- Email: [email protected]
Age-related changes in the body composition of mated and unmated blowflies Phormia terrae novae
Exp. Geront. Vol. 15, pp. 433-441. Pergamon Press Ltd. 1980, Printed in Great Britain.
AGE-RELATED
CHANGES
MATED AND UNMATED
IN THE BODY COMPOSITION
OF
B L O W F L I E S PHORMIA TERRAE NO VAE
K. G . COLLATZ a n d U . HOEGER Institut fur Biologie I, Universitat Freiburg, Albertstra6e 21 a, D-7800 Freiburg F. R. G. (Received 4 April 1980)
INTRODUCTION IN SPITE of the growing use of insects as experimental animals in the study of ageing, our knowledge of the physiological, histological and behavioural events that accompany this process is as limited as our knowledge of the mechanisms of ageing itself. Numerous factors influence the life-span of insects. Some are "intrinsic", such as genetic constitution, sex, egg laying, others "extrinsic", such as temperature, nutrition, population density and possibility to mate (summarized in Rockstein, 1973). Both intrinsic and extrinsic factors are often involved and it is difficult to relate timedependent changes in the constitution of an experimental animal to only one such factor. The selection of ageing mutants, as in Drosophila, may be, but does not necessarily have to be, useful in addressing this question (Ganetzky and Flanagan, 1978). Nevertheless, the sum of time-dependent changes of physiological and other conditions in a single species may bring about an idea of what the "syndrome" of ageing and senescence is. Since the early studies on Carabus and Drosophila (Krumbiegel, 1929, 1930) it is known that mating could have an influence on the life span of both males and females. Our intent in this study was to look for age dependent changes in the body composition of a short lived insect, the blowfly Phormia, and for the influence of mating on these changes. METHODS AND PROCEDURE Animals The subject under investigation was the fly Phormia terrae novae Rob.-Desv. (Diptera, Calliphoridae). The larvae were reared on meat up to the last instar and pupated in the laboratory. Only those flies were used which did not differ by more than 12 h in hatching time. The holding temperature was 25± 1°C, at a relative humidity of 75 ± 50?e. Light was given in a 12 : 12 h cycle. The flies were fed on standard Drosphila rearing medium, malt and minced meat adlibitum. Each cage (50 × 50 × 50 cm) contained about 150 flies. Four different groups of 5 individuals (mated and unmated males and females) were used and their body composition was analyzed at 10 different life stages: Stage 1: immediately after emergence; stage 2: one day, inactive, no feeding; stage 3; two days, very active, first feeding; stage 4: three days, very active, copulation, resp. the attempt by the single males; stage 5: four days, very active, oviposition of copulated females, virgin females bearing weU-developed eggs; stage 6: eight days, reduced activity; stage 7: twelve days, reduced mobility, copulated females after the second oviposition; stage 8: seventeen days, reduced activity, virgin females after the second oviposition, copulated females after the third oviposition; stage 9: thirty days, inactive; stage 10: immediately before death (this can be determined by uncontrolled leg movements and reflectory wing buzzing). Analysis o f body constituents Homogenates of whole flies were fractionated as described earlier (Collatz, 1973; Collatz and Mommsen, 1974) with slight modifications. Extraction of total lipids with chloroform-methanol, acid and alkaline treatment to extract total polysaccharides, precipitation of polysaccharides from acid and alkaline supernatant, separation of protein hydrolysate from unhydrolyzable residue. Colorimetric determination of lipids after oxidation with potassium bichromate (Amenta, 1964); determination of carbohydrates with anthrone reagent (Roe, 1955); protein with a modified biuret method (Koch and Putnam, 1971); amino acids with ninhydrin (Troll and Cannan, 1953); gravimetric measurement of dry weight and unhydrolyzable residue ("cuticle"). 433
434
K . G . COI.LATZ AND U. H(}E(iER
Calculation of standard deviation Five flies were usually pooled to give a sample. Therefore it was necessary to evaluate the variability of measurement of single animals within a group. For this purpose the body composition of ten single males in the same physiological condition, i.e. immediately after death, was analyzed and the standard deviation determined. The following standard deviations, given in percent of the mean value were found: low molecular carbohydrates 24070, amino acids 5°70, polysaccharides 14070, proteins 21070, lipids 12070, unhydrolyzable residue 1307o.An interpretation of data in "Discussion" is only given if the values differ more than the standard deviation. RESULTS
Life cycle Under good feeding conditions the larvae of Phormia need only 14 days to attain full growth from egg to pupation. Ten days later the adult flies emerge. During the first day after emergence the flies are totally inactive, crowd together in one corner of the cage and do not feed. The very next day, the flies show high flying activity, and begin to feed. They copulate one day later. The copulation frequency, as life activity in general, is highest during the first week, but copulations occur during the whole lifespan. The first batches of eggs are laid on the 4th day, others on the 12th-16th day. An additional oviposition on the 25th day was observed only by virgin females. Thereafter oviposition is nearly extinct. Some behavioural changes indicate senescence in the fly population, mainly a reduction of flying, walking, feeding and oviposition activity. Moreover, a partial loss of the wings and the tarsae occurs with increasing age. The mean duration of life is different in virgin females on one and single males and mated males and females on the other side (44 days in virgin females, about 30 days in the other groups). Differences between mated and virgin females also exist in the time of egg laying, being delayed in the latter (see above). Variations of body composition The variations of body composition during the life cycle are summarized in Figs. 1 and 2 for mated and unmated males and Figs. 3 and 4 for mated and virgin females respectively. Proteins In each of the four experimental groups, the protein decreases from the day of emergence to day one of adult life. The values are within the same range (about 115 m g / g wet wt. at day 0 and 85 mg/wet wt. at day 1). The minimum protein concentration at day 17 is also c o m m o n to all groups. It is followed by a rapid rise until the end of life, being most pronounced in the group of mated males (from 70 m g / g wet wt. at day 17 to 150 mg/g wet wt. immediately before death). Beginning with day 3, the protein concentration is generally higher in unmated than in mated flies. Lipids The lipids decline in all groups in a similar manner as observed for the proteins. The decrease lasts only until the second (9 9) or third (or or) day. Differences between mated and unmated males are not very remarkable except that unmated males have slightly higher lipid values throughout the "active p h a s e " of life (day 4 - d a y 30). Corresponding differences between the groups of females are more clearly visible, indicating that virgin females contain more lipids than mated females especially during the period of intensive egg laying of the latter (day 4 - d a y 12). Exceptionally high values were found in the virgin females at the last day of measurement.
