INTRODUCTION
Species throughout the animal kingdom actively aggregate into tight
clusters under certain conditions. This phencmenon, which I will refer
to as clumping, was studied by Allee (1931) in the early part of this
century in the water isopod Asellus communis, several species of land
isopods, and the brittle star Ophioderma brevispina; all species not
generally considered to be social. Natural aggregations of these animals
were seen to be primarily the result of the reaction of individuals to
the environment. Clumping behavior occurred most frequently under adverse
conditions and when the normally positive thigmotactic response was not
possible. In the laboratory, increased survival of clumps over individuals
was demonstrated in some situations. Despite the appeal of this early work.
in recent years little has been done in the area of animal aggregations,
Tigriopus californicus, a harpacticoid copepod inhabiting splash pools
of the intertidal zone from Baja California to Alaska, has been observed
to form clumps in the field as well as in the laboratory under conditions
of environmental stress. This is of particular interest in this species
which, except for periods of copulation, has shown no interaction between
members under normal conditions. To date, no investigation of clumping
behavior in Tigiopus or any other marine copepod has been made. Patterson
(1968), mentioned that Tigriopus forms clumps at high salinities under
laboratory conditions and is sometimes found to "swarm" in the field.
Burnett (1977), states that Tigriopus can form clumps in low temperatures
in the field.
The environment of the high splash pool is one of extremes of temperature
and salinity. Studies have suggested that Tigriopus is well suited to this
Page 2
greatly fluctuating environment. This paper investigates clumping in
Tigriopus and its possible adaptive significance in light of the animal's
habitat.
FIELD STUDIES
Field work was done on splash pools from 8 to 13 feet above mean low
tide at China Point, California. Pcols referred to as dessicated contained
no standing water with little or no moisture detectable in the bottom
sediment. Pools of extreme high salinity ranged from 120 o/oo to damp
salt crystals.
Tigriopus were found in 9 of 12 dessicated pools examined. 7 of these
9 pools had a substratum of sediment and loose rocks, the other 2 containing
only sediment. Clumps of Tigriopus were found on the underside of many of
the rocks with greater concentrations of clumps under rocks imbedded 1 to
2 cm. into the bottom sediment than those resting on dry sediment or
other rocks. A lower density of isolated Tigriopus was observed in the
bottom sediment of the pools containing rocks. No clumps were found in the
sediment of any pool. Several dry rocks with clumps were placed in fresh
sea water in the lab. After a period of several hours Tigriopus were
observed swimming in the container. Baxter (1977), reports similar findings
in which live adult Tigriopus did not appear until after a pericd of 3 weeks.
In tide pools above 120 o/o0
igriopus were found clumped in dark cracks
both above and below the waterline as well as under rocks at the bottom of
the pool. In a pool of 148 o/oo no animals were seen in the water column
or on the bottom, while an area of approximately 8 cme directly below
the waterline and in a constantly shaded crack was covered with a solid
mass of clumped Tigricpus. The animals in this massive clump appeared
in a near comatose state as dislodging animals produced very slow movement
Page 3
back to the clump or drifting to the bottom. Forty days later at a time
when the salinity was in excess of 220 o/oo, no animals were found in the
above location. However, behind a flat rock pryed loose from the wall of
the pool 2 cm. above the waterline were found hundreds of clumped Tigriopus.
LAB STUDIES
GENERAL METHODS
Tigriopus used in this study were collected from splash pools of
salinities from 35 to 45 o/oo at Asilomar Beach State Park and China Point.
During collection water containing the animals was strained through a fine
nylon mesh so only adult and late copepodid stages were retained. Plastic
containers of approximately 20 liters of sea water at 35-45 o/0o were used
to maintain populations in the lab. Every three days the water was
changed and the animals were fed Tetramin fish food.
Instant Ocean (trade name) dissolved in distilled water was used
where initial salinities greater than normal sea water was required.
Salinity was measured with a Goldberg refractometer. All experiments were
run at room temperature with constant room lighting unless otherwise
indicated.
Recovery from stress was in room temperature sea water for 24 hours.
Water was changed in salinity stress experiments by slowly drawing off the
