0
IIGRATION BEHAVIOR IN
IGRIOPUS CALIFORNICUS
(CRUSTACEA: COPEPODA: HARPACTICOIDA).
John R. Coope.
Spring, 1
Hopkins Marine Station
Bio. 175H
-1
INTRODUCTION
Tigriopus is a genus of harpacticoid copspods that inhabits the high
splash zone pools of rocky coasts along Europe, both coasts of North and
South America and Japan (Lang, 1948; Mori, 1938). It is thought to exist
as a breeding population in thase isolated pools and virtually every pool
which is a suitable habitat contains these animals. Yet individual pools
can evaporate to dryness for a sufficient time that the entire population
in that pool dies. (Patterson, 1968; Egloff 1966). Igarashi (1959)
showed that populations of Tigriopus japonicus are largely destroyed by
exposure to wave shock during spring tides. Vittor (1971) states that
"colonization of pools is probably accomplished via inshore current
transport of animals swept from pools which are essentially permanently
populated." However, Vittor was unable to document such an occurance,
Egloff (1966) found numbers of Tigriopus californicus clinging to Pachygrapsus
pas and hypothesized that the copepods are able to move among the
ass
tidepools by clinging to the highly mobile crabs.
Migration, the movement of individuals up a slow moving rivulet of
water, as opposed to transport via large animals or inshore currents, is
a phencmenon first observed in Tigriopus californicus by Robin Burnett
(personal communication) and may serve as an important mechanism by which
they colonize and maintain populations in the high tide pools. Dianne
Campbell (1977) observed movements of dyed Tigricpus californicus among
a series of nine sheltered tide pools which received only occasional
splash. She concluded that the movement of dyed copepods among these pools
was due to migration. Foster (1977) subsequently showed that the ability
igricpus califcrnicus to migrate in the lab is significantly less
of dyed Ti
than that of undyed individuals. Burnett (personal communication) also
found that a number of factors including the presence of tide pool rocks
and macerated Tigriopus californicus have profound effects upon migration.
In the spring of 1977, work was done at Hopkins Marine Station,
Pacific Grove, California, on the migration of T. californicus and on the
factors that influence it. The results of these studies are reported
here.
A. GENERAL OBS
SERVATIONS (LAB AND FIELD WORK)
Migration was induced in the field by trickling sea water over the
rocks into two different tide pools. A circular holding tank, six meters
in diameter, (hereafter called the courtyard pool or C.Y.P.) populated
with Tigriopus was used to examine sex ratios. (see Fig. 1) Fresh water
from the sea water system was trickled down a region of the tank's walls
and copepods were allowed to migrate up the rivulet. The animals aggregated
underneath a glass plate which serves to deflect water from a spigot down
the vertical wall (see Fig. 2). Samples were taken from this aggregation
and from the water column of the pool for sex determination according to
Fraser (1136).
RESULT
During a heavy rain all the pools in the study area were observed
to overflow. After the rains ceased, over forty individual Tigriopus,
including three copulating pairs, were seen in a long narrow channel
mnecting three tide pools. The movements of eleven closely watched animals
were judged to be migratory since they were not accompanied by the random
changes in direction common to normal
gricpus movement but rather
consisted of sustained movement against the slow flow of water coming
down from a higher pool.
The induced migrations occurred with substantially larger numbers.
Plow rates of approximataly 80 ml/mir. were used. Maximum migration
rate out of both pools was about 200 individuals/min.
An extreme difference was found between the sex ratios of random and
migrated Tigriopus californicus. No copulating pairs were observed to
have migrated while gravid females were the dominant migrators. (see Table I)
Of the ten times migration was observed in the courtyard pool, the
first migrating individual was observed to be a gravid female eight times.
Discussion
The sex ratios of migrated and random individuals suggests that females.
especially gravid ones, are either more sensitive to stimulus or physically
superior to males, larvae and copulating pairs.
