The effects of polyploidy on life span of Didymium iridis

EXPERIMENTALMYCOLOGY 6,71-76(1982)

The Effects of Polyploidy on Life Span of

JIM CLARK AND PERRY MULLEAVY’

T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506, ar?d Departmerzt of Botany, University of California, Berkeley, Caiifornia, 94720

Accepted for publication August 10, 1981

CLARK, J., AND MULLEAVY, P. 1982. The effects of polyploidy on life span of Didymium iridis. Experimental Mycology 6, 71-76. The life spans of Didymium iridis plasmodia, representing isogenic ploidal series, decrease with increasing ploidy levels. Polyploid nuclei, especially 8iV 2~3

above, are unstable and rapidly decrease in nuclear size. The survivors of this process reach a stable diploid to tetraploid level which they maintain until the start of programmed senescence. The termination of the life span is then accomplished by a distinct senescent phase which is charac- terized by progressively increasing nuclear size.

INDEX DESCRIPTORS: Didymium iridis: plasmodium: polyploid, senescence.

The presence of senescence in cilia& (Sonneborne, 1954; Nanney, 1980), fungi (Marcou, 1961; Esser et al., 1980), and the Myxomycetes (Poulter, 1969; Kerr and Waxlaw, 1968; Knott and Clark, 1980) has allowed these lower eukaryotes to be used in the search for general principles of aging. The potential usefulness of the Myxomy- cetes is enhanced by their heterothallic diplohaplontic life cycle (Collins, 1961) which make possible the construction of genetically identical individuals of different ages.

Poulter (1969) found that the sexually produced diploid plasmodium of Physavum po~ycep~alu~ Schw. could not be main- tained indefinitely by routine, serial sub- culture cm nutrient, solid medium. Vegeta- tive life span was minimally affected by en- vironmental factors and it was observed

at the genome of the individual was the key determinant of longevity. During the perisd of reduced growth prior to cell death in P. polycephalum, McCullough et al. (1973) reported that there was a progressive increase in the number of large polyploid nuclei. Kerr and Waxlaw (1968) reported that they had recovered a variant with a

’ Present address: Mushroom Research Laboratory, Produce Products Division, Ralston Purina Co., Checkerboard Square, St. Louis, MO., 63188.

limited life span from cycloheximide- resistant mutants of a nsrmally nonaging apogamic strain of Didymium iridis (Dit- mar) Fries (as D. nipvipes), while Lott and Clark (1980), using a sexual strain (HonB) of Didymium iridis, found that it had a geneti- cally determined, limited life span termi- nated by a senescent phase. Using the same isolate, Clark and Makim (1980) have also reported a progressive increase in the number of large nuclei during the senescent stage. The large nuclei could be t~rn~~rar~~y removed by allowing the plasmodia to mi- grate though a Nucleopore filter with 3- pm-sized pores and these re~~~~a~~ ‘%I- tered” ~lasmodia lived longer than um- treated subline plasmodia.

Since different ploidal sublines cam be isolated from a clonal ~~p~~~t~~~ of

ulleavy and Collins, 1979). plasmodia of different ploidal levels, having the same genetic information, cam be produced (N and 2N sublines of one clone crossed to N and 2N sublines of a second clone w d give 2N, 3N, and 4X plas- modia) I therefore wished to investigate the effects of ~~1y~lQidy on life span in view of the fact that ~~~yp~oidy was apparently associated with senescence of normal dip loids and the suggestion (Adler and Nolt, 1974) that po~yce~hu~~~ “polypioids” tended toward senescence rapidly.

71 0147-59751821010c71-o6$e)2.00/(9 Copyright 0 1982 by Acadenk Press. Inc. AU rights of rqroduction in any form reserved.

72 CLARK AND MULLEAVY

MATERIALS AND METHODS

Didymium iridis strains Honl, CR2, and Pan2 were used in these studies. Haploid myxamoeba derived from single-spore iso- lates will divide mitotically to form a myx- amoeba1 clone of identical genotype. Sta- ble spontaneously produced polyploid sub- lines were previously isolated from these strains (Mulleavy and Collins, 1979, 1981) and used in this study. The myxamoebae can also act directly as gametes and there- fore the mixing of clones of opposite mat- ing type (Collins, 1961) leads to the produc- tion of the plasmodial stage. This plasmo- dium is then maintained by weekly serial transfers (of 2-cm2 sections) to freshwater agar plates with a feeding of rolled oats.

