Studies on ageing in Turbatrix aceti

Mechanisms of Ageing and Development, 4 (1975) 81-88 © Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

81

STUDIES ON A G E I N G IN T U R B A T R I X A C E T I

M. J. KISIEL, J. M. CASTILLO, L. S. ZUCKERMAN and B. M. ZUCKERMAN Laboratory of Experimental Biology, College of Food and Natural Resources, University of Massachusetts, East Wareham, Mass. 02538 (U.S.A.)

S. HIMMELHOCH Section of Biological Ultrastructure, WeizmannInstitute, Rehovot (Israel)

(Received June 13, 1974; in revised form December 27, 1974)

SUMMARY Morphologic and physiologic changes which occur during senescence in the free-living nematode Turbatrix aceti are described. With age areas of the interchordal hypodermis containing nerve elements thickened, electron-dense aggregates formed within the pseudocoelom and age pigment granules accumulated within the intestinal epithelium. Specific gravity did not change with age. Old nematodes which had reproduced showed increased osmotic fragility, but this change was not observed in virgin females. The parameters characterizing senescence in T. aceti are compared with those of Caenorhabditis briggsae, another nematode being used as a model to study biological ageing.

INTRODUCTION Three free-living nematode species, Caenorhabditis briggsae, Panagrellus redivivus and Turbatrix aceti, are currently being used as models to study biological ageing. A number of changes characterizing senescence have been described in these nematodes. For C. briggsae these age dependent changes include an increase in specific gravity 1 and osmotic fragility 2, an increase in pigment granules within the intestinal epithelium 3, the progressive accumulation of electron-dense aggregates within the cuticle median layer 4 and the pseudocoelom a, degenerative changes within mitochondria and thickening of the interchordal hypodermis2, 4 and an increased susceptibility to fomaldehydeL Buecher and Hansen (unpublished results, 1970) were the first to investigatc the relation of age pigment and vitamin E to nematode ageing. Using P. redivivus, they showed that vitamin E extended the life span and partially reversed the inhibition of maturation caused by high oxygen tensions 6. In addition, an age dependent accumulation of lipofuscin was demonstrated by fluorescent spectroscopy. Reported studies of ageing in T. aceti have involved biochemical changes that ensue during ageingT-1L However, to obtain the required amount of nematode tissues

82 for analysis, in each of these experiments age synchrony was attained by exposing new-born larvae to a D N A synthesis inhibitor. Later studies showed that nematodes so treated were abnormal in many respectsS, lz, therefore casting doubt on the validity of concluding from the nematode experiments alone that ageing of specific biochemical processes had been demonstrated. The current study was undertaken to determine if certain of the parameters which characterize senescence in C. briggsae also develop in T. aceti during ageing, thereby hopefully providing evidence in support of broader generalizations concerning nematode ageing and enhancing the value of these organisms as models in the study of biological ageing. MATERIALS AND METHODS

Culture techniques Cultures of T. aceti were maintained axenically at 27°C in all experiments. Stock cultures were grown in a medium consisting of soy peptone 4 g, yeast extract 3 g, acetic acid 2 ml, heated liver extract 5 ml, and water 93 ml. For most studies T. aceti were reared singly in this medium until they reached the desired age as previously described ~. In tests of reproducing nematodes, each group of 10 nematodes was held in 1 ml medium until reproduction started, then transferred to fresh medium every 3 days until reproduction stopped, so that adults would not become confused with their progeny. Electron microscopy Electron microscope studies were performed on virgin females 35, 37, 38 and 42 days old. Sections were examined from 18 nematodes. Observations of young females (8-12 days old) were previously reported 13. For electron microscopy nematodes were killed and fixed as described by Zuckerman et al. ~, embedded in EPON 812 or Spurr's medium lz, sections cut at 50-90 nm with a diamond knife on an LKB Ultratome 1 or a glass knife on a Danon Ultramicrotome, and stained with uranyl acetate followed by lead citrate. Micrographs were taken either with a Philips 200, JEM-7 or JEM-T-7 electron microscope. Specific gravity determinations and osmotic fragility A set of water nonmiscible fluids (manufactured by Miles-Yeda Ltd., Rehovot, Israel and sold under the name of Gravikit, hereafter called the fluids) of predetermined specific gravity, consisting of a mixture of di-n-butyl phthalate and methyl phthalate, were used to obtain the specific gravity distributions of young and old T. aceti. The specific gravities of the fluids ranged from 1.062 to 1.138, with increments of 0.004. The tests were performed as follows: the tip of a capillary, 100 /~1 capacity and 1.1 mm inside diameter, was placed in the fluid and held there until a 5-7 mm column had penetrated. Then the tip was placed into culture medium until a 5-7 mm column of medium was drawn into the tube. Next, with the tip of the capillary viewed under a dissecting microscope, five nematodes were transferred into the

