SSTRACT
1. An inverse exponential relationship between ammonia excretion as ex-
pressed in ng NHa/mg dry body weight/hr when plotted as a function of
dry body weight (mg) has been established in Cirolana harfordi, a marine
isopod.
2. A diurnal pattern of ammonia excretion has been detected in specimens of
C. harfordi exposed to a normal light/dark photoperiod.
3. Non-fed members of a C. harfordi population exhibit a dampened pattern of
diurnal ammonia release and appear to excrete less total ammonia per unit
tissue weight over a 24-hour period than do fed individuals.
Conditions of constant light or constant dark do not remove the observed
pattern of diurnal ammonia release. Continuous light dampens the daily
excretion rhythm and appears to cause a diminution in ammonia excretion
rates observed over a 24-hour period.
(1)
TNTPODTI
INTRDO
TION
In most animals, the requirement to detoxify ammonia wastes represents
the principle reason behind making the transition from ammonotelism to ureo-
telism. In general, aquatic organisms excrete ammonia as the primary cata¬
bolic waste which arises from the oxidative deamination of amino acids. Nor-
mally, terrestrial organisms, which must conserve body water, have been re¬
quired to evolve alternative biochemical and physiological modes of nitrogen
excretion which usually involve the biosynthesis of urea and uric acid as pri¬
mary wastes. However, it has been found that some terrestrial isopods have
been able to retain ammonotelism by developing a process of gaseous ammonia
release (Dresel and Moyle, 1950; Wieser, et. al., 1969). This may represent
a significant terrestrial adaptation in itself in that retention of ammono-
telism may confer significant thermodynamic advantages to the organism.
Nitrogenous excretion in isopods has been investigated by a number of
individuals, but limited research has been performed which adequately char-
acterizes observed diurnal patterns of ammonia release. Such diurnal rhythms
may represent a contributing adaptation to the capacity for retention of am-
monotelism in terrestrial animals in that ammonia is excreted at times when
concomitant water loss from permeable surface membranes is minimized. Wieser
and his associates (1969) were the first to describe a daily excretion rhythm
in two species of terrestrial isopods. Kirby (1973) has found diurnal pat-
terns of ammonia release in a variety of marine species as well. In general.
it appears 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 conclusively if the pat¬
tern of ammonia release in isopods represents a truly endogenous rhythm or if
(2)
environmental cues play a significant role in modifying excretory processes.
This research examines the diurnal pattern of ammonia release in a marine
isopod, Cirolana harfordi, by initially establishing a guideline relationship
which shapes the interpretation of the subsequent data presented and by in-
vestigating the patterns of nitrogenous waste excretion in fed and non-fed
animals, and in organisms exposed to constant environmental conditions.
TERIALS AND METHODS
Generally found under rocks within intertidal areas along the Pacific
Coast of North America, Cirolana harfordi (Lockington, 1876), was chosen for
this investigation partially because it was thought that its high levels of
protein turnover due to its carnivorous eating habits would contribute to-
wards producing more pronounced excretory rate changes with time in response
to environmental variables, and because it is amenable to laboratory manipu-
lation. Specimens of C. harfordi were collected in baited traps along the
coast of Monterey Bay adjacent to Hopkins Marine Station, Pacific Grove,
California during the months of April and May, 1973. Squid wrapped in a per-
meable nylon material proved to attract adequate numbers of non-gravid in¬
dividuals. Specimens fed whole squid ad lib and a group of non-fed individuals
were kept in separate containers for approximately two weeks prior to experi-
mentation. During this time, animals were exposed to a normal light/dark
photoperiod (lights on 0600; lights off 2000). Incident light intensities
corresponded approximately to natural illumination levels.
Ammonia was determined by a modification of the phenol-hypochlorite
method as outlined by Solórzano (1969) who indicates the method to be in¬
sensitive to other nitrogenous compounds such as urea or amino acids. To a
1.0 ml ammonia sample were sequentially added 1.0 ml of a phenol-alcohol
solution, 1.0 ml of a sodium nitroprusside solution, and 2.5 ml of an oxi¬
dizing reagent containing sodium hypochlorite. The reaction mixture was al-
lowed to stand at room temperature for exactly one hour and a colorimetric
determination of ammonia content was performed on a Klett-Summerson Photo-
electric Colorimeter, model 800-3, using a red filter. Ammonia determinations
were generally done in duplicate. A standard curve was prepared using ammonium
sulfate as substrate. Control samples consisted of either deammonified water
or seawater.
Analyses available on a number of isopod species have shown ammonia to
be the predominant nitrogenous waste product (Dresel and Moyle, 1950). Because
of the extremely low production of fecal materials in C. harfordi (Johnson,
1973), and because it has been reported that the feces in other isopods con¬
tain only a small fraction of the total excreted ammonia (Dresel and Moyle,
1950), solid waste material were not specifically examined. Individual speci-
mens of C. harfordi were immersed in 4 ml of seawater, and at the specified
