DESCRIPTION AND HABITAT OF SOME MICRO-ORGANISMS
FOUND IN TEGULA FUNEBRALIS
Patricia Ann Black
Hopkins Marine Station
i
Ann
ology 17
robem
in Marine
Dr. Phillip:
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INTRODUCTION
Tegula funebralis (A. Adams, 1854), like many animals,
harbors a great many micro-organisms of different types
in its digestive tract. Very little is known about
these organisms and their relationship to their host,
their distribution in the tract, their density and type,
and their function within the organism. Galli (1956)
describes the eubacteria that he could culture and did
intensive studies on the digestive properties of five
selected rod-shaped bacteria. He mentioned little about
factors of distribution, density, or types actually seen
in the digestive tract.
This paper deals with the gross properties of the
micro-organisms found in the digestive tract of Tegula
funebralis, based on examinations of the material found
in the lumen as well as on observations of organisms
growing on plates. It also deals with one factor of the
environment in the digestive tract, namely the hydrogen
ion concentration.
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MEDIA
Two basic types of media were first used. One
contained 1.5% agar, 0.1% glucose, 0.05% yeast extract,
and 50% sea water; the other contained 1.5% agar, 1.0%
1.0% yeast extract, and 50% sea water. Both O.01%
aqueus extract of Tegula funebralis and a similar extract
of the alga, Iridaea, were added to some media as a
possible growth factor supplement. The enriched media
used by Galli (1956 ), salsod, was used.
METHODS AND RESULTS
Bacteria
1. Density and Distribution.
Alimentary contents were examined as wet mounts
under a phase contrast microscope to determine type and
relative density. This was supplemented by studies of
types growing from different parts of the digestive
tract on the media.
The anterior esophagus showed no obviously motile
forms with microscopic examination. Forms resembling
cocci were found either singly or in irregular clumps of
cells, but were not very numerous. One or two colonies
of different types of motile rods appeared on plates.
The lower esophagus showed the same kinds of
organisms and density under the microscope.
The population of bacteria increased sharply in the
stomach. The coccus form was very abundant. In addition,
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spiral shaped organisms were always found. Sometimes
only a few were found in a preparation, sometimes a
great number. Where they were numerous, three morpho-
logically different types could be distinguished, the
dominant one of which was found alone in less numerous
preparations. Two forms appear to be spirochaetes and
one may be a spirillum. Spirilla were found in great
numbers in some preparations. Several different kinds
of rods grew in abundance on plates from the stomach.
The spiral caecum also has one of the largest
populations of bacteria in Tegula funebralis' gut. One
can see rods and many cocci. In one animal several spiral
forms were also found, although usually this was not the
case. Numerous plates were made of spiral caecum material.
Many types of motile and non-motile rods were found. A
small motile short rod was isolated in large numbers.
Numerous colonies of the cocci were also found, but they
were extremely small, indicating that they did not thrive
under the conditions of culture.
The digestive gland contained rods and cocci, some in
chains of two or three. Plates showed a concentration
of motile rods of the same or similar types.
The thin walled hindgut had less bacteria than the
midgut area. It contained the same forms in general; in
one case it also contained some spirilla and spirochaete
forms. Plates revealed a few rods.
Motile rods were found in the rectum in good numbers.
Spirilla may also be present.
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2. Spiral Forms.
The spiral organisms found have not previously been
described. Two were thought to be spirochaetes on the
basis of their flexibility and tendency to form irregular
shapes. Smaller, more rigid forms, with a corkscrew
type of motility were distinguished in some cases and
thought to be spirilla. They were seen regularly in the
stomach and rarely outside. They have never been found
anterior to the stomach.
Two of the morphologically different types were placed
tentatively in the order Spirochaetales (Buchanan, 1918).
They belong to the family Spirochaetaceae (Swellengrebel,
1907), being placed there by size. They belong either
to the genus Spirochaeta (Ehrenberg, 1833) or the genus
Saprospira (Gross, 1911), which are differentiated by
Saprospira's periplast membrane and cross striations.
These features were difficult to ascertain in an
unstained wet mount.
One form that was in the minority was seen only
rarely. A few cells were found of about the same length
as the dominant one, the cell width thinner, with many
more waves along the cell. The spirilla form was about
half the length of the dominant form and had one to one
and a half waves along its body.
The dominant type ranged in size from 30 to 70 microns
with a mean at about 50 microns. It had 2 to 4 waves with
a mean wave length of 15 microns and mean spiral depth
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of 7 microns.
Yeasts
The intestinal contents were examined microscopically
for yeast. Yeast cells were observed in medium density in
the stomach, in high density with many budding cells in the
spiral caecum, and in medium density in the digestive gland.
They were only rarely observed in any other part of the
digestive tract.
Numerous unsuccessful attempts were made to culture
these yeasts. Cultures were inoculated from suspensions
of the intestinal contents streaked onto plates. The
pH of the medium was varied from 4 to 8 in an attempt to
discourage bacterial growth and encourage yeast growth.
Cultures were incubated botheaerobically and anaerobically.
Liquid media were tried in addition to agar plates.
In
addition, material was streaked on the medium used by
Galli (1956 ), who stated that he had obtained five
varieties of marine yeast from the intestinal tract of
Tegula funebralis.
There could be many reasons why no yeasts were
observed in culture. The yeast forms so commonly observed
may be different from those that Galli (1956 ) isolated.
He did not mention how frequently they were isolated.
Another relevant factor in this attempt is that the
number of micro-organisms in the pieces of the alimentary
tract was considerably less than those reported by
Galli (1956), as was indicated by two dilution series of
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ground intestinal tract prepared in a manner identical to
that prepared in his estimates.
