JOURNAL OF MORPHOLOGY 220:85-101 (1994)
Buoyancy Studies and Microscopy of Skin and Subdermal
Extracellular Matrix of the Antarctic Snailfish, Paraliparis devriesi
JOSEPH T. EASTMAN, ROBERT S. HIKIDA,
AND ARTHUR L. DEVRIES
Department of Biological Sciences, Ohio Uniuersity,Athens, Ohio 457012979 (J.T.E.,R.S.H.); Department ofPhysiology and Biophysics, University
of Illinois, Urbana,Illinois 61801 (A.L.D.)
ABSTRACT The Antarctic snailfish Paraliparis devriesi (Liparididae) occupies an epibenthic habitat at a depth of 500-650 m in the subzero waters of
McMurdo Sound, Antarctica. Although lacking a swim bladder, this species is
neutrally buoyant through the combined effects of reduced skeletal ossification
and expansion of a watery gelatinous subdermal extracellular matrix (SECM).
The SECM serves as a low density buoyancy agent. It comprises a mean of
33.8%of the body weight, the largest known proportion of any adult fish. The
SECM is loose connective tissue dominated by ground substance consisting of
glycosaminoglycans, especially hyaluronic acid, and immobilized water. Although the SECM is 97% water, elevated levels of NaCl provide an osmotic
strength greater than that of other body fluids. Only small amounts of antifreeze compounds have been identified in P . deuriesi; therefore, freezing
avoidance may result from the combined effects of antifreezes and the elevated
osmolality of body fluids. The skin overlying the SECM is thin (85-200 Fm)
and loose, and unlike most other fishes, the epidermis is several times thicker
than the dermis. The midepidermis, has a distinctive layer of vacuolated club
cells of unknown function. Light and electron microscopy indicate that the skin
is unspecialized for protection against entry of ice. c 1994 Wiley-Liss, Inc.
The snailfish family Liparididae has a
worldwide distribution and includes about
170 species living from the intertidal zone to
depths of 7,000 m (Stein and Andriashev,
'90). The family is best represented in polar
waters and probably originated and radiated
in the North Pacific region with subsequent
dispersal southward along the western coast
of the Americas (Andriashev, '86, '91).Liparidids underwent a secondary radiation in the
Antarctic region where all known species are
members of deepwater genera (Andriashev
and Prirodina, '90). The 31 species inhabiting the Southern Ocean, mostly members of
the genus Paraliparis, represent 11%of the
ichthyofauna (Stein and Andriashev, '90).
Since there are a n additional 20-30 undescribed species, the Liparididae will eventually surpass the endemic Nototheniidae as
the most speciose fish family in the Southern
Ocean (DeWitt et al., '90; Stein et al., '91).
Snailfishes are included in the euteleostean
order Scorpaeniformes, a predominantly marine group with > 1,100 species (Nelson, '84).
Many scorpaeniforms, e.g., scorpionfishes,
o 1994 WILEY-LISS, INC
searobins, sculpins, and lumpfishes, have
heavy bodies, possess spines or bony plates
on the head, and live on the bottom. However, many liparidids, especially deepwater
genera such as Paraliparis, have a bulbous
head and elongated trunk, dorsal and anal
fins (Fig. 1). Furthermore, they lack substrate contact adaptations, and beneath the
thin, scaleless skin, they possess a thick layer
of gelatinous material, the subdermal extracellular matrix (SECM). Many works on the
taxonomy of snailfishes refer to the thin,
loose skin and the extensive SECM. The flaccid skin and expanded SECM appear early in
larval life (Able et al., '84; Marliave and
Peden, '89) and are retained in some adult
liparidids. Glycosaminoglycans (GAGS),ubiquitous components of vertebrate connective
tissue, are primary constituents of the SECM
in mesopelagic salmoniforms and stomiiforms (Yancey et al., '89). These polyanionic
Address reprint requests to J.T. Eastman, Dept. of Biological
Sciences, Ohio University, Athens, OH 45701-2979.
J.T. EASTMAN ET AL.
86
compounds, hyaluronic acid, e.g., are greatly
hydrated with fluid domains 1,000 times
larger than the volume of the dry molecule
(Comper and Laurent, '78).
Paraliparis devriesi Andriashev ('BO), a n
epibenthic species living a t 500-650 m in
McMurdo Sound, has the most southerly distribution of any known liparidid. Although
the annual water temperature at McMurdo
Sound averages - 137°Cwithin the restricted
range of - 1.40"C to -2.15"C (Littlepage, '651,
P. deuriesi has a watery SECM comprising
about one-third of the body weight. P. devriesi lacks a swimbladder, but measurements
of buoyancy and observations of aquarium
specimens suggest it is close to neutral buoyancy. The SECM may serve as a buoyancy
agent similar to lipids in some Antarctic nototheniid fishes (Eastman, '88). The SECM
and its delicate overlying skin have not been
studied in any liparidid. Our investigations
of P. deuriesi emphasize: (1)measurements
of the buoyancy and an assessment of the
utility of the SECM as a buoyancy agent,
including its osmotic concentration and ion
levels, and (2) morphological studies relative
to buoyancy including light and electron microscopy of the skin and SECM.
MATERIALS AND METHODS
Field work and specimens
Field work and some phases of laboratory
work were conducted at the U.S. McMurdo
Station (77" 51' S , 166" 40' E) on Ross Island
in the southwestern Ross Sea. We captured
27 specimens of Paraliparis devriesi in the
Erebus Basin of McMurdo Sound during November, 1978,1991, and 1992. The collection
site (77" 47' 2134 S, 166" 09' 5400 E-GPS
coordinates) was -22 km from McMurdo
Station. Paraliparis were taken in large, conical wiremesh bottom traps fished a t a depth
of 635 m through holes drilled in the annual
sea ice. The traps were baited with chopped
fish, left on the bottom for 3-5 days, and
retrieved with an oceanographic winch.
