Ejfect o f liquid culture on survival
A possible complication of using liquid rather than plate culture was that the overall
concentration of food in liquid culture, although perfectly sufficient to maintain the worms, was probably less than that encountered by worms maintained on a dense lawn of OP50 on agar plates. Reduced food concentrations under several different conditions
extend lifespan in wild-type C. elegans (Klass 1977; Hosono et al 1989). Because it was
desirable to reproduce previous survival data from plates as closely as possible using the liquid culture method, it was decided to raise all worms on agar plates and transfer them to liquid culture at L4, the stage at which survival analyses began. In this way, any effects of liquid culture on larval development were avoided, with effects being limited to the adult phase of the life cycle.
Pilot lifespan studies (methodology described in Main Materials and Methods) indicated that for N2, both male and hermaphrodite survivals in liquid culture were comparable to those published previously on agar plates, although median and maximum lifespans were often slightly increased relative to agar plate culture. For example, while hermaphrodite median lifespan is typically 16 days at 22.5°C on agar plates, it ranged from 16 to 18 days in liquid culture at the same temperature. As in previous studies (Gems & Riddle 2000b), male survival was increased relative to that of hermaphrodites, but to variable extents. As noted previously, male median lifespan was particularly variable, sometimes being lower than that of hermaphrodites (Gems & Riddle 2000b). Male maximum lifespan, however, was almost always longer than hermaphrodite maximum lifespan, and male survival curves often displayed a "tail" indicating reduced mortality at later ages (e.g. Figure 1.1a). However, the degree of separation between male and hermaphrodite survival curves appeared to be less in liquid culture than previously recorded on agar plates (Gems & Riddle 2000b), and in some instances there was no significant overall difference between male and hermaphrodite survival (illustrated in Figure 1.1b below).
As experiments for the project were performed, it became clear that the variability in the extent of male longevity could be problematic. This was because in several trials where the dependence of intrinsic male longevity on certain genes was being determined, N2 males were not significantly longer lived than hermaphrodites. This resulted in an atypical wild-type baseline for increased male longevity with which to compare sex lifespan ratios of mutant strains.
The pilot survival curves shown in Figure 1.1 were obtained using a previously described transfer protocol for agar plate culture: that is, transferring hermaphrodites daily during egg-laying (to prevent confusion with second generation animals) and approximately weekly thereafter, while transferring males approximately weekly from Day 0. This had resulted in generally consistent greater male longevity (Gems & Riddle 2000b). It seemed possible, however, that the unequal treatment of the sexes with respect to transfer frequency could be affecting survival results in liquid culture. For example, it is possible that over time waste products build up in liquid culture wells which have detrimental effects on the worms. Alternatively, worms maintained in the same wells for longer periods may suffer from a degree of hypoxia that frequent transfers to fresh medium might alleviate. For this reason, pilot lifespan trials were repeated, this time transferring both males and hermaphrodites during hermaphrodite egg-lay, and both sexes approximately weekly thereafter (Figure 1.2 below). Care was taken to ensure males and hermaphrodites were always transferred on the same day.
It was found that by transferring both sexes at equal frequency resulted in greater separation of male and hermaphrodite survival, and gave more a consistent degree of difference between them. This meant that in trials where the underlying genetics of increased male longevity were being investigated there was now a more consistent wild- type survival sex ratio baseline for comparison. It was therefore decided to culture both sexes with daily transfer during hermaphrodite egg-lay for the remainder of the project. Since this effect only became apparent some way into the project, certain experiments were performed with unequal transfer of the sexes, and are highlighted in the relevant chapters. Male lifespan may therefore be slightly understated in such experiments.
Figure 1.1; Typical survival curves for N2 males (triangles) and herm aphrodites (squares) with unequal transfer of the two sexes showing (a) reduced male mortality at later ages, P= 0.19 and (b) very little difference between male and hermaphrodite survival P= 0.12 (22.5°C). P= probability that male and hermaphrodite survival differ by random chance (log rank test) (see M ain M aterials and Methods). (a) (b) 0 . 9 f > § 0 5 1 0 15 2 0 2 5 3 0 3 5 I > I 0.3 2 0 2 5 0 5 1 0 15 T i m e ( d a y s ) Ti me ( day s)
Figure 1.2: Typical survival curve for N2 males and herm aphrodites with equal transfer of both sexes (P= 0.0003) (20“C) 1 0 . 9 8 g. 0 > 0 . 7 £ 0 . 6
2
0 . 5 0 . 4 0 . 3 £ 0 . 2 0. 1 0 3 5 4 0 3 0 0 5 1 0 15 2 0 2 5 Time (days )E jfect o f liquid culture m ethod on brood size
Worms raise(i in liquid culture from eggs are usually longer and thinner and have significantly smaller brood sizes than those raised on plates (Klass 1977). As mentioned above, to minimise potential effects on fertility, a bacterial concentration of 1-5x10* cells mb' was used. In order to verify that the liquid culture method described above did not markedly affect brood size, counts of lifetime egg production were performed for N2 hermaphrodites raised on plates and transferred to standard liquid culture at L4 (Table 1.4). There was no significant difference in brood size between hermaphrodites cultured
brood size of the agar plate controls (260) was slightly lower than usual (-300)). By raising worms on plates until L4, it was therefore hoped that as little divergence as possible from results on plates culture had been achieved.
Table 1.4: Brood sizes of self-fertilised N2 hermaphrodites
Culture method after L4 Mean brood size ± s.e. N* F t
60mm Petri dish- agar 260.7± 15.1 10 ---
96-well microtitre plate- liquid (1) 266.2±5.1 20 >0.1 96-well microtitre plate- liquid (2) 288.6± 15.0 9 >0.1 * Number of hermaphrodites scored, f Probability that experimental liquid culture mean differs from control mean on plates by random chance (Student's t test).