Histochemistry and the structure of the skin of a murrel, Channa striata (Bloch,

1797) (Channiformes, Channidae). 11. Dermis and subcutis1

Department of Zoology,Banaras Hindu University, Varanasi221005, India

Received October 25.1974

MITTAL,A. K . , and T . K. BANERJEE. 1975. Histochemistry and the structure of the skin of a
murrel, Channa striata (Bloch, 1797) (Channiformes, Channidae). 11. Dermis and subcutis.
Can. J. 2001. 53: 844-852.
The structure and the cytochemistry of the dermis and the subcutis of Channa striata, which
buries in mud to survive droughts, is described and correlated with its habitat. The dermis
consists of an outer stratum laxum and an inner stratum compactum. The stratum laxum is mainly
Can. J. Zool. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/23/14

composed of well-developed scales lodged in connective tissue pockets which are characterized
by the presence of huge deposits of lipids. These lipids may play important roles: in supplying
energy during the period of fasting, acting as a barrier for water diffusion through the skin, and
serving as shock absorbers, protecting the body from mechanical injury during burrowing. The
presence of sulfated acid-mucopolysaccharides, the substantia amorpha in the stratum laxum,
has been described as an adaptation to prevent desiccation. Alkaline phosphatase, sulfated
acid-mucopolysaccharides, and calcium are closely associated in the osseous layer of the scales
and probably play an important role in calcification. Numerous fine collagen fiber strands
connecting the basement membrane to the scales provide firm attachment of the epidermis to the
dermis. The presence of a relatively thin subcutis may be correlated with the well-developed
layer of fat cells in the stratum laxum.

MITTAL,A. K . , et T . K. BANERIEE.1975. Histochemistry and the structure of the skin of a

murrel, Channa striam (Bloch, 1797) (Channiformes, Channidae). 11. Dermis and subcutis.
Can. J. 2001.53: 844-852.
For personal use only.

Chonnrr srriara se terre dans la boue durant les periodes de secheresse; on etudie la structure et
la cytochimie du derme et de la couche sous-cutanee en fonction de I'habitat. Le derme se
constitue d'une couche externe de stratum laxum et d'une couche interne de stratumcompactum.
Le stratum laxum est compose surtout d'ecailles bien developpees enfouies dans des poches de
tissu conjonctif caracterisees par la presence de depots enormes de lipides. Ces lipides peuvent
jouer des r8les importants: fournir de I'energie durant les periodes de jebne, sewir de barriere
pour la diffusion de I'eau a travers la peau et servir d'amortisseurs en protegeant le corps contre
les blessures lorsque I'animal s'enfouit. La presence de mucopolysaccharides acides sulfates, la
substance amorphe du stratum laxum, est une adaptation pour emptcher le dessechement. La
phosphatase alcaline, les mucopolysaccharides acides sulfates et le calcium sont en relation tres
etroite dans la couche osseuse des Ccailles et jouent probablement un rBle important dans la
calcification. On observe plusieurs minces faisceaux de fibres de collagene reliant la membrane de
base aux ecailles; ces faisceaux assurent la solidite des liens entre l'epiderme et le derme. La
couche sous-cutanee est relativement mince, compensee semble-t-il par la couche bien develop-
p6e des cellules graisseuses du stratum laxum. [Traduit par le journal]

