Egg Capsule Sheath Bacteria - 2
ABSTRACT
The egg capsule sheath of the squid Loligo opalescens is an
acellular matrix 1-2 mm thick which contains roughly 100 thin
striations in layers parallel to the capsule surface. In this
matrix is suspended a dense culture of bacteria in one to five
layers. Its probable source is the accessory nidamental gland.
Egg Capsule Sheath Bacteria - 3
INTRODUCTION
After mating, female Loligo opalescens deliver their eggs in
clusters of 100 to 300, covering them in a sheath secreted by
their nidamental glands (Arnold,'71). An ovulating female then
attaches this egg capsule, (Fig. 1), to a large, common egg-mass
built up by several females on sandy substrate (Recksiek and Frey
78). Local divers report little attrition of these egg masses,
and it appears the embryos escape animal, fungal, and microbial
predation during their three to five week incubation. An
interesting predation defense mechanism or mechanisms may exist.
while assessing microbial settlement on the egg capsule
surface in a search for this mechanism, we noticed bodies that
appeared to be bacteria far below the capsule's colonized
surface. We have investigated these bodies and here report the
first instance of an apparently natural deposition/colonization
of bacteria in the sheath of Loligo opalescens egg capsules. We
describe the morphology of the egg-capsule sheath, and detail the
distribution of its bacterial flora with electron and light
microscopy.
Egg Capsule Sheath Bacteria - 4
MATERIALS AND METHODS
General: Squid were captured in Monterey Bay, held in 2000
liter, circular holding tanks with constant flow, and induced to
mate by artificial egg masses (Yang, '83)
Electron Microscopy: After opening freshly laid (age «6 hrs)
capsules longitudinally and removing the chorions and embryos
with sterile forceps (Fig. 1), samples of the capsule-sheath were
fixed in glutaraldehyde (22 in sterile sea water for 2 hrs),
treated with osmium tetroxide (12 in sterile water adjusted to
oceanic salinity and pH), stained with uranyl-acetate (1 hr at
12, as for osmium tetroxide), serially dehydrated in acetone (20-
1002 in 102 steps), then set in Spurr's plastic (Spurr '69). This
block was then sectioned to 800 Angstroms in planes perpendicular
to the surface of the capsule, and examined on a Phillips 201
electron microscope at 80 ky.
Light Microscopy: The transmission EM protocol described
above was followed until the sample had been set in Spurr's
plastic. The sample was then sectioned to 3 microns in planes
perpendicular to the surface of the capsule, stained with Lee's
methylene-blue basic-fuchsin, and examined at 1000x total
magnification under oil.
Bacterial Distribution: The capsule sheath was separated
from the embryos and chorions (Fig. 1), then sectioned in 0.5 mm
planes perpendicular to the surface of the capsule. To minimize
extraneous bacteria all implements and surfaces in contact with
the capsule were sterilized for 10 minutes with 952 ethanol,
Samples were then stained with acridine orange (1 ug/ml: 10
minutes stain, 10 minutes wash in sterile sea water) or the DNA
specific stain DAPI (4', 6-diamidino-2-phenyl-indole diluted 1 to
1000 in sterile sea water, 20 min stain, 20 min wash in sterile
sea water), and examined at 1000x total magnification with UV
excitation. By photographing adjacent microscope fields while
traversing samples, photo-montages which transected the depth of
the capsule-sheath were prepared.
Egg Capsule Sheath Bacteria - 5
RESULTS
Sheath Structure
The sheath is a tough, acellular matrix one to two
millimeters thick which completely encapsulates the eggs (Fig,
In light and electron microscopy its most obvious structural
features are numerous, thin striations parallel to the capsule
surface (Fig. 2). These striations, which stain darkly with
Lee's-methylene-blue-basic-fuchsin in light microscopy, are also
stained by uranyl-acetate in samples prepared for transmission
electron microscopy. Both results suggest that these are areas
high in protein content. The striations are roughly 3 microns
thick, 10 to 20 microns apart. We estimate that the sheath, at
1000 to 2000 microns thickness, contains roughly 100 of these
striated layers.
Sheath Bacteria
In addition to striations, light microscopy also reveals
bacteria-sized bodies deep within the capsule sheath (Fig, 2).
Transmission electron microscopy further reveals morphologies
which strongly suggest bacteria (Figs. 3, 4). Fluorescence
microscopy of samples stained with the DNA-specific dye DAPI
confirms that these bodies contain genetic material and are
therefore most likely bacteria (Fig. 5b).
