Preliminary Tests of the Toxin Extracted from California 
Sea Hares of the Genus Aplysia 1 
Lindsay R. Winkler 2 
During A STUDY of the biology of California 
sea hares (Winkler, 1957), the large amounts 
of absorbed but unused substances from the sea 
hares diet which are to be found in the digestive 
gland were noted. On the assumption that if 
there are toxic sea weeds in the diet of the sea 
hare these toxins might possibly be present in 
the digestive gland, samples of the exudate from 
frozen digestive glands on hand were removed 
with a small pipette and were placed in test 
tubes. The test tubes were then placed in a boil- 
ing water bath for 10 min. After being centri- 
fuged, milliliter aliquots were injected intra- 
peritoneally into 20-gm. C57 black mice. The 
digestive glands of Helix aspersa O. F. Muller 
were similarly treated as controls. The animals 
injected with the experimental extract showed 
almost immediate respiratory symptoms fol- 
lowed by an excitement stage and death in 4 to 
6 min. Since neither the controls nor ashed sam- 
ples reconstituted and injected produced these 
effects, further study seemed desirable. Certain 
general aspects of the resulting study are re- 
ported here. 
It may or may not be significant that Latin 
and Medieval writers, beginning with Pliny ( ca . 
A.D. 60 ) considered the sea hare to be very poi- 
sonous and claimed its use in poisonings during 
the days of Imperial Rome. An excellent sum- 
mary of the beliefs and superstitions pertaining 
to the sea hare is given by Johnston (1850). 
MATERIALS AND METHODS 
Collections in March, April, and May were 
made at Doheney Beach near Dana Point, Cali- 
fornia. Later collections were made from May 
1 This investigation was supported in part by a re- 
search grant, RG-6445-A, from the National Institutes 
of Health, U. S. Public Health Service. Manuscript 
received February 6, I960. 
2 Department of Pharmacology, College of Medical 
Evangelists, Loma Linda, California. 
through August at Lunada Bay, Palos Verdes, 
Los Angeles County, California. The former 
were small specimens between 4 and 6 in., the 
latter were large breeding specimens measuring 
to 1 ft. in length. Animals were collected at low 
tides, packed in wet Pelvetia fastigiata , and 
transported back to the laboratory where they 
were either immediately dissected or were re- 
frigerated until the next day. In either case, the 
animals were alive when dissected and no dif- 
ference in the toxic effect was noted between 
those dissected immediately and those stored 
overnight. 
The digestive glands were dissected out by 
making a longitudinal, midpedal cut with scis- 
sors in the ventral surface of the foot from the 
tail to the lip area. The animal was then turned 
inside out. The digestive gland was removed, 
including the part of the intestine embedded 
in the gland and the ovotestis, which is an in- 
tegral part of the digestive gland complex. The 
percentage weight of the digestive gland com- 
plex to the weight of the intact animals was 
determined in a certain number of cases. Sam- 
ples of the crop and/ or intestinal contents were 
preserved in alcohol and studied where it seemed 
desirable in a rough attempt to ascertain if the 
diet was responsible for the toxicity. Specimens 
of Aplysia vaccaria Winkler were also collected 
and dissected for extraction. The digestive glands 
were stored in glass containers in a deep-freeze 
and were not thawed until ready for use. 
It early became apparent that a more refined 
method of extraction than the water methods 
used initially was necessary because of the large 
amounts of pigments, salts, and apolar materials 
present in the gland. After much trial and error 
the following method proved the most satis- 
factory for large-scale crude extract preparation. 
Thawed digestive gland (130-150 gm.) is 
placed in a Waring Blendor. When the gland 
is thoroughly liquified, 400 ml. of acetone is 
211 
