This protocol demonstrates standard measurements, reflective of fitness, in aedes aegypti mosquitoes, these fitness parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. To begin place egg papers into sterile water overnight in the insectory to hatch transgenic and wild type mosquitoes. Allow the larvae to feed ad libitum by providing several drops of ground fish food slurry.
To ensure the adults are virgins, separate the pupae by sex once they emerge, move the pupa to a glass microscope slide and remove excess water with tissue to immobilize the pupa. Under a stereoscope at two times to four times magnification, using a finely tipped paintbrush, position the pupa face up. Then visually analyze the tail for differences in genital lobe shape.
Place male and female pupae in separate water cups in proper containment. To cross wild type or transgenic gene drive one or gene drive two males on mass with virgin wild type females, add anesthetized mosquitoes in ratios of five males to one female into mosquito cartons containing approximately 150 mosquitoes. After two to three days, provide a blood source on the cage to feed the females following standard rearing practices.
Sort the blood fed females from unfed females by visually screening their abdomens for engorgement and discarding the non-fed partially, fed, and males. Place the engorged females back into their respective cages. After two to three days, place individual females in 50 milliliter conical tubes lined with filter paper pre-labeled with pencil.
Fill all the tubes with five milliliters of tap water. Place a small piece of raisin or sugar soaked cotton ball on each conical tube. Leave the females in the insectory for one to two days and allow them to oviposit.
Then count the number of eggs on each paper and calculate the average fecundity, as the number of eggs counted on each paper divided by the number of females screened. To dry the egg papers, drain water from the tubes and place them back into the insectory. To freeze the blood-fed females from the completed fecundity study, place the mosquito cages at minus 20 degrees Celsius for approximately 15 minutes.
Dissect the left wing of each mosquito, excising the joint of the wing and the thorax and removing the entire wing. Using forceps, place the wing flat on a glass slide, then add one drop of the 15 milliliter stock solution comprising 70%ethanol and one drop of dish soap to the wing on the slide using a transfer pipette. Next, add one drop of 80%glycerol to the cover slip and place it on top of the wing in the ethanol soap solution.
Photograph the wings mounted on the slides using a camera with a 65 millimeter lens. Use the TpsDig software to identify two dimensional Cartesian coordinates for the landmarks. Calculate the wing length and area using the R script provided in the manuscript.
Calculate the wing centroid size, which is a measure of size, statistically independent from shape defined as the square root of the sum of square distances of each landmark from the center point of the wing Hatch individual F1 eggs from the papers collected from single females into polypropylene clear deli containers containing 100 milliliters of freshly sterilized deionized water. At two to three days post hatching, remove the egg papers from the water and allow them to dry overnight. Re-hatch the egg papers in the same containers in which they were initially hatched.
At three to five days post initial hatching, visually inspect the larvae for a transgenic marker. Record the number of positive or negative transgenic larvae. Discard the negative larvae.
Calculate fertility as the number of transgenic or control larvae divided by the total number of eggs. To determine the sex ratio, add the number of females or the number of males collected across the study timeline per individual adult female mosquito. Divide this number by the number of pupae collected for the same mosquito line generated by a single female.
To determine larvae viability, add the total number of pupae collected across the study timeline per adult female mosquito. Divide this number by the number of larvae counted per individual adult female mosquito for the same line counted earlier. Calculate larvae to pupae development as the average time to pupa development post hatching.
To determine the male contribution, cross 50 transgenic or control males with 100 control females into 64 ounce cartons so that there are 50 males with 100 females. After mating, offer mosquitoes a blood meal, following standard rearing practices. Then separate individual fully engorged females into 50 milliliter conical tubes lined with pre-labeled white filter paper.
Leave females for one to two days in the insectory and allow them to oviposit. Allow the papers to dry in tubes in the insectory for at least five days. Use forceps to remove the papers and place them for hatching.
At two to three days post hatching, remove the egg papers from the water and allow them to dry. Re-hatch the egg papers in the same containers. At three to five days post initial hatching, visually inspect the larvae for a transgenic marker.
Calculate the male contribution as the number of F2 transgenic larvae divided by the number of total larvae per mosquito line. Transfer 50 male and 50 female wild type or transgenic age matched non-blood fed mosquitoes into a proper enclosure. Track the number of dead male and female mosquitoes each day.
Calculate longevity as the average number of days passed before mosquitoes die across the cages, omitting mosquitoes that die of non-informative causes. Larvae to adult pupae and pupae to adult development times were assessed with a Kruskal-Wallis'ANOVA and Dunn's post hoc comparisons. Wild type male mosquitoes had a shorter time to pupation than wild type females, While D7L1 one males did not significantly differ in their pupation time relative to D7L1 females.
D7L1 females took longer to reach pupation than wild type females, as do D7L1 males, relative to wild type males. Regarding pupae to adult development time, wild type and D7L1 females did not significantly differ, wild type males pupate faster than wild type females and D7L1 males. Wild type female mosquitoes never reached their median survival time during the study.
All other groups reached their median survival before 40 days, with a distinct temporal separation of the median survival times. D7L1 knockout mosquitoes had a significantly smaller wing area than their wild type counterparts, but there were no differences in wing or centroid size. Mosquitoes harboring one copy of a gene drive cassette under expression of the nanos or ZPG promoters did not have significantly different fitness compared to the Higgs wide eye line, with the notable exception of larva viability.
Fitness data collected here can be used in downstream applications such as mathematical modeling. For studies evaluating novel genetic control strategies, larger cage studies in semi field conditions would be necessary after the small skill fitness studies outlined here. Fitness studies in the lab help identify unintended effects of gene knockouts or knockins.
Knocking out a salivary protein can induce developmental stress, while the gene drive in gene drive one and two mosquitoes showed lower larvae survival rates. Measuring gene drive fitness strongly influenced its persistence in simulating populations after mathematical modeling.
