Fitness experiments

To determine the impact our GCaMP6s transgene insertion on mosquito fitness, a series of fitness experiments comparing the female fecundity, male fertility, larval hatchability, and duration between larval and pupal stages between our GCaMP6s +/+ line and the wild-type line the line was originally derived from. Female fecundity was determined by mating 100 virgin females of both the GCaMP6s +/+ and wild-type line to 50 wild-type males. Females were allowed to mate for 3 days after eclosion and were given access to anesthetized mice for 15 min on the 5th and 6th day after eclosion. Two days after blood feeding, single bloodfed females were individually captured into vials lined with moistened filter paper. Nonblood females were not collected. Bloodfed females were allowed 3 days in the vials to oviposit their eggs and were removed on the third day. Oviposited eggs were then counted. To determine male fertility, 25 males of both strains were mated to 100 virgin wild-type females and the same procedure for calculating female fecundity was used. To test egg hatching rate, eggs from single pair crosses of GCaMP6s +/+ (♀) X +/+ (♂) and +/+ (♀) X +/+ (♂) were counted and hatched 4 days after oviposited. Emerged larvae were then counted at the L2 stage. To calculate larvae to pupae development time, larvae of both strains were hatched and separate into 5 pans filled with 2.5L of water with 100 larvae per pan. The number of pupae emerged was counted everyday post hatching to estimate the number of days for larval developmental time.