"old" water through plankton netting, followed by slow addition of recovery
water with a mimimum of turbulence. Death was defined as an absence of re¬
sponse to prodding at the end of the recovery period. A copulating pair
was scored as dead if both members failed to respond.
Four working classifications of animals were defined: 1) females
with eggs - egg sack visible; 2) larvae - all solitary immature animals;
3) copulating pair - adult male grasping a larvae in the copulatory position;
4) adults - all other solitary adults.
Page 4
CLUMP FORMATION
1. Dessication Clumps. Approximately 100 animals in one ml. of normal
sea water were placed in the center of a 5.5 cm. Whatman's No. 1 filter
paper. As the water was absorbed clumps formed. The location of these
clumps relative to a directional light source of 8000-10,000 lux was
noted. Animals were run in the dark and a control using dead animals was
run to check for passive aggregation of animals due to cohesive forces of
water.
Results are shown in Table 1. 14 out of 14 of these "dessication
clumps" formed in the light were on the half of the filter paper furthest
from the most intense light source. The animals could be seen to move
in this direction before forming clumps. In the dark,clumps were randomly
distributed on the filter paper. The control with dead animals showed no
clump formation
2. Agitation Clumps. Strips of standard paper towel 8 cm.x 2 cm. were
placed along the sides of a l liter beaker of seawater containing approx¬
imately 5 animals per ml. When the water was agitated by stirring, clumps
formed on the paper towel. Clumps formed exclusively on the side of the
beaker furthest from the more intense window lighting. These agitation
clumps of from 10 to 40 animals were used in subsequent survival tests.
If periodic agitation (every 10 min.) was maintained, these clumps re¬
mained intact. Squirting with water from a pipette removed the individuals
from the toweling, leaving only clumps. From this procedure clumps were
determined to be more firmly attached to the substrate than individuals in
all of dozens of trials.
3. Slow Evaporation Clumps. 12 petri dishes containing 100 Tigriopus each
in 40 o/oo S.W. were allowed to evaporate to dryness over a 4 day period.
A single clump formed in 5 of the 6 dishes having paper towal substrates
with 2 clumps forming in one dish. All 7 of these clumps formed at the
Page 5
side furthest from the more intense window lighting. In the dishes lacking
substrates approximately 108 of the Tigriopus formed clusters of 3-5
animals clinging to one another. An average of 708 of the animals in the
no-substrate dishes were in the half of the dish away from the windows.
After one day of complete dessication, distilled water was added to all
12 dishes to return the salinity to normal. There was near 1008 mortality
at the end of the recovery period.
SURVIVAL T
1. High Salinity Stress. Tigriopus were placed into water of 140 c/0o
salinity for a 15 hour exposure period. The following groups of animals
were used: 1) clumps - animals in agitation clumps described earlier;
2) disrupted clumps - animals disrupted from agitation clumps by force¬
ful squirting with a pipette. This technique produced no mortality;
3) non-clumped - animals taken in a random sample from the same population
as the above groups.
When placed into the high salinity water, the non-clumped and
disrupted-clump animals greatly increased their activity for 1 to 2 minutes
before becoming torpid. Survival results are summarized in Table 2.
The sexual composition of the clumped and disrupted-clump animals compared
to that of the non-clumped is shown in Table 3. Larvae and copulating
pairs are omitted due to their occurrence in very low frequencies.
2. Cold Shock. Three separate experiments were run to test the differential
survival of clumped and disrupted-clump animals subjected to sudden exposure
to cold. The disrupted-clump animals were observed to remain active for less
than 30 seconds in trials 1 and 2 and for less than 2 minutes in trials
3 and 4. In all cases a 15 hour recovery period was allowed at room
temperature before assays were made. Conditions and results of each
trial are listed in Table 4.
Page 6
3. Gradual Cooling. 12 beakers of normal sea water containing Tigriopus
were cooled from 22°C to 0°C in 8 hours. All beakers had substrates.
In all cases no clumps formed. Test was conducted in total darkness.
OSMOREGULATION STUDIES
The osmotic concentration of clumped Tigriopus in 85 o/00 and 120 0/00
water and that of individual animals acclimated in these same ambient
salinities was determined with the help of Merchant (1977), who details
the method. Clumps in 85 o/oo were formed and maintained by gentle agitation,
Clumps in 120 o/oo were formed by an increase in salinity from 40 o/oo to