AGE-RELATED CHANGES IN BODY COMPOSITION OF BLOWFLIES
Meted
435
PR
140 15C 12C II0 I00
-I
90 •
80
~
7o
+ PR
5O
L
,dg:
.o
:,,.
I "I[" 20 I-
I
\ •
ll w°" '' :°w"
L
"" ~ o ~ u
DW*A C
lln! I ,0
A o
~
~
~
,~
,',
fill!! LC
/"
~o ",'"
II:,':
Jlll IIP~''~ nn
Doys
FIG. I. Variations of body constituents of mated male P h o r m i a terrae n o v a e during adult life. Values are in mg/g wet weight, dry weight in percent wet weight. It means PR--proteins, LC--low molecular carbohydrates, L--lipids, DW--dry weight, C--unhydrolyzable residue ("cuticle"), PS--polysaccharides, A--amino acids. Note the two breaks in the abscissa. On day 68 only those flies were collected which show the stage "immediately before death".
Polysaccharides The most striking variations of all are visible in the polysaccharide fraction and in the low molecular weight carbohydrates. At first there is a sharp maximum of polysaccharides in all four groups at day 2 followed by a second one at day 17. In both the unmated males and females, the concentration at maximum is higher than in the mated flies. Thereafter we observed a rapid decline in all four groups to extremely low values at the time of death (mated males: 1.8 mg/g wet wt.; unmated males: 3.2 mg/g wet wt.; mated females: 2.3 mg/g wet wt.; virgin females: 10.1 mg/g wet wt.). In the virgin females this last value is considerably higher than in the other groups.
Low molecular weight carbohydrates The time course of the low molecular weight carbohydrates is similar in mated and unmated groups, having maxima at days 3, 8 and 30. The last maximum at day 30 however, differs considerably within the groups; 36 mg/g wet wt. virgin females, 90 mg/g wet wt. mated males and 50 mg/g wet wt. mated females and unmated males respectively.
436
K.G. COLLATZANDU. HOEGER
130
0d Unmet-ed
PR PR
PlO
I00
+ PR
90 8C
..,.2 70
°1
40
201
~
Lci
,% iI ~.
,
r
.------.c
-C I I
L
V
'~ ~ i~~
oH
~ePS
!ow~
I ]
II
:: O 2
4
8
12
J
II • I lhl
i,'
17 r*, Days
30
FIG. 2. V a r i a t i o n s o f b o d y c o n s t i t u e n t s o f u n m a t e d m a l e P h o r m i a as in Fig. I. ~2o
A:I ~l
68
t e r r a e n o v a e d u r i n g a d u l t life. A b b r e v i a t i o n s
~ Mated
rio r / ~
PR
PR
I00 90 + PR 80 4c 70
~
6o
~'
5o
LC
40
C
•
c, ~°I'.~¢'.I.... r ~ . ~
~',~---
-~ ~w
I
°11 ,~W°,o
III, to ,,
,, n,
,, ,,
~s',
,,
\/".c
,,
,o
C
DW *X
tlo LC ill
I....,/ ,i
,
0
2
4
8
12
t7 Days
30
ol
PSl IIII,., 68
FIG. 3. V a r i a t i o n s o f b o d y constituents o f mated f e m a l e P h o r m i a terree n o v a e d u r i n g a d u l t life. A b b r e v i a t i o n s
as in Fig. 1.
AGE-RELATEDCHANGES IN BODYCOMPOSITIONOF BLOWFLIES
437
PR 160
Unmated
150 140 130 PR
120
PR
IIO
I00 I ~ .,.: 90 ~ 80
6O 5O "°
DW* CI'II
=a 0 2 4
i 8
i 12
K/, I7 -
30
DW % qIIl io ii i
li"
68
i!
Days FIG. 4. Variations of body constituents of unmated female Phormia terrae n o v a e during adult life. Abbreviations as in Fig. 1.
A mino acids Amino acid concentrations do not vary to a great extent between groups and time tests. All measurements fall into the range between 8 and 11 mg/g wet wt. except for those of the mated males, these being higher (up to 13 m g / g wet wt.). A minimum of 8 m g / g wet wt. is common to all groups at day 17. Again, one can observe a decrease between day 30 and the time of death.
Unhydrolyzable residue ("cuticle" ") In both groups of males we find low values of this fraction which starts to increase two days after emergence from 34 m g / g wet wt. up to 40 m g / g wet wt. on day 4. During the following days the levels differ remarkably being generally higher in the unmated males. The corresponding values in the virgin female group are higher than in the mated ones throughout the active life span.
