ACKNOWLEDGEMENTS
Special thanks to Dr. D.P. Abbott for his dedication
to providing a unique and personal learning experience for
students. Also, thanks to Chuckand Robin for both their
scientific and spiritual contributions to my project. In
general, thank you to the entire Hopkins Marine Station
staff and class; though this spring was a rigorous and
sometimes trying experience, it was indeed pleasant and
worthwhile.
a
2
I. Introduction
Investigations on planktonic copepods and other open water crustaceans
have shown that in some species phototaxis is affected by various environmental
conditions such as salinity (Loeb 1893, Rose 1925 in Waterman 1961), oxygen
tension (Herter 1927 in Waterman 1961), pH (Loeb 1906 in Waterman 1961),
temperature (Dice 1914, Loeb 1893, Rose 1925 in Waterman 1961) and turbulence
(Herter 1927 in Waterman 1961). Tigriopus californicus is an abundant
copepod found along the California coast in high splash pools. These pools
often remain out of communication with the open ocean for extended periods of
time, and undergo large fluctuations in salinity, oxygen and pH (Burnett 1977,
pers. comm.). In the face of these extreme conditions, the survival of the
animal may be enhanced by the ability to exhibit different responses under
different environmental conditions. This would include responses to such
stimuli as light and gravity. No previous work has been published on the
phototactic responses of Tigriopus californicus. Vertical movement is of
particular interest since most environmental parameters such as salinity,
oxygen, etc. exhibit vertical gradients in the habitat. This being the case,
vertical movement is more likely to bring about a change in local environment
than a horizontal one, and thus be the most adaptive short term strategy. In
the long term, habituation or acclimation may occur. The following study
examines short term upward and downward motion of Tigriopus californicus
associated with an abrupt change in salinity, oxygen tension or pH, under
a constant directional light source.
II. General Materials and Methods
The animals were collected from one high tidepool at Mussel Point,
Pacific Grove, CA. A suction bulb was used to obtain a sampling from all
portions of the water column, and the animals were strained through a nylon
netting which passed larvae but held the larger adults. The animals were
collected daily to avoid abnormal behavior cause by unnatural or stressful
laboratory conditions. Only females with egg sacs were used in the study to
avoid possible variability due to sex and age.
In all experiments, "normal" conditions (as of temperature, salinity,
oxygen, pH, etc.) are defined as those found in the running sea water system
of Hopkins Marine Station fed by bay waters (see fig. 2). The physical set-up
used in all experiments is diagrammed in figure 1. A series of 5 to 9 50ml
graduated cylinders was placed above or below the directional light source;
each cylinder contained sea water, 12cm deep, with one experimentally
adjusted parameter. Twenty females with egg sacs were sucked into a narrow
tube (inside diameter = 0.3cm) along with enough "normal" sea water to form
a column of water 12cm long. With its upper end closed by a finger, one such
tube containing 20 animals was gently lowered into each cylinder until its
lower end rested on the bottom of the cylinder. The tube and cylinder were
allowed to sit this way for 30 minutes. The 30 minute equilibration period
was chosen as a time for which the difference between normal conditions
within the tube and its surrounding cylinder could be maintained, yet allow
the animals maximal time to adjust to the light conditions and erase any
memory of turbulence or light-dark history. In most cases the base of the
inner tube fitted tightly enough against the bottom of the graduated cylinder
to prevent significant exhange of fluid or premature escape of Tigriopus.
In the few cases where the animals did escape, the results were not used in
calculating mean movements. At zero time the number of copepods in each 3cm
segment of the inner tube was recorded; then the animals were released from
the inner tube to the environment of the cylinder by slowly pulling the tube
out of the cylinder. Two minutes later the number of animals in each of
four 3cm segments of the cylinder was recorded. The copepods usually showed
marked short term changes in vertical distribution. Shorter exposure times
gave more variable results. For exposures greater than 5 minutes (depending
on the condition) the animals showed a grend toward random vertical distribution.
This system offers various advantages and disadvantages. It attempts
to mimic a possible realistic situation in which the animal swims into some
new local environment and experiences a change in some environmental parameter.
It allows manipulation of some parameter without confusing its effects with
those of turbulence or a change in light.
A dual control is provided by this test. First, for each tube, the
zero time count provides a normal environmental control contrasting with the
2 minute experimental value. Secondly, a normal control is included near the
middle of the test range, where no environmental change occurs upon the
animals' release from the inner tube.
III. Changes in Vertical Distribution with a Sudden Change in Salinity