83 medium. The opposite end was then flame-sealed and the capillary centrifuged at 3200 × g for 5 min at 20°C. After centrifugation, the tube was viewed under a dissecting microscope, and the number of nematodes which had penetrated the fluid or had not penetrated and remained in the medium was recorded. A figure designed to assist in visualizing these steps was given by Zuckerman et al. 1. Osmotic fragility was determined by placing nematodes in distilled water and after immersion intervals of 15 min and 1 h observing whether or not each nematode had burst. Since it was believed that physiologic ageing might in some ways be related to events following the termination of the reproductive cycle, two series of tests were performed. In the first virgin females 8-11 days old were compared with virgin females 44-52 days old. This series comprised 8 trials in which the specific gravity or osmotic fragility of 640 nematodes was determined. Reproducing 10 day old females were compared in the second series with females 25 or 42 days old which had completed reproduction. The specific gravity or osmotic fragility of 562 nematodes was determined during the course of 4 trials in this series. RESULTS

Electron microscopy Cuticle No structural changes were observed with age within the cuticle of T. aceti. In C. briggsae the median layer is translucent and transversed by supporting structures and during senescence, within the clear areas, aggregates of electron-dense materials accumulate 4. In contrast, the median layer of T. aceti consists of rows of rods orientated at right angles to each other to form a lattice 13. Hypodermis Areas of the interchordal hypodermis containing nerve elements frequently thickened greatly with age (Fig. 1A), so that the hypodermal tissue often increased to the extent of displacing the underlying bands of muscle fibers from their normal position. Hypodermal thickening also occurs during ageing of Panagrellus silusiae 15. Mitochondria Mitochondria in which the cristae had been wholly or partially replaced by membranous whorls frequently were observed in old nematodes (Figs. 1B, 2C). An age-related loss of structural integrity of mitochondria was also observed in C. briggsae 2,4, and has been reported from other micrometazoa 16 and higher animals, including man 17. Unusual cristae which are doughnut-shaped in cross section, previously reported from young T. aceti TM, occurred within nematodes of all ages examined (Fig. 1B). Pseudocoelom Electron-dense aggregates occurred with the pseudocoelom of old but not young T. aceti (Fig. 2B). These materials have the same appearance as do similarly located aggregates which are characteristic of aged C. briggsae 5. However, in C.

84

Fig. 1. Turbatrix acrti - Ageing Symptoms. A. 37 day old. Interchordal hypodermal bulges (B) displacing contractile elements (Co) of somatic muscle cells (S) from normal position contiguous to body wall (x 25 000). B. 37 day old. Membranous whorls within a giant muscle cell mitochondrion (M). Doughnut-shaped cristae occur within 2 smaller mitochondria (+). Also shown are the cuticle (C) and the muscle myofilaments (My) ( x 50 000).

Fig. 2. Turbatrix aceti - Ageing Symptoms. A. 37 day old. Dark pigmented inclusions (-) within the intestine epithelium (E). Also shown are the lumen (Lu), microvilli (Mi) and lipid droplets (L). Some of the lipid droplets show evidence of vacuolation (L - ). (x 13 500). B. 38 day old. Electron-dense aggregates (+) within the pseudocoelom (x 12 500); C. 42 day old. Encroachment of a lipid (L) droplet and a mitochondrion (M) between the terminal web (T-t) and the plasma membrane surrounding the lumen (Lu). Membranous whorls within the mitochondrion indicate that it is deteriorating. Also shown are the microvilli (Mi.) Arrows also point to openings which normally occur in the terminal web. (x 36 000)

86 TABLE I OSMOTIC FRAGILITY AND SPECIFIC GRAVITY OF YOUNG AND OLD TURBATRIX A CET11 Age and reproductive status 2

Osmotic fragility a

Specific gravity

Young virgin Old virgin Young reproducing Old - after reproduction ceased

0 0 2.2 14.4"

1.080 1.082 1.070 1.082

1 Figures represent the combined average of all trials. 2 Young were 8-11 days old and old 25-52 days old. a Based on the number of nematodes which burst after 1 h in distilled water. * P > 0.01 - - different from the young reproducing group.