We used 12 specimens for buoyancy measurements, two to six for osmolality, ion determinations and water content analyses, four
for histology and histochemistry, and one €or
electron microscopy. Both males and females
were included in the sample, and all were
adults averaging 179 mm TL (23 mm SEMI
and 64.90 gm (23.788 gm SEM). Two formalin preserved specimens were grossly stained
for lipid with oil red 0 (Humason, '79). After
24 hr, they were destained in several changes
of 60% isopropanol. The skeletons of two
specimens were ashed for 6 h r in a muffle
furnace at 600°C and skeletons of three additional specimens were cleared and stained
with alizarin red S . Most specimens were
radiographed after fixation in formalin. Radiographs were produced using a HewlettPackard Faxitron soft X-ray machine (model
43805N) with a dual cabinet. The machine
was operated a t 30 kVp and 2.75 mA, with an
exposure time of 2.0 min. Radiographs were
made on Kodak Industrex M film (M-5) contained in lead-backed cardboard cassettes.
Film to source distance was 122 cm.
Microscopy
Samples of skin and SECM were prepared
for light microscopy. They were fixed in
Bouin's solution, embedded in paraffin, sectioned a t 7 pm, and stained using a variety of
methods (Luna, '68; Humason, '79). We used
Mayer's hematoxylin and phloxine for general tissue structure, periodic acid-SchiffAlcian blue (PAS-AB) a t pH 2.5 and 1.0 for
nonsulfated and sulfated glycosaminoglycans (GAGS), Gomori's aldehyde fuchsin for
elastic fibers, bromphenol blue for proteins
and Gomori's trichrome, and Mallory's phosphotungstic acid hematoxylin for connective
tissue. Bodian's Protargol (Clark, '81) for 20
h r at 50°C proved to be effective in identifying cell nuclei in the epidermis and SECM.
We used hyaluronidase hgestion procedures (Sheehan and Hrapchak, '80, p. 174)to
refine the identification of GAGS in the
SECM. Prior to staining with Alcian blue at
pH 2.5, we incubated sections in buffer containing either 0.05% bovine testicular hyaluronidase (Sigma Type IV-S, EC 3.2.1.35) or
in hyaluronate lyase (1500 U 100 ml-l) from
Streptomyces hyalurolyticus (Sigma, EC
4.2.2.1). Hyaluronidase from Streptomyces is
specific for hyaluronic acid. Sections incubated in buffer without enzyme served as
controls.
Samples of dorsal trunk skin and SECM
were fixed for electron microscopy in either
picric acid-paraformaldehyde or paraformaldehyde-glutaraldehyde in 0.15 M phosphate
buffer and postfixed in 1% osmium tetroxide.
Samples were then dehydrated in ethanol
and propylene oxide and embedded in
Araldite-Epon. Standard procedures were employed in the subsequent processing of tissues.
SNAILFISH BUOYANCY AND SKIN
Fig. 1. Paraliparis deuriesi. Extensive subdermal extracellular matrix between dermis and muscle contributes to looseness and vertical wrinkling of skin. Darkly
pigmented parietal peritoneum is visible through body
wall. From Andriashev ('80),~0.7.
Fig. 2. Paraliparis deuriesi. Radiograph showing that
the majority of well-mineralized bone is confined to the
vertebral column. The subdermal extracellular matrix
87
appears whitish-gray and is most prominent in the anterior two-thirds of the body. X0.7.
Fig. 3. Paraliparis deuriesi. Anterior one-third of the
body of a n Alizarin stained and cleared specimen showing
delicate nature of skeletal ossification especially in the
dorsal aspect of the skull. Since the pectoral girdle is
cartilaginous (arrowhead), pectoral fin rays appear unattached to girdle. Dark parietal peritoneum is visible deep
and dorsal to pectoral rays. ~ 2 . 1 .
88
J.T. EASTMAN ET AL
Osmolality, ion concentrations,
and water contents
Using syringes, we withdrew blood and
coelomic fluid from five specimens. We obtained SECM by placing the fish on Parafilm,
removing the skin, and then scraping the
SECM into 20 ml centrifuge tubes. Care was
taken to avoid scraping the underlying
muscle. Under warm conditions, as in contact with human fingers, the SECM exudes a
watery fluid. In Tables 3 and 4, we refer to
this as SECM (not centrifuged). When ultracentrifuged for 5 h r a t 39,000 rpm in a Beckman SW 41 rotor, the SECM separates into a
clear, viscous supernatant (90-95%) and an
opaque pellet (5-10%) of collapsed matrix
material. The supernatant was used for osmometry and for determination of ions.
Cation concentrations in body fluids were
determined with a Corning model 455 flame
photometer. Chloride concentrations were obtained with a Buchler-Cotlove direct readout
chloridometer. Osmolality was measured with
a Wescor 5100C vapor pressure osmometer.
Pieces of SECM and trunk musculature
were weighed immediately after removal of
two to four samples from each of five (SECM)
or two (muscle) fish. After drying to a constant weight in a 60°C oven, they were reweighed. The difference in the weights was
taken as the water content.
Measurements of buoyancy
and calculation of density
Measurements of buoyancy, following the
procedure of Eastman and DeVries ('81,'821,
were conducted on specimens anesthetized in
a 1:1,000 concentration of 3-aminobenzoic
acid ethyl ester (MS-222, Sigma, St. Louis,
MO). Specimens were weighed while suspended in a large chromatography jar containing seawater from McMurdo Sound. The outside of the jar was packed with ice to maintain
the normal -1.9"C temperature. Care was
taken to remove air from the oral and branchid cavities while specimens were in water.
Specimens were suspended from a small bent
needle attached to the lower jaw. The needle
was connected to a piece of suture that was
secured to a hook on the beam of a RollerSmith micro-torque precision dial-reading
balance or to a wire side arm attached to the
balance pan of a Mettler electronic top loading balance. Readings taken to an accuracy of
0.01 gm. The entire suspension apparatus
weighed 0.01 gm and was calibrated periodically with balance weights. After being
weighed in water, specimens were weighed in
air. The method of Davenport and Kjorsvik
('861, employing Archimedes' principle, was
used to calculate the density of specimens
(Table 1).