B. EFFECTS OF FLOW RATE UPON MIGRATION
A small wooden enclosure overhanging the courtyard pool was lined with
black plastic to keep light and wind constant. (see Figs. 1 § 2). A
migration ramp made of frosted glass was laid flush against the inside
of the vertical cyp wall. Flow was confined to a uniform area on the
migration ramp by two parallel strips of plexiglass with a microscope slide
used below the source of water as a "spreader",
Migration rates were found to exhibit great temporal variation. In
order to counteract this variation the following procedure was employed;
A standard flow rate of 48 ml/min. was allowed to run for at least five
-1-
minutes. Counts were made to determine tha number of animals migrating per
minute past a given point. The flow was then instantanecusly changed and
counts were resumed after a one minute equilibration period. In each
case the average number of animals migrating per minute during a five
minute period immediately after the change in flow rate was divided by the
average number of animals migrating per minute during the five minutes
immediately before the change. Thus, a relative migration rate was calculated
and could be compared with values obtained on different days and at different
times.
See Fig. 3 § 4 for overall trends in migration rates. Indicated slope
values were obtained using linear regression, however the fit was extremely
pocr in each case.
Migration could not be induced in this study using solutions of
unfiltered synthetic sea salts (Instant Ocean) (34). Yet sea water at
the same flow rate induced significant migration with 5 minutes of the
Instant Ocean attempt. The fact that a solution of synthetic sea salts
would not induce migration while sea water would suggests that in addition
to the stimulus of moving water, some other factors, perhaps biotic, are
necessary for migration to occur.
C. HIGRATION IN ARTIFICTAL POOLS. THE AFFECTS OF BIOTIC STIMULI
terials and Method.
Two sets of artificial pools were constructed using cement and chicken
wire (see Fig. 6). Each set consisted of a series of three successively
lower pools connected by narrow channels. Both sats of pools were run
simultaneously with equal flow rates. Temporal variation in migration was
controlled for in the following manner: the ratio of simultaneously run
-5.
migrations without stimulus in either set (control) is compared to the ratio
of simultaneously run migrations with a stimulus in one set only. Animals
were placed in the pool of each set and allowed to acclimatize for eight
hours before water from the sea water system was flowed into each high
pool, for 5 to 7 hours. At the end of the experiment all individuals were
retrieved from the two higher pools using a portable vaccuum pump and taken
to the lab for counting.
A total of four migration runs were made. In the first run no stimulus
was placed in either set of pools (control I). A second and third run each
utilized a different stimulus. Stimulus 1 consisted of two large rocks that
were taken from a tide pocl occupied by Tigriopus. Stimulus 2 consisted
of about 500 individual Tigriopus that migrated into the high pools of the
series during the previous experiments. They were confined to the pool
within two interlocking tea strainers, having a mesh small enough to retain
adults. A fourth run, again without any stimulus in either pool, was done
to ensure that the relative migration lability of the pools had not changed.
Results
The data shown in table 2 shows a significant increase in migration
with the addition of either tide pool rocks or migrated Tigriopus californicus,
Discussion
Migration is enchanced by the presence, in the water, of factors
iopus and tide pool rocks. These factors are
associated with both Ti
not necessarily tha same but perhaps may be the result of the plant or
animal life that is associated with each stimulus.
-6.
D. AFFINITY STUDIES IN THE COUI
ARD POOL
Materials and Methods
Five 300 ml Frelenmeyer flasks, and eight 125 ml ground stoppered
bottles containing various physical and chemical stimuli were placed on the
bottom of the courtyard pool. After a period of time all the flasks were
retrieved. Numbers of individuals per flask were counted in the lab. (See
Table III for the contents of the various flasks and bottles.) The
living stimuli (flasks 2 to 5) were retained within plastic vials with fins
plankton netting covering the open end. These vials were then deposited
in their respective flasks. Each flask and bottle, unless otherwise noted,
was filled with 34 sea water before placement in the courtyard pool.