438
K. ( ; C O l I.ATZ A N D U. H O E ( I E R
Dry weight After a slight decline in all groups from day 0 to day 2, followed by a subsequent rise until day 4, the dry weights remain nearly constant during the life cycle. Beginning with day 4 the values for the virgin females are higher than those for the other groups. DISCUSSION
Phormia is a typical example of a short-lived insect with high activity and metabolic intensity. Therefore, short term metabolic variations can arise as the data show. A clear difference in longevity exists between virgin females and the other groups, the mean life span of the virgin females being longer (14 days on average). There is no difference between mated females and males. This is in contrast to the generally accepted rule that in most species females have a significantly longer life span. Corresponding studies exist for the dipterans Drosophila (Pearl and Parker, 1924) and Musca (Rockstein, 1973), yet it is not unequivocal, especially when both sexes are segregated and a "matching effect" concerned with copulation and egg laying (Nowosielski and Patton, 1965a) can take place. In Drosophila too, similar marked differences in the length of life between virgin and mated females were found (Bieganska-Pietrzakowa, 1961). In contrast to these observations, we found that unmated females of the mecopteran fly Panorpa vulgaris have a considerably shorter life span than the mated ones. Variation of body constituents Day O. Adult flies emerge with high protein and lipid portions similar in both sexes, but polysaccharides and low molecular weight carbohydrates, in contrast, are low. Females have slightly more polysaccharides than males. Up to day 2. Proteins and lipids decrease in only two days, whereas carbohydrates begin to rise. This obviously marks a form of biochemical development, known, especially in dipterans, as metachemogenesis (Rockstein, 1973). In the first four days of adult life of Phormia terrae novae, the pupal fat body disappears and the adult fat body develops (Harlow, 1956). Similarly the protein content of Phormia regina shows a rapid decline from the last instar to the newly emerged imago, having only 17% of the protein of the former (Chen, 1966). Comparable results are known from Drosophila (Maynard-Smith et al., 1970). Lipids also decrease as a result of their use as metabolic fuel. During the firsl two days of adult life, the food intake ceases and the respiratory quotient ranges from 0.70-0.71 (Collatz, unpublished data). In a three day starvation experiment after this time virtually no lipid reserves were used, most of the animals had died and their carbohydrate concentration fell into the range of day 68 (time of death). Carbohydrates are the mean if not the only source of energy supply for the flies after day 2. Between days 3 and 17. Day 3 is marked by high activity and mating. The sex-separated males try to copulate. In the body composition this is reflected by high values of sugar at the beginning and a depletion on day 4. The copulation of females leads to oviposition at the next day, virgin females laying fewer and infertile eggs on the 5th day. In both cases the oviposition was followed by a second rise in polysaccharides and low molecular weight carbohydrates. A similar difference in the time course of lipid variations was found. The lipids of m a t e d - - b u t not of virgin--females are reduced after oviposition. The generally higher concentrations of lipids and proteins of the virgin females are obviously due to their reduced rate of oviposition. Beginning with day 4, the level of low molecular carbohydrates in both the mated groups is significantly higher than in the unmated ones.
A G E - R E L A T E D C H A N G E S IN T H E BODY C O M P O S I T I O N O F B L O W F L I E S
439
Unmated males and females, in contrast, store more polysaccharides during the following days. The reason for this seems to be the higher activity of the mated groups. After one week the period of highest activity of both sexes is terminated. After this time and up to day 17 flying and walking activities are reduced. Until this point however, the feeding activity is not affected. The oviposition also lasts to day 17, thereafter eggs were only layed infrequently, with the exception of the virgin females mentioned above. Therefore at the end of this period of reduced activity and lasting food intake we observed high polysaccharide stores in both males and females, being more prominent in the unmated ones. The low lipid values of females at the end of this period indicate the termination of oviposition. The registered survival curves start to decline after the 17th day. Day 30. At this day, the population reached the mean duration of its life span. We had already noted a drastic reduction of food intake after the 25th day. Yet this reduction can be overcome by artificial feeding with glucose or sucrose; thus the sugar level of flies can be held constant throughout the whole life. The effect of this treatment on activity and longevity is under investigation. After studies on Drosophila funebris their flight ability reaches its maximum exactly at the same time at which glycogen concentration of the adults is highest (Williams et al., 1943). In contrast, this does not hold true for Phormia, where the highest glycogen contents are measurable at a time of already reduced activity. In our view, storage of glycogen is the result of normal food intake after periods of reduced activity. A decrease of sugar intake of the blowfly Phormia regina was found to begin with the 15th day of adult life (Gelperin and Dethier, 1967). A dramatic decrease of carbohydrates results from the changes in feeding behaviour. The polysaccharides of unmated males are already low at day 30 whereas the polysaccharides stored by virgin females are still more prominent than those of the mated ones. Day 68, immediately before death. The extremely low values of carbohydrates have to be mentioned first. These values are comparable to those of the first day of