times, aliquot samples were taken for ammonia analysis. Fresh seawater was
then introduced into the sample tubes and the specimens were kept ambient
with respect to ocean temperatures. Oviparous females were excluded from
all experiments and individuals utilized in the experimentation were selected
at random. Dry tissue weights were determined on a Mettler balance after in-
dividual specimens were dried to constant weight. Subsequently, mean hourly
ammonia excretion rates per milligram dry tissue weight were computed at the
specified times for a given sampling interval.
r
RSUL
Prior to the presentation of data which characterizes the diurnal rhythm
of ammonia excretion in C. harfordi, a significant relationship should be
established which has necessitated modifications in the statistical inter-
pretation of ammonia release patterns. Figure 1 illustrates log ammonia excre-
tion as a function of log dry body weight. One can note a significant nega¬
tively sloping regression line which exhibits a regression coefficient of
-1.116. When a linear plot for these values is constructed as in figure 2.
a diminishing exponential curve can be fitted to express the relationship.
These data strongly suggest that young, developing C. harfordi possess a much
higher rate of protein turnover than do older, more mature organisms. These
concepts are essentially summarized in table 1. One can note excretion values
ranging from a minimum of 1.3 ng NH,/mg/hr to a maximum of 38.5 ng NH./mg/hr
in different individuals over a 24-hour period, with significant excretion
rate differences between small and large animals. In terms of absolute excre-
tion rate values, organisms less than 14 milligrams dry body weight excrete
approximately 6 to 8 times as much ammonia per unit tissue mass than do larger,
mature organisms.
This inverted exponential relationship is important in that there exist
great variations in ammonia excretion measures as expressed on weight basis
among different individuals within a normal sample population. Thus, 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 in groups of C. harfordi. As far as we know, this concept
has been ignored by those who have investigated ammonia excretion rates in
isopod populations.
(5)
Figure 3 illustrates the diurnal pattern of ammonia release in fed C.
harfordi. The results presented here have been tabulated by a method which
corrects for the significant release rate variances among individuals at dif-
fering life stages. All excretion rate values obtained at specific time
points within a 24-hour period for a given specimen were averaged, with each
value in turn being compared against this 24-hour mean. The ratios of the
excretion rate to the mean value for each time point are indicative of the
general daily pattern of ammonia release for a given individual. Release rates
so normalized were averaged for a group of specimens at each specific time
and plotted as shown.
A critical analysis of figure 3 indicates that within this nutritionally
homogeneous population, there does exist a detectable, non-random diurnal
variation in release rates. A statistically significant maximum excretion
period centered around 0200 can be easily identified. In turn, a minimum ex¬
cretion period occurs in late afternoon. These findings are in marked con-
trast to those of Kirby (1973) and others (Wieser, et. al., 1969) who have
generally found daytime maxima in other isopods. However, many of the iso-
pods examined in this respect were terrestrial herbivores, a fact which may
limit comparison with C. harfordi, a marine carnivore,
Figure 4 displays the normalized release rates for non-fed specimens of
C. harfordi over a 24-hour period. The results are significant only in the
sense that they suggest that the nutritional state of the organism affects
the pattern of ammonia release. One will note that there is a marked dampen-
ing of the normal diurnal pattern to such a degree that it is not detectable
on a statistically significant basis. A rough quantitative comparison shows
that on the average over a 24-hour period, fed C. harfordi excrete about three
times as much ammonia as do non-fed individuals. Thus, in general, the diurnal
(6)
pattern of ammonia release in C. harfordi is a partial function of nutritional
status.
Figure 5 presents preliminary results on the effects of continuous light
and dark regimes upon normalized ammonia excretion rates over a 24-hour period,
Animals were pre-exposed to either constant light or dark for a 24-hour period
prior to the initiation of the appropriate experiment and were kept in con¬
stant conditions for the duration of the experiment. The graph indicates that
a continuous environment does not remove the previously observed diurnal pat¬
tern of ammonia excretion. This suggests that an endogenous release rhythm
is present in C. harfordi which is to a certain degree independent of environ¬
mental lighting cues. However, it does appear that constant lighting condi¬
tions do modify excretory response patterns. Both observed maxima have been
shifted towards a later time of day. This observation may support the con¬
cept that in the absence of environmental cues such as light/dark photoperiodism,
endogenous rhythms may become desychronized with time. However, more compre¬
hensive research is required in order to unequivocally demonstrate this prin¬
ciple at it applys to endogenous ammonia release patterns in C. harfordi.
Continuous dark also appears to enhance the diurnal release rhythm, and
a rough quantitative comparison indicates that dark adapted animals release
