SUMMAR
Diurnal rhythms in ammonia release were studied in the
following eight species of isopods found in Monterey, Cali¬
fornia: Porcellio scaber (Latreille, 1804), Alloniscus per¬
convexus (Dana, 1854), Armadillidium vulgare (Latreille, 1804)
Idothea montereye
is (Maloney, 1933), Idothea kirchanskii
(Lee, 1967), Idothea resecata (Stimpson, 1857), Cirolana
arfordi (Lockington, 1877), and Lironeca vulgaris (Stimpson,
1857). All species studied except C. harfordi showed con¬
spicuous peaks of release between noon and 4:00 p.m., and
ninima at various times during the night. The experiments,
carried out on fed male specimens in constant light are be¬
lieved to represent a general pattern in isopods.
Additional experiments revealed that ammonia release
is not correlated with fecal release in I. resecata, and
that sites of release on P. scaber seem to be located on
the ventral surfaces of the head and telson.
The relationship between ammonia release and locomotory
pattern is discussed.
ICTION
NTR
ROD
The ammonotelism of isopods has been documented by
Dresel and Moyle (1950) who found that ammonia was the major
nitrogenous end product in all species they studied, with a
few terrestrial species, particularly Armadillium vulgare,
producing significant amounts of uric acid, while urea was
totally absent. Hartenstein, Wieser, and Schweizer (1969)
found that the ammonia release of terrestrial isopods was
r greater than that reported by Dresel and Moyle owing to
a substantial gaseous evolution. Furthermore Hartenstein
et al found that gaseous ammonia release displays a diurnal
rhythmicity with a maximum during the afternoon, and they
concluded that the rhythm was inversely related to the pat¬
tern of locomotory activity of the animals. The animals
tested by Hartenstein et al were members of the Oniscoidaea,
and the species tested, Porcellio scaber and Oniscus assellus,
are primarily nocturnal scavengers. The Monterey, California
has a rich isopod fauna. Oniscoidaea, as well as other
amilies which display similar nocturnal locomotory patterns
are represented. The abundance of available species provided
an opportunity for a comparative study of diurnal rhythms in
marine and terrestrial species and reexamination of patterns
previously observed (Hartenstein et al 1969).
ALS AND METHODS
MATE
Whenever possible, freshly collected animals were used
to minimize any trauma from storage in the laboratory. When
storage was necessary, the Oniscoidaea were kept in plastic
bins with dirt or sand and litter from where the animals were
collected. The Valvifera and Flabellifera were stored in
tanks with running seawater, aeration, and a supply of food.
In all experiments, only males were used, and in all cases
arange of animal sizes was included.
The method of monitoring ammonia evolution was different
in the terrestrial and marine species. In terrestrial species,
individuals were placed in 25 ml Erlenmeyer flasks and the
flasks were continually flushed with air. The air was puri¬
fied by filtration through cotton, bubbling through a solu¬
tion of 1 N HoSO) 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 addified to 4.5 by the addition of dilute HCI.
Every four hours 1 ml samples were taken, and the trapping
solutions replaced. In marine species, individuals were
kept in test tubes with 10 ml seawater at ambient seawater
temperature and were aerated hourly with purified air.
Every four hours, the water was sampled and replaced, as
above. In all experiments, the animals were exposed to
constant light in the laboratory.
Ammonia was measured by the phenol/hypochlorite reac¬
tion as described by Solorzano (1969), and readings were
compared against a standard of ammonium sulfate, in a Klett¬
Somerson Photoelectric Colorimeter using a red filter.
Ammonia release was studied in terms of sites of re¬
lease by wrapping isopods in damp filter paper impregnated
with phenol red. The paper was impregnated by soaking in
1/5 dilution of standard indicator solution, 0.l g in 14.3 ml
0.02 M NaoH and allowed to partially dry. At 30 min. inter¬
vals, the animal was carefully unwrapped and the paper ex¬
amined.
EESULT
EESOEI
I. Rhythmicity in terrestrial species.
In experiments conducted in the month of May, the
release of gaseous ammonia was monitored through 24 hour
periods in three terrestrial species of isopods, Porcellio
scaber, Alloniscus perconvexus, and Armadillium vulgare with
the result that all species showed a significant and similar
rhythmicity. Fed males consistently released significantly
greater amounts of ammonia during the afternoon from noon
to 4:00 p.m. with release becoming more erratic and signifi¬
cantly diminished during the night. It was found that there
is no linear relationship between body weight and amount of
release, so each animal was recorded in percent of his own
mean hourly release, and compared on this basis, at each
reading with other animals.
In Porcellio scaber (Fig. 1) the results are very
similar to those reported by Hartenstein, Wieser, and
Schweizer (1969) for the same species and time of year in
Innsbruck, Austria. All animals produced ammonia during the
afternoon interval, in amounts that represented a maxima for
those animals. In other intervals, some animals released no
ammonia or reduced quantities. There is a significant mini¬
mum between midnight and 4:00 a.m. These experiments were
