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Diurnal patterns of ammonia release in marine and terrestrial isopods
cow+. Biochem. Physiol., 1974, Vol, 47A, pp. 1313 to 1321. Pergamon Press. Printed in Great Britain
DIURNAL PATTERNS OF AMMONIA RELEASE IN MARINE AND TERRESTRIAL ISOPODS PHILIP
K. KIRBY
and ROBERT
Hopkins Marine Station of Stanford University,
D. HARBAUGH
Pacific Grove, California 93950, U.S.A.
(Received 18 June 1973) Absmct-1. An inverse exponential relationship between the rate of ammonia excretion and dry body weight has been established in a selected isopod, Cirolana harfordi. 2. Similar diurnal patterns of rate of ammonia release have been demonstrated in eight species of isopods including members of the Oniscoidaea, Valvifera and Flabellifera. 3. C. harfordi exposed to either normal light/dark photoperiodism, continuous light or continuous dark retain a characteristic pattern of ammonia release. 4. Porcellio scaber and Idotea resecata were found to release ammonia during the afternoon in discrete events, with comparable rates of release. No correlation was found between fecal production and ammonia release.
IT HAS been found that a number of terrestrial isopods have been able to retain ammonotelism by developing a process of gaseous ammonia release (Dresel & Moyle, 1950; Wieser et al., 1969). It has been suggested that this may represent a significant terrestrial adaptation in itself in that retention of ammonotelism may confer significant thermodynamic advantages to the organism (Hartenstein, 1968). Nitrogenous excretion in isopods has been investigated by a number of individuals, but limited research has been performed which adequately characterizes observed diurnal patterns of ammonia release. Such diurnal rhythms may represent a contributing adaptation to the capacity for retention of ammonotelism in terrestrial isopods in that ammonia is excreted at times when concomitant water loss from permeable surface membranes is minimized. Wieser et al. (1969) found that volatile ammonia release in two species of terrestrial isopods displays diurnal rh~hmicity with a maximum excretory period occurring in the afternoon, and they concluded that the rhythm observed was inversely related to the pattern of locomotory activity of the animals. Thus, it would appear that the excretory process concerned with the release of nitrogenous wastes in isopods is at least, in a temporal sense, a non-random, carefully regulated function. However, it has not been shown if distinct or uniform diurnal patterns of ammonia release are exhibited within an array of isopod species, or if the pattern of nitrogenous excretion in isopods represents a truly endogenous rhythm. This research examines the diurnal pattern of ammonia release in eight species of marine and terrestrial isopods by initially 1313
1314
PHILIP K. KIRBY AND ROBERTD. HARBAUGH
establishing a guide-line relationship which shapes the interpretation of the subsequent data presented and by investigating the patterns and characteristic nature of nitrogenous waste excretion in organisms exposed to constant environmental conditions. MATERIALS
AND
METHODS
Whenever possible, freshly collected animals were used to minimize any trauma from storage within the laboratory. When storage was necessary, the Oniscoidaea were kept in plastic bins with dirt or sand litter from the collecting site. The Valvifera and Flabellifera were stored in tanks with running sea water and were fed ad lib. In all experiments, only males were used, and in all cases a representative range of animal sizes was included. The method of monitoring ammonia release differed between terrestrial and marine species. In the terrestrial species, individuals were placed in 25ml Erlenmeyer flasks, and the flasks were continuously flushed with purified air. The air was purified by filtration through cotton, bubbling through a solution of 1 N HaSO, to remove any ammonia, and then humidified by passage through distilled water. After passing through the experimental chambers, ammonia was trapped in 10 ml of distilled water acidified to pH 4.5 by the addition of dilute HCI, Every 4 hr l-ml samples were taken, and the trapping solutions replaced. In marine species, individuals were kept in test tubes with 10 ml sea water at ambient sea-water temperatures and were aerated hourly with purified air. The treatment of Cirolana harfordi was exceptional in that it was kept in 4 ml of sea water without aeration. Every 4 hr the water was sampled and replaced as above. Unless otherwise noted, animals were exposed to constant light in the laboratory. Ammonia was determined by a modification of the phenol-hypochforite method as outlined by Sol6rzano (1969). To a l-ml sample were sequentially added 1 ml of a phenolalcohol solution, 1 ml of a sodium nitroprusside solution and 2.5 ml of an oxidizing reagent containing sodium hypochlorite. The reaction mixture was allowed to stand at room temperature for exactly 1 hr and a calorimetric determination of ammonia content was performed on a Klett-Summerson photoelectric coIorimeter, model 800-3, using a red filter, A standard curve was prepared using Adonis sulfate as substrate. Control samples consisted of either deammonified water or sea water. Dry tissue weights were determined on a Mettler balance after individual specimens were dried to constant weight. Subsequently, mean hourly ammonia excretion rates per mg dry tissue weight were computed when appropriate. Sites of ammonia release were studied by wrapping isopods in damp filter paper impregnated with phenol red. The paper was impregnated by soaking in a 115 dilution of standard indicator solution, O-1 g in 14.3 ml O-02 M NaOH, and allowed to partially dry. At 30-min intervals the animals were carefully unwrapped and the paper was examined. RESULTS I. Relationship between body wezkht and ammonia excretion
A significant relationship was determined between the amount of ammonia excreted and body size as determined by dry weight measurements. Figure 1 illustrates this relationship by plotting ammonia excretion, expressed in ng NH,/ mg per hr, as a function of dry body weight for a marine isopod, C. harfordi. A diminishing exponentia1 curve can be fitted to express the relationship. These data strongIy suggest that young developing C. harfordi possess a much higher rate of protein turnover than do older, more mature organisms. This inverted exponential relationship is important in that there exist great variations in ammonia
1315
DIURNAL PATTERNS OF AMMONIA RELEASE IN ISOPODS zo-
0
I 20
I 40
I 60
Dry body weight,
I SO
I loo
I 120
mg
FIG. 1. Linear plot of ammonia excretion (ng NH,/mg per hr) for C. harfordi expressed as a function of dry body weight (mg). N = 32. Excretion rate values were obtained from total ammonia release over a 24-hr period.