RESULTS
Buoyancy and density
Table 1 summarizes measurements of
buoyancy and calculations of density for
Paraliparis. These data indicate that Paraliparis are close to neutral buoyancy. Buoyancy is expressed as a percentage weight in
water; the closer this figure to zero, the closer
is the fish to neutral buoyancy. The average
percentage weight for Paraliparis is 0.18%.
After the SECM is removed from the body,
TABLE 1. Calculation ofpercentage weight in water (buoyancy)and density for Paraliparis devriesi'
Specimen
no.
Wt. air
( m )(gm)
Wt. HzO
( m ' )(gm)
70 w t .
m'im x 100
PDE 2
PDE 3
PDE 4
PDE 5
PDE 6
PDE 7
PDE 8
PDE 9
PDE 10
PDE 11
PDE 13
PDE 14
MEAN
SEM
N
75.64
56.73
55.74
44.06
40.04
81.14
44.26
77.18
86.38
79.99
81.95
83.22
67.19
5.084
12
0.22
0.14
0.02
0.02
0.01
0.08
0.29
0.25
0.04
0.04
0.02
0.10
0.16
0.04
0.38
0.24
0.13
0.46
0.18
0.042
12
0.07
0.03
0.33
0.19
0.11
0.39
Upthrust
u=m-m'
(gm)
75.42
56.59
55.72
44.04
40.03
81.06
44.19
77.15
86.05
79.80
81.84
82.83
Volume
V = ui1.028
(cm-3)
73.37
55.05
55.20
42.84
38.94
78.85
42.99
75.05
83.71
77.63
79.62
80.58
Density
p = miV
(gm cm-3)
1.031
1.030
1.010
1.028
1.028
1.029
1.030
1.028
1.032
1.030
1.029
1.033
1.028
0.0017
12
'Density of McMurdo Sound seawater is calculated from the mean annual Sigma-t value of 27.96 (Littlepage, '65, p. 15) as follows: p
(Sigma-t x 10-3) + 1 = 1.028 gm cm-3.
=
SNAILFISH BUOYANCY AND SKIN
Paraliparis are less buoyant, with the average percentage weight in water increasing to
0.64% (3specimens). In a fish close to neutral
buoyancy, the weight of the fluid displaced,
the upthrust (u),closely approximates the
volume (V) when corrected for the density of
seawater (V = ui1.028). Using the formula p
= m/V and knowing weight and volume,
calculated densities of Paraliparis are identical to McMurdo Sound seawater at 1.028 gm
~ m (Table
- ~ 1).
Morphological evaluation of buoyancy
i n Paraliparis
I n the absence of a swim bladder, Paraliparis are neutrally buoyant through reduction in skeletal ossification and by elaboration of the subdermal extracellular matrix
(SECM), including expansion of the interstitial fluid compartment of the body. Before
considering these, however, it is necessary to
emphasize that neutral buoyancy has been
attained through static rather than hydrodynamic mechanisms requiring swimming. Although living in the water column, Paraliparis are slow swimmers and do not possess
large amounts of red muscle. When held in
aquaria, Paraliparis swim slowly and continuously using simultaneous anguilliform
and labriform locomotion, with the horizontal axis of the body tilted slightly upward. On
occasion we have also observed these fish
with their sides against the aquarium wall.
When disturbed, they swam away in typical
fashion.
Skeleton
The skeleton is the densest body component, with ash content providing a quantitative measure of the degree of mineralization.
Paraliparis have a light skeleton with a low
mineral content. The ashed skeletons of two
specimens comprise an average of only 0.25%
of the body weight compared with 2.5-5.8%
and 0.4-2.9% for other mesopelagic teleosts
with and without swim bladders, respectively
(Childress and Nygaard, '73). Radiography
(Fig. 2) and staining with alizarin (Fig. 3)
indicate little dense bone, especially in the
skull. Teeth on the premaxillae and dentaries
and otoliths are the densest structures in the
body. Although light and delicate, the vertebral centra are amphicelous and without a
persistent notochord.
With a density of 1.10 gm cm-3 (Alexander,
'67) cartilage is substituted for bone (density = 2.00 gm ~ m - in
~ )some parts of the
89
skeleton of Paraliparis. Large areas on the
dorsal aspect of the skull are cartilaginous.
The pectoral girdle is largely cartilaginous
(Fig. 3), with small centers of ossification
representing three round pectoral radials,
scapula and coracoid. These do not articulate
with each other or with the fin rays.
Subdermal extracellular matrix (SECM) of
high water content
Microscopy, location, andproperties. The
translucent gelatinous SECM is the most
remarkable morphological feature of Paraliparis. As the amorphous ground substance of
the loose hypodermal connective tissue, the
SECM is an example of a pliant composite
material, similar to the gelatinous mesoglea
of coelenterates and sponges (Wainwright et
al., '82; Vogel, '88). Among vertebrates it is
most similar to Wharton's jelly in the umbilical cord of mammals (Fawcett, '86) or to the
mucous connective tissues of teleosts (Benjamin, '88). Since the dominant component of
the SECM is the greatly hydrated gelatinous
matrix, there is little structural detail (Figs.
4-6). Dry weight of the matrix with contained cells and fibers is only about 3% of wet
weight (Table 2). The SECM also contains
other connective tissue elements including
nonfibrous collagen, fibroblasts, melanophores, capillaries and nerves. Collagen is
most prominent in the deepest portion of the
SECM near the axial musculature. Elastic
fibers are not present.
When removed from the fish and placed in
McMurdo seawater, pieces of SECM and attached skin float a t the surface and are therefore positively buoyant. The SECM comprises a n average of 33.8% of the body weight
(range = 26.2432%) and has a water content of 97.3% (Table 2). If close to neutral
buoyancy, the volume of a substance approximates its mass. Therefore, the SECM occupies nearly the same percentage of body volume as its weight. As a component of the
interstitial fluid compartment of the body,
the SECM of Paralipparis is more than double
the 13.5% of body weight typical for the entire interstitial fluid component of marine
teleosts (Thorson, '61).