In electron and light microscopy a fusiform bacteria is
consistently in high abundance, even in capsules less than six
hours old. It stains positively with acridine orange and DAPI,
and is present in the sheath of all the capsules we have examined
to date (Figs. 4, 5b).
The distribution of bacteria through the depth of the
capsule sheath, as assessed by optical sections stained
fluorescently for DNA, appears to be non-random. One to five
densely populated layers, in which bacteria may comprise as much
as ten percent of the volume of the sheath matrix, alternate with
layers which are nearly or completely free of bacteria.
Egg Capsule Sheath Bacteria - 6
By photographing adjacent microscope fields while traversing a
section cut through the depth of the sheath, a montage of the
bacterial distribution was prepared (Fig. 5a).
A composite drawing representing a square centimeter of
capsule-sheath viewed edge on, synthesizes the striations visible
in direct light with the layered bacterial distribution apparent
in fluorescence (Fig. 6).
Egg Capsule Sheath Bacteria - 7
DISCUSSION
Our observations indicate that the one to two millimeter
thickness of the egg capsule sheath contains roughly 100
striations, probably proteinaceous, and one to five densely
populated layers of bacteria. Bacteria in organized layers
abundant even in capsules less than six hours old, raise
questions of origin, function, and generality.
Origin
If work on Loligo pealei, an Atlantic squid, can be
generalized to the Pacific Loligo opalescens, a likely source for
the sheath bacteria is the female squid's accessory nidamental
gland. In many species of cuttlefish and squid this paired organ
of sexually mature females opens onto the mantle cavity, which
serves the organism as an oviduct (Williams '09), (Buchner '65).
Studies of L. pealei by Bloodgood ('77) reveal that the
accessory nidamental gland derives its mottled appearance from
internal convoluted tubules, which harbor one of three distinct
cultures of bacteria (one of which produces a sepiaxanthin-like
pigment responsible for the gland's color). Bloodgood's
investigations further show the gland has musculature permitting
it peristaltic movement and is contractile upon dissection from
the squid. His results also suggest that the gland secretes a
medium on which the bacteria grow.
while the capsule sheath is secreted primarily by the
nidamental glands, (Arnold '71), the duct opening of the
accessory nidamental gland suggests that it may also contribute
to formation of the capsule. Our results suggest that its
contribution could include bacteria. Perhaps the accessory
nidamental gland contracts during formation of the egg capsule,
periodically expressing its bacterial contents into the sheath,
This would account not only for the layered distribution, but
also for the abundance of bacteria in newly laid capsules.
Egg Capsule Sheath Bacteria - 8
This phenomenon is not unprecedented. Early investigators of
bacteria in the accessory nidamental glands of bioluminescent
squid (Pierantoni '14, '18), (Herfurth '36), (as cited in Buchner
65) noted bacteria in the egg capsule sheaths of three squid:
Loligo edulis, Loligo forbesi, and Sepia officinalis. These
investigators also looked on the accessory nidamental gland as
the most likely source for the sheath bacteria,
Function
Function of egg capsule sheath bacteria is perhaps the most
interesting question raised by this study. A likely pezibliig is
defense of the developing embryos. Loligo opalescens' eggs are
laid in large masses which appear to suffer little animal,
fungal, or microbial attrition. Sheath bacteria offer a possible
explanation for this predation resistance. Instances of natural
flora discouraging colonization by pathogenic organisms have long
been established (Zobell '46).
Sheath bacteria might populate layers of the capsule sheath
so heavily that all available resources are exhausted to the
detriment of pathogenic organisms. It is also possible that the
bacteria do not "passively compete" but actively produce an
antibiotic compound. A symbiosis of this type has been noted in a
crustacean's eggs, in which a bacteria of the genus Alteromonas
protects embryos by producing the fungicide isatin (Fisher '83,
Gil-Turnes, Hay, and Fenical '89).
If "embryo protection" is actually a role of sheath
bacteria, it may not be the only element in the capsule with this
function. Atkinson ('68, '73) has demonstrated an "immobilizing
factor" in the nidamental gland secretions which form the capsule
sheath. This factor agglutinates the cilia of motile metazoans,
and may inhibit protozoan predators of the egg capsule. Sheath
bacteria, in conjunction with "immobilizing factor" might
constitute an interesting system of embryonic defense.