carried through one 24 hour period, beginning at noon. It
appears unlikely that the observed maximum reflect a period
of adjustment to experimental conditions because experiments
started at 4:00 p.m. presented the same type of pattern.
Both Alloniscus perconvexus and Armadillidium vulgare (Figs.
2 and 3) produced the same pattern observed in Porcellio.
Release during the night was sporadic and at a low level for
individuals, and the same fairly uniform peak of release oc¬
curred from noon to 4:00 p.m.
II. Rhythmicity in marine species.
The marine species chosen for study represent two
families of isopods, the Valvifera and the Flabellifera.
From the former family, Idothea resecata, I. montereyensis,
(ii were selected, and from the latter family,
rchan!
and I. k
irolana harfordi and Lironeca vulgaris were chosen.
The species of Idothea have a habitat which is more
restricted than that of the terrestrial species, in the
sense that they are found living and feeding on specific
algae. While the terrestrial species are scavengers and
exhibit a diurnal oscillation between active scavenging
and retirement to burrows. Therefore, they were considered
to provide a contrast to the terrestrial forms beyond the
fact of their marine environment. However, the three species
of Idothea were found to have ammonia excretion patterns
closely resembling those of the Oniscoidaea (Figs. 4, 5, 6).
In each case, the maxima were located from noon to 4:00 p.m.,
with minima occurring either between midnight and 4:00 a.m.
or 4:00 a.m. and 8:00 a.m. In Idothea resecata (Fig. 6), the
amplitude of the fluctuation was decreased and no significant
difference between the peaks at 8:00 a.m. to noon and noon
to 4:00 p.m. was observed. However, the patterns of these
marine species are very similar.
The two Flabellifera observed are exceptional both in
activity and in excretory pattern. C
trolana harfordi and
ironeca vulgaris are primarily carnivorous, and Lironeca
which is a parisite on flatfish seems to be exclusively
carnivorous. Sloan (1967) has shown a relationship between
protein intake and ammonia release in isopods, and it was
considered desirable to see if high protein intake might
also affect release patterns. The pattern observed for fed
male Cirolana in constant light was obtained from Harbaugh
(1973) who was working on patterns of ammonia release in
Cirolana in the same lab that all other species were tested.
The pattern observed in Cirolana (Fig. 7) is less distinct
than that of the Idothea, but does reveal some interesting
features. First the typical maximum from noon to 4:00 p.m.
is if anything, a period of minimal release, and the maximal
period observed seems to occur between 4:00 a.m. and 8:00 a.m.
This apparent reversal of the patterns previously observed in
the other species is less pronounced because of the more
constant rate of ammonia release throughout a period of
24 hours. Harbaugh (1973) has reported a more distinct
pattern for animals maintained in the dark. The pattern
shown by Lironeca vulgaris (Fig. 8) combines features of
the patterns of Idothea, terrestrial isopods, and C
rolana
in a very striking rhythmicity. The individuals tested
produced most of their ammonia between noon and 4:00 p.m.
with a second substantial peak between midnight and 4:00 a.m.
The other readings were often characterized by a number of
animals producing no ammonia.
III. The nature of ammonia release.
Having observed patterns in release, the emphasis was
shifted to learning more about the nature of the release
itself. For this work, a terrestrial species, Porcellio
scaber and a marine species Idothea resecata were chosen.
These animals both have afternoon maxima, and were found to
be easy to work with. In addition, personal observations
of these animals revealed traits which made these animals
attractive. Specifically, fed specimens of I. resecata
are conspicuous producers of fecal pellets, making it pos¬
sible to test a relationship between fecal and ammonia re¬
lease. Porcellio had been observed to release a sufficient
amount of ammonia to cause a color change in phenol red
indicator paper.
However the basic aim of this set of experiments was
to increase the resolution on periods of significant release
and determine: (1) Is release a continuous process, or does
it occur as discrete events; and (2) is there a rhythm dis¬
cernible within the periods of maximum release? The animals
were monitored for ammonia production at half-hour intervals
from noon to 4:00 p.m. In addition, I. resecata was scored
for presence or absence of feces, and P. scaber for sites of
ammonia release. The results, see Figure 9 and Table I,
showed that release occurs as discrete events, within limits
of temporal resolution, and that the events occur randomly.
Despite the randomness of the events, there was a significant
consistency in event rate, both interspecifically and intra¬
specifically. I. resecata had a mean ammonia release rate of
O.737 events/hour with S.D. I 0.17, while P. scaber had the
comparable mean rate of 0.781 events/hour with S.D. I 0.16.
In I. resecata no correlation was found between fecal produc¬
tion and ammonia release, though significant amounts of feces
were produced. In P. scaber, no fecal release was noted, and
release sites were limited to the ventral surfaces of the
head and telson regions.