One-third of the body weight may be a low
estimate since it does not include extensions
of SECM medially into the musculature of
Paraliparis. The SECM is thickest in the
head and anterior trunk regions, but the
SECM also covers the rest of the body including the bases of the dorsal and anal fins (Figs.
4-5). Some SECM is also present in intennus-
90
J.T. EASTMAN ET AL.
Figs. 4,5. Parallparis deuriesi. Histological cross sections of the posterior one-fifth of the body showing (4)
positive (dark) staining reaction at pH 2.5 for GAGS in
the subdermal extracellular matrix (SECMJ at the bases
of the dorsal and anal fins. Loss of staining (5) at pH 1.0
indicates that GAGS in SECM are nonsulfated and that
only sulfated GAGS of bone, cartilage, and fin rays stain
positively (darkly) at this pH. The skin and SECM have
been removed from all areas except bases of fins. Dorsal is
to the left. Periodic acid-Schiff-Alcianblue. ~ 8 . 7 .
Fig. 6. Paralipparis deuriesi. Histological section of
trunk skm showing thick epidermis (l), thin darkly staining dermis (21, and extensive, amorphous subdermal
extracellular matrix (3). The midepidermis consists of
large vacuolated club cells ( C ) . Fully developed (bottom)
and nearly spent (top) sacciform cells (S) are also present.
Hematoxylin and phloxine. ~ 3 3 0 .
91
SNAILFISH BUOYANCY AND SKIN
cular locations, especially near the midline
vertical septum and around the vertebral
column. Because of the high water content
and amorphous nature of the SECM, radiographs of the anterior two-thirds of the body
have a whitish-gray radio-opaque tone without sharp boundaries (Fig. 2).
In Paraliparis trunk musculature has a
water content of 84.1% (Table 2), higher
than the 80.4% typical of the white muscle of
cod (Love, '70, p. 262). This may be attributable to extensions of the SECM into the
muscle.
Staining of the SECM for
glycosaminoglycans (GAGs)
Histochemistry provides a general identification of the major components of the SECM.
With PAS-AB a t pH 2.5, the SECM stains
blue-green, indicating a positive reaction for
acidic glycosaminoglycans (GAGS) (Fig. 4).
Adjacent to the musculature, the deepest portion of the SECM stains most intensely. Faint
staining with PAS-AB at pH 1.0 (Fig. 5)
suggests that the acidic glycosaminoglycans
are nonsulfated. Hyaluronic acid is a likely
component of the SECM since it is the only
nonsulfated GAG and is widely distributed in
loose connective tissue (Comper and Laurent, '78; Goldberg and Rabinovitch, '88).
TABLE 2. Values for characters influencing buoyancy
in neutrally buoyant Paraliparis compared to other
marine telosts'
Character
Wt. in water/wt. in
air x 100 (%)
Vol. of swim bladder
(abody vol.)
Wt. of ashed skeleton
(% body wt.)
Wt. of liver (% body wt.)
Wt. of SECM (% bodv
wt. & vol.)
Water content of SECM
(%wet wt.)
Water content of white
muscle (% wet wt.)
Serum osmolality
(mOsm kg-l)
No.
Paraliparis
devriesi
Other
marine
teleosts
12
0.18
(k0.042)
0.5-1.Z2
-
absent
5"
2
0.25
7
8
5
2
5
1.5
(20.26)
33.8
(22.03)
97.3
(20.12)
84.1
478
(223.6)
0.4-2.9*
1-55
966
807
3844768
'Numbers for Paralzparis are means (kSEMi.
"enton and Marshall ('58) for teleosts without swim bladders.
3MarshaU ('79).
4Childress and Nygaard ('73) for teleosts without swim bladders.
"eyev ('77).
'jYancey et d.('89).
'Love ('70).
8Holmesand Donaldson ('69)range for marine teleosts.
To refine the specificity of our histochemical observations, we employed enzymatic digestions prior to staining. After incubation in
buffers containing testicular or bacterial hyaluronidase, sections show considerable reduction in the intensity of alcian-blue staining at pH 2.5 compared to controls incubated
without enzyme. This strongly suggests the
presence of hyaluronic acid. Whereas testicular hyaluronidase digests a number of GAGs,
hyaluronidase from Streptomyces is specific
for hyaluronic acid (Sheehan and Hrapchak,
'80, p. 174).
Staining for lipids. Staining with oil red
0 reveals no grossly visible lipid deposits in
the SECM or in any of the somatic tissues of
Paraliparis. Positive staining of the liver,
mature ova, interstitial tissue of the ovary,
and the dorsal fat deposits of amphipods in
the stomach indicate that oil red 0 is sensitive for lipid in this species.
Osmolality and ionic content of SECM and
other body fluids. The osmolality of the body
fluids (Table 3) of Paraliparis is less than
one-half that of McMurdo seawater (1,053
mOsm kg-l; DeVries and Eastman, unpublished data), but elevated compared to values
for temperate marine teleosts. Assuming a
mean of 350 mOsm kg-l for temperate species, values for serum, coelomic fluid, and
uncentrifuged SECM of Paraliparis are 37%,
27%, and 47% higher, respectively.
The osmotic strength of these fluids is attributable primarily to inorganic ions, especially sodium and chloride (Table 4). The
ionic concentrations of all sampled fluids are
elevated and ionic content is similar (Table
TABLE 3. Osmolality (mOsm kg-') of body fluids and
subdermal extracellular matrix (SECM) as determined
bv vaoor m-essure osmometrv'
Fluid
SECM
from
Specimen
Coelomic
(not
centrifuged
no.
Serum
fluid
centrifuged)
SECM
PDE 15
PDE 16
PDE 17
PDE 18
PDE 19
PDE 20
PDE 15,
16 & 18
(pooled)
Mean
SEM
N
-
378
422
536
523
482
434
509
469
444
416
478
23.6
5
-
444
22.0
5
478
486
520
567
-
513
20.2
4
-
779
-
697
738
2
'Numbers are means of three determinations for each specimen.