briggsae these aggregates occupy much larger areas of the pseudocoelom and, presumably, are present in greater quantity. Zuckerman, Castillo and Kisie119 suggest that these masses may be insoluble aggregates of crosslinked molecules which are thought by Bjorksten 2° to form during ageing in all animals. Intestine Aged T. aeeti contained membrane enclosed pigment granules within the cells of the anterior region of the intestine (Fig. 2A). These granules were similar in appearance to the lipofuscin, or age-pigment, granules described from old C. briggsae a, but were of less abundance than in C. briggsae. Vacuolated lipid droplets occurred within the intestinal epithelium and hypodermal chords in some sections (Fig. 2A), but in other sections all of the lipid droplets were intact. Lipid droplets and structures containing many membranes occurred between the terminal web and the bacillary layer lining the lumen of old T. aceti (Fig. 2C). Cristae were observed within areas of a few of the lamellar bodies, so that, apparently, at least some of these structures were degenerating mitochondria. Specific gravity and osmotic fragility The specific gravities of the several combinations of young, old, virgin and non-virgin T. aeeti did not differ significantly (Table I). Virgin nematodes did not undergo an age-related increase in osmotic fragility (Table I). Old nematodes which had reproduced were more labile to osmotic stress than were young, reproducing females (P > 0.01); however, non-virgin T. aceti underwent much smaller changes in osmotic fragility than did C. briggsae during ageing z.

DISCUSSION Several of the age-related structural changes observed in T. aceti were also characteristic of ageing in C. briggsae, though differences exist in the degree to which some of the changes were manifested between species. For example, age pigment formation within the intestinal epithelium and the deposition of electron-dense aggreg-

87 ates in the pseudocoelom were each greatly less in T. aceti than in C. briggsae. On the other hand, the number of deteriorating mitochondria and the amount of thickening of nerve tissues in the interchordal hypodermis appeared similar in both species. Similar hypodermal hypertrophy in C. briggsae was thought to interfere with the function of the somatic muscles, possibly explaining, in part, the slower movement of old nematodes2. The lipid vacuoles and the encroachment of lipid droplets and other structures between the terminal web and the plasma membrane of the T. aceti intestine were not observed in C. briggsae. Two facts mitigate against the lipid vacuoles being artifacts formed during processing for electron microscopy: (1) lipid vacuoles were not observed in sections from young nematodes and (2) lipid vacuoles are characteristic of ageing of human diploid cells in culture 21. Similar lamellar bodies were described as being associated with the apical plasma membrane of a Metastrongylus sp. and it was proposed that these structures have lysosomal activity and can move through the bacillary layer into the lumen 22. To our knowledge lipid encroachment between the terminal web and the bacillary layer has not been previously described. Since this phenomenon was not observed in young nematodes, our observations suggest that it is probably a part of the degeneration process accompanying ageing. The comparative differences exhibited by T. aceti and C. briggsae in respect to increases in specific gravity and osmotic fragility during ageing cannot be interpreted with certainty, but observations from our studies and those of others which may bear on these differences will be considered. (1) The C. briggsae cuticle contains within its median layer rigid supporting rods, whereas the comparable area of the T. aceti cuticle is occupied by a lattice of rods. These differences suggest a greater flexibility of the T. aceti cuticle, which may explain in part, the differences in osmotic fragility with age between these two species. (2) Age pigment is formed principally through the peroxidation oflipids 2~, and in C. briggsae this pigment appeared to accumulate to a far greater extent than in T. aceti. The replacement of lighter lipids by heavier, insoluble lipofuscin would lead to the marked increases in specific gravity exhibited by C. briggsae during ageing. This speculation receives support from Prankerd's 25 studies from which he concluded that lipid loss accounts for increased gravity in human red blood cells. Conversely, it is possible (D. Danon, personal communication) that the increased specific gravity of old red blood cells is due to loss of water, and such may also prove to be the cause of the age-related increase of specific gravity in C. briggsae. The small increase in osmotic fragility and specific gravity of non-virgin T. aceti during ageing may be related to the preceding factors. In comparison, when C. briggsae was cultured at the same temperature (27°C) and tested for osmotic fragility under the same conditions, 40 ~ burst at 14 days and 100 ~ at 28 daysL CONCLUSIONS

The current studies demonstrated that degenerative changes during ageing of T. aceti were in some ways similar, and in some ways different, to those occurring during senescence of C. briggsae. However, even where similar, the ageing response by

88

C. briggsae was more clearly defined in regard to the parameters evaluated. In this respect, C. briggsae would be a more satisfactory model in ageing studies. These differences shown by T. aceti may be related, in part, to the adaptations evolved while adjusting to an environment alien to that of other nematodes, i.e. vinegar.