SCUSSION
As has been previously stated, the primary objectives
in undertaking this study were to investigate the possible
generality of the pattern observed by Wieser (1969) and, by
examining species with contrasting activity patterns, 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 release
was found which, with only one exception, closely approximated
that reported by Wiser in Porcellio scaber and Oniscus assellus
with a large maximum in the afternoon. The general presence
of a pattern or rhythm seems fairly well established, but the
underlying causes for this rhythm is more obscure. Basic to
all theories advanced by Hartenstein to explain rhythmicity,
is the existence of inverse relationship of excretion to lo¬
comotory pattern. Rhythmicity reflects energy releasing
metabolism of amino acids during activity and excretion
during periods of rest in humid burrows where membrane perme¬
ability to ammonia release can best be permitted.
In both Porcellio and Alloniscus, 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
-. 7
species Armadillidium, the nocturnal pattern, though common,
may vary from population to population (Paris, 1962) and in
local populations near the laboratory, individuals were ob¬
served to be foraging by day. These Armadillidium still show
a pattern distinguished by pronounced excretion maxima and
minima.
In the marine species, the problems in relating the
release pattern to activity are even greater. The species
of Idothea 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 level.
However the same rhythm of release was found. Clearly, no
humidity or moisture considerations 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 con¬
trol release. Cirolana harfordi is a distinctly nocturnal
animal (Harrold, 1973) which has its maximum in activity
corresponding with its maximum period of release, or an ap¬
parent direct correlation between activity and excretion.
Lironeca vulgaris, in contrast, is an almost sessile parasite,
which seems incapable of locomotion in its fully matured
male 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
in each species, the very similarity of pattern suggests that
there might be a more basic common rhythm in isopods, control¬
ling ammonia excretion.
The mechanism of ammonia release in isopods also re¬
mains unclear. Preliminary investigation in P. scaber indi¬
cated that the telson might play a role in gaseous release
with the anus and respiratory pleopods considered to be
likely sites of release. Accordingly, the relation of the
telson to release was examined. The technique of wrapping
animals in soaked filter paper bearing the non-toxic indi-
cator phenol red was found to be a satisfactory method for
10
recording general areas of release, though resolution is not
great. The finding that both the head and telson seem in¬
volved in ammonia release poses questions. Is the observed
release an artifact of the experimental method, or a response
to stress or fright, resulting in 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 burrowed
animal. If the release is not an artifact, then the possi¬
bility of multiple sites must be considered. It has been
noted that some crustaceans excrete by means of an antennal
gland located on the head (Hartenstein, 1970), and isopods
have this gland present. It is also possible that the diges¬
tive tract, which opens at both apparent sites of release
may be used as the excretory organ. To investigate this
possibility, I. resecata was chosen and investigated to see
if the gut might produce simultaneous bursts of feces and
ammonia. No correlation could be found.
The observation that ammonia excretion occurs in fairly
discrete bursts rather than as a continuous process suggests
that there might be a build up, either of ammonia or à pre¬
cursor, prior to release. A search for this precursor in