92
J.T. EASTMAN ET AL
4), with the exception of greatly elevated Na+
values in fluid from centrifuged SECM. These
may be attributable to the binding of cations
by the anionic GAGS. Ion concentrations are
unusual in that values for Na+ are typically
less than those for C1-. The ratio of Na+ to
C1- (Na+:Cl-) is 0.86 for seawater and 1.1
for plasma of most teleosts (McDonald and
Milligan, '92). In Paraliparis, however, the
ratio in serum is 0.91 (Table 4). Low ratios
(0.85-0.87) are also characteristic of various
species of Liparis from Spitzbergen, Norway
(A.L. DeVries, unpublished data).
The relatively low values for K+ indicate
that the SECM of Paraliparis, like serum
and coelomic fluid, is a n extracellular fluid
with few cells.
Microscopy of the skin
The skin of Paraliparis is scaleless with
the exception of a few 35-45 pm long thumbtack prickles (see Fig. 7). These modified
scales are embedded in the superficial epidermis. The skin is 85-200 pm thick and, unlike
most other fishes, the epidermis is several
times thicker than the dermis (Fig. 6). Regional variation in thickness is attributable
to differential development of the club cells
in the midepidermis. These are especially
prominent in the anterior trunk region.
Means for 10 measurements of skin thickness at each of three sites on one fish are:
skin over pectoral girdle-92 pm (25.1 p m
SEMI, head skin around nasal aperture121 pm (24.8 km SEM), and trunk skin
dorsal to pectoral region-159 pm (55.5 pm
SEM). Paired t-tests indicate that all comparisons between mean differences in thickness
a t these three sites are significant a t the level
of a t least P < 0.007.
Basal epidermis
The epidermis is a noncornified stratified
epithelium that is six t o eight cells thick. It is
arranged in distinct basal, middle, and superficial layers (Figs. 6, 7). The basal layer consists of a single layer of cuboidal epithelial
cells. There are no specialized adhesive junctions such as hemidesmosomes to hold cells
to the prominent basement membrane. Adjacent basal cells, however, are connected to
each other by tight junctions and extensive
lateral membrane interdigitations (Fig. 8).
Desmosomes connect the basal cells to more
apical cells. The cytoplasm of the basal cells
has a prominent convoluted ring of microfilaments around the nucleus and most organelles (Figs. 9-10]. In most epidermal cells of
Paraliparis, these filaments measure 6-7 nm
in diameter and are therefore microfilaments. Filaments measuring 9-11 n m in diameter and clearly associated with desmosomes are tonofilaments (Fig. 9). In many
cells the ring of microfilaments encloses most
of the organelles, and only sparse microfilaments and mitochondria occupy the peripheral cytoplasm. Microfilaments also occur in
less organized arrays external to the convoluted ring. Many basal cells also contain large
vacuolated regions caused by dilations of the
endoplasmic reticulum and perinuclear space.
These do not usually occur in other epidermal cells.
Midepidermis
Apically the basal cells interdigitate with
cytoplasmic extensions of large vacuolated
cells comprising the midepidermis. Not reaching the free surface of the epidermis, the two
TABLE 4 . Ionic content (mMol liter 1
' ofbody fluids and subdermal extracellar matrix fSECM)as determined by
flame photometry and chloridometry'
SECM
Specimen
no.
PDE
PDE
PDE
PDE
PDE
PDE
PDE
15
16
17
18
19
20
15,16,
& 18 (pooled)
Mean
SEM
N
Serum
Coelomic fluid
CI-
Na+
K+
208
196
240
256
220
212
12
20
18
6
12
12
263
258
233
218
258
232
218
222
9.0
6
13
2.0
6
243
10.6
4
226
8.7
5
-
Na+
212
212
-
K+
6
6
-
6
4
10
6
1.1
5
C1220
212
-
257
-
226
229
9.8
4
(not centrifuged)
Na+
Kt
232
234
10
10
-
258
Na+
K+
C1-
352
12
383
336
344
12
12
348
366
2
2
2
240
-
-
6
-
-
-
-
241
8.4
3
el-
Fluid from
centrifuged SECM
8.7
1.3
3
290
300
277
18.6
3
'Numbers are single determinations (Na+, K+)or means of two determinations (C1 1 for each specimen
SNAILFISH BUOYANCY AND SKIN
93
to three layers of vacuolated cells are the Superficial epidermis
most conspicuous and unusual component of
The superficial epidermis consists mainly
the epidermis of Paraliparis (Figs. 6, 7). of cuboidal epithelial cells in a layer three to
These cells are similar to the vacuolated cells five cells thick. Other cell types include macof gobiids of the genus Periophthalmus (Whi- rophages and cells that superficially resemble
tear, '88; Yokoya and Tamura, '92). We iden- mucous goblet cells, but are actually saccitify them as club cells based on their location form cells (Mittal et al., '80; Whitear, '86).
in the midepidermis; interdigitated lateral These relatively large cells measure 30-60
cell membranes; large central vacuole with km along their major axis (Fig. 61, reach the
low electron density and twisted or coiled epidermal surface, and do not stain for mufilaments (called tonofilaments by some au- cosubstances. Furthermore, unlike mucous
thors) associated with desmosomes in the cells, the secretion product is not contained
cortical cytoplasm (Henrikson and Matoltsy, in small membrane-bound globules. Sacci'68; Whitear, '86; Whitear et al., '91). As 'form cells possess a large central vacuole
mentioned previously, the filaments in Parali- surrounded by small membrane-bound vacuparis appear to be microfilaments, although oles (Figs. 11, 13).The darkly staining cytotonofilaments are associated with the desmo- plasm is rich in rough endoplasmic reticulum
somes.
and is arranged as a ring around the periphery of the centrally placed secretion. The ring
Club cells. The most basally located club contains mitochondria, many vacuoles, mulcells are the largest, measuring 40-100 km tivesicular bodies, and irregularly arranged
in height and 25-40 km in width. The club microfilaments. The nucleus is basally locell layer has narrow pillars extending be- cated in the ring. The inner border of the ring
tween basal and superficial epidermal cells. is lined with small, irregularly shaped vacuThe pillars are narrow cytoplasmic portions oles that are probably emptying into the large
of adjacent club cells and the connecting mem- central vacuole.