adult life. In the virgin females, however, we see that the polysaccharides are still about five times as high as the corresponding values in the mated females. This fact strongly suggests that the reduction of carbohydrates alone cannot account for the termination of life. On the other hand it is interesting to note that at the day of collection of the "immediately before d e a t h " samples, nearly 15o70 of the virgin females were still alive, but only 2°7o of all the other populations. Nevertheless, we must expect that the loss of these important metabolic fuels has severe influence on the whole metabolism of Phormia. The poor movements as well as moderate flight ability of aged flies indicate this. However, the cause-and-effect relationship between life activity and the loss of glycogen is not clearly established. The flight performance of aged mosquitos (Aedes aegyptt) is not related to the glycogen content but to the failure to utilize existing glycogen reserves (Rowley and Graham, 1968). Our feeding experiments point in the same direction. An age-dependent decrease of glycogen was also found in Drosophila (Samis et al., 1971), the reduction, however, amounting to only 50°/o of the glycogen content of young flies. The observed high protein and lipid values does not seem to be a consequence of net synthesis. Moreover they increase relatively, due to the rapid fall of the carbohydrates. This is consistent with the observation that the wet weight of ageing flies decreases without a change in the percentage of dry weight. An increasing protein synthesis in 60-day old Drosophila was discussed (Clarke and Maynard-Smith, 1966), yet other experiments with Drosophila and Phormia strongly suggest the opposite (Bauman and Chen, 1968; Levenbook and
440
K.G. COLLATZANDU.HOEGER
Krishna, 1971). In the hemolymph of adult house crickets, proteins and lipids as well as amino acids decrease with increasing age (Nowosielsky and Patton, 1965b). Evidently, it is hardly possible to get insight into the mechanisms of ageing simply by measuring the changes of the body compositions. However, it was desirable to look for metabolic expression of ageing and for that purpose we first had to elucidate in which part of the metabolic network such expressions take place, before we start to study their regulations. The results show that in the case of P h o r m i a carbohydrates and carbohydrate metabolism play an important role in the ageing events. SUMMARY 1. The variations of the organic body constituents of the blowfly P h o r m i a terrae novae, i.e. lipids, polysaccharides, low molecular carbohydrates, and free amino acids were analyzed at ten different life-stages as well as the unhydrolyzable "cuticle" residue and the dry weight of mated and unmated males and females. 2. The flies emerge with high protein and lipid contents, and during the first two days of adult life the values of proteins, lipids, and amino acids decrease while the carbohydrates increase. This points to a special form of biochemical development (metachemogenesis). 3. With the beginning of sexual activity on the 3rd day, all groups have a maximum of low molecular weight carbohydrates, but the polysaccharide stores begin to deplete after their maximum at the second day. During the adult life-span remarkable differences are detectable in the body composition of both male and female, mated and unmated flies. From the 4th day the level of low molecular weight carbohydrates is significantly higher in the mated groups than in the unmated. In contrast, unmated males and females store more polysaccharides during the following days. A rapid decline of the lipid values in both mated and virgin females between the 12th and the 17th day corresponds to the high rate of oviposition during this time. The amino acid concentration shows only slight variations and is higher in the mated than in the unmated flies. 4. The last days of life are characterized by a dramatic decrease of carbohydrates. The loss of these important metabolic fuels is assumed to have a severe influence on the whole metabolism, but alone cannot account for the termination of life because of the considerably higher carbohydrate values of the virgin females at their last day of life. A c k n o w l e d g e m e n t - - T h i s work was partly supported by a grant from the "Deutsche Forschungsgemeinschaft".
REFERENCES AMENTA, J. S. (1964) J. lip. Res. 5,270. BAUMAN, P. and CHEN, P. S. (1968) Revve. suisse Zool. 75, 1051. BIt:C;ANSKA-P~ETRZAKOWA,M. (1961)Zool. Polon. 10,265. CHEN, P. S. (1966)Adv. Insect Physiol. 3, 53. CLARKE, J. M. and MAYNARD-SMITH, 3. (1966) Nature Lond. 209,627. Col LATZ, K. G. (1973) In: Effects o f Temperature on Ectothermic Organisms (Edited by W. WIE:SER), p. 195. Springer, Berlin. COLLATZ, K. G. and MOMMSEN, T. (1974) J. comp. Physiol. 91, 91. GANETZKY, B. and FLANAGAN, J. R. (1978) Exp. Geront. 13, 189. GELPtqRKN, A. and DETHIER, V. G. (1967)Physiol. Zool. 40,218. HARLOW, P. M. (1956) J. exp. Biol. 33, 777. HOEGER, U. (1976) Diplomarbeit, Freiburg i. Br. KocH, A. L. and PUTNAM, S. L. (I 971)Anal. Biochem. 44,239. KRUMBIEGEL, 1. (1929) Zool. Jb. Abt. Anat. Ontog. 51, 111. KrUMBJt-CiEL, 1. (1930)Forsch. Fortschr. 2, 85.
AGE-RELATEDCHANGESINTHE BODYCOMPOSITIONOF BLOWFLIES
441
LEVENBOOK,L. and KRISHNA,I. (1971) J. Insect Physiol. 17, 9. MAYNARD-SMITH,J., BOZCUK,A. N. and TEBBUT, S. (I 970)J. Insect Physiol. 16, 601. NOWOSlELSKY,J. W. and PATTON, R. L. (1965a) J. Insect Physiol. l l , 201. NOWOSIELSKY,.I.W. and PATTON, R. L. (1965b)J. Insect Physiol. 11,263. PEARL, R. and PARKER,S. L. (1924) Amer. Nat. 58, 71. ROCKSTEIN, M. (1973) In: The Physiology of Insecta (Edited by M. ROCKSTEIN), vol. l, p. 371. Academic Press, London. ROE, J. R. (1955) J. biol. Chem. 212, 335. ROWLEY, W. A. and GRAHAM,C. L. (1968)J. Insect Physiol. 14, 719. SAMIS, H. V. Jr., ERK, F. C. and DAIRD, M. B. (1971) Exp. Geront. 6, 9. TROLL,W. and CANNAN,R. K. (1953) J. biol. Chem. 200, 803. WILLIAMS, C. M., BARNESS,L. A. and SAWYER,W. H. (1943) Biol. Bull. 84, 263.