excretion measures as expressed on a weight basis among different individuals within a normal sample population. When this concept is applied to various species of isopods, unless very large or carefully matched samples are utilized, internal standards are required in order to evaluate in a meaningful manner the diurnal patterns of ammonia excretion. As far as we know, no significant note of this concept has been made by those who have investigated ammonia excretion rates in isopod populations. Because of the relationship between weight and ammonia release observed in C. harfordi, the data presented which describe the diurnal patterns of ammonia excretion in isopods have been tabulated by a method which corrects for the significant release rate variances among individuals at different life stages. All excretion rate values obtained at specific time points within a 24-hr period for a given specimen were averaged, and each individual value expressed as a percentage of this 24-hr mean. Release rates so normalized were averaged for a group of specimens at each reading and plotted as shown in Figs. 2-5, which include the standard deviations of this estimate. II. Comparison of patterns of ammonia release
Figure 2 displays a very uniform diurnal pattern of ammonia release in the family, Oniscoidaea. Porcellio scaber, Alloniscus perconvexus and Armadillidium vulgare all had maxima of ammonia release rates from 1200 to 1600, and minima from 2400 to 0400, a pattern which is identical to that found by Wieser et al. (1969) in members of the same family of terrestrial isopods.
PHILIP K. KIRBY AND ROBERTD. HARBALJGEI
1316
1200-1600 1600-2000 2000-2400 2400-0400 0400-0800 0800-1200 Time intwvoI FIG. 2. The diurnaI pattern of ammonia release in fed male specimens of A. dgare (light grey), P. scaber (white) and A. perconetexus (dark grey), in constant light. Per cent mean hourly excretion is plotted against a 24-hr cycle where 1200 = noon. N = 10 for P. scaber, and N = 8 for A. vulgare and A. perconvexus. Standard deviations are indicated.
I. KirchhonsKii
ZOO-1600
16GO-2000
ZOCQ-2400
Ttme
2400-0400
0400-0800
0.300-1200
Servo I
FIG. 3. The diurnal pattern of ammonia release in fed male I. montereyasis (light grey), I. resecata (white) and I. Kirchanskii (dark grey), in constant light. Per cent mean hourly excretion is plotted against a 24-hr cycle where 1200 = noon. N = 10 for each species, and standard deviations are indicated.
1317
DIURNAL PATTERNS OF AMMONIA RELS3ASEIN ISOPODS
Figure 3 sbows that the members of the family Valvifera, Idotea resecata, Idotea montereyensisand Idotea kirchanskii, show the same pattern as that found in the Oniscoidaea, with a maxima of ammonia release rates from 1200 to 1600 and minima at night. The night minima were found to be either between 2400 and 0400 or between 0400 and 0800. As shown in Fig. 4, the members of the Flabel~~era, Cirolanu hQ~f~di and Lironeca vufgur&, were exceptional in their patterns of ammonia release. C. karfordi showed a maximum of release rates between 0400 and 0800 and a minimum between 1200 and 1600, a pattern which is anomalous in that it is a reversal of the type of pattern observed in the other isopod families. L. z~Zg& had its maximum from 1200 to 1600, with a minimum during the night, but showed a second peak of release between 2400 and 0400. 401 3-
81 P PI
30
o-
20
o-
4 aIOO-
1200-1600
1600-2OCQ
2OUO-2400
Time
2400-0400
0400-0800
0600-1200
interval
FIG. 4. The diurnal pattern of ammonia release in fed male C.
harfordi(white) and
L. vulgar& (grey), in constant light. Per cent mean hourly excretion is plotted against a 24-k
cycle where 1200 = noon. N = 10 for each species, and standard deviations are indicated.
III. Esfects of environmentallighting upon the diurnal pattern of ammonia excretion in a selected isopod, C. harfordi Figure 5 presents preliminary results on the effects of continuous light, continuous dark and normal light~dark regimes upon st~d~dized ammonia excretion rates over a 24-hr period in a selected isopod, C. harfordi. Groups of animals were maintained on a normal light/dark cycle or were pre-exposed to either constant light or dark for a 24hr period prior to the initiation of the appropriate experiment and were kept in constant conditions for the duration of the experiment. The graph
PHILIP K. KIRBY ANDROBERTD. HARBAUCH
1318
AII
1200-
Normal
p$j@jj
Continuous
py.j
Continuous
1600
light
I
1600-2000
/dark dark
light
T
: :: :; ., ; ,.. ;;;,;‘. ;, :: :: :. .:
. :: .: .:;.:. ....