branes (Figs. 11, 12). The membranes of the
There are two structural types of superfiadjacent cells are interdigitated and con- cial epithelial cells (Fig. 14). The first appears
nected by desmosomes (Figs. 11, 121, al- to be the remnant of club cells of the midepithough there are fewer desmosomes connect- dermis. These have a large central nucleus,
ing club cells with adjacent sacciform cells. lightly staining cytoplasm, many cytoplasmic
The narrow band of cytoplasm between the vacuoles and a convoluted ring of microfilalateral membrane and the edge of the large ments similar to basal cells. The other struccentral vacuole contains few organelles and tural type of cell has a small nucleus, darker
has an amorphous, irregularly arranged fila- cytoplasm, relatively few cytoplasmic vacumentous appearance. These filaments are oles, and a ruffled free surface. These cells
characteristic of club cells (Henrikson and may be precursors of sacciform cells. Both
Matoltsy, '68; Whitear, '86). Mitochondria epithelial cell types have features indicative
and nuclei often protrude into the large cen- of cell secretion including a well-developed
tral vacuole (Figs. 6, 7, 11, 12). Occasional Golgi apparatus and many vacuoles and relipidlike inclusions and lysosomes are also sidual bodies.
present. The vacuoles are membrane limited
Apical cells adjacent to club cells contain
and appear empty, although they may con- large bands of vacuoles, but cells closer to the
tain profiles of isolated mitochondria, nuclei, epidermal surface have progressively fewer
cell debris, and multivesicular bodies that are vacuoles. Those closest to the club cells also
surrounded by the vacuole membrane (Fig. contain convoluted contractile rings, similar
12). The contents of the vacuole exhibit no to those in basal cells. Closer to the surface,
affinity for stains and have no structure a t the cells are more rounded, less vacuolated,
the level of light or electron microscopy. Con- and lack filamentous rings. Many cells have
tents do not appear to be proteinaceous or lipidlike inclusions and an extensive network
lipoidal and, given the ultrastructure of the of microfilaments that may not be organized
cell, do not seem destined for secretion. Club into a convoluted ring. The free surface of
cells and their vacuoles become progressively most of the darker apical cells is often slightly
smaller toward the superficial epidermis ruffled and the apical cytoplasm contains high
where there is a transition to a more typical concentrations of microfilaments. The apical
surfaces of adjacent cells are connected by a
type of epithelial cell.
94
J.T.EASTMAN ET AL
Figures 7-9
SNAILFISH BUOYANCY AND SKIN
junctional complex that includes a tight junction and a desmosome.
Basement membrane
The basal epithelial cells are not attached
to the basement membrane by hemidesmosomes. Instead, a layer of large fibrils lies in
the lamina lucida of the basement membrane
(Fig. 8). These 20-30 nm diameter structures have a density greater than collagen
and the lamina densa, which is 100-250 nm
thick.
Dermis
The dermis is a homogeneous reticular
layer of collagen fibrils oriented at right angles
to each other. Each layer is thin, consisting of
one to three layers of fibrils (Figs. 8, 10).
Cells do not occur in this region, but the
border between the dermis and hypodermis
consists of a continuous layer of cells made
up of modified fibroblasts and melanophores.
These cells appear to be interconnected by
junctions or by very closely opposed membranes (Fig. 15). Although unlike a true endothelium-a simple squamous epithelial layer
lining the lumina of blood vessels and chambers of the heart-these modified fibroblasts
are sometimes referred to as the endothelium of the skin (Whitear et al., '80).
DISCUSSION
Modifications for neutral buoyancy
in Paraliparis
Liparidids are a benthic group without a
swim bladder, but some species occupy habitats in the water column. Observations made
at 656 m from a submersible research vehicle
Fig. 7. Paraliparis deuriesi. Histological section of
epidermis of trunk skin showing detail of superficial,
middle, and basal layers of epidermis and a darkly stained
thumbtack prickle (modified scale). Nuclei protrude into
the central vacuole of large club cells (C) in the midepidermis. D, dermis. Gomori's trichrome. ~ 6 7 0 .
Fig. 8. Paralipparis deuriesi. Basal portion of the epidermis. Two cells are joined by a tight junction and the
cell borders are interdigitated. Hemidesmosomes are absent, and the basement membrane's lamina lucida contains dark fibrils (arrowhead) associated with the prominent lamina densa. The dermis (D) consists of collagen
fibrils. Microfilaments fill the cytoplasm of the lower cell.
~35,000.
Fig. 9. Paralipparis deuriesi. Detail of microfilaments
(F) from the convoluted ring of a basal epithelial cell. Also
shown are desmosomes (J)and associated tonofilaments
(TI. x 120,400.
95
in the Atlantic indicate that Paraliparis garmani lives 1-3 m above the bottom (Wenner,
'79). The absence of substrate contact adaptations and our catch records from bottom
traps and observations of aquarium specimens suggest that Paraliparis devriesi occupies a similar epibenthic habitat in McMurdo
Sound. Without a swim bladder, Paraliparis
have achieved neutral buoyancy through evolutionary alteration of density, especially
through a n increase in light and a reduction
in heavy body constituents.
High water content tissues are known to
enhance buoyancy in mesopelagic teleosts
without swim bladders (Blaxter et al.,'71). In
the absence of significant deposits of somatic
lipid, the SECM of Paraliparis is the primary
low density buoyancy agent. An extensive
high water content SECM has never been
reported in a fish from a subzero environment where the chance of encountering ice
with subsequent freezing of the body is always a possibility. Notothenioid and zoarcid
fishes from McMurdo Sound possess glycopeptide and peptide antifreezes, respectively,
and these compounds protect them from
freezing (DeVries, '58; Cheng and DeVries,
'91). Liparidids occupy similar ice-free deepwater habitats and were originally thought
to lack antifreeze, remaining instead in a
supercooled state (DeVries and Lin, '77). Recent work, however, indicates the serum of P.
devriesi contains modest amounts of antifreeze based on the presence of a measurable
(0.4"C)thermal hysteresis (A.L. DeVries, unpublished data). The antifreeze does not sufficiently lower the freezing point of the serum to protect Paraliparis from freezing a t
normal ambient surface temperatures in McMurdo Sound. However, the in situ freezing
point (- 1.86"C)is much lower a t 500-650 m
because of the effect of pressure, and this
may be sufficient to protect Paraliparis from
freezing in their normal habitat.
