be derived. (See Figure 3) After, midguts can be dissected and imaged utilizing the scanning electron microscope.   TablesUnfed Mosquitos               300x                                            4000x                                                   7000×24 hours post blood meal acquisition             300x                                                   4000x                                               7000x 72 hours post blood meal acquisition               300x                                                    400x                                                7000xResultsResults thus far have shown that the acquisition of a blood meal to uninfected aedes aegypti mosquitoes is accompanied with increased permeability of the basal lamina. Images can be qualitatively analyzed for wholistic breaks and as shown from the sample of unfed images there is not shown to be any true “tears” in the basal lamina rather it is a bumpy surface of muscle. The images from 24 hours post blood meal show clear tears in the basal lamina at 4000x and 7000x the same. At 300x the entire basal lamina is shown to be gridded and also small cracks are apparent. After 72 hours post blood meal restoration of the basal lamina occurs as shown by the unapparent tears and the fact that gaps aren’t seen within all magnifications 300x, 4000x, and 7000x. (See appendix)Discussion Results from phase one thus far have supported the hypothesis that if multiple blood meals are administered the integrity of the basal lamina will be compromised. From the unfed midguts it is clear that there are no real breaks in the basal lamina and it is seemingly unscathed. Images from 24 hours post blood meal can be qualitatively analyzed to show clear breaks in the basal lamina.  (Figure 4).  Due to the fact that previous literature  shown that the basal lamina is a dynamic structure (Houk, 1981), it can be inferred that this is why the basal lamina “tears” when taking a blood meal. A proposed explanation is that the midgut actually expands after being fed which can constitute as the reason for the basal laminas imperfections since it is being stretched through this grid like structure (Figure 5). Other research has shown that with a second non infectious blood feed, mosquitos that had been infected with a virus had increased virus dissemination (Brackney, 2017). When the mosquito feeds the midgut inherently expands and this can result in stress to the basal lamina and in response it expands, but in this process creates “stretch marks” a term coined to describe the stress that is shown on the midgut. These “stretch marks” can become too stretched and create breaks. The restoration of the basal lamina that is shown can be due to the fact that the mosquitos were not given any other blood meals for 72 hours. This means that the basal lamina had no added physiological stressors and was able to regenerate itself. Previous studies have shown that the midgut epithelial cells are able repair itself bringing in healthy cells through a cone like response that utilizing the protein actin (Gupta, 2005).  Other studies have shown that the basal lamina is able to restore itself after 7 days (Dong, 2017), but contrastingly in this study after only 72 hours the basal lamina was able to restore itself. New insights can be justified that the period of facilitated virus escape is between 24 hours and 72 hours post blood meal, due to the fact that there are more possible escapes through the basal lamina. Results thus far have shown that the basal lamina is compromised after taking a single non-infectious blood meal. Future Work This project will continue to move on to phase two in the future and determine if apoptosis is a contributor to basal lamina breaks. By suppressing apoptosis and imaging them on a scanning electron microscope it can be determined if there are more breaks with or without apoptosis. It is hypothesized that if apoptosis is inhibited then the integrity of the basal lamina will be compromised due to the fact that decreased cell death constitutes more chance for the cells to replicate and expand the basal lamina. When the midgut is expanded the basal lamina will consequently break and tear. Results of this experiment provide a mechanistic understanding of how the virus is able to escape the basal lamina and travel back through to the salivary glands to be disseminated to a larger population. Findings of compromised integrity after 24 hours of a blood feed can provide a way to understand novel virus control mechanisms. Limitations in this study include the small sample sizes for experimental groups, only 7 midguts for each different feeding background was utilized. The increased amounts of midguts could provide more certainty to the project and make sure that it was a repeated occurrence. Due to the fact that this was a qualitative experiment there is no way to tell if the results  were due to chance since no p-value was utilized. But, regardless these results show a clear effect of another blood meal on the basal lamina integrity.