120 o/oo over a 4 hour period. Body fluid osmotic concentrations are given
in Table 5.
OXYGEN CONSUMPTION
Oxygen consumption rates were determined in conjunction with Senko
(1977), for 3 groups of Tigriopus: 1) individuals in 34 o/00; 2) agitation
clumps in 34 o/oo; 3) individuals in 90 o/00. Senko notes that in 90 o/oo
nearly 100 of the animals were in tightly clustered masses. This was not
seen for individuals in 34 o/00. Results are summarized in Table 6.
DISCUSSION
CLUMP FORMATION
It appears from the results of this study that clumping is the net
effect of a combination of responses induced by environmental stress. The
primary reactions of Tigriopus to stress are postulated to be:
1. positive thigmotropism
negative phottaxis
3. increased attraction to other Tigriopus
In all conditions of stress examined, Tigriopus are seen to leave
the water column if possible. In the lab, formation of agitation clumps
involves rapid movement toward the substrate with subsequent clinging.
Page 7
Findings by Foster (1977), indicate that the response of Tigriopus to
wave shock in the tidepool and to turbulence in the lab is one of a strong
positive thigmotropism. Animals deprived of a substrate were noted in this
study to cling to one another in both the evaporation and oxygen corsumption
experiments, presumably as an attempt to satisfy this response.
A negative phototactic effect under stress is well substantiated
by laboratory and field work. Combined with the thigmotropic response,
a reaction to stress is seen wherein Tigricpus will move towards the dark
areas of the tidepool and cling to the substrate. These locations tend
to be the damp cracks and under sides of rocks, areas which are the last
to be completely dessicated.
That clumps are formed under stress does not necessarily follow from
the above explanation. Some other response must arise that induces the
formation of distinct clumps rather than solitary clinging in shaded places.
Cooper (1977) and Bozic (1975) suggest that Tigriopus are attracted
to one another and that there may be a chemical stimulus involved.
This attraction might be expected to increase in both magnitude and effect
under stress due to: 1) The greater concentration of "attractant" in
areas to which Tigriopus have been directed by the phototactic and
thigmotactic responses; 2) a change in the response to the attractant under
stress. This model has Tigriopus tending to be attracted by other
animals to a particular area and cued to clump there under stress. This
theory does not preclude the formation of a number of clumps if an animal
is assumed to move towards the strongest attraction it perceives, this
being a different location for some animals, and once fixed to the substrate,
remain there. That clumps do form in the dark when the directional cue of
lighting is not present is also well suited to this model.
An experiment suggested hare is to test the effect of extreme high
densities of Tigriopus under non-stressful conditions. Indications are that
Page 8
clumps would not form, suggesting that there is a different response to
the postulated attractant under stress.
COMPOSITTON OF CLUMPS
It can be seen in Table 3 that females with eggs comprise a higher
percentage of the population in agitation clumps than in the sampling
population. Their survival strengths were not found to be statistically
different from those of all adult Tigriopus. Further work on the sensi-
tivity of females with eggs to stress may offer an explanation for these
results.
ADAPTIVE SIGNIFICANCE
The benefits accrued members of a clump are threefold and appear to
arise from changes in the physical and physiological state of the animal.
Most readily explained is the increased resistance to wave shock. Lab¬
oratory evidence indicates that when subjected to equal water velocities,
animals in a clump are less prone to being dislodged from the substrate
than are individual Tigriopus. Combined with the occurrence of clumps
primarily in protected regions of the tidepool it follows that when a
dessicated or high salinity pool is inundated by wave splash, animals
in a clump are less likely to be washed out.
Increased resistance to high salinities is of obvious adaptive im-
portance. A mechanism suggested by the closely packed crientation of
the animals in a clump is one in which a decrease in body surface area
exposed to high salinity produces a corresponding decrease in the total
osmotic gradient impinging on the animal. Thus, a member of a clump would
be able to tolerate salinities beyond the normal osmoregulatory capacity
of an individual. Osmoregulation studies on Tigriopus indicate that this
mechanism is probably not the case. Tigriopus in a clump have a higher
Page 9
osmotic concentration of the body fluid than do torpid animals at 120 o/00.
