The alkaline Comet assay was done as described by
Singh et al. (1998) with several modifications for
hemolymph (Ianistcki et al., 2009). Images of 100 randomly
selected cells (50 cells from each of two replicate
slides) were analyzed for each individual. To calculate a
damage index (DI), cells were visually classified into one
of five classes based on tail size (0 = no tails and 4 = maximum-length
tails) as assessed by light microscopy. This
classification resulted in a single DNA damage score for
each individual and consequently for each group studied.
The DI for each individual could range from 0 (completely
undamaged = 100 cells x 0) to 400 (maximum damage =
100 cells x 4). The damage frequency (DF, in %) was calculated
for each sample based on the number of cells with tails
vs. those without tails (Heuser et al., 2002). All sides were
coded for blind analysis.