Life Extension in the Rotifer Mytilina brevispina Var Redunca by the Application of Chelating Agents.
Andrew M Sincock PhD
Extensions of life span and reproductive period were achieved in the rotifer Mytilina brevispina var redunca by regular immersions n solutions of one of the following chelating agents, sodium citrate, sodium tartrate, EDTA and EGTA. It was shown by means of radiolabelling with 45 calcium that the rate of calcium accumulation in the chelation treated rotifers was markedly lower than in the untreated controls. Furthermore, significant quantities of calcium, which increased throughout the life-span, were withdrawn from rotifers at chelation. These results add further evidence to a mechanism of aging in this organism that is related to a detrimental accumulation of calcium during the life period.
Previous experiments which have investigated the effects of calcium on the life-span of rotifer (Lansing, 1942a,b; Sincock, 1974) have shown that over a certain range of concentration, life is shortened by an increase in the environmental level of calcium. Measurements of the calcium present in rotifer (Sincock, 1974) have revealed an accumulation throughout adult life which is more rapid and substantial at higher calcium concentrations in culture regime. Lansing (1942b) demonstrated that immersions in sodium citrate solutions, which he believed would remove calcium from rotifers (Lansing & Scott, 1942), significantly increase their life expectancy. The present study shows that life extension is produced by chelating agents other that sodium citrate and that the calcium level in rotifers is reduced by the chelation procedure.
Materials and Methods
Rotifers of the specie Mytilina brevispina var redunca were cultured axenically at 24 C. on a dietary regime of Chlamydomonas rheinhardtii as described for controls in Sincock (1974).
In the first experiment, groups of 30 rotifers were immersed for 45 sec. either in .05% sodium citrate or a 0.25% solution of one of the following -- sodium tartrate, ethylene diamine tetra-acetic acid (di-sodium salt), i.e., EDTA, 1,2-di(2 aminoethoxy) ethane tetra-acetic acid, i.e, EGTA. The treatment was given to each rotifer individually in 0.04 ml drops of control medium before being returned to the culture slide. Two groups wee not exposed to the chelating agents. One of these, a parallel control, was washed three times in 0.04 ml of control culture medium on the same days as the chelation-treated rotifers, while the other, the control, was transferred only to fresh culture on each day along with all other groups in the experiment.
In the second experiment direct measurement was carried out on the calcium levels of rotifers treated with 0.5%% sodium citrate. The rotifers were cultured in control medium supplemented with 45 calcium in the manner described by Sincock (1974). Ten animals from the control group were removed from their culture drops on each day of life, washed three times in 3 ml volumes of control culture medium without 4 calcium, and each individual dried onto a separate filter disc. The same number of animals was washed in 0.5 sodium citrate and rinsed, as previously described, before being dried onto separate filter discs. A third series of rotifers was washed in calcium 40 culture medium before the citrate treatment and each animal processed in the same manner, while the citrate washings and rinsings from each individual were dried on separate filter discs and retained to establish the amount of calcium removed and the amount remaining in each rotifer. Al filter discs were renewed for each individual and their activities were measured ten times on the same day by beta-ray liquid scintillation counting.
In the first experiment, the four groups receiving treatment with a chelating agent showed significant life expectancy and fecundity increases over the untreated controls. (Fig. 1 and Table 1). Close correlation between the survival and egg-laying values of the parallel controls and controls indicated that the extra mechanical handling of individuals produced no ill effects. The greatest survival increase of 75.9% was recorded for the group treated with EGTA, the most calcium specific chelating agent in terms of log beta formation value. However, lower survival increases of 51.7, 49.4 and 43.7% recorded for the sodium citrate, EDTA, and sodium tartrate treated groups were not related to the log beta values of these chelating agents (see table 3). The onset of egg-laying was delayed in all the chelation-treated groups by one day, but the values of subsequent reproductive periods and fecundities were all greater than in the control group.
The results of the second experiment (see fig. 2 and Table 2) showed that the rate of calcium accumulation in the untreated control samples was markedly in excess of the calcium accumulated in the citrate treated rotifers. Indeed, at Day 3, when the first counts were obtained for the untreated controls, no counts were evident in citrate-treated rotifers, while the counts obtained from the citrate washings indicated 100% withdrawal of the 45 calcium present in rotifers before washing treatment. At day 9, when the last count of 1060 cpm was obtained for the untreated controls, a count of only 198 cpm was recorded for the citrate-treated rotifers. Taken in conjunction with the counts obtained from the sodium citrate washings at this time (140 cpm), this corresponded to a reduction in the fraction of calcium removed to 41%. After Day 9, the rate of calcium accumulation in the citrate treated samples began to rise steadily. However, even at day 11, the count recorded for this group was less than 1/3 of the final count registered for the untreated samples, and the percentage of calcium removed as a fraction of the total calcium present before washing remained almost unchanged at 40%. The final count of 782 cpm recorded for the citrate treated rotifers at day 15 was significantly lower than that of the untreated rotifers, and it was accompanied by a reduction in the fraction of calcium removed to 34%.