AGE-RELATED
CHANGES
MATED AND UNMATED
IN THE BODY COMPOSITION
OF
B L O W F L I E S PHORMIA TERRAE NO VAE
K. G . COLLATZ a n d U . HOEGER Institut fur Biologie I, Universitat Freiburg, Albertstra6e 21 a, D-7800 Freiburg F. R. G. (Received 4 April 1980)
INTRODUCTION IN SPITE of the growing use of insects as experimental animals in the study of ageing, our knowledge of the physiological, histological and behavioural events that accompany this process is as limited as our knowledge of the mechanisms of ageing itself. Numerous factors influence the life-span of insects. Some are "intrinsic", such as genetic constitution, sex, egg laying, others "extrinsic", such as temperature, nutrition, population density and possibility to mate (summarized in Rockstein, 1973). Both intrinsic and extrinsic factors are often involved and it is difficult to relate timedependent changes in the constitution of an experimental animal to only one such factor. The selection of ageing mutants, as in Drosophila, may be, but does not necessarily have to be, useful in addressing this question (Ganetzky and Flanagan, 1978). Nevertheless, the sum of time-dependent changes of physiological and other conditions in a single species may bring about an idea of what the "syndrome" of ageing and senescence is. Since the early studies on Carabus and Drosophila (Krumbiegel, 1929, 1930) it is known that mating could have an influence on the life span of both males and females. Our intent in this study was to look for age dependent changes in the body composition of a short lived insect, the blowfly Phormia, and for the influence of mating on these changes. METHODS AND PROCEDURE Animals The subject under investigation was the fly Phormia terrae novae Rob.-Desv. (Diptera, Calliphoridae). The larvae were reared on meat up to the last instar and pupated in the laboratory. Only those flies were used which did not differ by more than 12 h in hatching time. The holding temperature was 25± 1°C, at a relative humidity of 75 ± 50?e. Light was given in a 12 : 12 h cycle. The flies were fed on standard Drosphila rearing medium, malt and minced meat adlibitum. Each cage (50 × 50 × 50 cm) contained about 150 flies. Four different groups of 5 individuals (mated and unmated males and females) were used and their body composition was analyzed at 10 different life stages: Stage 1: immediately after emergence; stage 2: one day, inactive, no feeding; stage 3; two days, very active, first feeding; stage 4: three days, very active, copulation, resp. the attempt by the single males; stage 5: four days, very active, oviposition of copulated females, virgin females bearing weU-developed eggs; stage 6: eight days, reduced activity; stage 7: twelve days, reduced mobility, copulated females after the second oviposition; stage 8: seventeen days, reduced activity, virgin females after the second oviposition, copulated females after the third oviposition; stage 9: thirty days, inactive; stage 10: immediately before death (this can be determined by uncontrolled leg movements and reflectory wing buzzing). Analysis o f body constituents Homogenates of whole flies were fractionated as described earlier (Collatz, 1973; Collatz and Mommsen, 1974) with slight modifications. Extraction of total lipids with chloroform-methanol, acid and alkaline treatment to extract total polysaccharides, precipitation of polysaccharides from acid and alkaline supernatant, separation of protein hydrolysate from unhydrolyzable residue. Colorimetric determination of lipids after oxidation with potassium bichromate (Amenta, 1964); determination of carbohydrates with anthrone reagent (Roe, 1955); protein with a modified biuret method (Koch and Putnam, 1971); amino acids with ninhydrin (Troll and Cannan, 1953); gravimetric measurement of dry weight and unhydrolyzable residue ("cuticle"). 433
434
K . G . COI.LATZ AND U. H(}E(iER
Calculation of standard deviation Five flies were usually pooled to give a sample. Therefore it was necessary to evaluate the variability of measurement of single animals within a group. For this purpose the body composition of ten single males in the same physiological condition, i.e. immediately after death, was analyzed and the standard deviation determined. The following standard deviations, given in percent of the mean value were found: low molecular carbohydrates 24070, amino acids 5°70, polysaccharides 14070, proteins 21070, lipids 12070, unhydrolyzable residue 1307o.An interpretation of data in "Discussion" is only given if the values differ more than the standard deviation. RESULTS
Life cycle Under good feeding conditions the larvae of Phormia need only 14 days to attain full growth from egg to pupation. Ten days later the adult flies emerge. During the first day after emergence the flies are totally inactive, crowd together in one corner of the cage and do not feed. The very next day, the flies show high flying activity, and begin to feed. They copulate one day later. The copulation frequency, as life activity in general, is highest during the first week, but copulations occur during the whole lifespan. The first batches of eggs are laid on the 4th day, others on the 12th-16th day. An additional oviposition on the 25th day was observed only by virgin females. Thereafter oviposition is nearly extinct. Some behavioural changes indicate senescence in the fly population, mainly a reduction of flying, walking, feeding and oviposition activity. Moreover, a partial loss of the wings and the tarsae occurs with increasing age. The mean duration of life is different in virgin females on one and single males and mated males and females on the other side (44 days in virgin females, about 30 days in the other groups). Differences between mated and virgin females also exist in the time of egg laying, being delayed in the latter (see above). Variations of body composition The variations of body composition during the life cycle are summarized in Figs. 1 and 2 for mated and unmated males and Figs. 3 and 4 for mated and virgin females respectively. Proteins In each of the four experimental groups, the protein decreases from the day of emergence to day one of adult life. The values are within the same range (about 115 m g / g wet wt. at day 0 and 85 mg/wet wt. at day 1). The minimum protein concentration at day 17 is also c o m m o n to all groups. It is followed by a rapid rise until the end of life, being most pronounced in the group of mated males (from 70 m g / g wet wt. at day 17 to 150 mg/g wet wt. immediately before death). Beginning with day 3, the protein concentration is generally higher in unmated than in mated flies. Lipids The lipids decline in all groups in a similar manner as observed for the proteins. The decrease lasts only until the second (9 9) or third (or or) day. Differences between mated and unmated males are not very remarkable except that unmated males have slightly higher lipid values throughout the "active p h a s e " of life (day 4 - d a y 30). Corresponding differences between the groups of females are more clearly visible, indicating that virgin females contain more lipids than mated females especially during the period of intensive egg laying of the latter (day 4 - d a y 12). Exceptionally high values were found in the virgin females at the last day of measurement.