REFERENCES 1 B. M. Zuckerman, B. Nelson and M. Kisiel, Specific gravity increase of Caenorhabditis briggsae with age, J. Nematol., 4 (1972) 261-262. 2 B. M. Zuckerman, S. Himmelhoch, B. Nelson, J. Epstein and M. Kisiel, Aging in Caenorhabditis briggsae, Nematologica, 17 (1971) 478-487. 3 J. Ep3tein, S. Himmelhoch and D. Gershon, Studies on ageing in nematodes. II. Electronmicroscopical studies on age-associated cellular damage, Mech. Age. Dev., 1 (1972) 245-255. 4 B. M. Zuckerman, S. Himmelhoch and M. Kisiel, Fine structure changes in the cuticle of adult Caenorhabditis briggsae with age, Nematologica, 19 (1973) 109-112. 5 M. Kisiel, S. Himn~lhozh and B. M. Zuzkerm~n, Caen~rhJS:litisbriggsae: Effects of aminopterin., Exp. Parasitol., 36 (1974) 430-438. 6 E. J. Buecher and E. L. Hansen, Aging studies using axenic nematode cultures, Proc. 2rid Int. Cong. Plant Pathol., Abstr. No. 210 (1973). 7 D. Gershon, Studies on aging in nematodes. 1. The nematode as a model organism for aging research, Exp. Gerontol., 5 (1970) 7-12. 8 M. Erlanger and D. Gershon, Studies on aging in nematodes. II. Studies on the activities of several enzymes as a function of age, Exp. Gerontol., 5 (1970) 13-19. 9 H. Gershon and D. Gershon, Detection of inactive enzyme molecules in ageing organisms, Nature 227 (1970) 1214-1217. 10 M. S. Reitz and D. R. Sanadi, An aspect of translational control of protein synthesis in aging: changes in the isoaccepting forms of t R N A in Turbatrix aceti, Exp. Gerontol., 7 (1972) 119-129. 11 P. Zeelon, H. Gershon and D. Gershon, Inactive enzyme molecules in aging organisms. Nematode fructose-l,6-diphosphate aldolase, Biochemistry USA, 12 (1973) 1743-1750. 12 M. Kisiel, B. Nelson and B. M. Zuckerman, Effects of D N A synthesis inhibitors on Caenorhabditis briggsae and Turbatrix aeeti, Nematologica, 18 (1972) 373-384. 13 S. Himmelhoch, M. Kisiel, J. Lavimoniere and B. M. Zuckerman, Fine structure of young, adult Turbatrix aceti, Nematologica, 19 (1973)449-454. 14 A. R. Spurr, A low-viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res., 16 (1969) 31-43. 15 M . R . Samoiloff and J. Pasternak, Nematode morphogenesis: fine structure of the cuticle of each stage of the nematode, Panagrellus silusiae (de Man 1913) Goodey 1945, Can. J. Zool., 46 (1968) 1019-1022. 16 M. A. Brock, Ultrastructural studies on the life cycle of a short-lived metazoan, Campanularia flexuosa. 11. Structure of the old adult, J. Ultrastruct. Res., 32 (1970) 118-141. 17 R. R. Kohn, Principles of mammalian aging, Prentice Hall, Englewood Cliffs, N. J., (1971). 18 B. M. Zuckerman, M. Kisiel and S. Himmelhoch, Unusual mitochondrial cristae in the vinegar eelworm, J. Cell Biol., 58 (1973) 476-480. 19 B. M. Zuckerman, The effects of procaine on aging and development of a nematode. In Theoretical Aspects of Aging (ed. M. Rockstein), Academic Press, New York, 1974, pp. 177-186. 20 J. Bjorksten, The crosslinkage theory of aging, Finska Kemistsam fundets. Medd., 80 (1971) 23-38. 21 J. Lipetz and V. J. Cristofalo, Ultrastructural changes accompanying the aging of human diploid cells in culture, J. UItrastruet. Res., 39 (1972) 43-56. 22 T. Jenkins and D. A. Erasmus, The ultrastructure of the intestinal epithelium of Metastrongylus sp. (Nematoda: Strongyloidea), Parasitol., 59 (1969) 335-342. 23 K. S. Chio, U. Reiss, B. Fletcher and A. L. Tappel, Peroxidation of subcellular organelles: formation of lipofuscinlike fluorescent pigments, Science, 166 (1969) 1535-1536.