Nuclear DNA measurements were made on plasmodia hydrolyzed for 60 min in 5 N

HCl at 23°C immersed in Schiff reagent for 90 min, dehydrated through a graded ethanol series, cleared in xylene, and then mounted in refractive index oil (1.540 for myxamoebae and 1.544 for plasmodia). DNA values were determined by measuring light absorption in Feulgen-stained nuclei using a Zeiss 03 or CYDAC scanning mi- crospectrophotometer. In most instances 50 individual nuclei were measured to obtain a mean DNA value for a plasmodium. Before and after each group of measurements, 10 replicate measurements were made, from

which an estimate of random measuring error could be computed. A stable diploid clone CR2-25(O) was used as an internal standard since all samples were not stained simultaneously.

Nuclear size determinations were made using an ocular micrometer in a Leitz phase contrast microscope at 1000x magnifica- tion. Determinations were made on unfixed plasmodial squash preparations.

RESULTS

Life Spans of Polyploid Plasmodin

The myxamoebal clones used in this study and their Feulgen-DNA-measured ploidal levels are given in Table 1. These reisolated clonal ploidal levels were deter- mined by Mulleavy and Collins (1979) and maintained as encysted stocks. Although Feulgen-DNA levels were not rechecked prior to crossing the clones, nuclear size was measured (Table 1, Fig. 3) and found to fit expectations. When these 7 clones are crossed in all possible combinations, within the constraints of mating type, they pro- duce 14 different plasmodia with a number of presumptive ploidal levels (Feulgen- DNA measurements were made for 7 of these plasmodia and nuclear size mea- surements on all 14, Table 2). The average life spans of these 14 plasmodia, derived from 20 replicates of each of the crosses,

TABLE 1 Designation, Nuclear Size, Feuglen-DNA Measurement, Ploidy Level, and

Mating Type of the Myxamoebal Clones

Amoebal clone Nuclear sizea DNA unitsb Ploidy Mating type Other designationC

Honl-7(l)d Honl-7(2) CR2-25(O) CR2-25( 1) (X2-63(7) CR2-63(9) Pan2-16(11)

3.7 c 0.1 2.6 5 0.0 3.7 f 0.1 2.6 + 0.1 5.0 k 0.2 5.9 IO.2 5.2 t 0.1

10,459 k 72 2N 5,271 +- 42 1N 9,452 + 59 2N 4,183 k 45 1N

20,651 % 181 4N 39,980 t 419 8N 20,321 + 224 4N

A2 Honl-7.RC-1 A’ Honl-7.RC-2 A4 CR2-25 AS CR2-25(3-30-76) A5 A5 A?

n Average nuclear diameter, in pm, and standard error determined for 20 nuclei. * Average Feuglen-DNA measurement, in CYDAC units, and standard error for 150 nuclei. c In Mulleavy and Collins (1979). d The number in parentheses indicates a subline isolated from the original clonal population, such as Honl-7.

POLYPLOIDY AND LIFE SPAN 73

were determined (Table 2) and were found to be generally shorter and more variable

0

0

than those of most normal diploids (Lott and Clark, 1980). 87

These 14 plasmodia can be divided into 2 e

five groups: Honl-7 x CR2-25’s (plasmodia Z6 I L

1,2,6, and 7 = x in Fig. l), Honl-7 x CR2- b! is

63’s (3,4,8, and 9 = 0 in Fig. l), Honl-7 x .s Pana-16’s (5 and 10 = o), Pan 2-16 x CR2- ,”

94 25’s (11 and 12 7 Cl), and Pan2-16 x CR2- 63’s (13 and 14 = e), with each group con- sisting of different ploidy levels of the same genetic information. In Fig. 1, the average life spans are graphed against ploidy levels to show that within each genotypic group there is a nearly universal trend toward shorter life spans with increasing ploidy.

PLOIDY

FIG. 1. Graph of average life span, in days, against ploidal level for the 14 different plasmodia. Honl-7 x CR2-25 series (x), Honl-7 x CR2-63 series (0). Honl-7 x Pan2-16 series (V), Pan2-16 x CR2-25 series (0). and Pan2-16 x CR-63 series (0).

Since the chromosome number contrib- uted to the plasmodia by the different clones are not always balanced (i.e., Honl-7(2) x CR2-63(9) is a 1N x 8N cross) it was possible that the short life spans were due to chromosomal imbalance. However, when chromosomal dosage ratios are com- pared to average life span there does not

appear to be a major correlation between them (Fig. 2). There is fairly broad dis- tribution of life spans within each dosage ratio and within each ratio there also is a

TABLE 2 Mean Life Spans of the 14 Plasmodia of a Variety of Ploidal Levels Which Result from Crossing the

7 Myxamoebal Clones in All Possible Combinations within the Constraints of Mating Type

Number Cross Nuclear size” DNA units0 Ploidy” Life span”

