*1
Nippon
Suisan
Gakkaishi
57(4),
Histochemical
613-618
(1991)
Localization
Alimentary
of
Canal
of
Kazuma
Using
a freeze-substitution in
olicola
bifidus,
Alkaline Acid the
the
phosphatase
nidase midgut digestive midgut
canal
Ostrincola
phosphatase striated
koe,
of
and ƒÀ-glucuronidase No
enzymes
parasitic
and 12,
examined
light
we
detected
microscopic
localizations
copepods:
striated
japonicus
and
border
of the
secretary
vacuoles
of the
midgut
cells.
Non-specific
detected
only
observed
copepods
in are
in
the
of
Conchyliurus
in the
were were
Ko*2
Neoergasilus
in the
the
1990)
of parasitic
yamagutii,
in
Copepods
Yoshio
September
species
non-vacuolar
reactions
these
six
detected
activities
positive of
was was
the
of
Panaietis
activity activity
border
cells.
method,
alimentary
Activities
Parasitic
Yoshikoshi*1 (Received
•@activities
Hydrolase
Some
hydrolase
quintus, Ergasilus
non-vacuolar
Modiorientalis.
midgut
vacuolar
midgut
cells.
cells
and
esterase, ƒÀ-N-acetylglucosami-
the
secretory
labral
lysosomal
vacuoles
glands.
These
enzymes
secreted
of
the
results
vacuolar
suggest
by
the
that
vacuolar
cells.
The production of digestive enzymes and the digestion and the absorption of nutritive substances are major subjects in the digestive physiology of animals. Voluminous literature on the gut structure of copepods has described the presence of vacuolar and non-vacuolar midgut cells in both free-living and parasitic copepods.1-9) Many investigators inferred that the vacuolar cells are the secretory cells producing digestive enzymes. However, light microscopic examinations using conventional methods and early electron microscopic observations were not always successful because of the limited resolution in light microscopy and poor tissue preservation for electron microscopy. Recent ultrastructural examinations have shown that the vacuolar cells participate in the intracellular digestion of the gut contents.10-18) Most of these studies have also suggested that the nonvacuolar cells participate not only in absorbing nutritive substances but also in secreting digestive enzymes. However, literature dealing with the histochemistry and ultrastructure of the copepod alimentary canal is still meager, particularly for parasitic copepods, and much remains to be solved. In order to extend our knowledge of the digestive physiology of copepods, we carried out histochemical and ultrastructural examinations on the alimentary canals of some parasitic copepods. Faculty of Fisheries,
Nagasaki
University,
Hongochimachi,
Nagasaki
This
paper
describes
examinations
to
the
results
visualize
of
Materials
and
activities.
Methods
Animals We
used
six
copepods. names
species
Their of
chyliurus
hosts
of
(paren
quintus (short-necked
necked
clam),
theses)
as
follows:
japonicus
Ostrincola
koe
and
brackish in
Con-
(short-
(top
(bluegill)
collected
common
Modiolicola
yamagutii
(common
shell), Ergasilus
goby).
All
Nagasaki
the
Prefecture.
Histochemistry A
freeze-substitution
preparation from
foil
frozen
and
nitrogen were
tissue the
-60•Ž
for
4
Sections
of
6 ƒÊm
ed
on
glass
following activities: the
metal
phosphatase
were
in
and
for azo
salt
dye
method (EC
Japan(高
ethanol
detection
of
of
for
the
852,
越 一 馬:長崎
Japan(吉
良夫:長崎市
本 河 内 町).
azo
the
enzymic
Burston
3.1.3.1)19,20);
Nagasaki
mountused
of
Gomori
about
paraffin.
and We
method
specat
cut
water. the
liquid
in
were
the
aluminium with
frozen
embedded
thickness without
methods
on
The in
for specimens
chilled
dehydrated
slides
the
placed
-160•Ž.
days
used
The
isopentane
about then
was
sections.
hosts in
at
imens
method
of
obtained
Bunkyomachi,
850,
the
are
clam),
were
cyclopoid
and
clam),
Panaietis
Neoergasilus
specimens
female
names
(short-necked
bifidus
orientalis
adult
specific
産 学 部).*2 Present adress:
histochemical
hydrolase
and alkaline dye
and
学 水
metal
salt
acid
methods
method
of
3.1.1.2
and/or
of
al.
non-specific
3.1.1.6)20);
for
azo
azo
the
azo
dye
(EC
dye
method
of
Hayashi
(EC
experiments
consisted
medium by
Table
the
in
1.
3.2.2.31)23). in
either
adding
enzymes
(EC
method
by
specific
The
modifying
omitting
the the
inhibitor
or
subinacti-
sections.
Percentage
ary canal
in length
of each
Results
dye
esterase
the
for ƒÀ-glucuronidase
or
Anderson the
for ƒÀ-N-acetylglucosaminidase
incubation
vating
and
3.1.3.2)21);
for
EC
and
control
strate
Barka
(EC
Gomori
Hayashi
3.2.1.30)22) et
of
phosphatase
part
Gross Anatomy of the Midgut The midgut occupied the greatest part of the alimentary canal (Table 1). It consisted of a wide and sac-like anterior zone and a narrow, shorter posterior zone. The anterior zone consisted of vacuolar cells and non-vacuolar cuboidal or columnar cells. The vacuolar cells containing large secretory vacuoles protruded into the gut lumen. The glandular epithelium containing the
of the alimentary
canal
with respect
to the total
length
Table
2.
Hydrolase
activities
detected
in the midgut
of parasitic
copepods
aliment-
vacuolar cells occupied from bifidus) to 71.1% (Panaietis total alimentary canal length. dbnsisted of a single type of mous cells.
