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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calcium, which increased throughout the life-span, were withdrawn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.
Results
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%.
Discussion
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.
Summary
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
dependant 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.
References
Lansing, A. I. Some effects of the
hydrogen ion concentration, total salt concentration , calcium and
citrate on longevity and fecundity of the rotifer. Journal of
Experimental Zoology, 1942, 91, 195-211 (a)
Lansing, A. I. Increase of cortical
calcium with age in the cells of a rotifer Euchlanis dilatata, a
planarian, Phagocata sp. and a toad Bufo fowleri, as shown by the
micro-incineration technique. Biological Bulletin, 1942, 2,
228-238, (b)
Lansing, A. I. & Scott, G. H. The
effect of perfusion with sodium citrate on the content and
distribution of the minerals in various cells of the cat as shown by
electron microscopy and microincineration. Anatomical Record, 1942,
84, 91-96.
Lansing, A. I. A nongenic factor in
the longevity of rotifers. Annals of the New York academy of
Sciences, 1954, 57, 455-464.
Sincock, A. M. Calcium and Aging in
the Rotifer Mytilina brevispina var redunca. Journal of
Gerentology, 1974, 29 514-517.
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