:: ::
i.. :
1;:’ :: ::.
~ 2000-2400 Time
2400-0400
0400-0800
0600-1200
interval
FIG. 5. The diurnal pattern of ammonia release in C. harfordi exposed to normal light/dark (white), constant dark (dark grey) and constant light (light grey) regimes. Per cent mean hourly excretion is plotted against a 24-hr cycle where 1200 = noon. N = 10 for each sample group, and standard deviations are indicated.
indicates
that an endogenous ammonia release rhythm persists which is to a certain degree independent of environmental lighting cues. However, when compared to animals exposed to a normal light/dark photoperiod, it does appear that constant light and constant dark regimes do modify excretory response patterns as both observed maxima have been shifted towards a later time of day. However, further investigations are required in order to clarify fully the nature of ammonia excretion rhythmicity in C. harfordi. IV.
The nature of ammonia release
Having observed patterns in ammonia release, the emphasis was shifted to learning more about the nature of the release itself, by focusing on the period of maximal release. P. scaber and I. resecata were monitored through their maximal period of ammonia excretion by taking samples and making observations every half hour between 1200 and 1600. I. resecata produces conspicuous amounts of fecal matter and at each reading the presence or absence of fecal pellets was noted. The results showed that ammonia release is not a continuous process during the 4-hr observation period, but occurs in discrete and random events. The mean hourly rate of release events was calculated at 0,737 events/hr with S.D. + 0.17 as is shown in Table 1. No correlation was found between fecal production and ammonia release. P. scaber had been observed to release sufficient ammonia to cause color change in phenol red indicator paper, and was monitored by wrapping individuals in moist
DIURNAL PATTERNS OF AMMONIA
TAB= ~-EXCRETORYEVENTS IN P. scuber AND I. resecatu P. scaber, N = 8 Ammonia release Fecal release Fecal and ammonia release
1319
IWLJWSE IN ISOPODS FROM
1200 TO 1400
I. resecata, N = 20
0.781 f 0.09 0 0
0.737 f 0.17 O-512 + O-47 O-162 + O-21
Values express mean events/hr, and standard deviations are indicated.
indicator paper. At each reading, in addition to noting release, the location of the release was recorded. As displayed in Table 1, the results show that release occurs as random and discrete events, with a mean release rate of 0.781 events/hr with S.D. + 0.09. These results are very similar to those found in 1. resecatu. In addition, release was found to be primarily associated with the ventral surface of the telson and, to a lesser degree, with the ventral surface of the head region as is indicated in Table 2. In no instance was any fecal production noted. TABLE ~--EVENTS
AND SITES OF AMMONIA
RELEASE IN P.
Scuber 12W1600
Animal Time
1
2
1230 1300 1330 1400 1430 1500 1530 1600
Telson Telson
Telson Telson Telson
Head
3
Telson Telson Telson
4
5
6
7
Telson
Telson Head Head
Head
Telson
Head
Telson
8
Head
Head Telson Telson
Head
Telson
Head Telson
DISCUSSION As
has been previously stated, the primary objectives in undertaking this study were to investigate the possible generality of the diurnal pattern of ammonia release observed in two species of terrestrial isopods by Wieser et al. (1969) and to examine species with contrasting activity to gain more information about the hypothesis that ammonia release is inversely related to locomotory activity. In eight species belonging to three families of isopods, a pattern of ammonia release was found which, with only one exception, closely approximated that reported by Wieser et al. (1969) in P. scaber and Oniscus aselks with a large maximum in the afternoon. The general presence of a pattern or rhythm appears fairly well established, but the underlying causes for this rhythm are more obscure. Basic to all theories advanced to explain rhythmicity is the proposal that there exists an inverse relationship between ammonia excretion and locomotory activity (Wieser et al., 1969). Such rhythmicity reflects energy-releasing metabolism of amino acids during activity and
1320
PHILIP K. KIRBY AND ROBERT D. HARBAUGH
excretion of volatile nitrogenous wastes during periods of rest in humid burrows when concomitant water loss through permeable surface membranes is minimized. In both P. scaber and A. ~~co~vex~ there are established patterns of activity which conform to nocturnal scavenging and retreat by day, as documented by Brereton (1957) and Pratt (1973) respectively. However, in the related terrestrial species A. vulgare, the nocturnal pattern, though common, may vary from population to population (Paris, 1962), and in a local population near the laboratory, individuals were observed to be foraging by day. When these specimens of A. vulgare were examined they still showed a pattern distinguished by pronounced excretion maxima and minima in accordance with the general pattern of ammonia release as observed in other isopod species. In the marine species, the problems in relating the release pattern to activity are more complex. The species of Idotea studied may be found on their characteristic algal substrata at any time of the day with filled intestines, and with no observable fluctuations in activity levels. However, the same characteristic pattern of ammonia release as found in the Oniscoidaea was detected in these species of marine isopods. Clearly, no