about 75 percent more ammonia over 24 hours than do normal light/dark or
light adapted organisms. Due to the great individual variations in release
rates among these relatively small sample populations, a precise quantitative
comparison cannot be made here. However, this would suggest that light is
associated with decreased ammonia release, and this may in turn be correlated
with the observation that C. harfordi are less active when exposed to light
(Harrold, 1973).
(7)
SCUSSION
Harrold (1973) has suggested from his observations that C. harfordi are
most active in the early morning hours between 0200 and 0400, a time which
would correspond to the observed period of maximum diurnal ammonia release.
Since protein represents the principle source of food energy in C. harfordi,
increased protein catabolism with a subsequent rise in nitrogen excretion
could be expected to correlate with elevated activity levels.
Wieser and his associates (1969) have found that a starved terrestrial
herbivore, Porcellio scaber, excretes approximately three times as much ammonia
as a normally fed animal. A number of researchers have suggested that a
variety of crustaceans, isopods included, utilize protein as a source of
energy reserve (Neiland and Scheer, 1953; Holdich, 1971). When overall ex-
cretion rates are compared between species of isopods which are presumably
utilizing protein as an energy source, similar values are obtained. During
spring months, P. scaber (Wieser, et. al., 1969) and fed C. harfordi each ex¬
crete over a 24-hour period approximately 11 ng NH,/mg/hr. Thus, ammonia
excretion may represent a parameter by which protein metabolism may be es¬
timated in a variety of isopod species. Sloan (1967) has shown a significant
correlation between the level of protein in the diet and excretion of volatile
ammonia, and no correlation between the level of protein in the diet and ex¬
cretion of urea, uric acid and ammonium in the fecal materials. In addition,
Wieser and Schweizer (1970) have found a significant correlation between am¬
monia excretion and variation in body metabolism due to abrupt temperature
changes in P. scaber. Therefore, in at least several species of terrestrial
isopods, amino acid catabolism and the release of ammonia appear to represent
qualitatively and quantitatively integrated phenomena.
(8)
In conclusion, the data presented here suggest that in C. harfordi, a
non-random, carefully regulated process of ammonia release exists which ex-
hibits properties of endogenous rhythmicity. Hartenstein (1968) has suggested
that in the colonization of land, terrestrial isopods retained ammonotelism
because of the thermodynamic advantages conferred by not being required to
convert toxic ammonia wastes to more inert products. It appears that a num¬
ber of terrestrial isopod species excrete ammonia by releasing it in a volatile
form. However, these isopods have been required to develop patterns of
volatile ammonia release so that release takes place during exposure to rela-
tively high humidities when concomitant body water loss is also minimized.
Wieser and 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." In a sense, it appears possible
that the capacity to "program" ammonia release is not necessarily a terrestrial
adaptation, but a fundamental uniform physiological characteristic of a
number of isopod species, marine included.
0
OENO TPROEM
ACKNOWLEDGETE
I would like to gratefully acknowledge the assistance of Dr. John Phillips.
Philip Kirby, and Kathryn Storch for their invaluable help in conducting the
research outlined within this paper and for their advice in the preparation
of this manuscript. I would also like to thank the entire staff and faculty
associated with the Biology 175H course for making it such a rewarding ex-
perience.
(10)
FERENCES
Dresel, E. & Moyle, V. (1950). Nitrogenous excretion of amphipods and iso-
pods. J. Exp. Bio. 27:210-225
Harrold, C. (1973). Environmental factors affecting patterns of activity in
Cirolana harfordi (Lockington, 1876). research report 175H, Hopkins Mar-
ine Station
Hartenstein, R. (1968). Nitrogen metabolism in the terrestrial isopod.
Oniscus asellus. Am. Zoo. 8:507-519
Holdich, D. (1971). Changes in physiology, structure, and histochemistry
occurring during the life-history of the sexually dimorphic isopod
Dyamene bidentata (Crustacea: Peracarida). Marine Biology. 8:35-47
Johnson, W. (19
). Population dynamics, energetics, and biology of the mar-
ine isopod, Cirolana harfordi (Lockington, 1876) in Monterey Bay, California,
Ph.D. thesis (in preparation,
Kirby, P. (1973). Diurnal pattern of ammonia release in marine and terrestrial
isopods. research report 175H, Hopkins Marine Station
Neiland, K. & Scheer, B. (1953). The hormonal regulation of metabolism in
crustaceans, V. The influence of fasting and of sinus
gland removal on
body composition of Hemigrapsus nudus.
('-Grav.).
ysiol. comp.
3:321-326
Sloan, W. (1967). unpublished observations
Solorzano, L. (1969). Determination of ammonia in natural waters by the
phenol-hypochlorite method. Limnology & Oceanography. 14:799-801
Wieser, W., Schweizer, G., & Hartenstein, R. (1969). Patterns in the release
of gaseous ammonia by terrestrial isopods. Oecologia (Berl.). 3:390-400
Wieser, W., & Scheizer, G. (1970). A re-examination of the excretion of nitro-
gen by terrestrial isopods. J. Exp. Bio. 52:267-279
0
Figure 1. Log ammonia excretion (ng NHa/mg dry body weight/hr) for C. har
fordi plotted as a function of dry body weight (mg). The significant
linear regression has the equation y = -1.116 x + 2.374. N = 32.
0
0
0
4 0
4
I
u