under a Constant Directional Light Source
The salinities tested ranged from 1Oppt to 110ppt, approximately 1/3
to 3 times normal sea water. All salinity solutions except normal (34ppt)
were made up with Instant Ocean sea salts and tap water. The parts per
thousand of each solution was determined by an American Optical Co,
refractometer calibrated for salinity determinations. In the high salinity
tests the "normal" salinity water tended to rise to the surface. Occasionally
copepods entered this layer and stayed there. Therefore, after raising the inner
tube the top one milliliter was aspirated off the water column of the cylinder
to eliminate a less dense, normal salinity layer as an alternate environment
to the test condition of the rest of the cylinder. Removal of a possible
denser normal layer on the bottom of the low salinity tests was not possible
without introducing disruptive turbulence.
A. Responses to Changes in Salinity with a Constant Light Source from Above
The results of four separate trials are shown in Table 1. In all
trials, regardless of the time of day they were carried out, some consistent
trends are observed, justifying combining the data sets to yield a composite
picture of the response, shown numerically in Table 1, graphically in fig. 3.
The trends are as follows:
1) Counts taken before the animals were released into the test condition
show no marked tendency for the animals to be in either upper or lower
half of the column.
2) Upon encountering low salinities (lOppt - 25ppt) the animals tend strongly
downward, away from the light, resulting in most of the animals occupying
the bottom quarter of the column.
3) Upon encountering high salinities (80ppt - 110ppt) the animals tend
strongly upward, towards the light, resulting in most of the animals
grouped in the top quarter of the water column.
4) In the intermediate salinity range (34ppt - 60ppt) there is a small
shift downward.
This behavior seems quite adaptive. If it is advantageous for an
animal to avoid high or low extremes of salinity it should swim downward on
encountering lower salinities since the denser, higher salinities would be
found towards the bottom; conversely, upon encountering a higher salinities
an animal ought to swim upward, the most likely direction of a lower salinity.
In a tidepool in the field, two major stimuli provide cues as to the directions
up and down. Light always enters from above the horizon, ranging from near
horizontal to directly above. Gravity provides another cue as to the
directions up and down. In the above experiments, at low salinity, Tigriopus
show a negative phototaxis and a positive geotaxis. At high salinities they
«how a positive phototaxis and negative geotaxis. At unusually low and
high salinities the physical properties of the medium itself would tend to
reinforce the above taxes; animals neutrally buoyant at a given salinity
would tend to sink down in the less dense water at lower salinities and
float upwards in the denser water at high salinities. Such physical response
may contribute slightly to the foregoing results, but does not explain
them as will be seen in the next experiments.
The experiments with light from above offer no clue as to what
environmental cues Tigriopus is using to distinguish up from down. One
approach to separating the effects of light and gravity is to experimentally
provide illumination from below instead of from above. If the response of
Tigriopus is made mainly on the basis of gravitational cues, the vertical
distribution of copepods should not change from that seen in the experiment
with light from aboye. If light provides the major directional cue, a
reciprocal distribution would be generated. If both cues are operating
strongly, some intermediate results would be expected.
B. Responses to Changes in Salinity with a Constant Light Source from Below
In all three triabperformed, the animals moyed toward the bottom of
the tube at all salinities, though the tendency is weakest at both high and
low salinity extremes (Table 2, fig. 2). A row-by-column contingency test
indicates that the distribution found at these extremes is significantly
different from the distribution at the intermediate salinities to a confidence
level of p.005. The results at the extremes immediately dispel any possibility
that buoyancy effects are a major factor, since eyen at the densest salinity
animals can be found on the bottom, and even at the lowest salinity animals
can be found at the top.
The results also cannot be explained by response to gravity or to
light alone. Usually at high salinities the adaptively advantageous behavior
should be to swim upward, reflecting a negative taxis to gravity and a
positive taxis to light. With illumination from below, light and gravitational
cues are contradictory. Adaptively speaking the animals should have moved
upwards yet they are found on the bottom, so light must be exerting a
stronger directional effect than gravity. At low salinities, the adaptive
response should be to swim down; normally this means away from light and
toward gravity, but here again with light from below, the cues conflict. More
animals swam down than up, so perhaps the gravitational cue here is the more
important. However, the tendency to go down is much less clear cut here than