AGE-RELATED CHANGES IN BODY COMPOSITION OF BLOWFLIES
Meted
435
PR
140 15C 12C II0 I00
-I
90 •
80
~
7o
+ PR
5O
L
,dg:
.o
:,,.
I "I[" 20 I-
I
\ •
ll w°" '' :°w"
L
"" ~ o ~ u
DW*A C
lln! I ,0
A o
~
~
~
,~
,',
fill!! LC
/"
~o ",'"
II:,':
Jlll IIP~''~ nn
Doys
FIG. I. Variations of body constituents of mated male P h o r m i a terrae n o v a e during adult life. Values are in mg/g wet weight, dry weight in percent wet weight. It means PR--proteins, LC--low molecular carbohydrates, L--lipids, DW--dry weight, C--unhydrolyzable residue ("cuticle"), PS--polysaccharides, A--amino acids. Note the two breaks in the abscissa. On day 68 only those flies were collected which show the stage "immediately before death".
Polysaccharides The most striking variations of all are visible in the polysaccharide fraction and in the low molecular weight carbohydrates. At first there is a sharp maximum of polysaccharides in all four groups at day 2 followed by a second one at day 17. In both the unmated males and females, the concentration at maximum is higher than in the mated flies. Thereafter we observed a rapid decline in all four groups to extremely low values at the time of death (mated males: 1.8 mg/g wet wt.; unmated males: 3.2 mg/g wet wt.; mated females: 2.3 mg/g wet wt.; virgin females: 10.1 mg/g wet wt.). In the virgin females this last value is considerably higher than in the other groups.
Low molecular weight carbohydrates The time course of the low molecular weight carbohydrates is similar in mated and unmated groups, having maxima at days 3, 8 and 30. The last maximum at day 30 however, differs considerably within the groups; 36 mg/g wet wt. virgin females, 90 mg/g wet wt. mated males and 50 mg/g wet wt. mated females and unmated males respectively.
436
K.G. COLLATZANDU. HOEGER
130
0d Unmet-ed
PR PR
PlO
I00
+ PR
90 8C
..,.2 70
°1
40
201
~
Lci
,% iI ~.
,
r
.------.c
-C I I
L
V
'~ ~ i~~
oH
~ePS
!ow~
I ]
II
:: O 2
4
8
12
J
II • I lhl
i,'
17 r*, Days
30
FIG. 2. V a r i a t i o n s o f b o d y c o n s t i t u e n t s o f u n m a t e d m a l e P h o r m i a as in Fig. I. ~2o
A:I ~l
68
t e r r a e n o v a e d u r i n g a d u l t life. A b b r e v i a t i o n s
~ Mated
rio r / ~
PR
PR
I00 90 + PR 80 4c 70
~
6o
~'
5o
LC
40
C
•
c, ~°I'.~¢'.I.... r ~ . ~
~',~---
-~ ~w
I
°11 ,~W°,o
III, to ,,
,, n,
,, ,,
~s',
,,
\/".c
,,
,o
C
DW *X
tlo LC ill
I....,/ ,i
,
0
2
4
8
12
t7 Days
30
ol
PSl IIII,., 68
FIG. 3. V a r i a t i o n s o f b o d y constituents o f mated f e m a l e P h o r m i a terree n o v a e d u r i n g a d u l t life. A b b r e v i a t i o n s
as in Fig. 1.
AGE-RELATEDCHANGES IN BODYCOMPOSITIONOF BLOWFLIES
437
PR 160
Unmated
150 140 130 PR
120
PR
IIO
I00 I ~ .,.: 90 ~ 80
6O 5O "°
DW* CI'II
=a 0 2 4
i 8
i 12
K/, I7 -
30
DW % qIIl io ii i
li"
68
i!
Days FIG. 4. Variations of body constituents of unmated female Phormia terrae n o v a e during adult life. Abbreviations as in Fig. 1.
A mino acids Amino acid concentrations do not vary to a great extent between groups and time tests. All measurements fall into the range between 8 and 11 mg/g wet wt. except for those of the mated males, these being higher (up to 13 m g / g wet wt.). A minimum of 8 m g / g wet wt. is common to all groups at day 17. Again, one can observe a decrease between day 30 and the time of death.
Unhydrolyzable residue ("cuticle" ") In both groups of males we find low values of this fraction which starts to increase two days after emergence from 34 m g / g wet wt. up to 40 m g / g wet wt. on day 4. During the following days the levels differ remarkably being generally higher in the unmated males. The corresponding values in the virgin female group are higher than in the mated ones throughout the active life span.