Introduction dermis and subcutis of an air-breathing fresh-

past work on fish skin little attention has
been paid to the functional organization of the
dermis and the subcutis (Jakubowski 1958, 1959,
1960a, 19606; ~i~~ 1967; Mittal ,968; Mitta]
and Munshi 1970, 1971 ; Roberts et al. 1971 ;
Mittal and Banerjee 1974). The present work
examines the structure and cytochemistry Of the
'This investigation was supported by Research Grant
No. 38(131)/72 GAU-I1 from the Council of Scientific
and Industrial Research, Government of India.
water fish, Channa striata, which can survive
extreme conditions of drought buried in the mud.
attempt has been made to "Bestthe
adaptive significance of various skin structures.
Materials and Methods
The technical details of the materials, procedures, and
various histochemical techniques used for the demonstra-
tion of carbohydrates, lipids, calcium, alkaline phos-
phatase, and connective tissues have been outlined in the
previous publication (Mittal and Banerjee 1975).
T o overcome the problems of lipid diffusion from one
site to another, layers of the skin were dissected out under
 MITTAL AND BANERJEE: SKIN OF MURREL. 11. DERMIS AND SUBCUTIS
a binocular microscope, stained for lipids, and then
whole-mount preparations made in glycerine jelly.
Liang's (1947) method for nerves was also used.
Observations
Dermis
The dermis is composed of two layers, an outer
stratum laxum and an inner stratum compactum.
Stratum Laxum
This was a thick (ca. 780 pm), areolar connec-
tive tissue layer distinguished by the presence of
well-developed scales, lodged in characteristic
connective tissue pockets (Fig. 1). This layer was
richly infiltrated with large globular, polyhedral,
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unilocular fat cells each having a flat nucleus
surrounded by a small amount of cytoplasm
pressed against the cell membrane by a single,
large, almost colorless fat droplet. In the loose
tissue immediately below the basement mem-
brane only a few small fat cells were discernible.
In routine haematoxylinleosinpreparations (par-
affin sections), these cells appeared as empty
rings (Fig. 1). The lipoidal contents gave strong
color reactions for neutral lipids (Fig. 3), stained
weakly for phospholipids, and were extracted
when treated with organic solvents (95% alcohol,
For personal use only.
acetone, and others) for short durations at room
temperature (Table I). In cryostat sections (even
at 40 pm thick) the lipoidal contents of these
cells showed heavy diffusion, giving a false local-
ization in other tissue constituents. Whole-
mount preparations of a part of this tissue gave
a more specific localization of fat droplets.
Delicate connective tissues, mainly composed
of collagen fiber bundles, which stained blue in
Mallory's triple-stain preparation, intermingled
with a few fine elastic fibers, which stained black
in Verhoff's elastin stain preparations, were be-
tween the fat cells and served as a bed for
numerous capillaries (Fig., 3).
In addition to lipid droplets, an amorphous,
acellular, slightly basophilic material, the sub-
stantia amorpha (Fig. 2), was also observed.
This material gave positive color reactions with
various histochemical techniques (Table 1) spe-
cific for sulfated acid-mucopolysaccharides (Fig.
5) but did not give positive reactions for calcium
(Table 1).
The thin, loose connective tissue layer lying
immediately below the basement membrane was
richly supplied with capillaries, stained black
with Sudan black B (Fig. 9), and deep blue in
845
Acid haematin and Nile blue sulfate preparations ;
the nerves stained magenta with Liang's method
(Fig. 7). Both were profusely branched, forming
a complicated network (Fig. 8) connected with
the blood vessels and nerves of the stratum com-
pactum and the subcutis through the branches in
the connective tissue scale pockets. Collagen
strands were also observed connecting the base-
ment membrane to the surface of the underlying
scales (Fig. 2).
A layer of pigment cells, supplied with capil-
laries (Fig. 9) and nerves, was found in the loose
tissue below the basement membrane and also in
the connective tissue pockets on the surface of
the scales (Fig. 1). These pigment cells are of two
types : large stellate brownish-black melano-
phores which were irregularly distributed in the
connective tissue pockets and arranged in paral-
lel rows in the loose tissue below the basement
membrane (Figs. 4, 9), and minute, irregularly
round, yellowish-red or orange lipophores, ir-
regularly distributed between the melanophores.
Lipophores stained bright red with Oil red 0 ,
Sudan 111, and Sudan IV, and black with Sudan
black B (Fig. 4); they could not be located in
paraffin sections and did not stain with Sudan
dyes after the treatment of tissues with organic
solvents (acetone, 95% alcohol, and others).
The scales of Channa striata are composed of
two distinct layers: a thin, upper basophilic
osseous layer and a thick, lower eosinophilic
fibrillary plate. The annuli on the free surface of
the osseous layer appeared serrated in cross
section (Fig. 6). The upper, osseous layer of the
scales gave strong positive color reaction for
calcium and sulfated acid-mucopolysaccharides
(Fig. 5), showed high alkaline phosphatase
acitvity (Fig. 6), and stained a deep aniline blue
color in Mallory's triple stain and deep black
with Verhoff's elastin stain (Table 1). The
fibrillary plate did not give a positive reaction for
calcium, stained blue in Mallory's triple stain,
gave weak reactions for acid-mucopolysaccha-
rides, and showed very weak or no alkaline phos-
phatase activity (Table 1).
Stratum Compactum
This layer was thin (ca. 168 pm) (Fig. 1) and
mainly composed of coarse collagen fiber bundles
compactly arranged in several layers. A few col-
lagen fiber bundles ran vertically at intervals.
The collagen fibers appeared blue in Mallory's
Can. J. Zool. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/23/14
For personal use only.