in experiments with light from above, so light may well play a modifying role.
At intermediate, nonstress salinities, the animals shifted consistently
downward with illumination from above and more strongly downward with
illumination from below. No adaptive advantage is postulated here and no
marked responses were expected. Perhaps with no salinity stress gravity
and light exert antagonistic effects on the animals with illumination above.
Thus, the animals might be found in either half of the column. Whereas, with
illumination from below, the two cues work together to yield the strong
downward shift. Perhaps crowding interferes with the animals' moyement
toward the bottom within the narrow tube, thus upon release into the whole
cylinder, the downward shift is made.
C. Responses to Changes in Salinity with No Light Source
In hopes of gaining further insight on the respective roles of light
and gravity as directional cues for copepod movements accompanying changes in
salinity, a trial was run in darkness. However, without the aid of an
infrared system to view the animals in the dark, light had to be introduced
to make the counts. Except for darkness, the experiments were conducted as
before. However, after 30 minutes in the dark a light from below was turned
on (for less than a minute) just to check for any marked tendency for the
copepods in the central tubes to be in either upper or lower half of the
column. The animals were released and the light turned off. They were
checked again, after 2 minutes, with the same light from below. At low
salinities the animals were found on the bottom; at high salinities they
moved up to the top (Table 3). These results are in accordance with those
obtained with the light from above. In addition, the animals moved in the
adaptive direction, an important ability if the animals are to respond
correctly in the absence of light as in night.
IV. Changes in Vertical Distribution with a Sudden Change in Oxygen Concentration
under a Constant Directional Light Source
Oxygen concentration in these experiments ranged from 1.5ppm to off
scale 20"ppm. Low oxygen sea water was made by bubbling with nitrogen; high
oxygen water was made by bubbling with oxygen. Oxygen concentrations of the
test media were determined, following completion of the 2 minute count, with
an oxygen meter (Yellow Springs Instrument Co., Inc. Model 54). The listed
values are probably reliable to +.5ppm. Each graduated cylinder was covered
with parafilm during the tests to minimize gaseous equilibration with the
atmosphere.
A. Responses to Changes in Oxygen Concentration with Constant Light from
Above.
The various trials show some consistent trends (Table 4, fig. 4):
1) At low oxygen concentrations (1.5ppm to 2.5ppm) with illumination from
above, the animals shifted strongly up towards the light resulting in
almost all of the animals in the top quarter of the column at the 2
minute count.
2) Through the rest of the range, intermediate to high oxygen content,
only a slight downward shift occurred, with animals still distributed
throughout the water column.
Again, the responses appear to be adaptive. At low oxygen concentrations
the best direction to swim in search of higher dissolved oxygen is upward,
where greater gaseous exchange could occur at the surface. However, at high
oxygen concentrations, behavior does not differ from that exhibited in
normal conditions. It is unlikely that in nature dissolved oxygen levels
would get sufficiently high to be toxic or provide stress to the animals, and
therfore one would not expect the animals to show any marked response. These
copepods are routinely found in pools with high photosynthetic activity and
supersaturated waters (dissolved oxygen = 12ppm - 16ppm).
When introduced to low oxygen water the animals were positively
phototactic and negatively geotactic. Again, experiments with light from
above do not supply information on the respective roles of gravity and
light. In the next set of experiments light was offered from below.
B. Responses to Changes in Oxygen Concentration with Constant Light from
Below
With illumination from below, in tests at all oxygen tensions, the
animals showed a marked movement downward (Table 5, fig. 4). At the 2
minute count almost all animals were found within the bottom quarter of the
column (fig. 4). If gravity were the predominating cue at low oxygen levels
no change in distribution from experiments with light from above would be
expected, but this result was not obtained. If light were the predominating
cue at low oxygen tensions, the animals would be found at the bottom of the
column in these tests. The results fall in accordance with this model. Thus,
despite the adaptive advantage of going up upon encountering low oxygen
concentrations, the animals went down with light from below, suggesting light
as the predominating directional cue.
As in nonstress salinity changes, upon encountering intermediate and
high oxygen tensions, the animals shifted markedly downward with light
from below but only slightly dowward with light from above.
V. Changes in Vertical Distribution with a Sudden Change in pH under a
Constant Directional Light Source
pH values ranged from 4.5 to 10.5. Solutions were acidified with
IN HCl and made basic with IN NaOH. The precipitate formed upon addition of