Environment
In order to understand at least one variable of the
internal environment of these micro-organisms, attempts
were made to measure the hydrogen ion concentration. This
was accomplished by dissecting each section of the tract
out dry, placing it on a slide, cutting it open, and
inserting Nitrazine Paper (E.R. Squibb & Sons). The
results are given in Table I. It was not possible, however,
to determine the pH of the digestive gland by this
method.
DISCUSSION
Some new information about these micro-organisms has
been noted here. The data indicates, first, that most of
the micro-organisms are found in the midgut region of the
stomach, spiral caecum, and digestive gland. Second, the
most abundant organism seen microscopically was a coccus.
The role these organisms play in relation to their host is
difficult to interpret. It is also difficult to decide
whether all represent constant components of the micro¬
organism flora. Some may simply have been ingested by a
Tegula funebralis.
The constant occurrence of the spiral organisms in
large numbers found primarily in the stomach suggests
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that they may be regular inhabitants of the digestive
tract. Spirochaetes of the genus Cristispira (Gross, 1910)
have been described from the intestinal tracts of
lamellibranchs. These are fairly large spirochaetes
with a characteristic ridge or crista which serves as the
axial filament, running longitudinally and making a couple
of turns around the body. Their normal habitat is the
crystalline style of lamellibranchs, although they have
been seen elsewhere in the tract (Bergey's Manual, 1957).
They do not appear to harm their hosts (Hawker, et.al;
1960). The Cristispira must have one or several unique
requirements, since they usually live isolated from the
rest of the gut in the style sac. Their nutritional
requirements are unknown. They have never been cultured;
in this respect they are like most other spirochaetes,
which are very difficult to culture.
The spirochaetes in Tegula funebralis may represent
similar commensals. They too are reasonably restricted
to one part of the digestive tract and live in an area
of rich, semi-digested material.
The internal environment of Tegula funebralis is
very complex. A wide variety of food materials pass
through the gut, giving a collection of varied nutrients
difficult to access. Another factor, the hydrogen ion
concentration, varies over a range of one pH unit and is
generally higher than sea water. The bacteria cultured
grow well on media ranging in pH from 5.5 to 8.0.
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SUMMARY
The digestive tract of a gastropod was examined to
determine type, location, and density of micro-organisms.
Digestive contents were examined microscopically and
streaked onto media. The center of density was the stomach,
spiral caecum and digestive gland area, where abundant
yeast cells were seen and numerous rod-shaped bacteria
cultured. Cocci were seen most abundantly throughout,
although rarely cultured. Spiral organisms, thought to be
two spirochaetes and a spirillum, were found in the
stomach and described. The pH of the gut varied from 6.5
to 7.5.
Breed, Robert S., E.G.D. Murray and
Nathan R. Smith. 1957
Bergey's Manual of Determinative
Bacteriology.
Baltimore, The Williams & Wilkins
Company, xviii + 1094 pp.
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SUMMARY
The digestive tract of a gastropod was examined to
determine type, location, and density of micro-organisms.
Digestive contents were examined microscopically and
streaked onto media. The center of density was the stomach,
spiral caecum and digestive gland area, where abundant
yeast cells were seen and numerous rod-shaped bacteria
cultured. Cocci were seen most abundantly throughout,
although rarely cultured. Spiral organisms, thought to be
two spirochaetes and a spirillum, were found in the
stomach and described. The pH of the gut varied from 6.5
to 7.5.
*. ..
Galli, Donald R. 1956
Carbohydrate digestion in a
herbivorous marine snail, Tegula
funebralis.
Masters Thesis, Stanford University,
Stanford, California, viii + 153 pp.
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SUMMARY
The digestive tract of a gastropod was examined to
determine type, location, and density of micro-organisms.
Digestive contents were examined microscopically and
streaked onto media. The center of density was the stomach,
spiral caecum and digestive gland area, where abundant
yeast cells were seen and numerous rod-shaped bacteria
cultured. Cocci were seen most abundantly throughout,
although rarely cultured. Spiral organisms, thought to be
two spirochaetes and a spirillum, were found in the
stomach and described. The pH of the gut varied from 6.5
to 7.5.
Hawker, Lilian E., A.H. Linton, B.E.
Folkes, and M.J. Carlile. 1960.
An Introduction to the Biology
of Micro-organisms. London.
Edward-Arnold (Publishers) Ltd.
452 pp.
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SUMMARY
The digestive tract of a gastropod was examined to
determine type, location, and density of micro-organisms.
Digestive contents were examined microscopically and
streaked onto media. The center of density was the stomach,
spiral caecum and digestive gland area, where abundant
yeast cells were seen and numerous rod-shaped bacteria
cultured. Cocci were seen most abundantly throughout,
although rarely cultured. Spiral organisms, thought to be
two spirochaetes and a spirillum, were found in the
stomach and described. The pH of the gut varied from 6.5
to 7.5.
Stanier, Roger Y., Michael Doudroff
& Edward A. Adelberg. 1963
The Microbial World, Second
Edition. Edgewood Cliffs, N.J.,
Prentice-Hall, Inc., xiii + 753 pp.
Table I.
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THE HYDROGEN ION CONCENTRATION OF THE ALIMENTARY
TRACT OF TEGULA FUNEBRALIS
REGION
DH
6.5
Mouth
6.5
Anterior esophagus
Lower esophagus
6.5
Stomach
6.5
7.0
Spiral caecum
7.0
Anterior hindgut
Middle hindgut
6.5
Rectum