The apparent disparity between survival values and osmotic concentrations
suggests that the clumped state may differ from simple torpor. It may
be that there are physiological changes other than those of torpor
associated with clump formation. This is suggested by the results of the
salinity shock experiments. Clumped animals had a small but significantly
greater survival value than did animals that had been in a clump briefly
with both groups being much more resistant than non-clumped animals. If
these physiological changes are not instantaneous, than these results
might be expected.
More work on the time of formation of disrupted clumps and its effect
on survival as well as the osmoregulation and the oxygen consumption of
clumps is highly suggested.
The most curious results of this study are those of the increased
rasistance to cold shock of clumped Tigriopus. Being an ectotherm, there
is no possibility of increased heat retention by a clump. Increased
membrane permeability is known to be a phenomenon associated with low
temperatures, (Prosser, 1973). It is conceivable that the decreased
surface area of a clumped Tigriopus could lower the detrimental ion
flow between the animal and its environment. Determination of the
ionic constitution of clumped and solitary Tigriopus is suggested.
SUMMARY
Tigriopus californicus is seen to form clumps in the field and in
the lab under conditions of high salinity and dessication. There is an
indication that clumps may form in response to wave shock as well. Clump
formation serves to increase the resistance of Tigriopus to high salinity
and low temperature shock. A definite negative phototactic response is
associated with clump formation and an increased thigmotaxis is suggested.
Page 10
LITERATURE CITED
Allee, W.C. 1931. Animal Aggregations.
Baxter, C., 1977. Personal Communication.
Bozic, B., 1975. Detection actometrique d'un facteur d'interattraction chez
ligriopus, (Crustacaces, Copepodes, Harpacticoides). Bull. Soc. Zool.
France. 100: 305-311.
Burnett, R., 1977. Personal Communication.
Cooper, J., 1977. Unpublished paper on file at Hopkins Marine Station,
Pacific Grove, Ca.
Foster, L., 1977. Unpublished paper on file at Hopkins Marine Station,
Pacific Grove, Ca.
Merchant, T., 1977. Unpublished paper on file at Hopkins Marine Station,
Pacific Grove, Ca.
Patterson, R., 1968. Physiology Ecology of Figriopus californicus, a har-
pacticoid copepod. Master's Thesis, University of California,Berkeley.
C.L., 1973. Comparative Animal Physiolcgy, Vol. 1, W.B. Saunders
Prosser,
Co., Philadelphia.
Senko, T., 1977. Unpublished paper on file at Hopkins Marine Station,
Pacific Grove, Ca.
ACKNOWLEDGEMENT!
Thanks to the entire faculty and staff of Hopkins
for a very enjoyable and instructive experience. Especially
thank you Robin for your encouragement, advise, and help
with numbers and statistics. Without you, the Burnett lab
wouldn't have been the same.
Table 1. Comparison of dessication clump
formation in room lighting and darkness.
CONDITION
ROOM LIGHT
DARKNESS
NUMBER
OF ANIMALS
80
81
71
4
96
88
99
186
158
TABLE 1
% CLUMPED
93
46
88
83
56
69
76
53
3
NUMBER OF
CLUMPS
4
3
MEAN CLUMP
SIZE
18.5
37.3
24
II.3
13.5
61
25
19,8
0
C
Table 2. Comparative survival values of
clumped, not-clumped, and disrupted-clump Tigriopus in
sudden exposure to a salinity of 140 0/00.
STATE OF
ANIMALS
CLUMPED
NOT
CLUMPED
DISRUPTED
CLUMP
TABLE 2
NUMBER OF
ANIMALS
66
53
85
64
166
16
112
52
83
171
110
144
186
170
85
18
87
SURWVAL
97
96
92
87
90 X=73.0
12.7
91
14
98
92
62
64 X-64.7
17.5
68
88
89
82 X=83.3
+5.9
7½
86
81
Table 3. Comparison of the sexual
composition of agitation clumps and not-clumped Tigriopus
from the same population. Also a comparison of the survival
strengths of females with eggs from these two groups.
STATE OF
ANIMALS
NOT CLUMPED
CLUMPED
LIVE
DEAD
NUMBER
ADULTS
113
1139
902
132
TABLE 3
NUMBER
OF 9
WITR E65S
1
57
13
13
TOTAL
117
1276
1041
145
h PWIn
EG6S
.61
12.1
13.4
8.7
P0
P N.S.
Table 4. Comparative survival values of
clumped and disrupted-clump Tigriopus in sudden ex¬
posure to low temperature. Conditions of exposure are:
Trial 1, 4 to 0 degrees; Trial 2, 4 to 2 degrees; Trials
3 and 4, 6 to 0 degrees.
5
a

a
R

8

.

a
2



2


7
2-


5
Table 5. Comparison of the osmotic
concentration of the body fluid of clumped and not¬
clumped Tig
pus acclimated in salinities of 85 and
120 0/00.
STATE OF
ANIMALS
CLUMPED
SOLITARY
CLUMPED
SOLITARY
TABLE 5
AMBIENT
SALINITY
85%
85%0
120%
20%o
OSMOTIC
CONC. OF
BODY FLUID
76%0
74%o
73%0
60%
75%o
83%0
71%0
0
Table 6. Comparison of oxygen consumption
of clumped and free swimming Tigriopus in 34 0/00 and
torpid-clustered Tigriopus in 90 0/00. Q 02 values in
ul. /mg./hr.
STATE OF
ANIMALS
FREE
SWIMMING
CLUMPED
TORPID
CLUSTERED
TABLE 6
SALINITY
340/00
340700
90 0/00
00.
4.76
2.90
1.23