humidity or moisture variables can account for the fluctuation, and the afternoon does not seem to be a period of reduced activity. The Flabellifera studied present even stronger evidence that factors apart from activity may control ammonia release. C. h~~f~~d~has been reported to be predominantly nocturnal in its activity patterns (Harrold, 1973), with its maximum activity levels corresponding with its maximum period of ammonia release. This suggests that a direct correlation between activity and ammonia excretion exists in C. harfordi. In contrast, Limneca vulgaris is an almost sessile parasite, which seems incapable of locomotion in its fully matured female form (Whiteside, 1973). This animal lives most of its life in the gills of fish and yet shows a very marked and relatively complex pattern of release, with maxima in both afternoon and early morning. These observations are comparative, and while it is realized that a similar pattern of release could meet the environmental pressures imposed on each species, the very similarity of pattern suggests that there might be a more basic common rhythm in isopods controlling ammonia excretion. In the comparison of the patterns of ammonia release rates, all species were subjected to constant light and a relatively constant environment in order to detect endogenous rhythmicity. It has been shown that the rhythms are not dependent on the presence of a light/dark cycle, and in the lighting cue experiment performed on C. hurfordi it was shown that the pattern of ammonia release is not abolished by the presence of a normal light/dark photoperiod, However, the variation of lighting conditions was found to modify the pattern by shifting the times of the observed maxima and minima. This observation may support the concept that in the absence of environmental cues such as light/dark photoperiodism, endogenous rhythms may become desynchronized with time. The investigation of the period of maximal release in 1. resecata and P. scuber showed ammonia release to occur in discrete and random events, unrelated to fecal production. The discrete nature of the events of ammonia release suggests that
DIURNAI.PATTERNS OF~ONIA~~E
IN ISOPODS
1321
there might be a build-up of ammonia or precursors in the tissues at the site of release prior to an event. A search for this precursor may shed further light on the pathway and mechanism of ammonia excretion in these animals. The fact that the tissues associated with release of ammonia in P. scaler seem to be located in the head and telson poses questions. Is the observed release an artifact of the experimental methods, or a response to stress or fright, resulting in the release from the integumental glands as noted by Gorham (1973) ? All animals survived the experiment apparently unharmed, and the position of an animal wrapped in moist filter paper is not unlike that of a burrowing animal. If the release is not an artifact, then the possibility of multiple sites must be considered. It is possible that the digestive tract could be used as an excretory organ for ammonia, but the absence of correlation between fecal production and ammonia release does not support this theory. In conclusion, the data presented here suggest that ammonia release in isopods is a carefully regulated, non-random process which exhibits properties of endogenous rhythmicity. No simple correlation between ammonia release and activity pattern was observed, though these phenomena may interact as parts of a more complex cycle. Wieser & Schweizer (1970) have stated that “isopods have adapted protein metabolism to terrestrial conditions by programming the excretion of nitrogen in such a way that it takes place mainly during periods of inactivity when the animals are in their moist retreats”. We conclude that the capacity to “program” ammonia release is not necessarily a terrestrial adaptation, but a fundamental, uniform physiologic characteristic of a number of isopod species, marine included. Acknowledgements-We would like to gratefully acknowledge the assistance of Dr. John Phillips for his invaluable help in conducting the research outlined in this paper and for his advice in the preparation of this manuscript. REFERENCES BRERETONJ . (1957) The distribution of woodland isopods. O&OS8,85-l 06. DRE~EL E. & MOYLE V. (1950) Nitrogenous excretion of amphipods and isopods. r, exp. Biol. 27, 210-225. GORHAMW. (1973) Personal communication. HARROLDC. (1973) Environmental factors affecting patterns of activity in Cirolana harfordi (Lockington). Research Report 175H, Hopkins Marine Station, Pacific Grove, California. HARTESSTEINR. (1968) Nitrogen metabolism in the terrestrial isopod, Otziscusaseilus. Am. Zoologist 8, 507-S 19. PARIS, 0. H. F%TELKA F. A. (1962) Population characteristics of the terrestrial isopod Armadillidium vu&are in California grass-lands. Ecology 14, 229-248. PRATT S. (1973) Factors affecting the activity patterns of the sandy beach isopod, Alloniscus pe~convexus. Research Report 17SH, Hopkins Marine Station, Pacific Grove, California. SOL&ZANO L. (1969) Determination of ammonia in natural waters by the phenol-hypochlorite method. Limnol. Oceunogr. 14,799-801. WHITESIDE C. (1973) Feeding mechanics and food preference in Lironeca vulgaris. Research Report 175H, Hopkins Marine Station, Pacific Grove, California. WIENER W. & SCHW~IZERG. (1970) A re-examination of the excretion of nitrogen by terrestrial isopods. J. exp. Biol. 52, 267-279.