animals prior to the times of maximum release may shed further
light on the pathway and mechanism of ammonia excretion in
these animals.
BIBL
GRAE
Dresel, E. and U. Moyle. 1950. Nitrogenous excretion of
amphipods and isopods. J. Exp. Biol. 27: 210-225.
Harbaugh, R. 1973. Diurnal pattern of ammonia release in
a marine isopod Cirolana harfordi. Research paper,
Hopkins Marine Station, Pacific Grove, Calif.
Harrold, C. 1973. Environmental factors affecting patterns
of activity in Cirolana harfordi. Research paper,
Hopkins Marine Station, Pacific Grove, Calif.
Paris, O. H. and F. A. Petelka. 1962. Population charac¬
teristics of the terrestrial isopod Armadillidium
vulgare in California grassland. Ecology 43: 229-248.
Pratt, S. 1973. Personal communication.
Sloan, W. C. 1967. Unpublished.
Solorzano, L. 1969. Determination of ammonia in natural
waters by the phenol hypochlorite method. Limnology
and Oceanography 14: 799-801.
Wieser, W., G. Schweizer, and R. Hartenstein. 1969. Patterns
in the release ofogaseous ammonia by terrestrial isopods,
Oecologia 3: 390-400.
Whiteside, C. 1973. Research paper, Hopkins Marine Station,
Pacific Grove, Calif.
Gorham, W. 1973. Personal communication.
ACKNOWLEDGEMENTS
I would like to thank Dr. John H. Phillips for his
advice on experimental methods, and on preparation of this
paper. In addition the tireless work of Bob Harbaugh and
Rose Thorvaldson has made this paper possible.
FIGURE KE
Figure 1. The diurnal pattern of ammonia release in
fed male Porcellio scaber in constant light. The vertical
xis is percent of mean hourly excretion, and the horizontal
axis is a 24 hour cycle where 1200 = noon; N - 10, and stan¬
dard deviation is indicated.
Figure 2. The diurnal pattern of ammonia release in
fed male Alloniscus perconvexus in constant light. Percent
mean hourly excretion is plotted against a 24 hour cycle
where 12 = noon; N - 8, and S.D. is indicated.
Figure 3. The diurnal pattern of ammonia release in
fed male Armadillidium vulgare in constant light. Percent
mean hourly excretion is plotted against a 24 hour cycle
where 12 = noon; N = 8, and S.D. is indicated.
Figure 4. The diurnal pattern of ammonia release in
fed male Idothea montereyensis in constant light. Percent
mean hourly excretion is plotted against a 24 hour cycle
where 12 = noon; N = 10, and S.D. is indicated.
Figure 5. The diurnal pattern of ammonia release in
fed male Idothea kirchanskii in constant light. Percent
mean hourly excretion is plotted against a 24 hour cycle
where 12 = noon; N = 10, and S. D. is indicated.
Figure 6. The diurnal pattern of ammonia release in
fed male Idothea resecata in constant light. Percent mean
hourly excretion is plotted against a 24 hour cycle where
12 = noon; N = 10, and S.D. is indicated.
Figure 7. The diurnal pattern of ammonia release in
fed male Cirolana harfordi in constant light. Percent mean
hourly excretion is plotted against a 24 hour cycle where
12 - noon; N - 10, and S.D. is indicated.
Figure 8. The diurnal pattern of ammonia release in
fed male Lironeca vulgaris in constant light. Percent mean
hourly excretion is plotted against a 24 hour cycle where
12 - noon; N - 10, and S.D. is indicated.
Figure 9. Mean release rates in Idothea resecata and
Porcellio scaber, measured at half hour intervals from noon
to 4:00 p.m. Values given for fecal release or release from
given sites are means calculated during the same interval.
N = 20 for I. resecata and N = 8 for P. scaber.
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FIGURE
EXCRETORY EVENTS FROM 1200-400
IN
P SCABER AND I. RESECATA
I.RESECATA
N - 20
NHRELEASE
O.737 EVENTS/HOUR
S.D. O.17
FECAL RELEASE
0.512
S.D. O.47
1
NH, AND FECAL
0.162
8.D. 0.21

P SCABET
NHa RELEASE
O 781 EVENTS/HOUR S0. 0.16

FECAL RELEASE

.
NHa RELEASE


FROM HEAD
0.284
S.D. 0.22


1
O.46.
FROM TELSON
S.D. 0.22





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