846

.L
Dr-
CAN. J. ZOOL. VOL. 53, 1975
 MITTAL AND BANERTEE: SKIN OF MURREL. 11. DERMIS AND SUBCUTIS 847

triple stain and stained weakly with periodic
acid - Schiff's (PAS) techniques (Table 1).
Elastic fibers could not be located in this layer.
Capillaries and nerves were also discernible in
this layer. The lipoidal contents which might
have diffused from stratum laxum or from sub-
cutis in cryostat sections could not be demon-
strated in whole-mount preparations. A layer of
sparsely distributed chromatophores was seen
on the inner aspect of this layer.
Subcutis
This was a very thin layer (ca. 25.5 pm) lying
between the muscle bundles and stratum com-
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to Giinther (1880) can survive droughts living in
a semifluid mud or lying torpid below the hard-
baked crusts at the bottom of a tank from which
all water has evaporated. Lipid provides the most
favorable form of temporary energy storage
because it weighs less and occupies less volume
per calorie than either carbohydrate or protein
(Bloom and Fawcett 1968). During the period of
fasting, when the fish is buried in mud under
adverse conditions such as droughts, the lipids
stored previously in the stratum laxum during
conditions of excess food, may play an important
role in the maintenance of a stable energy supply.
A thick layer of lipids, which are water-repellent

pactum (Fig. l), and mainly composed of fat
cells containing neutral lipids extractable with
exposure to organic solvents for short durations
at room temperature (Table 1). Fine collagen
fiber bundles intermingled with elastic fiber
bundles were discernible continuing on the proxi-
mal side with those of the myosepta, and on the
distal side with those of stratum compactum.
Main branches of nerves and blood vessels were
invariably found in this layer.
Discussion
For personal use only.
substances, in the stratum laxum in C. striata
may also act as a barrier to water loss through
the skin. When C. striata burrows in mud, the
presence of stored lipids in the stratum laxum
may act as shock-absorbing pads, protecting the
body from mechanical injury.
Histochemical investigation revealed the pres-
ence of sulfated acid-mucopolysaccharides be-
tween the loose connective tissues in the stratum
laxum of C . striata. Mittal and Munshi (1971)
and Mittal and Banerjee (1974) made similar
observations while studying the skin of Amphip-

The presence of large amounts of lipids in the
stratum laxum of the dermis of Channa striata is
very interesting. Other workers in the field have
made no reference to such fat deposits. The
presence of lipids may be correlated with the
peculiar mode of life of the fish, which according
nous cuchia (Synbranchiformes, Amphipnoidae)
and Notopterus notopterus (Oesteoglossiformes,
Notopteridae) respectively, and interpreted their
presence as an adaptation to prevent desicca-
tion, since mucus has a remarkable power to
bind or release copious amounts of water (Letterer