438
K. ( ; C O l I.ATZ A N D U. H O E ( I E R
Dry weight After a slight decline in all groups from day 0 to day 2, followed by a subsequent rise until day 4, the dry weights remain nearly constant during the life cycle. Beginning with day 4 the values for the virgin females are higher than those for the other groups. DISCUSSION
Phormia is a typical example of a short-lived insect with high activity and metabolic intensity. Therefore, short term metabolic variations can arise as the data show. A clear difference in longevity exists between virgin females and the other groups, the mean life span of the virgin females being longer (14 days on average). There is no difference between mated females and males. This is in contrast to the generally accepted rule that in most species females have a significantly longer life span. Corresponding studies exist for the dipterans Drosophila (Pearl and Parker, 1924) and Musca (Rockstein, 1973), yet it is not unequivocal, especially when both sexes are segregated and a "matching effect" concerned with copulation and egg laying (Nowosielski and Patton, 1965a) can take place. In Drosophila too, similar marked differences in the length of life between virgin and mated females were found (Bieganska-Pietrzakowa, 1961). In contrast to these observations, we found that unmated females of the mecopteran fly Panorpa vulgaris have a considerably shorter life span than the mated ones. Variation of body constituents Day O. Adult flies emerge with high protein and lipid portions similar in both sexes, but polysaccharides and low molecular weight carbohydrates, in contrast, are low. Females have slightly more polysaccharides than males. Up to day 2. Proteins and lipids decrease in only two days, whereas carbohydrates begin to rise. This obviously marks a form of biochemical development, known, especially in dipterans, as metachemogenesis (Rockstein, 1973). In the first four days of adult life of Phormia terrae novae, the pupal fat body disappears and the adult fat body develops (Harlow, 1956). Similarly the protein content of Phormia regina shows a rapid decline from the last instar to the newly emerged imago, having only 17% of the protein of the former (Chen, 1966). Comparable results are known from Drosophila (Maynard-Smith et al., 1970). Lipids also decrease as a result of their use as metabolic fuel. During the firsl two days of adult life, the food intake ceases and the respiratory quotient ranges from 0.70-0.71 (Collatz, unpublished data). In a three day starvation experiment after this time virtually no lipid reserves were used, most of the animals had died and their carbohydrate concentration fell into the range of day 68 (time of death). Carbohydrates are the mean if not the only source of energy supply for the flies after day 2. Between days 3 and 17. Day 3 is marked by high activity and mating. The sex-separated males try to copulate. In the body composition this is reflected by high values of sugar at the beginning and a depletion on day 4. The copulation of females leads to oviposition at the next day, virgin females laying fewer and infertile eggs on the 5th day. In both cases the oviposition was followed by a second rise in polysaccharides and low molecular weight carbohydrates. A similar difference in the time course of lipid variations was found. The lipids of m a t e d - - b u t not of virgin--females are reduced after oviposition. The generally higher concentrations of lipids and proteins of the virgin females are obviously due to their reduced rate of oviposition. Beginning with day 4, the level of low molecular carbohydrates in both the mated groups is significantly higher than in the unmated ones.
A G E - R E L A T E D C H A N G E S IN T H E BODY C O M P O S I T I O N O F B L O W F L I E S
439
Unmated males and females, in contrast, store more polysaccharides during the following days. The reason for this seems to be the higher activity of the mated groups. After one week the period of highest activity of both sexes is terminated. After this time and up to day 17 flying and walking activities are reduced. Until this point however, the feeding activity is not affected. The oviposition also lasts to day 17, thereafter eggs were only layed infrequently, with the exception of the virgin females mentioned above. Therefore at the end of this period of reduced activity and lasting food intake we observed high polysaccharide stores in both males and females, being more prominent in the unmated ones. The low lipid values of females at the end of this period indicate the termination of oviposition. The registered survival curves start to decline after the 17th day. Day 30. At this day, the population reached the mean duration of its life span. We had already noted a drastic reduction of food intake after the 25th day. Yet this reduction can be overcome by artificial feeding with glucose or sucrose; thus the sugar level of flies can be held constant throughout the whole life. The effect of this treatment on activity and longevity is under investigation. After studies on Drosophila funebris their flight ability reaches its maximum exactly at the same time at which glycogen concentration of the adults is highest (Williams et al., 1943). In contrast, this does not hold true for Phormia, where the highest glycogen contents are measurable at a time of already reduced activity. In our view, storage of glycogen is the result of normal food intake after periods of reduced activity. A decrease of sugar intake of the blowfly Phormia regina was found to begin with the 15th day of adult life (Gelperin and Dethier, 1967). A dramatic decrease of carbohydrates results from the changes in feeding behaviour. The polysaccharides of unmated males are already low at day 30 whereas the polysaccharides stored by virgin females are still more prominent than those of the mated ones. Day 68, immediately before death. The extremely low values of carbohydrates have to be mentioned first. These values are comparable to those of the first day of