1322
PHILIP K. KIRBY AND ROBERTD. HARBAUGH
WIESERW., SCHWEIZERG. & HARTENSTEIN R. (1969) Patterns in the release of gaseous ammonia by terrestrial isopods. Oecologia (BerZ.). 3, 390-400. Key Word Index-Ammonia; resecata.
isopods;
Cirolana harfordi;
Porcellio scaber; Idotea
DIURNAL PATTERNS OF AMMONIA RELEASE IN MARINE AND TERRESTRIAL ISOPODS PHILIP
K. KIRBY
and ROBERT
Hopkins Marine Station of Stanford University,
D. HARBAUGH
Pacific Grove, California 93950, U.S.A.
(Received 18 June 1973) Absmct-1. An inverse exponential relationship between the rate of ammonia excretion and dry body weight has been established in a selected isopod, Cirolana harfordi. 2. Similar diurnal patterns of rate of ammonia release have been demonstrated in eight species of isopods including members of the Oniscoidaea, Valvifera and Flabellifera. 3. C. harfordi exposed to either normal light/dark photoperiodism, continuous light or continuous dark retain a characteristic pattern of ammonia release. 4. Porcellio scaber and Idotea resecata were found to release ammonia during the afternoon in discrete events, with comparable rates of release. No correlation was found between fecal production and ammonia release.
IT HAS been found that a number of terrestrial isopods have been able to retain ammonotelism by developing a process of gaseous ammonia release (Dresel & Moyle, 1950; Wieser et al., 1969). It has been suggested that this may represent a significant terrestrial adaptation in itself in that retention of ammonotelism may confer significant thermodynamic advantages to the organism (Hartenstein, 1968). Nitrogenous excretion in isopods has been investigated by a number of individuals, but limited research has been performed which adequately characterizes observed diurnal patterns of ammonia release. Such diurnal rhythms may represent a contributing adaptation to the capacity for retention of ammonotelism in terrestrial isopods in that ammonia is excreted at times when concomitant water loss from permeable surface membranes is minimized. Wieser et al. (1969) found that volatile ammonia release in two species of terrestrial isopods displays diurnal rh~hmicity with a maximum excretory period occurring in the afternoon, and they concluded that the rhythm observed was inversely related to the pattern of locomotory activity of the animals. Thus, it would appear that the excretory process concerned with the release of nitrogenous wastes in isopods is at least, in a temporal sense, a non-random, carefully regulated function. However, it has not been shown if distinct or uniform diurnal patterns of ammonia release are exhibited within an array of isopod species, or if the pattern of nitrogenous excretion in isopods represents a truly endogenous rhythm. This research examines the diurnal pattern of ammonia release in eight species of marine and terrestrial isopods by initially 1313
1314
PHILIP K. KIRBY AND ROBERTD. HARBAUGH
establishing a guide-line relationship which shapes the interpretation of the subsequent data presented and by investigating the patterns and characteristic nature of nitrogenous waste excretion in organisms exposed to constant environmental conditions. MATERIALS
AND
METHODS
Whenever possible, freshly collected animals were used to minimize any trauma from storage within the laboratory. When storage was necessary, the Oniscoidaea were kept in plastic bins with dirt or sand litter from the collecting site. The Valvifera and Flabellifera were stored in tanks with running sea water and were fed ad lib. In all experiments, only males were used, and in all cases a representative range of animal sizes was included. The method of monitoring ammonia release differed between terrestrial and marine species. In the terrestrial species, individuals were placed in 25ml Erlenmeyer flasks, and the flasks were continuously flushed with purified air. The air was purified by filtration through cotton, bubbling through a solution of 1 N HaSO, to remove any ammonia, and then humidified by passage through distilled water. After passing through the experimental chambers, ammonia was trapped in 10 ml of distilled water acidified to pH 4.5 by the addition of dilute HCI, Every 4 hr l-ml samples were taken, and the trapping solutions replaced. In marine species, individuals were kept in test tubes with 10 ml sea water at ambient sea-water temperatures and were aerated hourly with purified air. The treatment of Cirolana harfordi was exceptional in that it was kept in 4 ml of sea water without aeration. Every 4 hr the water was sampled and replaced as above. Unless otherwise noted, animals were exposed to constant light in the laboratory. Ammonia was determined by a modification of the phenol-hypochforite method as outlined by Sol6rzano (1969). To a l-ml sample were sequentially added 1 ml of a phenolalcohol solution, 1 ml of a sodium nitroprusside solution and 2.5 ml of an oxidizing reagent containing sodium hypochlorite. The reaction mixture was allowed to stand at room temperature for exactly 1 hr and a calorimetric determination of ammonia content was performed on a Klett-Summerson photoelectric coIorimeter, model 800-3, using a red filter, A standard curve was prepared using Adonis sulfate as substrate. Control samples consisted of either deammonified water or sea water. Dry tissue weights were determined on a Mettler balance after individual specimens were dried to constant weight. Subsequently, mean hourly ammonia excretion rates per mg dry tissue weight were computed when appropriate. Sites of ammonia release were studied by wrapping isopods in damp filter paper impregnated with phenol red. The paper was impregnated by soaking in a 115 dilution of standard indicator solution, O-1 g in 14.3 ml O-02 M NaOH, and allowed to partially dry. At 30-min intervals the animals were carefully unwrapped and the paper was examined. RESULTS I. Relationship between body wezkht and ammonia excretion
A significant relationship was determined between the amount of ammonia excreted and body size as determined by dry weight measurements. Figure 1 illustrates this relationship by plotting ammonia excretion, expressed in ng NH,/ mg per hr, as a function of dry body weight for a marine isopod, C. harfordi. A diminishing exponentia1 curve can be fitted to express the relationship. These data strongIy suggest that young developing C. harfordi possess a much higher rate of protein turnover than do older, more mature organisms. This inverted exponential relationship is important in that there exist great variations in ammonia
1315
DIURNAL PATTERNS OF AMMONIA RELEASE IN ISOPODS zo-
0
I 20
I 40
I 60
Dry body weight,
I SO
I loo
I 120
mg
FIG. 1. Linear plot of ammonia excretion (ng NH,/mg per hr) for C. harfordi expressed as a function of dry body weight (mg). N = 32. Excretion rate values were obtained from total ammonia release over a 24-hr period.