2 3 4 5 6 7 8 9

10 11 12 13 14

Honl-7(l) x CR2-25(O) 5.2 2 0.1 Honl-7(l) x CR2-25(l) 4.3 2 0.1 Honl-7( I.) x CR2-63(7) 5.8 2 0.3 Honl-7(l) X CR2-63(9) 6.0 i 0.1 Honl-7(l) x Pan2-16(11) 5.8 i 0.2 Honl-7(2) x CR2-25(O) 4.6 + 0.1 Nonl-7(2) x CR2-25( 1) 4.0 +- 0.1 Honl-7(2) x CR2-63(7) 5.6 -c 0.2 Honl-7(2) x CR2-63(9) 5.8 -+ 0.1 Honl-7(2) x Pan2-16(11) 5.2 It 0.2 Pan2- 16( 11) x CR2-25(O) 5.6 k 0.1 PanZ-16(11) x CR?+-25(l) 5.6 ir 0.2 Pan2-16(11) X CR2-63(7) 6.4 -t 0.4 PanZ-16(11) x CR2-63(9) 6.9 i 0.3

- 639 i 11 917 - 13

1256 ir 24 -

606 + 12 408 i- 8 836 _c 18 356 -t- 15

- - - - -

4N 59.1 '- 3.9 3N 60.9 s 5.0 6N 80.7 -c 9.1

ION 30.7 F 3.2 6N 45.5 2 2.2 3rd 82.3 It 5.8 2N 83.5 i 4.1 5N 66.6 +- 4.9 9N 34.6 F 3,6 SN 49.7 f 2.3 6N 44.1 t 7.7 5w 85.3 F 6.6 8M 48.7 + 2.9

12N 40.7 i 2.6

a Average nuclear diameter, in pm, and standard error determined for 10 nuclei of a young plasmodium. b Average Feuglen-DNA measurements, in Zeiss 03 units, and standard error for 50 nuclei of a re!a&iveiy

young plasmodium. Values determined for only 7 of the 14 types. f Presumed ploidai levels derived from adding gametic ploidal levels. d Mean life spans and standard errors derived from 20 replicate crosses.

74 CLARK AND MULLEAVY

Go-

.S .2

SO- ::

30- .10

O- I I I I Ii, IQ 1’4 14

CHROMOSOME DOSAGE

FIG. 2. Graph of average life span, in days, against chromosome dosage ratio derived from the two amoeba1 clones of the cross. The number by the point indicates the ploidy of that plasmodium.

gradient of decreasing life span with in- creasing ploidy.

Nuclear Size over Life Span

Since it was known that polyploid plas- modia can eliminate extra chromosomes (Collins et al., 1978) and that they appar- ently increase in chromosome number dur- ing senescence (McCullough et al., 1973; Clark and Hakim, 1980), we decided to fol- low nuclear size throughout the life span. Figure 3 is a graph of ploidal level against average nuclear diameter for the 7 amoeba1 (+) clones and the initial (Day 14) nuclear sizes of the 14 plasmodial(0) types. These values show the expected exponential rela- tionship and indicate that nuclear size can be used as an approximation of ploidal level. Nuclear DNA measurements for the nuclei of 7 of the plasmodial types are also shown in Fig, 3. In this case, the expected linear relationship is not quite achieved and in one case (9N) the amount of DNA is much less than expected. A comparison of the presumed 9N plasmodium, Honl-7(2) X CR2-63(9), with a 2N plasmodium, Honl- 7(2) x CR2-25(l), shows that the 2N plas- modium (Fig. 4A) has a tighter histogram with little dispersion while the presumed 9N plasmodium (Fig. 4B) has a major dis-

. 0

4 + .

02 3 4 5 6 0 zw 400 GO0 800 1000 1200 wm

NUCLEAR DIAMETER iv m 1

NUCLEAR DNA LEVEL (ARBITRARY “NITS)

FIG. 3. Graph of ploidy number against nuclear size or DNA content. Amoebal clone nuclear diameters (+), plasmodial nuclei diameters (O), and plasmodial nuclear DNA content (0).

persed group at the 2N (400 units) level be- sides minor groups at higher ploidy levels. The presence of these higher levels indi- cates that the Honl-7(2) x (X2-63(9) plas- modium was originally 9N but has already shed many chromosomes from most of its

25r

FIG. 4. Histograms of nuclear DNA content. (A) Plasmodium Honl-7(Z) x CR2-25(l), a normal 2N diploid. (B) Plasmodium Honl-7(2) x CR2-63(9), a presumptive 9N polyploid which has shed much DNA from most of its nuclei.

