Survival in the absence of sugar water
All adults successfully eclosed and were alive in the cage on day 1. On day 2, 12 males were dead and on day 3 the remaining 36 males had died. We concluded that a minimum of 2 nights of feeding is necessary to ensure all males have imbibed the dyed sugar water or that they would die under the experimental environmental conditions. As a precaution, we routinely marked males for 3 nights.
Acute dye toxicity
Rhodamine acute toxicity
Of the three cages of males exposed to 0.8% rhodamine, after 3 nights of exposure, all males in two of the cages had died and this dose was not pursued further. In observations of the three cages of 20 males treated at 0.2%, 11, 4, and 5 males average mortality = 33%) were dead after 2 nights of exposure and we continued observations at this and lower doses.
Uranine Acute Toxicity
After 4 days, little mortality was observed at any concentration (n = 0, 3, 3, 2 / 50 at 0.1, 0.2, 0.4 or 0.8% uranine, respectively) and there was no indication that mortality varied with concentration (F=1.65, d.f.=8,11, p=0.18).
Detecting marker when fed at different concentrations
Marking of males after 3 nights of feeding on 0.025% rhodamine was difficult to detect with certainty and no further observations were made at that concentration. Marking was easily detectable at 0.05 and 0.1%.
Uranine was tested for marking at concentrations of 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8% for 2 nights. No marking was detectable at the two lowest concentrations, 0.05 or 0.025%, and those were discontinued.
Detection by various methods
Both markers were more sensitively detected using the fluorescence microscope and, consistent with the other observations made elsewhere, rhodamine could generally be detected at lower concentrations regardless of the method (Fig. 1).
Durability of marking
When adults were marked with 0.1 and 0.2% rhodamine, marker was detected every week for 3 weeks after marking in all individuals that were alive (0.1%, 93 females, males 79; 0.2%, 89 females, 76 males). In the 0.1% treatment cages, 33 females and 19 males remained alive at week 3. By the final week of the 0.2% cages, 29 females and 16 males were alive. No marker was identified among the living rhodamine controls (69 females, 53 males). Because all living treated adults remained marked, no statistical analysis was warranted.
Uranine produces a yellow-green color that is similar to natural autofluorescence and, at low treatment doses, these can be difficult or impossible to distinguish (Fig. 2). The proportion identified as uranine-marked in control cages did not vary among the different trials and all control cages were considered as one level (F=0.03, d.f.=55,61, p=0.99). The level of false positives in control treatments led us to ask at what dose would treatment effects be clearly distinguishable from these. In both sexes, the proportion identified as marked did not differ from controls in either dose levels 0.1 or 0.2% (F=0.88, d.f.=61,63, p=0.42 and F=2.28, d.f.=67,68, p=0.14 respectively).
A higher proportion of male mosquitoes were identified as ‘marked’ (F=14.45, d.f. 66,67, p=0.0003), but male and female mosquitoes responded similarly to the dose received and through time (sex:week F=2.72, d.f.=65,66, p=0.10; sex:dose F=1.22, d.f.= 66,68, p=0.30). There was, however, an interaction between dose and week (F=4.48, d.f.= 68,70, p=0.015); the proportion identifiable as marked declines differently through time as a function of dose.
At no dose was the marking visible in all individuals for the full 3 weeks in either sex though all mosquitoes marked at 0.8% were detectable after 1 and 2 weeks. All males marked at 0.4% were detectable for 2 weeks.
Detection of mating by marked males
Results of the detection of mating are shown in Table 1. Only one test at the dose rate of 0.05% rhodamine was conducted after it was determined that the marker could not be reliably detected in males when males were fed at this rate. In spite of this, the marker could be detected in all matings from males marked with 0.05% rhodamine; though the number was small, as a mating marker it appears useful at concentrations that are not high enough for detecting the marker in the male body. At all doses of rhodamine, matings with the marker could be easily detected in almost all females.
At the lowest dose of uranine (0.2%), the marker could be detected in the spermathecae although the marker could not be reliably detected in the adult males themselves.
Spermatheca marking durability
Spermathecae that were positive for sperm were all positive for rhodamine at 3, 11, 17 and 24 days after mating (positive/negative, 8/14, 6/4, 8/2, 1/0). In the other experiment using uranine marking, spermathecae that were positive for sperm were all positive for uranine when dissected and observed at 4, 11, 18 and 25 days (positive/negative, 7/15, 9/1, 9/1, 8/2 respectively).
A slightly greater proportion of females were mated by marked males than with unmarked males (mean=0.57, 95%CI=0.51-0.63, χ 2=23.93, d.f.=14, p=0.047, Fig. 3).
The treatments, however, did not affect this proportion. The effect of dosage did not depend on the marker used (F=2.79, d.f.=10,11, p=0.13). The dose received itself did not affect choice of male type (F=1.97, d.f.=11,13, p=0.19) nor did the marker used (F=1.18, d.f.=13,14, p=0.30).
In neither marker trial, did the marker status of the accompanying mosquitoes in the cage affect survival patterns (uranine: L.Ratio=0.48, d.f.=9,10, p=0.49; rhodamine: L.Ratio=0.10, d.f.=9,10, p=0.76).
With uranine, the two mosquito sexes responded differently to dosage; males generally had shorter lives than females, but were not as sensitive to increasing dye concentrations as were females (Table 2, L.Ratio=58.38, d.f.=8,9, p<0.001).
With rhodamine, longevity in control males was also shorter than for control females and, although the range of doses was low, longevity also declined less precipitously with increasing dye concentration than seen in females (Table 2, L.Ratio=14.37, d.f.=8,9, p<0.001).