excretion measures as expressed on a weight basis among different individuals within a normal sample population. When this concept is applied to various species of isopods, unless very large or carefully matched samples are utilized, internal standards are required in order to evaluate in a meaningful manner the diurnal patterns of ammonia excretion. As far as we know, no significant note of this concept has been made by those who have investigated ammonia excretion rates in isopod populations. Because of the relationship between weight and ammonia release observed in C. harfordi, the data presented which describe the diurnal patterns of ammonia excretion in isopods have been tabulated by a method which corrects for the significant release rate variances among individuals at different life stages. All excretion rate values obtained at specific time points within a 24-hr period for a given specimen were averaged, and each individual value expressed as a percentage of this 24-hr mean. Release rates so normalized were averaged for a group of specimens at each reading and plotted as shown in Figs. 2-5, which include the standard deviations of this estimate. II. Comparison of patterns of ammonia release
Figure 2 displays a very uniform diurnal pattern of ammonia release in the family, Oniscoidaea. Porcellio scaber, Alloniscus perconvexus and Armadillidium vulgare all had maxima of ammonia release rates from 1200 to 1600, and minima from 2400 to 0400, a pattern which is identical to that found by Wieser et al. (1969) in members of the same family of terrestrial isopods.
PHILIP K. KIRBY AND ROBERTD. HARBALJGEI
1316
1200-1600 1600-2000 2000-2400 2400-0400 0400-0800 0800-1200 Time intwvoI FIG. 2. The diurnaI pattern of ammonia release in fed male specimens of A. dgare (light grey), P. scaber (white) and A. perconetexus (dark grey), in constant light. Per cent mean hourly excretion is plotted against a 24-hr cycle where 1200 = noon. N = 10 for P. scaber, and N = 8 for A. vulgare and A. perconvexus. Standard deviations are indicated.
I. KirchhonsKii
ZOO-1600
16GO-2000
ZOCQ-2400
Ttme
2400-0400
0400-0800
0.300-1200
Servo I
FIG. 3. The diurnal pattern of ammonia release in fed male I. montereyasis (light grey), I. resecata (white) and I. Kirchanskii (dark grey), in constant light. Per cent mean hourly excretion is plotted against a 24-hr cycle where 1200 = noon. N = 10 for each species, and standard deviations are indicated.
1317
DIURNAL PATTERNS OF AMMONIA RELS3ASEIN ISOPODS
Figure 3 sbows that the members of the family Valvifera, Idotea resecata, Idotea montereyensisand Idotea kirchanskii, show the same pattern as that found in the Oniscoidaea, with a maxima of ammonia release rates from 1200 to 1600 and minima at night. The night minima were found to be either between 2400 and 0400 or between 0400 and 0800. As shown in Fig. 4, the members of the Flabel~~era, Cirolanu hQ~f~di and Lironeca vufgur&, were exceptional in their patterns of ammonia release. C. karfordi showed a maximum of release rates between 0400 and 0800 and a minimum between 1200 and 1600, a pattern which is anomalous in that it is a reversal of the type of pattern observed in the other isopod families. L. z~Zg& had its maximum from 1200 to 1600, with a minimum during the night, but showed a second peak of release between 2400 and 0400. 401 3-
81 P PI
30
o-
20
o-
4 aIOO-
1200-1600
1600-2OCQ
2OUO-2400
Time
2400-0400
0400-0800
0600-1200
interval
FIG. 4. The diurnal pattern of ammonia release in fed male C.
harfordi(white) and
L. vulgar& (grey), in constant light. Per cent mean hourly excretion is plotted against a 24-k
cycle where 1200 = noon. N = 10 for each species, and standard deviations are indicated.
III. Esfects of environmentallighting upon the diurnal pattern of ammonia excretion in a selected isopod, C. harfordi Figure 5 presents preliminary results on the effects of continuous light, continuous dark and normal light~dark regimes upon st~d~dized ammonia excretion rates over a 24-hr period in a selected isopod, C. harfordi. Groups of animals were maintained on a normal light/dark cycle or were pre-exposed to either constant light or dark for a 24hr period prior to the initiation of the appropriate experiment and were kept in constant conditions for the duration of the experiment. The graph
PHILIP K. KIRBY ANDROBERTD. HARBAUCH
1318
AII
1200-
Normal
p$j@jj
Continuous
py.j
Continuous
1600
light
I
1600-2000
/dark dark
light
T
: :: :; ., ; ,.. ;;;,;‘. ;, :: :: :. .:
. :: .: .:;.:. ....