POLYPLOIDY AND LIFE SPAN 75

TABLE 3 Average Nuclear Size of Representatives of the 14 Piasmodial Types at Weekly intervals

Week l(4N) 2ON) XW 4(10N) 5(6N) 6(3N) 7(2N)

2 5.2 t o.aa 4.3 + 0.1 5.8 +- 0.3 6.0 t 0.1 5.8 t 0.2 4.6 i- 0.2 4.0 + 0.1

3 4.7 2 0.1 4.3 f 0.1 5.1 -c 0.1 6.2 k 0.2 5.6 -c 0.2 4.0 27 0.2 3.4 ” 0.1

4 4.4 t 0.1 4.0 c 0.0 4.5 + 0.2 4.6 t 0.1 3.8 t @.l 3.3 + Q.1

5 4.2 ?z 0.1 4.3 c 0.2 3.5 i 0.1 4.1 2 0.1 3.5 i_ 0.1

6 4.2 -+ 0.1 4.0 ” 0.1 3.9 i 0.1 3.8 ” 0.1 3.6 rfr 0.1

7 4.4 i 0.1 4.2 -c- 0.1 3.6 + 0.1 3.6 i- 0.1 3.4 IO.1

8 4.4 k 0.4 4.6 + 0.4 4.0 2 0.2 4.4 t 0.2 4.0 c 0.3

9 4.5 t 0.1 4.0 +- 0.0 3.4 + 0.:

IQ 4.9 ‘-’ 0.1 3.7 4 0.1 3.6 t 0.1

il 3.5 i 0.1 3.1 k 0.1

I2 3.8 i 0.1 3.7 F 0.1

i3 4.0 + 0.0 3.7 t 0.43

14 3.7 2 0.1 3.3 ” 0.1

1.5 3.6 k 0.1 3.6 L 0.1

16 3.5 f 0.1 3.8 ” 0.1

17 4.2 + 0.1 4.0 -c 3.1

WN) 9(9N) lO(5N) 1 l(6N) 12(5N) B3@) l4(!2M) -

2 5.6 + 0.2 5.8 f 0.1 5.2 * 0.2 5.6 + 0.1 5.6 t 0.2 6.4 i 0.4 6.9 rt 0.3

3 4.6 k 0.1 5.2 I? 0.2 5.0 + 0.1 5.3 k 0.1 5.4 + 0.1 5.4 + 0.2 6.1 1 c.9

4 4,4 c 0.1 5.0 k 0.1 4.5 ? 0.2 5.4 i: 0.1 5.4 t 0.1 4.6 rt 0.1

5 4.6 -c 0.1 4.8 k 0.1 5.0 + 0.2 4.9 IT 0.2 5.1 Tr 0.2

6 4.7 t 0.1 5.2 t ‘3.2 7 5.5 i 0.2 8 5.1 + 0.3 9

10 11 12 13 14 1s 16 17

c Average size, in pm, of 10 nuclei.

nuclei (Collins et al., 1978). The DNA mea- surements were made on relatively young plasmodia and in most cases while there were no reductions from the expected levels, for the lower ploidies, the presump- tive levels, for the higher ploidies, were never detected. These expected levels may never have been achieved in the cross or the low levels may be due (as we suggest) to rapid early loss of chromosomes. Also, the pllasmodial types, except for No. 9, main- tain their expected rank order and thus in-

dicate that polyploids are being formed m the crosses even if they are not completely additive for the gametic levels.

However, there appears to be extensive changes over time when nuclear diameter is used as a measurement of ploidy in follow- ing representatives of the 14 ~~asrno~~a~ types (Table 3). There is a general rapi crease in diameter that ends aroun fourth week where it stablizes, if the modium survives, at a diploid to tetr~~lo~~ level. The surviving plasmodia will tben

76 CLARK AND MULLEAVY

maintain these levels until the last several REFERENCES

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The life spans of Didymium iridis plas- modia are considerably affected by nuclear polyploidy. Within a polyploid series having the same genetic information, increasing ploidy causes a decreasing life span. Also these polyploids, especially the higher levels, are apparently very unstable in the plasmodial state and rapidly shed chromo- somes (Collins et al., 1978). This decrease in nuclear size appears to be critical in maintaining a viable plasmodium since many die during this process. The rest stablize at a diploid to tetraploid level and apparently live most of their programmed life span (Lott and Clark, 1980). Termina- tion of the life span is accomplished by a distinct senescent phase which is partially characterized by increasing nuclear size (Clark and Hakim, 1980).

The apparent relaxation of nuclear size controls during senescence and the detri- mental effect of initial polyploidy on the plasmodium may provide insight into the mechanisms of aging. Polyploidy often ac- companies vertebrate liver cell aging, hypertrophy repair, and drug responses (Brodsky and Uryvaeva, 1977) and seems to indicate a loss of control functions. The large, coenocytic plasmodium may have its nuclear size highly integrated with function such that changes interfere with normal cytoplasmic regulation. Therefore, a pro- grammed breakdown of this integration control could result in cellular malfunction.

ACKNOWLEDGMENTS

This investigation was supported in part by National Institutes of Health Grant NIA AG00809-01 to J. Clark. We wish to express our appreciation to 0. Col- lins for the use of his scanning microspectrophotome- ter and Sarah Gage for her technical assistance.

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