In the first experiment, it is very obvious that treatment with chelating agent significantly extends life-span of the rotifer Mytilina brevispina var redunca. This extension is noticeably marked in the case of the chelating agent having the highest affinity for calcium in terms of its 1st Order log beta value, but the order of efficiency of the other chelating agents is not related to their log beta values (Table 3). Indeed, if one were to relate the efficiency of calcium removal with life extension, the inefficiency of EDTA in the face of a high 1st Order log beta value for calcium would have to be explained in terms of additional physical and chemical properties, possibly of a toxic nature.
The delayed onset of egg-laying associated with subsequently increased fecundity and reproductive period in all populations that showed life extension (Table 1) was also a feature of the group whose life-span was extended by low calcium conditions. (Sincock, 1974). The significance of delayed first egg production may be better understood in terms of Lansing's (1954) pediaclones where it constituted a recognizable feature of orthoclones whose life-spans were in the process of extending over generations of selection. In this experiment, the delayed egg-laying effect would have to be exhibited as a first-generation feature of a 4-day orthoclone.
In the first experiment, only supposition connects the extension of life with the removal of calcium. Direct measurement of the effect of citrate washing on the calcium content of the rotifers was therefore carried out by adding the radioisotope 45 calcium tot he culture medium. The results (Fig. 2) clearly demonstrated that the rate of calcium accumulation in the untreated samples greatly exceeded the rate of accumulation by the citrate treated group. Furthermore, significant quantities of the radionuclide 45 calcium appeared in increasing amounts throughout life in the citrate washings of the treated rotifers, which suggests that direct removal of the radiolabel was brought about by the chelation procedure.
If one considers the sum of the calcium withdrawn and the calcium present on each day of citrate treatment, the values do not equal the corresponding values for the untreated controls after day 3, (see table 4). This suggests that, quite apart from the calcium removed at each chelation, there is a reduction in the rate of calcium accumulation in the treated rotifers between days of treatment. Furthermore, examination of the percentage of calcium removed at chelation as a fraction of the total calcium already present in each rotifer (Table 2) shows that there is a fall in the fraction of removable calcium from 100% to 34% during the life-span. The inferred increase in a permanently bound calcium fraction in the rotifer may impose a limit of efficiency of the chelating agent in permanently offsetting the attainment of a lethal level of accumulated calcium.
If one postulates that the given calcium content of the control group on a given day may be taken as a measure of its physiological age, then the physiological ages of the treated group may be computed by comparison. For instance, the physiological age of the treated group at Day 9 is 4 and 1/2 days, which represents a 4 and 1/2 day difference from its chronological age. The distinction between physiological and chronological age (fig. 3) enables a comparison to be made between the rates of accumulation of calcium in the control and treated groups on the basis of the calcium already present in the animal (i.e., physiological age). From this comparison, it may be concluded that the rate of net calcium accumulation is proportional to the physiological rather than the chronological age of the rotifer studied.
On Day 15 of life, the physiological age of the chelation treated rotifers is 8 days, and it must be supposed that at this time, the rate of calcium accumulation is so high that the physiological life-span of the rotifer is exceeded before the next chelation treatment. Even if more frequent treatment with the chelating agent proved possible, it is still likely that the increase in the fraction of inexchangeable calcium referred to earlier would impose a limit on the increase of the physiological life-span that could be obtained.
In the absence of all genetic variability saving mutation between individuals homologated with respect to maternal age, one may be confident that the rotifers within the finely controlled culture conditions of the experiments exhibited a detrimental accumulation of calcium throughout adult life that could be slowed with accompanying life extension by treatment with chelating agents that remove calcium.
Rotifers of the species Mytilina brevispina var redunca that had been homologated with respect to maternal age, were cultured at 24C. n artificial saline medium water under standard and aseptic culture conditions. Observations on the survival times of individuals exposed by brief immersion on alternate days of adult life to one of the following chelating agents --- EGTA, EDTA, sodium tartrate and sodium citrate, revealed significant life extension in all cases. The greatest life extension of 75.9 was recorded for the group exposed to the chelating agent having the highest calcium affinity, namely EGTA. However the order of efficacy of the other chelating agents was not related to calcium affinities. Observations on egg production in the chelation-treated populations revealed a single day's delay in the onset of egg-laying but a subsequent increased fecundity and reproductive period relative to the controls.
Direct measurement of the effect of citrate washing on the calcium content of the rotifers cultured in the presence of the radionucleotide 45 calcium revealed a significantly lowered rate of 45 calcium accumulation throughout the life period, which was accompanied by an increasing amount of the radiolabel in the citrate washing medium. The sum of the calcium accumulated and the calcium withdrawn in the treated populations was below the corresponding day's totals of the calcium accumulated in the untreated controls, which indicates a lower rate of accumulation between treatments. Similarly, an increase in the fraction of inexchangeable as evident during life in the treated rotifers, since the percentage calcium removed as a fraction of the total calcium present before the washings fell from 100 to 34%. A comparison of calcium accumulation between untreated and treated samples on the basis of physiological age revealed a rate of accumulation that was dependent on physiological rather than chronological age.
The over-all result of the experiments was to demonstrate that a reduction in the age related accumulation of calcium in rotifers by the application of chelating agents with a high affinity for calcium produced significant increases in life expectancy.
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