Egg Capsule Sheath Bacteria - 9
Generality
Already noted in the egg capsule sheaths of three
bioluminescent squid (Buchner '65), and now a non-bioluminescent
squid (Biggs and Epel '90), egg capsule sheath bacteria may be a
more general phenomenon than previously suspected. Marine
bacteria have also been found in association with the eggs of a
scaphapod (Geilenkirchen, Timmermans, Van Dongen, and Arnolds,
70), and even a crustacean (Fisher, '83).
In the cephalopods investigated so far, it appears sheath
bacteria are derived from the accessory nidamental gland. Since
this organ is common in squid and cuttlefish, it is conceivable
that further investigation will reveal other instances of
accessory nidamental gland-derived sheath bacteria. If so, the
function of accessory nidamental gland bacteria, which has
remained elusive in adults (Bloodgood '77), might become evident
in the context of the capsule sheath.
LITERATURE CITED
Atkinson, B., and N. Granholm (1968). A ciliary activity
inhibitor extracted from the nidamental gland of Loligo
pealei. Biol. Bull., 135: 413.
Atkinson, B. (1973). Squid nidamental gland extract: isolation of
a factor inhibiting ciliary activity. J. Exp. Zool, 184:
335-340
Arnold, J. (1971). Cephalopods. In: Experimental Embryology of
Marine and Fresh-water Invertebrates. G. Reverberi, ed.
North Holland, pp. 265-311
Biggs, J., and Epel, D. The egg capsule sheath of Loligo
opalescens: structure and association with bacteria. J. Exp.
Zoo.
Bloodgood, R. (1977). The squid accessory nidamental gland:
ultrastructure and association with bacteria. Tissue and
Cell, 9(2): 197-208.
Buchner, P. (1965). Endosymbiosis of Animals with Plant
Microorganisms. Interscience, New York, pp. 543-571.
Fisher, W. (1983). Eggs of Palaemon macrodactylus: II.
association with aquatic bacteria. Biol. Bull. 164: 201-213.
Geilenkirchen, W.L.M., Timmermans, L.P.M., Van Dongen, C.A.M.
and W.J.A. Arnolds. Experimental Cell Research. 67: 477.
Gil-Turnes, M., M. Hay, and W. Fenical. (1989). Symbiotic marine
bacteria chemically defend crustacean embryos from a
pathogenic fungus. Science, 246: 116-117.
Herfurth, A. H. (1936) Bietrage zur Kenntnis der
Bacteriensymbiose der Cephalopoden. Z. Morphol. Okol Tiere,
31.
Pierantonni, U. (1918). Organi luminosi, organi simbiotici e
glandola nidamentale accessoria nei Cefalopodi. Boll, Soc.
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Recksiek, C., and H. Frey. (1965). The structure, development,
reproduction, and life history of the squid Loligo
Opalescens Berry. Calif. Dept. of Fish and Game Fish
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Spurr, A. (1969). A low-viscosity epoxy resin embedding medium
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Lesueur. Brill, Leiden, Am. Mus. Nat. Hist. pp. 51-55.
Yang, W.T., R.T. Hanlon, M.E. Krejci, R. F. Hixon, and W. H.
(1983). Laboratory rearing of Loligo opalescens, the
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FIGURES
Fig. 1. Egg capsules: c-capsule, e-eggs, s-sheath. The capsule
on the right has been opened and its contents pulled away from
the sheath.
Fig. 2. Histological staining of 3 micron sections of freshly
laid capsule's sheath show regular striations at 1000x total
magnification. Their spacing at 10 to 20 microns implies roughly
100 layers in total.
Fig. 3. Bacillus bacteria midway through the depth of the sheath
covering a freshly laid capsule - 45,000x.
Fig. 4. A shorter rod (a), and the common fusiform morphology (b)
midway through the depth of the sheath - 45,000x.
Fig. 5a - The vertical bar represents a typical bacterial
distribution through the 2 mm depth of the capsule sheath. The
water is above/outside, and the eggs below/inside. Distribution
of bacteria is distinctly layered. The dark rectangle corresponds
to figure 5b. 5b - A typical fluorescence photomicrograph used to
map the bacterial distribution depicted in whole in 5a. This is a
border between regions of high and low bacterial density,
Fusiform sheath bacteria are evident below, stained with DAPI and
viewed at 1000x magnification in this optical section through the
depth of the sheath.
Fig. 6. Striations visible in light microscopy, combined with the
fluorescence distribution transect, yield a composite structure:
a matrix of fine striations bearing thick, bacteria-laden layers,
The dark rectangles, A and B, correspond to figure 2 and figure
5b respectively.