AN AT
TEMPT TO PRODUCE ACTIVE IMMUNITY
IN THE HERMIT CRAB PAGURUS GRANOSIMANUS
George H. F
attison
from
rd Universit
Hopkins Marine Station of Stanfo
rove.
INTRODUCTION
There has been very little immunological work done on
invertebrates relative to the large amount done on vertebrates.
and especially on mammals. The discovery by Cantacuzéne (1912.
1925, 1934) that the European hermit crab, Eupagurus prideauxii,
apparently develops immunity to the toxin of the sea anemone,
Adamsia palliata, through close biological association with it
suggests that the phenomenon of immunity might account for the
ability of the local hermit crab, Pagurus granosimanus (Stimpson,
1859), to walk unharmed over large beds of anemones. To ascer-
tain if immunity was actually protecting them, an attempt was
made to immunize P. granosimanus to the toxic effects of an
homogenate of the sea anemone, Anthopleura elegantissima.
The manner of assay for immunity involved the determination
of the median lethal dose (LD50) of a standard homogenate for a
sample of the P. granosimanus population by the method of Finney
(1962). This LDgo was then compared to the LDgo of the same
homogenate determined for two groups of P. granosimanus previously
injected with different sublethal doses of the homogenate. In
the two latter cases median lethal doses significantly larger
than that for the normal sample would be considered evidence for
the development of immunity to the toxic effect of the homogenate,
Complementary to the above experiment an attempt was made to
increase resistance of P. granosimanus to anemone toxin by placing
them for a period of time in an aquarium containing a high
concentration of anemones. Again the median lethal dose was
(2)
computed and used as an indication of the tolerance of the group.
MATERIALS & METHODS
All the P. granosimanus used were collected at one time at
Stillwater Cove, just north of Carmel beach, Carmel, California.
The specific area of the cove in which the animals were collected
had a low concentration of anemones of all kinds relative to
other areas of the coast.
About twenty-five small Anthopleura elegantissima were mas-
cerated in 150 ml. of Ringer's solution and then homogenized.
This standard homogenate was frozen for later use in separate
bottles.
A. The LDgo for normal crabs was determined as follows:
seventy P. granosimanus were cracked out of the shells they were
inhabiting and were divided into seven groups of ten individuals
each irrespective of size. Each group was injected with a dif-
ferent dilution of the standard homogenate, one group serving as
a control. The control group was injected with only Ringer's
solution which was used as the vector for the homogenate. The
injections were made into the right side of the abdomen through
a portion of the columellar muscle with a Yale one quarter ml.
syringe with needle size +26. Each individual was weighed and a
dose proportional to weight was given, namely, .Ol ml. of homogenate
at the appropriate dilution per .2 grams body weight. Broken but
usable shells were returned to the hermit crabs and the number dead
in each group was recorded between twelve and twenty-four hours
0
(3)
later. An individual which was moribund but moving either its
first or second antennae was classified as alive; if no movement
of antennae was observable the individual was recorded as dead.
B. Two other groups of P. granosimanus were injected with
sublethal doses of the standard homogenate which was diluted to
1:160 and 1:320 with Ringer's solution, i.e. they contained 144
Ag N/.200 ml and 72 Ag N/200 ml, respectively. After an incubation
period of six days each group was challenged in the manner des-
cribed above and the median lethal doses were computed.
C. Seventy P. granosimanus were placed in an aquarium with
approximately forty-five small Anthopleura elegantissima for
seven days. At the end of this time they were challenged as
previously described and the LD50 computed.
RESULTS
The doses, numbers of animals used, and number killed are
presented in Tables 1 - 4.
The values of the median lethal doses and their approximate
95% confidence intervals for the various groups are presented in
Table 5. The values are expressed in micrograms of nitrogen per
200 mg. of body weight.
If we regard the values of the median lethal doses as the
mean tolerances, tests of significance can be performed between
the mean of the normal sample and the means of each of the other
samples. In each case the test was performed at as.05 and con-
firmed the obvious fact that there is no apparent difference
(4)
between the means, i.e., the null hypothesis was accepted each
time. Therefore, no indication of inducible immunity was demon¬
strated.
DISCUSSION
Using the method described there is no evidence for an in¬
crease in the mean tolerance of P. granosimanus to the toxic
effects of sea anemone homogenate after exposure to these animals
or their extracts.
Since the null hypothesis has been rejected the power of
the tests becomes important. The power was approximately computed
for the test between the mean of the normal sample and the sample
immunized at 114 g N2/.2 grams body weight. If it is assumed.
for instance, that the actual difference between the means was 145
gN the power is about .18. The cause of this relatively low
power for the relatively large sample size lies in the fact that
a natural population heterogeneous for size, age, and physiological
condition was sampled. While an attempt was made to use animals
having a minimal exposure in the past, the actual history of these
animals was not known and one can assume that this accounts for
some of the variability of natural resistance.
It seems most probablg that P. granosimanus is protected from
anemone nematocysts in natural circumstances by the chitinous
exoskeleton which covers the exposed portions of its body.
Results which suggested that P. samuelis does not produce
active immunity to anemone homogenate were obtained but they lacked
(5)
completeness and so could not be treated statistically.
SUMMARY
1) An attempt was made to establish active immunity in
Pagurus granosimanus to an homogenate of the sea anemone, A.
elegantissima, with no success.
2) Keeping P.
granosimanus in close contact with A. elegan-
tissima did not increase its resistance to anemone homogenate.
DOSE IN
MICROGRAMS ON
460
307
230
184
164
144
0
DOSE IN
MICROGRAMS OEN
+60
307
230
134
164
4 4
0
TABLE F
NUMBER OF
ANIMALS USED
10
10
10
10
10
10
10
+2
TADLE
NUMBER OE
ANIMALS USED
5
7
10
NUMBER OF
ANIMALS KILLED
10
3
3