base was removed by filtration through Whatman No. 1 paper. The pH was
determined using a Beckman pH meter.
A. Responses to Changes in pH with a Constant Light from Above
With light from above, the animals shifted toward the bottom at
the pH's differing from normal, 8.4 (Table 6, fig. 5). However, eyen the
pH test choices closest to the normal were rather extreme in view of very
slight pH fluctuation found in the field (Burnett 1977, pers. comm.). Also
Adaptive responses under stressing pH's may not have been selected for as
strongly as for other environmental stresses such as salinity or low oxygen
concentrations. Furthermore, there is no obvious direction (up or down) to
swim to effectively avoid stress-exerting pH conditions. At the altered pH's
tested the animals were positively geotactic and negatively phototactic with
light from above.
B. Responses to Changes in pH with a Constant Light from Below
With light from below, animals at all pH's shifted strongly dowward
(Table 7, fig. 5). If light were the predominating cue at altered pH's
0
the animals would be found at the top with light from below. However, the
animals move to the bottom with light from below as they did under light
from above, the result predicted if gravity were the stronger cue. As in
salinity and oxygen experiments at normal conditions, the animals shifted
slightly downward with light from above and strongly downward with light
from below.
VI. Summary
1) The short term responses to sudden changes in salinity, oxygen
concentration, and pH, in terms of tendency to swim upward or downward,
were studied using a light coming either from above or below, to
investigate the role of light as a directional cue to vertical moyement.
2) At low salinities, the adaptive response should be to swim down; nommally
this means away from the light and toward gravity. Accordingly, with
light from above the animals were negatively phototactic and positively
geotactic. However, with light from below, most animals swam down
toward gravity and fewer animals were found at the top, away from light.
This result suggests gravity over light as the predominant directional
cue for vertical movement, with light playing a modifying role.
3) At high salinities, the adaptive response should be to swim up;
normally this means toward the light and away from gravity. Accordingly.
with light from above, the animals were positively phototactic and
negatively geotactic. However, with light from below, most animals swam
down toward the light, and fewer found on top away from gravity. This
result suggests light over gravity as the predominant directional cue
for vertical movement at high salinities, with gravity playing a
modifying role.
4) The animals are capable of responding in an adaptive manner when confront
2
with a change of salinity in the absence of light, with gravity providing
the only directional cue.
5) At low oxygen tensions, the adaptive response should be to swim up;
normally this means toward the light and away from gravity. Accordingly,
with light from above the animals were positively phototactic and
negatively geotactic. However with light from below the animals swam
down, again positively phototactic, but in this case positively geotactic.
This result suggests light as the predominant cue for vertical moyement
at low oxygen concentrations.
6) At pH's other than normal (8.4) the animals shifted in the direction of
gravity, with little effect seen with a change in the direction of the
light source.
7) In all cases, upon release of the animals into the normal or nonstress
conditions tested, they shifted slightly downward with light from
above and markedly downward with light from below.
8) In conclusion, reproducible trends to move with or against gravity and
towards or away from a directional light source offered from above or
below are seen in response to a sudden change in environmental conditions,
suggesting the role of both light and gravity in governing directional
movement within a vertical column.
EFERENCES
Dice, L.R. 1914. The Factors Determining Vertical Movement of Daphnia, J.
Animal Behavior 4:229-265.
Herter, K. 1927. Taxieu und Tropismen der Tiere, Tabulae Biol, 4:348-381.
Loeb, J. 1893. Über künstliche Umwandlung positiv heliotropischer Tiere in
negativ un umgekehrt, Arch. ges. Physiol. Pflüger's 54:81-107.
Loeb, J. 1906. Ueber die Erregung von positiven Heliotropismus durch Säure
insbesondere Kohlensäure und von negativen Heliotropismus durch
ultraviolette Strahlen. Arch. ges. Physiol. Pflüger's 115:564-581.
Rose, M. 1925. Contribution à l’etude de la biologie du plankton, Arch. Zool.
expte et gen 64:387-542.
Waterman, T.H., The Physiology of Crustacea-Sense Organs, Integration, and
Behavior, Academic Press, New York, 1961, v.2.
Table I presents the data on changes in vertical distribution
with a sudden change in salinity with a constant light
source from above. The time of day that each series
of tests was carried out is listed with the trial
number. The number of animals counted in each of the
segments (1-4) of the cylinder at 0' and 2' is listed
for each salinity tested. The shaded areas indicate
cases in which animals escaped the inner tube prior to
the 0' and thus these data points were not included in
the composite tables.
L