Orientation within the replicate groups was constant and is shown in Figure
6 along with the location of each group.
Results: Run Number 1
An R by C contingency test on the results (see appendix 1) gives a
value of 99, indicating that variations between the replicate groups existed
and were not controlled. Therefore only test cases run adjacent to each
other were examined for significant differences. Such test cases are listed
in Table IV.
Discussion: Run Number 1
There are significantly less animals in A. elegantissima and tiger juice
than in control. The specific stimulus for each reaction may be the same
since the individual anenomes had, just prior to the experiment, been fed
-7-
Tigriopus. Or, the copepods may be detecting the presence of Anthopleura
itself.
The fact that migrated Tigriopus were a positive stimulus when compared
to the random individuals is interesting. Perhaps this is a result of the
increased number of gravid females in the stimulus sample (see I results).
Possibly the general population cues on the presence of gravid females who
are more sensitive to a variety of environmental conditions. This hypothesis
can and should be investigated by examining the sex ratios of animals
responding to the different chemical stimuli,
Tigriopus preferred the darkened bottle to the control under the
conditions of this experiment. Possible explanations for such a behavior are
too varied and a hypothesis is not here presented. (See Glaser, 1977;
Miyakawa, 1977; Koch, 1977). However, the results were dramatic and rot
only indicate that lighting may have important effects upon migration but
that it should be carefully controlled in future experiments.
The tide pool and terrestrial rocks had similar texture yet the copepods
were found more with the tide pool rocks. Tigriopus preferred courtyard
pool water to isotonic unfiltered synthetic sea salts. The animals may be
responding positively to some organic component of the courtyard water or
negatively to some factor in the sea salts,
laterials and Methods: Run Number 2
Thirty-five 250 ml Erelenmeyer flasks were placed randomly within a
1 meter square area on the bottom of the C.Y.P. See Table V and Figure 6
for the contents and location of the flasks. Six replicates of the filtered
synthetic sea-salt control and five replicates of the C.Y.P. water were
used. All other variables were run in triplicate. Flasks containing live
specimens as stimuli were prepared in the same manner as in Run Numbery,
-8-
Pach flask was filled with 55 o/00 filtered synthetic sea-salts bafore place¬
ment in the C.Y.P. unless otherwise indicated.
Results: Run Number 2
The average values obtained for each flask and the rasults of statistical
analysis on noteworthy test cases are listed in Table VI.
Discussion: Run Number 2
The results of the first three test cases support those obtained in
Run Number 1.
The second three test cases listed in Table VI support the hypothesis
that the animals are reacting to some type of organic material. This material
is partially heat labile and partially solvent soluble. It may be that the
copepods are reacting to a number of organic constituents in the water.
The difference between filtered synthetic sea salts and courtyard water
is not significant. This may be an indication that the copepods wera
reacting negatively to some factor in the unfiltered sea salts of run Number 1.
Millipore (.45u) filtered and unfiltered courtyard pool waters were
not significantly different. This result is not surprising since this type
of filtration will remove only relatively small particles and not chemical
compounds.
Finally, it is interesting to note in run Number 2, that Tigriopus
juice is not significantly different from control. However, it was a
significant difference in Run Number 1. These experiments need to be run
again simultaneously to determine whether or not the different results are
due to methodology.
-9.
GENERAL DISCUSSION
The extraordinary results obtained by flowing sea water into a tide
pool inhabited by Tigriopus suggest that migration is an important aspect
of their ecology. The fact that no copulating pairs were found to migrate
in the courtyard pool and the observation that copulating pairs were found
in the channel with naturally occurring migrators suggests that the individuals
in the channel had been washed out of the high tide pool and some (eleven
gravid females) were in the process of returning to that pool. If this is
so, then perhaps migration is a mechanism by which Tigriopus maintains
populations in the high tide pools in spite of rain or splash.