0
NUMBER OF
ANIMALS KILLED
4
4
—

DOSE IN
MICROGRAMS GEN
+60
907
230
194
153
1 3
D
DOSE IN
MICROGRAMS OET
160
207
230
184
153
O
TABLE
HUMBER
OF
ANIMALS USED
8
6
8
ABLE I
NUMBER OF
ANIMALS USED
10
10
/0
10
10
/1.
NUMBER OF
ANIMALS KILLED
6
2
5

2
0
O
NUMBER OF
ANIMALS KILLEO
6
5
2
2
O
NORNAL
CRABS
CRABS TAAUNIZED
A AA ERTRACT
NITROCEN PER 28 WEIORI
CRABS TAADDI2BO
AT 72% EYTRReT
NITROCEN PER 2SWEERT
CRABS EYPDSED
TO ANEMONES
NATUR ALLY
TABLE 5
LDeo
341
233
314
238
959. CONFIDEUCE
ENTERVALS
186 - 623
173 - 316
205 -48
192- 132
*
LITERATURE CITED
Cantacuzéne, J. (1912) Sur certains anticoup naturels observés
pagurus prideauxii. C. R. Soc. Biol. Paris 73:663-664.
chez
. (1925) Action toxique des poisons d'Adamsia palliata
sur les Crustacés Decapoda. C. R. Biol. Paris 92:1131-1133,
1133-1136, 1464-1466.
(1934) L'action precipitante specifique du serum
d'Eupagurus prideauzii. C. R. Biol. Paris 117:269-271.
Finney, D. J. (1962) Probit Analysis - A Statistical Treatment of
the Sigmoid Curve. Cambridge University Press, London.
4
LEGENDS
Table 1.
Table showing doses, number of animals used, and number
of animals killed in the determination of the ID50 for
the normal crabs.
Table showing doses, number of animals used, and number
Table 2.
of animals killed in the determination of the LD5o for
crabs immunized at 144 micrograms of extract nitrogen
per .200 ml. body weight.
Table showing doses, number of animals used, and number
Table 3.
of animals killed in the determination of the LD50 for
crabs immunized at 72 micrograms of extract nitrogen per
.200 ml. body weight.
Table showing doses, number of animals used, and number
Table 4.
of animals killed in the determination of the LD5o for
crabs naturally exposed to anemones.
Table 5. Table showing values of LDgo and approximate 95% confi¬
dence intervals.