:: ::
i.. :
1;:’ :: ::.
~ 2000-2400 Time
2400-0400
0400-0800
0600-1200
interval
FIG. 5. The diurnal pattern of ammonia release in C. harfordi exposed to normal light/dark (white), constant dark (dark grey) and constant light (light grey) regimes. Per cent mean hourly excretion is plotted against a 24-hr cycle where 1200 = noon. N = 10 for each sample group, and standard deviations are indicated.
indicates
that an endogenous ammonia release rhythm persists which is to a certain degree independent of environmental lighting cues. However, when compared to animals exposed to a normal light/dark photoperiod, it does appear that constant light and constant dark regimes do modify excretory response patterns as both observed maxima have been shifted towards a later time of day. However, further investigations are required in order to clarify fully the nature of ammonia excretion rhythmicity in C. harfordi. IV.
The nature of ammonia release
Having observed patterns in ammonia release, the emphasis was shifted to learning more about the nature of the release itself, by focusing on the period of maximal release. P. scaber and I. resecata were monitored through their maximal period of ammonia excretion by taking samples and making observations every half hour between 1200 and 1600. I. resecata produces conspicuous amounts of fecal matter and at each reading the presence or absence of fecal pellets was noted. The results showed that ammonia release is not a continuous process during the 4-hr observation period, but occurs in discrete and random events. The mean hourly rate of release events was calculated at 0,737 events/hr with S.D. + 0.17 as is shown in Table 1. No correlation was found between fecal production and ammonia release. P. scaber had been observed to release sufficient ammonia to cause color change in phenol red indicator paper, and was monitored by wrapping individuals in moist
DIURNAL PATTERNS OF AMMONIA
TAB= ~-EXCRETORYEVENTS IN P. scuber AND I. resecatu P. scaber, N = 8 Ammonia release Fecal release Fecal and ammonia release
1319
IWLJWSE IN ISOPODS FROM
1200 TO 1400
I. resecata, N = 20
0.781 f 0.09 0 0
0.737 f 0.17 O-512 + O-47 O-162 + O-21
Values express mean events/hr, and standard deviations are indicated.
indicator paper. At each reading, in addition to noting release, the location of the release was recorded. As displayed in Table 1, the results show that release occurs as random and discrete events, with a mean release rate of 0.781 events/hr with S.D. + 0.09. These results are very similar to those found in 1. resecatu. In addition, release was found to be primarily associated with the ventral surface of the telson and, to a lesser degree, with the ventral surface of the head region as is indicated in Table 2. In no instance was any fecal production noted. TABLE ~--EVENTS
AND SITES OF AMMONIA
RELEASE IN P.
Scuber 12W1600
Animal Time
1
2
1230 1300 1330 1400 1430 1500 1530 1600
Telson Telson
Telson Telson Telson
Head
3
Telson Telson Telson
4
5
6
7
Telson
Telson Head Head
Head
Telson
Head
Telson
8
Head
Head Telson Telson
Head
Telson
Head Telson
DISCUSSION As
has been previously stated, the primary objectives in undertaking this study were to investigate the possible generality of the diurnal pattern of ammonia release observed in two species of terrestrial isopods by Wieser et al. (1969) and to examine species with contrasting activity to gain more information about the hypothesis that ammonia release is inversely related to locomotory activity. In eight species belonging to three families of isopods, a pattern of ammonia release was found which, with only one exception, closely approximated that reported by Wieser et al. (1969) in P. scaber and Oniscus aselks with a large maximum in the afternoon. The general presence of a pattern or rhythm appears fairly well established, but the underlying causes for this rhythm are more obscure. Basic to all theories advanced to explain rhythmicity is the proposal that there exists an inverse relationship between ammonia excretion and locomotory activity (Wieser et al., 1969). Such rhythmicity reflects energy-releasing metabolism of amino acids during activity and
1320
PHILIP K. KIRBY AND ROBERT D. HARBAUGH
excretion of volatile nitrogenous wastes during periods of rest in humid burrows when concomitant water loss through permeable surface membranes is minimized. In both P. scaber and A. ~~co~vex~ there are established patterns of activity which conform to nocturnal scavenging and retreat by day, as documented by Brereton (1957) and Pratt (1973) respectively. However, in the related terrestrial species A. vulgare, the nocturnal pattern, though common, may vary from population to population (Paris, 1962), and in a local population near the laboratory, individuals were observed to be foraging by day. When these specimens of A. vulgare were examined they still showed a pattern distinguished by pronounced excretion maxima and minima in accordance with the general pattern of ammonia release as observed in other isopod species. In the marine species, the problems in relating the release pattern to activity are more complex. The species of Idotea studied may be found on their characteristic algal substrata at any time of the day with filled intestines, and with no observable fluctuations in activity levels. However, the same characteristic pattern of ammonia release as found in the Oniscoidaea was detected in these species of marine isopods. Clearly, no humidity or moisture variables can account for the fluctuation, and the afternoon does not seem to be a period of reduced activity. The Flabellifera studied