O

0
8
Figure 2. Linear plot of ammonia excretion (ng NH,/mg/hr)
expressed as a function of dry body weight (mg). N = 3
80
IS
2u

28
0
I

00
0
—
O
Table 1. Mean ammonia excretion rate values for selected size groupings o-
C. harfordi, with average, maximum, and minimum rate measures.
IT NUMBER
WEIGE
14 mg
10
15-25
26-55
256
AVERAGE
RANGE:
MAXIMUM
MINIMUM
VOPETTON
MEAN NH AORTTOI
RATE
17.84 ng NH3/mg/hr
12.19
2.97
2.26
10.90
38.5
1.3
Figure 3. Diurnal pattern of ammonia release in fed C. harfordi. All ex-
cretion rate values obtained at specific time points within a 24-hour
period for a given specimen were averaged, with each value in turn
being compared against this 24-hour mean. The ratios of the excretion
rate to the mean value for 10 samples were then averaged and plotted
for each time interval. Standard deviations are indicated for each
sample group.
2
O
P8

1
e
Figure 4. Diurnal pattern of ammonia release in non-fed C. harfordi. All
excretion rate values obtained at specific time points within a 24-hour
period for a given specimen were averaged, with each value in turn
being compared against this 24-hour mean. The ratios of the excretion
rate to the mean value for 10 samples were then averaged and plotted
for each time interval. Standard deviations are indicated for each
sample group.
I

zzuamp;
Figure 5. Diurnal pattern of ammonia release in fed C. harfordi exposed to
continuous light and continuous dark regimes. Animals were kept in
constant conditions for 24 hours prior to the initiation of experimen-
tation. All excretion rate values obtained at specific time points with-
in a 24-hour period for a given specimen were averaged, with each value
in turn being compared against this 24-hour mean. The ratios of the ex¬
cretion rate to the mean value for 10 samples were then averaged and
plotted for each time interval. Standard deviations are indicated for
each sample group.
—
Z
1
VII
L
V
—
E
2

Z
L
+
D
5

z21