ABBREWAT~ONS: BM, basement membrane; CTP,connective tissue pocket: EPD,epidemis; FC, fat atlls;
IT, fibrillary plate; MEL, rnelanophore: MUS, muscle; OSL, osseous layer; PC, pigment cell: S, scale;
SA, substantia amorpha; SCT, subcuris; STC, stratum compactum; STL, stratum laxurn.
FIG. 1. Cross section of the skin of Channa striafa showing the general organization of the dermis and
the subcutis. (Ehrlich's haematoxylin/eosin.) x 182. FIG.2. The stratum laxum and part of the epidermis
showing collagen fiber strands (arrows) (stained blue in original) connecting the basement membrane with
the surface of the scales. Note the presence of weakly stained substantia amorpha between the basement
membrane and the scale. (Mallory's triple stain.) x 1824. FIG.3. The stratum laxum showing polygonal
fat cells (stained red in original). Note the presence of a capillary (arrow) between the fat cells. (Sudan 111.
whole-mount preparation.) x 720. FIG.4. Part of a scale from which epidermis has been separated showing
small, irregularly distributed lipophores (arrows) (stained black in original) between melanophores ar-
ranged regularly in parallel rows in the connective tissue. (Sudan black B, whole-mount preparation.)
x 354. FIG. 5. The stratum laxum and part of the epidermis showing substantia amorpha (arrows) (stained
purple in original) in the loose tissue enclosing the scale. Note a strong positive color reaction in the osseous
layer of the scale. (Alcian bluelperiodic acid - Schiff technique.) x 2088. FIG.6. Part of a scale showing
strong alkaline phosphatase activity (arrows) in the osseous layer. (Calcium cobalt method, frozen section.)
x 1716. FIG. 7. Part of a scale from which epidermis has been separated showing nerve fibers (stained
magenta in original) in the loose connective tissue. (Liang's method, whole-mount preparation.) x 180.
FIG. 8. Part of a scale from which the epidermis has been removed showing capillary network (arrows) in
the loose connective tissue. (Liang's method, whole-mount preparation.) x 560. FIG. 9. Part of the scale
from which the epidermis has been removed showing profusely branched capillaries supplying the loose
connective tissues. (Sudan black B, whole-mount preparation.) x 182.
 Can. J. Zool. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/23/14
For personal use only.

TABLE 1
A summary of the histochemistry of the various components of the dermis and the subcutis of Charlna sfriala
Stratum laxum
Connective tissue pockets Scale Subcutis
Comcerivc Conrwctive
Serial Histochemical Suhanfia tirsue Fibrillsry Stratum tigsuc
No. technique Fixative Section Reference Fat e l l s emomha fibers layer vIa1c compacturn Fat mlls fibtrs

1 Sudan black B 1 % lwutral Frozen, Cassclman 1959

brrdin. whole mount
r o r m l calcium
2 Sudan 111 107: neutral Frozen, Casselman 1959
rormalin. whole mount
formol calcium
3 Sudan IV t E oculr~l Frozen. Cassclman 1959
formalin, whole mount
Cotmol caleiurn
4 Oil red 0 IWoneutral Frozen. Casselman 1959
Formalin. whole mount
Iormol cakium
5 Nile blue 10% neutral Frozen, Casselman 1959
sulfate formalin, whole mount
formof cakium
6 Organic lox neutral Frozen, Casselman 1959
solvents/ formalin. whole mount
Sudan dyes furrnol calcium
7 Acid Formol calcium. Frozen, Baker 1946
hacmatin test post-chromd whole mount
8 Pyridinelacid Formol calcium. Frozen, Pearse 1968
haematin test port-rhromcd whole mount
9 Mallory's Hclly's nuid Paraffin Jones 1950
triple stain
in v-botf's Htlly's Ruid Lillie 1954
elartrn stain
11 Sc+
lTs' 10% Pearsc 1968
wrthout Tmmalin
oxidation muinpaAuid
12 Periodic acid - 1 neutral Paraffin McManus 1946
Schiff's formalin
(PAS) Ro~lm'ah i d
 Can. J. Zool. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/23/14
For personal use only.

TABLE 1 (Concluded)