present even stronger evidence that factors apart from activity may control ammonia release. C. h~~f~~d~has been reported to be predominantly nocturnal in its activity patterns (Harrold, 1973), with its maximum activity levels corresponding with its maximum period of ammonia release. This suggests that a direct correlation between activity and ammonia excretion exists in C. harfordi. In contrast, Limneca vulgaris is an almost sessile parasite, which seems incapable of locomotion in its fully matured female form (Whiteside, 1973). This animal lives most of its life in the gills of fish and yet shows a very marked and relatively complex pattern of release, with maxima in both afternoon and early morning. These observations are comparative, and while it is realized that a similar pattern of release could meet the environmental pressures imposed on each species, the very similarity of pattern suggests that there might be a more basic common rhythm in isopods controlling ammonia excretion. In the comparison of the patterns of ammonia release rates, all species were subjected to constant light and a relatively constant environment in order to detect endogenous rhythmicity. It has been shown that the rhythms are not dependent on the presence of a light/dark cycle, and in the lighting cue experiment performed on C. hurfordi it was shown that the pattern of ammonia release is not abolished by the presence of a normal light/dark photoperiod, However, the variation of lighting conditions was found to modify the pattern by shifting the times of the observed maxima and minima. This observation may support the concept that in the absence of environmental cues such as light/dark photoperiodism, endogenous rhythms may become desynchronized with time. The investigation of the period of maximal release in 1. resecata and P. scuber showed ammonia release to occur in discrete and random events, unrelated to fecal production. The discrete nature of the events of ammonia release suggests that
DIURNAI.PATTERNS OF~ONIA~~E
IN ISOPODS
1321
there might be a build-up of ammonia or precursors in the tissues at the site of release prior to an event. A search for this precursor may shed further light on the pathway and mechanism of ammonia excretion in these animals. The fact that the tissues associated with release of ammonia in P. scaler seem to be located in the head and telson poses questions. Is the observed release an artifact of the experimental methods, or a response to stress or fright, resulting in the release from the integumental glands as noted by Gorham (1973) ? All animals survived the experiment apparently unharmed, and the position of an animal wrapped in moist filter paper is not unlike that of a burrowing animal. If the release is not an artifact, then the possibility of multiple sites must be considered. It is possible that the digestive tract could be used as an excretory organ for ammonia, but the absence of correlation between fecal production and ammonia release does not support this theory. In conclusion, the data presented here suggest that ammonia release in isopods is a carefully regulated, non-random process which exhibits properties of endogenous rhythmicity. No simple correlation between ammonia release and activity pattern was observed, though these phenomena may interact as parts of a more complex cycle. Wieser & Schweizer (1970) have stated that “isopods have adapted protein metabolism to terrestrial conditions by programming the excretion of nitrogen in such a way that it takes place mainly during periods of inactivity when the animals are in their moist retreats”. We conclude that the capacity to “program” ammonia release is not necessarily a terrestrial adaptation, but a fundamental, uniform physiologic characteristic of a number of isopod species, marine included. Acknowledgements-We would like to gratefully acknowledge the assistance of Dr. John Phillips for his invaluable help in conducting the research outlined in this paper and for his advice in the preparation of this manuscript. REFERENCES BRERETONJ . (1957) The distribution of woodland isopods. O&OS8,85-l 06. DRE~EL E. & MOYLE V. (1950) Nitrogenous excretion of amphipods and isopods. r, exp. Biol. 27, 210-225. GORHAMW. (1973) Personal communication. HARROLDC. (1973) Environmental factors affecting patterns of activity in Cirolana harfordi (Lockington). Research Report 175H, Hopkins Marine Station, Pacific Grove, California. HARTESSTEINR. (1968) Nitrogen metabolism in the terrestrial isopod, Otziscusaseilus. Am. Zoologist 8, 507-S 19. PARIS, 0. H. F%TELKA F. A. (1962) Population characteristics of the terrestrial isopod Armadillidium vu&are in California grass-lands. Ecology 14, 229-248. PRATT S. (1973) Factors affecting the activity patterns of the sandy beach isopod, Alloniscus pe~convexus. Research Report 17SH, Hopkins Marine Station, Pacific Grove, California. SOL&ZANO L. (1969) Determination of ammonia in natural waters by the phenol-hypochlorite method. Limnol. Oceunogr. 14,799-801. WHITESIDE C. (1973) Feeding mechanics and food preference in Lironeca vulgaris. Research Report 175H, Hopkins Marine Station, Pacific Grove, California. WIENER W. & SCHW~IZERG. (1970) A re-examination of the excretion of nitrogen by terrestrial isopods. J. exp. Biol. 52, 267-279.
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PHILIP K. KIRBY AND ROBERTD. HARBAUGH
WIESERW., SCHWEIZERG. & HARTENSTEIN R. (1969) Patterns in the release of gaseous ammonia by terrestrial isopods. Oecologia (BerZ.). 3, 390-400. Key Word Index-Ammonia; resecata.
isopods;
Cirolana harfordi;
Porcellio scaber; Idotea