Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

The overall goal of the following experiment is to examine the effects of an epigenetic drug on various life stages of malaria mosquitoes. This is achieved by dissolving the drug in the immature mosquitoes habitat to determine its effects on larvae, survivorship, and body size. The drug is also added to mammalian blood and fed to adult female mosquitoes, which will test the effect of the drug on reproduction.

Finally, an enzyme assay is conducted in order to test inhibition of mosquito enzyme by the drug. The results show the effects of epigenetic changes on the malaria mosquito survivorship and for Conti based on bioassay analysis, We first had the idea for this protocol. When we learn about dine being developed as a promising anti-cancer epigenetic drug, this drug induces apoptotic death in human cancer cells as a mechanisms for histo modification are conserved hormone animals, we sort of tasting as a effection app on the malaria mosquito.

To begin, select the Novelis Gambi mosquito larvae at second in star and put them on a paper towel for two to three seconds to remove excess water. Then gently add 15 larvae per well of a six well nuclease free cell culture plate containing 10 villa liters of distilled water per well. Label the wells with color coded tapes to randomize the experiment.

Then label two wells per test concentration on each plate. Next, prepare a one millimolar deasy NEP hydrochloride stock solution by dissolving 0.3 milligrams of deasy nep. In one milliliter of distilled water, add the stock solution of Z NEP hydrochloride to the labeled wells to achieve a final concentration of 0.5 micromolar and 5.0 micromolar.

Addition of the stock solution for aforementioned concentrations will not change the volume of the water in the well significantly. However, in case of much higher concentrations, it is advisable to dissolve the compound in the larva media to avoid any unnecessary dilution of the compound. After addition of the compound feed and incubate the larvae for 24 hours at 28 degrees Celsius.

Then record the mortality for the DZ NEP treated and untreated larval mosquitoes. Repeating this recording every day until all larvae die or pupate and emerge. As adults, remove and discard any dead larvae each day.

Also, record the size of larvae every second day until they pate to perform the fecundity assay. First, assemble the artificial feeder system connecting the feeders and heating element by the tubes. Once completed, switch on the system and select SP one.

Press enter to start the system. Monitor the temperature of water using a digital thermometer in the heating system. The connectors and tubes help maintain a constant circulation of water through the system, so the temperature of the blood remains.

At 37 degrees Celsius. Fill a flat bottom tray with 10%bleach solution that will be used to decontaminate any blood spills. Next, add two milliliters of defibrillated sheep's blood to a micro centrifuge tube and label as control.

Repeat the process for the experimental tube labeled as five micromolar DZ nep. Then add six microliters of DZ NEP stock solution to the experimental tube labeled five micromolar DZ nep. Gently mix the blood and drug by inverting it several times before incubating the tube for 10 minutes at room temperature.

Using a pipette, add two milliliters of control and test blood to the top of correspondingly labeled feeders. Discarding the pipette in the bleach solution. Breathe air on the cage periodically to encourage females to feed.

Cover the cage with a dark bag. Let the mosquitoes feed for approximately 30 minutes. Once feeding is complete, switch off the heating element.

Take the feeders out and strip the para film after soaking the feeder and bleach solution for five minutes. Rinse well in deionized water. Next, put cotton balls with sugar water on the cages for 48 hours.

Place egg dishes labeled with the respective test or control concentration that contain water and filter paper in the cages overnight. To facilitate egg laying, remove the egg dish the next day and examine the number and structure of eggs under a stereo microscope. Obtaining images, count the number of eggs and analyze the difference in the number of eggs obtained from test and control cages.

This portion of the video shows an enzyme activity assay using DTNB as an indicator to determine the effect of dz NEP on SAH Hydro Lace inhibition in adult mosquitoes. To measure the SAH Hydro Lace inhibition by DZ nep, prepare accrued enzyme extract of mosquitoes homogenized 10 non-blood fed adult mosquitoes in one milliliter of ice cold 0.1 molar mono basic sodium phosphate containing 0.3%tritton X 100. Using a glass tissue homogenizer, transfer the homogenate to a 1.5 milliliter micro centrifuge tube.

After centrifuging the homogenate for five minutes at 10, 000 G and four degrees Celsius, transfer the supinate to a clean 1.5 milliliter micro centrifuge tube. Use the supinate as the enzyme source for the SAH biochemical assay. For the blank, add 50 microliters of SAH 50 microliters of DTNB and 100 microliters of diba sodium phosphate to make a master mix and add to the individual wells of a 96 well flat bottom microplate.

Next, for the control add 50 microliters of SAH 50 microliters of DTNB 50 microliters of diba sodium phosphate, and 50 microliters of SAH hydro lace enzyme to the individual wells. Finally, for DZ NEP treatments, add 50 microliters of SAH 50 microliters of DTNB 50 microliters of enzyme and 50 microliters of the selected DZ NEP concentration to the individual wells. Prepare for replicates per treatment and control.

Read the optical density or OD of the SAH enzyme samples at 405 nanometers for five minutes at 22nd intervals using a 96 well microplate reader. Finally subtract the blank OD from the control and treatment od obtained from each well and calculate the percent remaining SAH hydro lace activity Using the equation found in the text protocol, these results demonstrate that the epigenetic drug DZ NEP suppresses growth and development and induces mortality in immature mosquitoes. The majority of the mosquitoes exposed to 0.5 micromolar died by day 10 of the experiment, whereas a large number of mosquitoes exposed to five micromolar died by day eight.

In contrast, the mortality of the mosquitoes in the control treatment remained unaffected and several emerged as adults on day eight of the experiment. The effect of DZ NEP on mosquito fecundity is illustrated here. Adult female mosquitoes fed with blood containing DZ NEP exhibited a significant reduction in number of viable eggs when compared to the control.

A large number of eggs obtained from the test cage were darker in color and lacked floats and rosette like assemblies when compared with eggs obtained from normal blood fed female mosquitoes. The absence of floats along with darker colored eggs indicates the potential role of epigenetics in exo corion formation. The effect of DZ NEP on SAH hydro lace activity in adult male and female malaria mosquitoes is demonstrated here.

A DZ NEP dependent decrease in OD is observed for each drug treatment as compared to the control treatment indicating that dz NP inhibits SAH hydro lace activity After its development. This technique paved the way for researchers in the field of vector biology to explore the effects of epigenetic drugs on the malaria mosquito.