Stratum laxum
H
Connective tissue pockets Scslt Sukuib r
>
Connective Cullnoetlm
Serial Histoehemical Substantia tiasue Osamlla Fibrlttary Stratum time %
No. technique Fixative Wion Reference Fat cells amorpha fibers lever plate compactum FBIcelb fikm
m
13 DiastaseIPAS neutral
Ivrgrmalin. Paraffin Lillie and - ++M fM ++M f M f M -
Grew 1947
Bouin's fluid
I4 SalivaIPAS t(rL ncutml Paraffin Pearse 1968 - ++M f M ++M fM f M -
Cormaim. *M t$g
Bou~n'. fluid ::,
I5 Alcian blue 107: nctrlrel Paraffin Lev and - .I. .I I GB - ++ +GB - - - -
(AB) at formalin. Spicer 1964
pH 0.5-1.0 Ilouin's fluld E
16 PASIAB 10"; neutraT Paraffin Pearse 1968 - +++Pur +M +++Pur +M f M f M 2
formslln
Bourn's iuid
17 ABIPAS 1K2 nculral Paraffin Pearse 1968 - ++#Fur fM +++Pur f M f M - f M g
formal~n,
tlourn's fluid
18 Thionin I K neutral Paraffin Pearse 1968 - + + +meta tortho ++ +meta fortho +ortho - +ortho
formalln,
19 Toluidine blue
Bou~n'sfluid
lo:, neutru I Paraffin Tock and
'
Er
Formalin. Pearse 1965 ?
Bou~n'sfluid
- t mcta - +meta - - -
- - -
--
;:: + +mela - + +meta - R
pH 4 f J mrra f ortho -
0
- f + +meta fortho - fortho
PH ! .I l 4 mclu fortho ++ +meta +ortho +ortho +ortho
-
20 Allzar~nred S 10% neutral Paraffin Lillie 1954 -- - -- -
- + 1-+S -
formalin.
$>
Bouin's fluid
21 Von Kosa's 1 W neutral Paraffin Lillie 1954 - - - +++B - - - a
method for fgnnalin m
calcium Bouin's huid
22 Calcium cobalt Fresh, cold Frozen Gomori 1952 - f 3 - +++B f B - - -
method for formalin.
sZ!
alkaline cold d o n e 4
phosphatase
SYMBOLS: B, black; Bb, bluish black; B1, blue;,GB, greenish blue; M, magenta; Pur, purple; R, red; S, scarlet; meta, metachromasia; ortho, orthochromasia; -, negative; f . very weak; +, 2
moderate; + +,strong; + very strong reactions.
+ +,
 850 CAN. J. ZOOL. VOL. 53. 1975
1959; Rogers 1961). The presence of these sub-
stances in the skin of C. striata may also prevent
desiccation.
Elkan (1968) reported calcium mixed with acid-
mucopolysaccharide deposits in the stratum
spongiosum (stratum laxum) of the skin of many
anurans. The present investigation, however, did
not reveal calcium associated with acid-muco-
polysaccharides between the loose connective
tissues in the stratum laxum of C. striata. The
amorphous, non-glandular substances in the
stratum laxum of N. notopterus also do not stain
with alizarin red S, indicating the absence of
calcium (Mittal and Banerjee 1974).
Can. J. Zool. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/23/14
The presence of alkaline phosphatase activity
in the osseous layer of scales in C. striata is very
significant. Yamada (1956) also observed a high
alkaline phosphatase activity in developing fish
scales and thought that the deposition of calcium
phosphate, a principal bone salt, is a result of
alkaline phosphatase activity. Kiguel (1964,
1965) reported the presence of this enzyme in
developing teeth and suggested that its primary
function during odontogenesis is the synthesis of
mucopolysaccharides. Thus the presence of this
enzyme in the osseous layer of the scale of C.
striata may be correlated with the synthesis of
For personal use only.
mucopolysaccharides, which are present in large
quantities in this layer, as indicated by strong
positive color reactions with different histo-
chemical techniques specific for mucosubstances.
Bevelander and Johnson (1950) and Irving (1960)
suggested that acid-mucopolysaccharides may
be important for in vivo calcification. Sobel
(1955) reported that acid-mucopolysaccharides
together with sulfate can bind calcium for local-
ized calcification. Maekawa and Yamada (1970),
using electron microscopy, reported a gradual in-
crease of dense particles which may be related to
a sulfated acid-mucopolysaccharide or a protein-
polysaccharide complex within the dense amor-
phous PAS-positive ground substance in the
osteoid and osseous layer of the scale of Salmo
gairdnerii irideus. They observed crystals ap-
pearing in these dense particles and pointed out
that they act as nucleation seeds in calcification
of the scale matrix. Mittal and Banerjee (1974)
also reported sulfated acid-mucopolysaccharides
in the osseous layer of the scales of N. notopterus
and suggested that these mucopolysaccharides
are important building materials of the bony
layer. The presence of a high sulfated acid-
mucopolysaccharide content in the osseous layer
of the scales in C. striata along with appreciable
amounts of calcium supports this view. It may
thus be concluded that alkaline phosphatase,
sulfated acid-mucopolysaccharide, and calcium
in the osseous layer of C. striata are closely in-
terrelated and may be correlated with the process
of calcification.
Sulfated acid-mucopolysaccharides are absent
in the fibrillary plate of the scales of C. striata.
Maekawa and Yamada (1970) and Mittal and
Banerjee (1974) also made similar observations
in the skin of Salmo gairdnerii irideus and N.
notopterus respectively. The absence of sulfated
acid-mucopolysaccharides may be correlated
with the absence of calcium, suggesting that this
layer does not undergo calcification.
The presence of small amounts of neutral
mucopolysaccharides, as indicated by a very
weak PAS reaction, along with collagen fibers in
the fibrillary plate and also in the stratum com-
pactum in C. striata, may be regarded as an
important interfibrillary cementing substance.
The present investigation reveals fine collagen
fiber strands connecting the basement membrane
and the scales in the skin of C. striata. Mittal
and Banerjee (1974) made similar observations
in the skin of N. notopterus and correlated their
presence with providing firm anchorage of the
epidermis to the dermis.
Mittal and Munshi (1971) suggested that there
is an inverse relationship between the thickness
of the stratum compactum and squamation. In
C. striata, where the scales are well developed,
the presence of a thin stratum compactum is
consistent with this view.
The subcutis binds the stratum compactum
with the underlying muscles and is mainly com-
posed of fat cells. According to Rabl(1931), the
subcutis is present in all the fishes except Amia
calua. No reference to this layer was made by
Bhatti (1938), Becker (1941), Jakubowski (1959,
1960a, 1960b), and Szabo (1965) in their descrip-
tions. This layer is thin and poorly developed as
in Misgurunusfossilis (Cypriniformes, Cobitidae)
(Jakubowski 1958), Bagarius bagarius (Siluri-
formes, Sisoridae) (Mittal and Munshi 1970),
Mastacembelus pancalus (Perciformes, Masta-
cembelidae), and A. cuchia (Mittal and Munshi
1971), but well developed in Monopterus albus
(Synbranchiformes,Synbranchidae)(Liem 1967),
Rita rita (Siluriformes, Bagridae) (Mittal 1968),
Heteropneustes fossilis (Siluriformes, Heterop-
 MITTAL AND BANERJEE: SKIN OF MURREL. 11. DERMIS AND SUBCUTIS 851