Compared to other adult fishes with gelatinous tissue, Paraliparis have the largest
known proportion of body weight, about onethird, represented as SECM. An expanded
SECM is a convergent feature in a number of
teleostean lineages. For example, 18%of body
weight is SECM in the lumpfish Cyclopterus
lumpus (Davenport and Kjorsvik, '861, also a
scorpaeniform, and a gelatinous SECM is
present in representatives of phyletically diverse groups, including zoarcids (McAllister
and Rees, '631, mesopelagic salmoniforms and
stomiiforms (Yancey et al., '89), and various
96
J.T. EASTMAN ET
Figures 10-12
AL.
SNAILFISH BUOYANCY AND SKIN
larval anguilliforms and elopiforms (Pfeiler,
'86, '91).
The body fluids of marine teleosts provide
static lift because they are about one-third
the osmotic strength of seawater. The contained ions also lower the freezing point of
body fluids. This depression is vital for survival under subzero conditions, and compared to other temperate teleosts, Paraliparis show a 27-47% elevation of the serum
osmolality (assuming a mean of 350 mOsm
kg-' for temperate species) due to increased
levels of NaCI. Elevated osmolality of the
body fluids is a physiological characteristic
that Paraliparis share with Antarctic notothenioids (O'Grady and DeVries, '82). Body
fluids of Paraliparis provide static lift since
they are only 42-48% of the osmolality of
seawater from McMurdo Sound. Paraliparis
does not have the dilute body fluids typical of
some low latitude teleosts living at the greatest depths of the mesopelagic realm (Childress and Nygaard, '73; Yancey et al., '89;
Phleger, '91).
Muscle water content is slightly elevated in
Paraliparis compared to other teleosts. This
may be attributable, however, to inclusion of
SECM in samples of muscle. Muscles with a
low protein and high water content, characteristic of some meso- and bathypelagic fishes,
might need to be fortified with antifreezes in
Antarctic waters. The energetic cost of synthesizing and maintaining these compounds
might be greater than any saving realized
from the weight reduction attributable to
watery muscles.
Fig. 10. Paraliparis deuriesi. Basal cuboidal epithelial cells show prominent filamentous rings that surround most of the cytoplasmic organelles including the
nucleus. The interdigitating cellular borders connect similar adjacent cells, while desmosomes connect the ring
cells to the club cells (C) that have pale cytoplasm. D,
dermis. ~ 3 , 3 0 0 .
Fig. 11. Paralipuris deuriesi. The cell with the dark
cytoplasm is a sacciform cell, associated with two lighter
club cells (C) by interdigitating membranes. The sacciform cell has small vacuoles lining the large central
vacuole (V), and the cytoplasm is filled with microfilaments. The club cells are connected to each other by
desmosomes (J),and have organelles projecting into the
central vacuole (CV). x 14,000.
Fig. 12. Paraliparis deuriesi. A group of club cells
connected to each other by both interdigitating membranes (arrowhead) and desrnosomes (J). The nucleus
and part of the cytoplasm extend into the central vacuole,
as do single mitochondria (M). ~4,800.
97
Skin and SECM i n Paraliparis
Skin
The skin of phyletically diverse tropical,
temperate and polar teleosts ranges from 100
to 1,000 k m in thickness (Eastman and
Hikida, '91). Some tetraodontiforms and
sharks have skin that is nearly 1 cm thick
(Harder, '75). Unlike benthic nototheniids
from McMurdo Sound, the thin, delicate skin
of Paraliparis falls at the low end of this
range. The looseness of the skin is attributable to the thin dermis and its separation
from the trunk musculature by the extensive
SECM.
The role of the distinctive layer of club cells
in the midepidermis of Paraliparis is unknown. Club cells have been identified in the
skin of phyletically &verse teleosts. They
probably serve different functions in different species and may have multiple functions
within the same species. Contents of anguilliform club cells may contribute t o the formed
elements in the mucous covering of the skin
(Henrikson and Matoltsy, '68). In amphibious gobiids like Periophthalmus, the club
cells may store and release water to prevent
desiccation of the skin (Yokoya and Tamura,
'92). In ostariophysans the contents of club
cells, when released in the water, elicit a
fright reaction in conspecifics (Henrikson and
Matoltsy, '68). Club cells in some cobitid
ostariophysans and carapid ophidiiforms, but
not congrid anguilliforms, show serotoninlike immunoreactivity (Zaccone et d.,'90).
I n other ostariophysans, club cells exhibit
immunoreactivity for chondroitin and keratan sulfate (Ralphs and Benjamin, '92). I n
these species club cells may protect damaged
epithelial surfaces through release of gellike
secretions containing GAGS (Whitear et al.,
'91). In skin prepared for microscopy, the
empty appearance of the vacuoles is probably
artifactual as fixation is sufficient trauma to
cause club cells to release their contents to
the intercellular space (Henrikson and Matoltsy, '68).
Tonofilaments are variously arranged
within the epithelial cells of the epidermis
(Whitear, '88). Since mitotic activity is not
confined to the basal epidermis in fishes,
tonofilaments may be reformed and structurally modified as cells move toward the free
epithelial surface. Tonofilaments offer structural support to soft epithelial tissues. This
may be especially important in Paraliparis
because club cells, the major component of
Fig. 13. Paralipparis deuriesi. Sacciform cell with its
central vacuole lined by smaller vacuoles. The cytoplasm
contains abundant rough endoplasmic reticulum, and
the cell is located among other epidermal cells that have
microfilamentous rings. x 6,800.