8

16
TABLE 1
TABLE 1


2'
2'
2'
17
ATRIAL

3
2.0
2400
34
PPT
134
34
110


—
—
TRIAL
+2
0100
e
34
54
34
80
34


——

TRIAL
+3
13
1700
PPT34
34
60 34
34
34
110

—


2'
ATRIAL
3
2
+4
20
0200
134
34
34
PPT
25 A34
34
—





2'
15
70
TOTAL
COUNTS
60
134
Pe
—

—

2
27
45
22
135
33
8
100
34
PPT34
6O
110
34
L
8
46
14
Table 2 presents the data on changes in vertical distribution
with a sudden change in salinity with a constant
light source from below. The time of day that each
series of tests was carried out is listed with the
trial number. The number of animals counted in each
of the four segments (1-4) of the cylinder at O
and 2' is listed for each salinity tested.
L

I
L
TABLE 2


2'
1
AERIAL
4
3
12
0300
PPT
34
34
34
—

—
2'
0
ITRIAL
*2
o
1500
34
PPT
134
—
—


2'
ATRIAL
+
+3
0

20
1600
31
25
10 34
PPT 1134

2
6

TOTAL
COUNTSE
o
16
10
53
122
34
34
34
PPT
L
2'
10
516
2
2
%
0
21
30
2
10
4 1337
58
88

31
25
APPT
10
—
60

O
2
31
—
2'
O
60
—

60
31

2 1
10
30
85 28
—
31
34

114
2
32
110
Table 3 presents the data on changes in vertical distribution
with a sudden change in salinity with no light source.
Just prior to releasing the animals the light was
turned on just long enough to check for any marked
tendency to be in either upper or lower half of the
water column. No such distribution was noted. The
light was not left on long enough to take a count:
consequently, only the 2' counts were obtained.
(
Z
TRIAL
1800
%
TABLE 3
—
PPT
—


20

10
34
PPT

34
—
—
3+

—
2'
20
25
50
—
110
110
7
20
Table 4 presents the data on changes in vertical distribution
with a sudden change in oxygen concentration with a
constant light source from above. The time of day
that each series of tests was carried out is listed
with the trial number. The number of animals counted
in each of the four segments (1-4) of the cylinder at
0' and 2' is listed for each oxygen concentration
tested.
TABLE 4
2





—
—

—L
13




0
—



o

+



—




O -

+


—
+

1o
+

L


-
—4—



0
+ 10
SL
2

—brmhndm




91


immerhme


2
20

&a *
X*
3


lsodwoo
Aogv IH





—
Table 5 presents the data on changes in vertical distribution
with a sudden change in oxygen concentration with a
constant light source from below. The time of day
that each series of tests was carried out is listed
with the trial number. The number of animals counted
in each of the four segments (1-4) of the cylinder
at 0' and 2' is listed for each oxygen concentration
tested.
TABLE

—
8
3



i
—





—
-O
0
1
OO









S
g
E4


M9 1H517

o
o
0

0
+
Sin

9
o
—
:

—
+

—.
N
—
2
0




—




—
iisodwoo

—
Table 6 presents the data on changes in vertical distribution
with a sudden change in pH with a constant light source
from above. The time of day that each series of tests
was carried out is listed with the trial number. The
number of animals counted in each of the four segments
(1-4) of the cylinder at 0' and 2' is listed for each
pH tested.
L
O

