Source of mosquito colonies and mass rearing procedures
Experiments were carried out with mosquitoes from three established colonies. Anopheles arabiensis (Dongola strain), were sourced from field collections in the Northern State of Sudan and transferred to the Food and Agricultural Organisation/ International Atomic Energy Agency (FAO/IAEA) Insect Pest Control Laboratory (IPCL) in Seibersdorf, Austria by the Tropical Medicine Research Institute in Khartoum in 2005. Aedes albopictus (Rimini strain) were sourced in Rimini, Italy and transferred to the IPCL by the Centro Agricoltura Ambiente “G. Nicoli” in Crevalcore, Italy in 2010. Aedes. aegypti (Brazil strain) were sourced in Juazeiro, Brazil and transferred to the IPCL by Moscamed, Brazil in 2012. All strains have been subsequently maintained at the IPCL under controlled temperature, relative humidity (RH) and light regimes (27 ± 1°C, 70 ± 10% RH, 12:12 hour light:dark (L:D) photoperiod with 1 hour periods of simulated dawn and dusk). Eggs used for these experiments were generated following the An. arabiensis and Aedes rearing guidelines developed at the IPCL [19,20]. An. arabiensis larvae were mass-reared in plastic trays (100 x 60 x 3 cm) containing 4 litres of deionized water. Four thousand eggs were added per tray within a plastic ring floating on the water surface. Larvae were fed daily with 1% (wt/vol) IAEA diet developed and described in [21]. Aedes larvae were mass-reared in the same way as An. arabiensis larvae, but with 5 litres of water per tray and at a larval density of 18, 000 first-instar (L1) and provided with 7.5% IAEA diet as detailed in [22].
Pupae collection
An. arabiensis pupae were manually separated from larvae using a cold-water vortex technique as described in [23] and males separated from females by observing the terminalia under a stereomicroscope [24]. Aedes pupae were sexed mechanically using a Fay-Morlan glass plate separator [25] as redesigned by Focks (John W. Hock Co., Gainesville, FL [26]). Male pupae were left to emerge inside 30 × 30 × 30 cm cages (BugDorm, Taipei, Taiwan) and provided with either a 5% (An. arabiensis) or 10% (Aedes) sucrose solution.
Dust colour, optimized dust quantity and marking technique
The initial dust amounts tested with An. arabiensis were 1000 and 500 mg of dust per 100 male mosquitoes, based on the amounts used by [7] to mark Culicoides midges but these severely impacted immediate post-dusting survival. Therefore, a subsequent series of dust weights were investigated per 100 males and mortality assessed after 24 hours, i.e. 100, 75, 50, 15, 10, 7.5, 6.3 and 5 mg of dust, with the lowest dust amount (5 mg) chosen as the optimal weight for all subsequent experiments. This amount was chosen as it provided an even coating of dust that was visible with both the naked eye and under UV light. A lower series of dust weights were chosen for determining the optimal amount to use for Ae. aegypti and albopictus males, (1.5, 1, 0.75 and 0.5 mg per 100 males), as it was discovered during the first marking session that 5mg, despite marking adequately, a surplus of dust remained. It was postulated that this may be due to their smaller body size as was noted when comparing the weight and volume occupied by 1000 males of all three aforementioned species in earlier laboratory tests, with both batches of male Aedes species weighing less than that of An. arabiensis (NJC personal observation) .
Initially, three colours of fluorescent pigment were investigated - A-11 Aurora pink, A-17-N Saturn yellow and A-19 Horizon blue, all from the DayGlo® series as it is a brand routinely used to mark various other insects within the IPCL laboratory. Plastic urine cups (x9 100 ml) were zeroed on an analytical balance and 5 mg of dust added to a cup, with 3 replicates per colour. After the addition of a plastic lid, the cups were shaken vigorously to coat the interior evenly. 12 batches of 100 male mosquitoes were immobilized at 4°C. All batches were transferred to the pre-dusted cups via a mouth aspirator. The cups were then gently rotated for 30 seconds, equating to 25 full rotations, to ensure all mosquitoes were evenly coated. The remaining 3 batches were rolled inside an undusted cup and served as controls. All males were returned to their original Bugdorms, maintained within the lab, whilst still inside their cups. The lid was removed, the cup placed on its side and the mosquitoes given sufficient time to recover before the cup was removed.
Marking and adult male longevity
The impact of marking on male An. arabiensis longevity was assessed by comparing marked experimental with unmarked control males. Six batches of 100 male pupae were sexed under a stereomicroscope and allowed to emerge in Bugdorm cages with access to a sugar solution. All batches were immobilized and dusted as previously described. Three batches were marked with 5 mg of dust with the remaining 3 left undusted and serving as controls. Survival was monitored by removing dead individuals daily until all cages were empty on day 47. Aedes males were marked as described above for An.arabiensis but with 1.5, 1, 0.75 or 0.5 mg per 100 adults. Survival was monitored for 28 days post-dusting.
Dust persistence over time
To investigate the persistence of dust over time in An. arabiensis, 3 groups of 500 males were immobilized and marked as previously described and released into large field cages (1.8 m2) with a sugar solution provided. Two black plastic cups (500 ml) were placed inside each cage. One in a horizontal and one in a vertical position. Every second day, a lid was placed over each cup prior to removal to determine how many mosquitoes were inside. A photograph of each cup was taken to assess the persistence of the mark over time. All mosquitoes were then released into a fourth cage, to prevent resampling of the population, until few or no mosquitoes were collected in the black cups for several subsequent days.
Dust transfer between marked and unmarked adults
Marked males were caged with unmarked males and females to determine whether they are capable of transferring dust. Pupae were sexed under a stereomicroscope into sets of 100 to populate 9 large Bugdorm cages (30 x 30 x 30cm) containing 100 of each sex. A further 9 sets of 100 males were sexed and allowed to emerge in small Bugdorm cages (15 x 15 x 15 cm). All cages contained a sugar solution. The 9 small cages of males were immobilized and dusted with 5 mg of dust as previously described. Each set of dusted males was then transferred to a large Bugdorm cage containing 100 undusted males and females. After 1 day, all marked males were then carefully aspirated out of 3 randomly selected cages before the cages were placed in a -20°C freezer to kill all remaining mosquitoes. Males and females were then screened under a stereomicroscope to check for the presence of dust particles. On day 3 post-dusting, this step was repeated with an additional 3 cages and again on day 7 with the remaining 3 cages.
Statistical analysis
Binomial linear mixed effect models were used to analyse the impact of the various dust treatments on survival (response variable). The dust treatments were used as fixed effects whilst the replicates were set as random effects. The significance of fixed effects was tested using the likelihood ratio test [27,28]. Fixed-effects coefficients of all models and their corresponding p-values are reported in Tables S1-S5.