adult life. In the virgin females, however, we see that the polysaccharides are still about five times as high as the corresponding values in the mated females. This fact strongly suggests that the reduction of carbohydrates alone cannot account for the termination of life. On the other hand it is interesting to note that at the day of collection of the "immediately before d e a t h " samples, nearly 15o70 of the virgin females were still alive, but only 2°7o of all the other populations. Nevertheless, we must expect that the loss of these important metabolic fuels has severe influence on the whole metabolism of Phormia. The poor movements as well as moderate flight ability of aged flies indicate this. However, the cause-and-effect relationship between life activity and the loss of glycogen is not clearly established. The flight performance of aged mosquitos (Aedes aegyptt) is not related to the glycogen content but to the failure to utilize existing glycogen reserves (Rowley and Graham, 1968). Our feeding experiments point in the same direction. An age-dependent decrease of glycogen was also found in Drosophila (Samis et al., 1971), the reduction, however, amounting to only 50°/o of the glycogen content of young flies. The observed high protein and lipid values does not seem to be a consequence of net synthesis. Moreover they increase relatively, due to the rapid fall of the carbohydrates. This is consistent with the observation that the wet weight of ageing flies decreases without a change in the percentage of dry weight. An increasing protein synthesis in 60-day old Drosophila was discussed (Clarke and Maynard-Smith, 1966), yet other experiments with Drosophila and Phormia strongly suggest the opposite (Bauman and Chen, 1968; Levenbook and
440
K.G. COLLATZANDU.HOEGER
Krishna, 1971). In the hemolymph of adult house crickets, proteins and lipids as well as amino acids decrease with increasing age (Nowosielsky and Patton, 1965b). Evidently, it is hardly possible to get insight into the mechanisms of ageing simply by measuring the changes of the body compositions. However, it was desirable to look for metabolic expression of ageing and for that purpose we first had to elucidate in which part of the metabolic network such expressions take place, before we start to study their regulations. The results show that in the case of P h o r m i a carbohydrates and carbohydrate metabolism play an important role in the ageing events. SUMMARY 1. The variations of the organic body constituents of the blowfly P h o r m i a terrae novae, i.e. lipids, polysaccharides, low molecular carbohydrates, and free amino acids were analyzed at ten different life-stages as well as the unhydrolyzable "cuticle" residue and the dry weight of mated and unmated males and females. 2. The flies emerge with high protein and lipid contents, and during the first two days of adult life the values of proteins, lipids, and amino acids decrease while the carbohydrates increase. This points to a special form of biochemical development (metachemogenesis). 3. With the beginning of sexual activity on the 3rd day, all groups have a maximum of low molecular weight carbohydrates, but the polysaccharide stores begin to deplete after their maximum at the second day. During the adult life-span remarkable differences are detectable in the body composition of both male and female, mated and unmated flies. From the 4th day the level of low molecular weight carbohydrates is significantly higher in the mated groups than in the unmated. In contrast, unmated males and females store more polysaccharides during the following days. A rapid decline of the lipid values in both mated and virgin females between the 12th and the 17th day corresponds to the high rate of oviposition during this time. The amino acid concentration shows only slight variations and is higher in the mated than in the unmated flies. 4. The last days of life are characterized by a dramatic decrease of carbohydrates. The loss of these important metabolic fuels is assumed to have a severe influence on the whole metabolism, but alone cannot account for the termination of life because of the considerably higher carbohydrate values of the virgin females at their last day of life. A c k n o w l e d g e m e n t - - T h i s work was partly supported by a grant from the "Deutsche Forschungsgemeinschaft".
REFERENCES AMENTA, J. S. (1964) J. lip. Res. 5,270. BAUMAN, P. and CHEN, P. S. (1968) Revve. suisse Zool. 75, 1051. BIt:C;ANSKA-P~ETRZAKOWA,M. (1961)Zool. Polon. 10,265. CHEN, P. S. (1966)Adv. Insect Physiol. 3, 53. CLARKE, J. M. and MAYNARD-SMITH, 3. (1966) Nature Lond. 209,627. Col LATZ, K. G. (1973) In: Effects o f Temperature on Ectothermic Organisms (Edited by W. WIE:SER), p. 195. Springer, Berlin. COLLATZ, K. G. and MOMMSEN, T. (1974) J. comp. Physiol. 91, 91. GANETZKY, B. and FLANAGAN, J. R. (1978) Exp. Geront. 13, 189. GELPtqRKN, A. and DETHIER, V. G. (1967)Physiol. Zool. 40,218. HARLOW, P. M. (1956) J. exp. Biol. 33, 777. HOEGER, U. (1976) Diplomarbeit, Freiburg i. Br. KocH, A. L. and PUTNAM, S. L. (I 971)Anal. Biochem. 44,239. KRUMBIEGEL, 1. (1929) Zool. Jb. Abt. Anat. Ontog. 51, 111. KrUMBJt-CiEL, 1. (1930)Forsch. Fortschr. 2, 85.
AGE-RELATEDCHANGESINTHE BODYCOMPOSITIONOF BLOWFLIES
441
LEVENBOOK,L. and KRISHNA,I. (1971) J. Insect Physiol. 17, 9. MAYNARD-SMITH,J., BOZCUK,A. N. and TEBBUT, S. (I 970)J. Insect Physiol. 16, 601. NOWOSlELSKY,J. W. and PATTON, R. L. (1965a) J. Insect Physiol. l l , 201. NOWOSIELSKY,.I.W. and PATTON, R. L. (1965b)J. Insect Physiol. 11,263. PEARL, R. and PARKER,S. L. (1924) Amer. Nat. 58, 71. ROCKSTEIN, M. (1973) In: The Physiology of Insecta (Edited by M. ROCKSTEIN), vol. l, p. 371. Academic Press, London. ROE, J. R. (1955) J. biol. Chem. 212, 335. ROWLEY, W. A. and GRAHAM,C. L. (1968)J. Insect Physiol. 14, 719. SAMIS, H. V. Jr., ERK, F. C. and DAIRD, M. B. (1971) Exp. Geront. 6, 9. TROLL,W. and CANNAN,R. K. (1953) J. biol. Chem. 200, 803. WILLIAMS, C. M., BARNESS,L. A. and SAWYER,W. H. (1943) Biol. Bull. 84, 263.