I
11

TABLE 6
ITRIAL

2300
38.4
18.4

TRIAL
42
O130
8.4
p
194

—
TRIAL
+3
15
1300
8.4

4.5
1e
TOTAL
COUNTS
117
52
p
8.4
84
—
28
%
1333
87
4
184
0
1484
—
2'
2

6.O
8.4
8.4
9.5
10.5


17
10.5
8.+
8.4
6.O

3
+
10.5
84
9.5
6.0
8.4


2
2'
11
8
12
35
10.5
9.5
8.4
8.4

18:
2
3
20
58
10.5
6.0 8.4
8.4
9.5
8.4
25
2
Table 7 presents the data on changes in vertical distribution
with a sudden change in pH with a constant light source
from below. The time of day that each series of tests
was carried out is listed with the trial number. The
number of animals counted in each of the four segments
(1-4) of the cylinder at 0' and 2' is listed for each
pH tested.
—
TRIAL
1500



+
TRIAL
L
72
1700
TOTAL
COUNTS
11

d
%
TABLE 7
2'
2'
1
3

184
184
6.O
8.4
8.4
—

20
8.4 6.0 18.4
8.4

8
—

11
112
p
8.4
8.1 84
8.4
584

o
5
90
18.4
8.4
p 184
8.4
8.4
9.5
2'
15
10.5
8.“

8.4
10.5
2'
84
85
10.5
27
Figure 1 diagrams the physical aspects of the experimental
set-up. The 50ml graduated cylinder was placed
in a constant directional light offered from above
or below. Light intensities were measured with a
Li-Cor photometer (model LI-185) with the sensors
oriented facing the light source and parallel to
the axis of the graduated cylinder.
Figure 2 diagrams the protocol used in all experiments.
2
Zem
LIGHT
ANIMALS
ENVIRONMENT
TIME
0.3 cm
— normal
sea water
20 females
vith egg sat
FIGURE 1
+ LIGHT SOURCE
0
above or below
/1
15 watt
fluorescent bulb
diam=21cm
30cm  distance between
light &a water column
120 ux
zom
Mendemn
S
photometer
sensors

u
50m
graduated cylinder
FIGURE 2
pH 8.4
NORMAL: 34%o
6.5-7.OppmO2. 20-22?c.
ZOMIN
COUNT


----
2
2P


MIN

TEST
ARELEASE COUNT

2
—
R
—
30
0
"igure 3a presents a composite of 4 trials on changes in
vertical distribution with a change in salinity with
light from above. Each horizontal bar within the
outlined shapes represents the percentage of animals
tested found in each of the 4 segments (1-4) of the
cylinder at 0' (top) and 2' (bottom). Analogously,
figure 3b presents a composite of 3 trials with changes
in salinity with light from below.
L

L
P




N
E

MSV THO
FIGURE 3


e

42
—2

D
—

s
L






L



L
NO



1
MOTT.
Figure 4a presents a composite of 3 trials on changes in
vertical distribution with a change in oxygen
concentration with light from above. Each horizontal
bar within the outlined shapes represents the
percentage of the animals tested found in each of the
4 segments (1-4) of the cylinder at 0' (top) and
2' (bottom). Analogously, figure 4b presents a
composite of 2 trials with changes in oxygen concentra
tion with light from below.
39
FIGURE 4




6.5
6.5
.
55
5
b.5

55
5






L















PPM

5.5

6.5 8.0
15 25
15.U
11.0
A.U

—







CT
as
—


L







aaaakaa
ai e    e et ei e e e i se.


PPN
6.5
6.5
6.5
6.5
6.5
5.5

6.5

a

b.









L


a



.
en
110
80
204



srern


0
Figure 5a presents a composite of 3 trials on changes in
vertical distribution with a change in pH with light
from above. Each horizontal bar within the outlined
shapes represents the percentage of the animals tested
that were found in each of the 4 segments (1-4) of
the cylinder at 0' (top) and 2' (bottom). Analogously,
igure 5b presents a composite of 2 trials with changes
in pH with light from below.
4
FIGURE 5

L

L
J
D
L

L



o

I

P
L Erie






D


L
J
a
—


L


—
—

MORa THST
36