The finding that Tigriopus would not migrate in a solution of unfiltered
synthetic salts clearly indicates that the copepods are reacting to some
factors in addition to the stimulus of moving water. Both the affinity
studies and those which observed migration in artificial pools involved
the ability of animals to detect and react to certain factors in the water,
The results using the stimulus of migrated individuals or tide pool rocks
were duplicated in both types of experiments. It seems reasonable therefore,
to apply the results of the affinity studies to the biology and ecology of
migration.
Some of the factors act clearly as deterents. Burnett has found that
migrating Ti
piopus have a dramatic negative response to macerated individuals.
This supports the results of Run Number 1 and seems to indicate that the
results of Run Number 2 are either artifactual or due to methodology. A.
elegantissima may also be a deterent. However, the fact that copepods are
responding negatively to macerated individuals indicates that they may be
able to detect predation in a tide pool and thus avoid either the predator,
the tide pool or both. This would be an extremely valuable adaptarion to
life in high tide pools ard deserves further examination.
A number of factors were observed to act as attractants. The results
from tide pool rocks with different treatments and from tide pool and terrestrial
rocks indicate that Tigriopus can detect whether or not water in a rivulet
is coming from a tide pool. Such an adaptation may prevent individuals from
migrating to a pool that is non-existent.
Although his work was scmething less than rigorous, Bozic ((175) found
that Tigriopus fulvus are mutually attracted. Tigriopus californicus are
attracted to migrated individuals. Perhaps the migrated individuals are
more sensitive to a variety of stimuli and serve as leaders which the general
population is then able to cue upon. If correct, this would also be a
way in which migrations are directed to suitable ervironments.
These types of experiments exhibit a great potential for use in
uncovering the various biological and chemical processes involved in
migration, as well as explaining the heterogeneous distributions of
iopus within a tide pool, and should therefore be exploited.
0
-11-
ACKNOVLEDGEMENTS
I would like to express my appreciation for the incredible students
and staff of Hopkins Marine Station. Special thanks go to D.P.A., the
Little Tomatoe and her friend, the Little Cabbage, the H.A.M., and most
of all, Dr. Zelmo Burnett, for his constant inspiration and guidance.
"I thank the P.G.J.H. for
Use of their gym
And God for the
Sunlight.
And Larry for being around to insult
And Tigriopus for going into bottles'
-R.B.
-12-
LITERATURE CITED
Bozic, B. 1975. Detection Actometrique d'un Facteur d'Interaction
chez Tigriopus (Crustaces, Copepodes, Harpactecoides). Bull.
Soc. Zool. de France. 100: 305-311.
Coping with Copepods. Unpub. stud. rep.; Bio. 175H
197
Campbell, D.
Hopkins Marine Station, Stanford University.
Egloff, D. A. 1966. Ecological Aspects of Sex Ratio and Reproduction
in Experimental and Field Populations of the Marine Copepod, Tidri
opus californicus. Thesis-Stanford University.
Foster, C. 1977. Effects of Turbulence on the Behavior of Tigriopus
californicus. Unpub. stud. rep.: Bio. 175H Hopkins Marine Station,
Stanford University.
Fraser, J. H. 1936. The Distribution of Rock Pool Copepoda accord-
ing to tidal levels. J. Anim. Ecol. 5: 23-28.
Glaser. T. 1977. Phototaxis and Behavioral Responses to Changes in
Light Intensity in the High Tide Pool Copepod Tigriopus califor¬
nicus. Unpub. stud. rep.: Bio 175H Hopkins Marine Station,
Stanford University.
Igarashi, S. 1959. On the Relationship Between the Environmental
Conditions of a Tide Pool and the Tigriopus Population. Bull.
Mar. Biol. Sta. Asamushi 9: 167-171.
G. 1977. Clumping Behavior in the High Tide Pool Copepod
Koch,
Tigriopus californicus. Unpub. Stud. rep. : Bio. 175H Hopkins
Marine Station, Stanford University.
