Localization and Proof of Chitin in the Opisthobranch Mollusks 
Aplysia calif ornica Cooper and Bulla gouldiana (Pilsbry), 
with an Enzymochromatographic Method for Chitin Demonstration 
Lindsay R. Winkler 1 
Chitin has been generally considered as almost 
synonymous with the arthropod exoskeleton. 
However, as Richards (1951) points out, other 
substances enter into the structure of the ar- 
thropod exoskeleton and, conversely, some ar- 
thropods are known which have no detectable 
chitin. It is also true that chitin is found in 
other invertebrate phyla and in the plant king- 
dom as well. Wester (1909) and Kunike (1925) 
surveyed the animal kingdom for chitin and 
reported it present in at least some part of the 
body of members of most of the phyla below 
the echinoderms. These and the other authors 
mentioned below found chitin present in four 
of the five molluscan orders (Scaphopoda was 
not studied ) . Besides these works Sollas ( 1907 ) , 
Spek (1919), Pantin ( 1925 ), and Toth (1940) 
have contributed to the study of chitin in the 
Gastropoda. Chitin has been reported present in 
only two structures, the radula and the jaws. 
Toth (1940) reported that the organic portion 
of the shell of Helix pomatia did not contain 
chitin. Kunike (1925) found no chitin in the 
organic shells of the gastropods and lamelli- 
branchs which he tested. Roche (1951) hy- 
drolyzed the organic residues of the gastropod 
shells of Turbo, Meleagrina , and two species of 
Pinna, and reported them to be scleroproteins. 
Spek tested the radulae and jaws of a number 
of common prosobranchs and pulmonates and 
of the opisthobranch Aplysia sp., and found 
chitin present in all cases. The radulae and jaws 
of the genera which have been tested in these 
works include Helix, Arion, Patella, Haliotis, 
Natica, Conus, Buccinium, Littorina . Tergipes, 
and Aplysia. 
As Richards (1951) points out, it is essential 
1 School of Tropical and Preventive Medicine, Loma 
Linda, California. Manuscript received January 2, 
1959- 
that we establish a criterion for the demonstra- 
tion of the polysaccharide chitin. He accepted a 
positive chitosan color test as the most valid 
qualitative test for the presence of chitin. This 
test is also the standard used in this paper, al- 
though the chromatographic demonstration of 
N-acetylglucosamine, the basic saccharide of 
chitin, would also be an adequate demonstration 
of the presence of chitin. 
Besides the two chitinous structures previ- 
ously studied, the opisthobranch Aplysia, com- 
monly known as the sea hare, possesses two 
other "chitinous” structures, the stomach teeth 
and a vestigial shell, both of which are com- 
posed almost entirely of organic material. The 
shell-bearing opisthobranch, Bulla, possesses 
stomach teeth and a small amount of organic 
periostracum on its shell. These structures have 
been tested by the chitosan color test method. 
In addition, an enzymochromatographic method 
for chitin demonstration was adapted and tested 
and is herewith presented. The shell of the 
prosobranch Oncomelania was also tested for 
comparison. 
MATERIALS AND METHODS 
Stomach teeth were excised from growing 
specimens of Aplysia californica Cooper and 
frozen until ready for testing. Shells of adult 
specimens were removed and preserved in 70 
per cent ethyl alcohol until tested. 
Adult specimens of the shell-bearing opis- 
thobranch, Bulla gouldiana ( Pilsbry ) , were col- 
lected and preserved in neutral formalin (which 
was later changed to 70 per cent ethyl alcohol). 
When the test was made the shells were decal- 
cified in 10 per cent HC1, rinsed well, and 
blotted. The dry shells of Oncomelania nos- 
ophora (Robson) were similarly decalcified. 
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