Fig. 14. Paralipparis deuriesi. Cross section through
the apical epidermis showing the surface light and dark
cells, cells with microfilamentous rings (R), and lower in
the cell layers, club cells (C)of the mid-epidermis. x 1,700.
Fig. 15. Paraliparis deuriesi. The cellular border separating the dermis (D) from the hypodermis (H) contains
modified fibroblasts that appear to be connected by tight
junctions (J).A process of a melanophore contributes to
this lining. ~9,600.
SNAILFISH BUOYANCY AND SKIN
the epidermis, are thought to have a gelatinous consistency.
Although epidermal cells of teleosts have
been described as having tonofilament skeins
(Whitear, '88;Yokoya and Tamura, '921, most
of the filaments within the cells of Paraliparis measure 6-7 nm in diameter and are
therefore microfilaments. Tonofilaments (intermediate filaments) are usually 8-1 1nm
in diameter, whereas microfilaments are 6
nm (Fawcett, '86). Whitear ('88)reports the
presence of several classes of filaments, some
10-11 nm and most 7-8 nm, but called both
types tonofilaments. It would seem reasonable to refer to the former as tonofilaments
and the latter as microfilaments. Since the
diameters of the two filament types are so
similar, immunocytochemical identification
would be required to verify the presence of
these two classes of filaments. Tonofilaments
probably anchor the junction to the cytoplasm, whereas the microfilament rings serve
either a contractile function, perhaps constricting the cell during secretion, or as a
means of support for the large and tall cells.
Subdermal extracellular matrix (SECM)
Vertebrate connective tissues are combinations of cells, fibers, and ground substance,
with one component usually predominant.
The SECM ofparalipparis is a mucous connective tissue because the ground substance and
its immobilized water are responsible for the
physical properties of the SECM. Various
types of mucous connective tissue serve as
packing and filling material in phyletically
diverse teleosts, but have a limited distribution in the body (Benjamin, '88). Furthermore, those described to date have more cells
and fibers and less ground substance than
the SECM of Paraliparis.
Expansion of the SECM begins early in
larval life in many liparidids. This is evidenced by the flaccid skin forming a pronounced bubble over the anterior portion of
the body (Able et al., '84). At 6-9 mm standard length, e.g., larvae of some species of
Rhinoliparis (Kido and Kitagawa, '86) and
Liparis (Marliave and Peden, '89) begin to
exhibit this appearance. Since hyaluronic acid
is found in highest concentrations in embryonic tissues (Comper and Laurent, '781, persistence of an expanded SECM into adult life
may be an example of paedomorphic heterochrony.
Histochemical, monoclonal antibody, and
electrophoretic techniques have been used to
determine the glycosaminoglycan(GAG)com-
-
99
position of connective tissues in teleosts
(Banerjee and Yamada, '85; Benjamin, '88;
Benjamin and Ralphs, '91; Pfeiler, '91). The
GAG composition of elopiform leptocephali is
mostly keratan sulfate, whereas anguilliform
leptocephali contain a mixture of GAGS
(Pfeiler, '911. In these thin-bodied larvae,
hydrated GAGs provide structural support
and buoyancy, but also serve as an energy
source during metamorphosis (Pfeiler, '86).
The GAGs of high water content subdermal
connective tissue in other teleosts have not
been specifically identified, although hyaluronic acid is typically present in these tissues (Benjamin and Ralphs, '91).
Since the SECM does not stain with
PAS-AB at pH 1.0, shows reduction in intensity of alcian-blue staining after digestion
with bacterial hyaluronidase, and has viscous properties, we suspect that hyaluronic
acid is the primary constituent of the SECM
in Paraliparis. A polymer of the disaccharide
N-acetyl-D-ghcosamine and o-glucuronic
acid, hyaluronic acid is fully ionized under
physiological conditions, with one negative
charge associated with the carboxyl group of
each disaccharide (Comper and Laurent, '78).
The negative charge of the molecule is responsible for the alcian-blue staining as well as
the water and cation binding we observed in
the SECM of Paraliparis. More specifically,
the concentration of negative charges accounts for the high levels of Na+ in the fluid
centrifuged from the SECM. Since hyaluronic acid has a n elongated or extended
configuration, it has a large fluid domain and
forms three dimensional hydrated networks
in the SECM (Toole, '81). Water is held in the
matrix by hydrogen bonding to the hydroxyl
groups of hyaluronic acid (Comper and Laurent, '78).
The osmotic behavior of GAGs is extremely
nonideal. The osmotic pressure of hyaluronic
acid in NaC1, e.g., rises rapidly in a nonlinear
fashion. The colligative properties are presumably determined by the activities of Na+
and GI- (Comper and Laurent, '78). InPuruliparis, the binding of cations, especially Na+,
is partly responsible for the elevated osmolality of the SECM relative to serum. Chloride
ions, possibly attracted to the bound Na+,
also contribute to the osmolality.
Conclusion
Trunk skin is a barrier to entry of ice
crystals into the body of some polar fishes. In
vitro experiments on the skin of winter flounder (Pseudopleuronectes americanus) from
100
J.T. EASTMAN ET AL
Newfoundland support the hypothesis that
epithelia exclude the entry of ice, probably
through the action of antifreeze contained in
the interstitial fluid and by the narrowness of
the intercellular space (Valerio et al., '92).
However, we see no obvious relationship between the structure of the skin and SECM in
Paraliparis and the Antarctic environment.
Based on light and electron microscopic appearance and its thinness, the skin of Paraliparis presents a less substantial barrier to ice
entry or propagation than the skin of Antarctic notothenioids, a group protected by antifreeze (Eastman and Hikida, '91).
ACKNOWLEDGMENTS
We are grateful to Dave Petzel and Hans
Ramlov for technical assistance at McMurdo
Sound. Professor Emerita Mary Whitear,
University College, London, provided helpful
comments on the microscopy of the skin. We
thank William Winn for photographing Figures 1-5. This work was supported by NSF
grants DPP 79-19070 (to J.T.E.) and DPP
90-19881 (to A.L.D.), by a grant from the
Ohio University Research Committee, and
by funds from the Ohio University College of
Osteopathic Medicine.
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