Lang, K. 1948. Monographie der Harpacticiden. Ohlssons. Lund.
2 vols.
1977. Changes in Vertical Distributions in Tigriopus
Miyakawa, J.
californicus (Crustacea: Copepoda: Harpacticoida) with Changes
pH: Responses in Light from Above, from
in Salinity, Oxygenand
Unpub. stud. rep.: Bio. 175H Hopkins
Below, and in Darkness.
Marine Station, Stanford University.
T. 1938. Tigriopus japonicus, a new species of neritic
Mori.
Copepoda. Zool. Mag. 50: 294-295.
Vittor, B. A. 1971. Effects of the Environment on Fitness-related
californicus. Thesis¬
Life History Characters in Tigriopus
University of Oregon.
Figure 1
Figure 2
Figure
Figure 4
Figure 5
Figure 6
Table 1
Table II
Table III
Table IV
Table V
Table VI
Appendix 1
Appendix 2
-13
FICURES AND TABLES
Photograph of the courtyard pool
Photograph of the courtyard pool showing detail
of spigot and doghouse
Relative movement rates
Net relative migration rate
Photograph of the artificial pools
Schematic diagram depicting location and
orientation of Run No. 2 and Run No. 2
(cross-hatched area)
Sex ratio of random and migrated animals
Migration results from artificial pools and data
analysis (difference between two percentage
points - Sokal 8 Rohlf Box 16.12)
Flasks and bottles used in Run No. 1 with a
description of their contents.
Valid test cases from Run No. 1 with results of
chi-square analysis
Flasks used in Run No. 1 with a description of
their contents.
Noteworthy test cases from Run No. 2 with results
of chi-square analysis
Tabulated results for Run No.1
Tabulated results for Run No. 2
—-




N.4




FIGURE
FIGURE

a  e.
2
L
2
L
I
L
3-
2
M=—.00
O
—
20 40
60 80 109
M=.015
—

190
80
20
40
61
FLOW RATE - ML/MIN
3
FIGURE
Z
I
L
2
Z

O

L

0
M= —.016
C
C

t-
—
—
40 60 80 190
20
FLOW RATE - ML/MIN
4
FIGURE
FIGURE
20
30
40
O-BOTTLE
D -FLASK

3 n  .
FIGURE6

JHOUSE,
111
TABLE
GRAVID COP
PAIR
9
RANDOM
493
142
1.34
SAMPLE N=150
MIGRATEL
257
367
07
CLUMP N=200
ANALYSIS FOR
ROO
PO0
POO
DIFFERENCE
TABLEI
MIGRATION
RATIO
- A/TOTAL
CONTROL
60 N=428
TIDE POOL ROCKS
77 N -368
IN A
LEADERS IN B
47 N- 403
CONTROL
57 N- 169
ARVAE
8
407
47
367
32
NS
NS
ANALYSIS
FOR
DIFFERENCE
POOI
POO
NS
TABLE
I11
RUN NO.
1) CONTROL
-one empty plastic vial
-one small individual, aggregating type
2) A. ELEGANTISSIMA
3) PAGURUS SAMUELIS
-two individuals in T. funebralis shells
-150 individuals from the water column
4) RANDOM TIGRIOPUS
of the courtyard
pool
5) MIGRATED TIGRIOPUS
-150 individuals from the migrated clump
on the vertical wall in the courtyard pool
6) CONTROL
-covered with black tape
7) DARK
-filled with an unfiltered solution of 44%
8) INSTANT OCEAN
synthetic sea salts
-filled with 44% water from the courtyard
9) COURTYARD POOL
pool
-three granite pebbles from a high tide
10) TIDE POOL ROCKS
pool inhabited by
T. californicu:
-three granite pebbles form the station
11) TERRESTRIAL ROCKS
grounds with surface texture similar to
the tide pool rocks
-three uniform pieces of paper towel blotted
12) TIGER JUICE
in a solution made by macerating about ten
igriopus in fifteen mls. of sea water
-three uniform pieces of paper towel
13) TIGER JUICE CONTROL
——----———
Numbers 1 through 5 correspond to 300 ml. erlenmeyer flasks.