neustidae) (Mittal and Munshi 1971), and N. JONES,R. M. 1950. McClung's handbook of microscopical
notopterus (Mittal and Banerjee 1974). In C. technique. Paul B. Hoeber, Inc., New York. Med. Bk.
Dep. of Harper and Row.
striata this layer is comparatively inconspicuous. KIGUEL,E. 1964. Alkaline phosphatase activity in de-
Because in the skin of C. striata the fat cells have veloping molars of vitamin D deficient rats. I. High
mostly aggregated in the scale pockets of the calcium-phosphorus ratio diet. J. Dent. Res. 43: 71-77.
stratum laxum, the presence of another well- 1965. Fibrillary elements forming dentine and min-
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developed fatty layer at or about the same level 267-274.
may not have much functional value. LETTERER,E. 1959. Allgemeine pathologie. Thieme,
Stuttgart.
LEV,R., and S. S. SPICER.1964. Specific staining of sul-
Acknowledgments phated groups with alcian blue at low pH. J. Histochem.
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LIANG,H. N. 1947. A new method for staining nerves and
Postgraduate Department of Zoology, Bhagalpur their endings using the Schiff s reagent. Anat. Rec. 99:
University, Bhagalpur, India, for encouragement 511-521.
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and taking keen interest in the work. Our thanks
are also due to Prof. J. P. Thapliyal, Department
of Zoology, Banaras Hindu University, for pro-
viding necessary laboratory facilities.
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