Numbers 6 through 13 correspond to 125 ml. ground stoppered bottles.
IV
TABLE
RUN NO.
A ELEGANTISSIMA
CONTROL
RANDOM
MIGRATED
DARK
CONTROL
INSTANT OCEAN
COURTYARD POOL
TIDE POOL ROCKS
TERRESTRIAL ROCKS
TIGER JUICE
TIGER JUICE CONTROL
TOTAL NO
1188
383
1775
2030
1396
594
72
435
1277
799
694
412
P4,005
P4.005
P4,005
P4.005
P6.005
P4.005
ABLE
RUN NO 2
1) CONTROL
2) CONTROL (with vials)
-one empty plastic vial
3) RANDOM TIGRIOPU
-150 individuals from the water column
of the courtyard pool
-150 individuals from the migrated clump
4) MIGRATED TIGRIOPU.
on the vertical wall in the courtyard pool
-five rocks from a tide pool containing
5) SOLVENT-TREATED
igriopus were washed in petroleum ether
for fifteen minutes and then finsed thor-
oughly in distilled water
-five rocks from a tide pool containing
6) HEAT-TREATED
ligriopus were boiled in sea water for
one hour and rinsed in distilled in dis¬
tilled water
-five rocks from a tide pool containing
7) UNTREATED
Tigriopus
- 44% water from the courtyard pool was
8) FILTERED COURTYARL
filtered through a.45u millipore filter
POOL
9) COURTYARD POOL
-44% water from the courtyard pool
10) TIGRIOPUS JUICE
-30 ml. of a solution made from about
200 Tigriopus macerated in 90 ml. sea water
——---—
All of the above were run in 250 ml erlenmeyer flasks.
VI
TABLE
RUN NO.2
RANDOM TIGRIOPUS
CONTROL
MIGRATED TIGRIOPUS
CONTROL
RANDOM TIGRIOPUS
TIGRIOPUS
MIGRATEL
SOLVENT-TREATED ROCKS
CONTROL
HEAT-TREATED ROCKS
SOLVENT—TREATED ROCKS
UNTREATED ROCKS
HEAT-TREATED ROCKS
CONTROL
COURTYARD POOL
FILTERED COURTYARD POOL
COURTYARD POOL
TIGER JUICE
COURYARD POOL
—
AVG NO
HER FLASK
162
147
227
147
162
227
24
147
329
24
416
329
47
156
130
156
15
156
X
NS
PC.005
P6.005
P.005
P.005
PK.005
NS
NS
NS
1) CONTROL
2) A. ELEGANTISSIMA
3) PAGURUS SAMUELI!
4) RANDOM TIGRIOPUS
5) MIGRATED TIGRIOPUS
6) CONTROL
7) DARK
8) INSTANT OCEAN
9) COURTYARD POOL
10) TIDE POOL ROCKS
11) TERRESTRIAL ROCKS
12) TIGER JUICE
13) TIGER JUICE CONTROL
APPENDIX
115
48
378
917
II
668
501
645
168
317
101
382
171
161
265
III
172
171
120
174
60
311
240
155
230
0
APPENDIX
97
1) CONTROL
2) CONTROL (with vials)
3) RANDOM TIGRIOPUS
204
4) MIGRATED TIGRIOPUS
209
5) SOLVENT-TREAT
394
6) HEAT-TREA
7) UNTREATED
524
8) FILTERED COURTYARD POOL
131
9) COURTYARD POOL
122
120
10) TIGER JUICE
2
158
170
264
248
279
126
16
177
231
179
196
213
268
416
444
133
155
148
120 126
224 175