Figs.
1-5.
were
lumen; Fig.
2.
shown
of hydrolases as
dark
stain.
in
the
Arrows
midgut
of
indicate
the
the
parasitic
copepods.
vacuolar
cells.
Fg,
Positive foregut;
reac-
L,
midgut
in the
striated
Magnification •~400.
1.
Alkaline
border; Fig.
Alkaline Phosphatase (Enzyme-I) The striated border of the non-vacuolar cells in both anterior and posterior zones of the midgut
Histochemistry
tions
Histochemistry The results obtained were summarized in Table 2.
34.7% (Modiolicola yama;uitii) of the The posterior zone non-vacuolar squa-
Acid
japonicus; secretory method.
phosphatase
b,substrate-free phosphatase d,
Ergasilus
vacuoles.
activity control. activity. orientalis.
a, c, d, Naphthol
in Neoergasilus Naphthol a, Conchyliurus Positive AS-BI
japonicas. AS-BI quintus;
reactions phosphate,
a, positive
phosphate,
Garnet
b, Panaietis were
seen
hexazonium
reaction GBC.
yamagutii;
in the
striated
pararosanilin;
c, Neoergasilus border
and
b, metal
the salt
of the three species (Table 2 and Fig. 1). considerably among were also observed in epithelial cells.
showed positive reactions The staining intensity varied species. Positive reactions the cytoplasm of the foregut
ƒÀ-N-Acetylglucosaminidase The the
secretory
five
species
(Table
2 and
The the 2
and
tivity as
Fig.
the
vacuolar
cells
positive
of
reactions
4).
5).
case
of
showed The
staining
the
the
positive
vacuolar
localization
intensity
cells
reactions of
were
of
(Table
enzymic
almost
ac-
the
same
of ƒÀ-N-actylglucosaminidase.
Discussion
Acid
Non-specific Esterase (Enzyme-III) The secretory vacuoles of the vacuolar cells of the four species associated with marine shellfish showed intense reactions, while those of the two species of fish parasites showed no positive reactions (Table 2 and Fig. 3).
weak
vacuoles
species
and
in
showed
(Enzyme-V)
secretory
five
of
Fig.
ƒÀ-Glucuronidase
Acid Phosphatase (Enzyme-II) The secretory vacuoles of the vacuolar cells of all the six species and the striated border of the non-vacuolar cells in both anterior and posterior zones of the midgut of the five species showed positive reactions (Table 2 and Fig. 2). The staining intensity of the secretory vacuoles varied among species, as well as specimens.
(Enzyme-IV) vacuoles
been the
and
alkaline
detected
in
intestinal
and
animals.
In
phatase
of
the
phosphatase
only
in
striated
border
renel
has
brush
and
cytoplasmic
midgut has
in gland been
vacuoles the
midgut
of alkaline
detected
of
various phos-
the
striated
cells.24-26) detected
but and
have
border
epithelia
crustaceans, been
activity
of
activities or
tubule
midgut
Acid
the
striated
decapod
activity
border
phosphatase the
also midgut
Fig. 3. Non-specific esterase activity. a, Conchyliurus quintus; b, Modiolicola bifidus; c, Ostrincola koe; d. Panaietis yamagutii. Positive reactions were seen in the secretory vacuoles. Numerous dark granules in the non-vacuolar cells of Ostrincola koe (c) are pigment granules . Naphthol AS acetate, Garnet GBC.
not in
the
gland
Fig.
4. ƒÀ-N-Acetylglucosaminidase secretory
vacuoles;
azonium Fig.
activity
b, substrate-free
in
control.
Neoergasilus
japonicus.
Naphthol
AS-BI
a, positive
reaction
in
N-acetyl-ƒÀ-glucosaminide,
the hex-
pararosanilin.
5. ƒÀ-Glucuronidase reactions
activity.
were
seen
in
the
a,
Conchyliurus
secretory
vacuoles.
quintus;
b,
Naphthol
Neoergasilus
japonicus.
AS-BI ƒÀ-glucuronide,
Positive hexazonium
pararosanilin.
cells by light microscopy.24-27) scopically, acid phosphatase
Electron microactivity has been
known to occur in both lysosomal and such extralysosomal sites as in the basal tuhular system, Golgi lamellae extralysosomal activity
have
and mitochondrial cristae.27) Such localizations of acid phosphatase been
shown
cells.28,29) We could study whether acid in the striated
in mammalian
not confirm phosphatase
border
It a
is
well
established
lysosomal
that
marker
in the present demonstrated
of the midgut
parasitic copepods is lysosomal brane associated enzymes. acid
somes.
are
ent
study
are
lysosomal
aminations
*
also
Enzymic thus
known
Submitted
to
occur
detected
that
that
is es-
and ƒÀ-glu-
vacuoles. showed
mem-
Non-specific
activities suggest
or
phosphatase
terase, ƒÀ-N-acetylglucosaminidase curonidase
cells of the
origin
enzyme.
tissue
the
to this Journal
.
glands
are,
site
of
digestive
the cytoplasmic cells of the typicus. ticipate
only
(mouth in the
production
in
these
of the midgut vacuolar copepod Centropages
This type of vacuoles is known to parin intracellular digestion of food and, in
lyso-
this regard,
from the secretory copepods in the
ex-
enzyme
vacuoles free-living
pres-
are
the
parasitic copepods. Arnaud et al.13,14) detected acid phosphatase and arylsulfatase activities in
the
vacuoles
as is known,
present study were small glands showing no positive enzymic reactions. It is highly probable, therefore, that the vacuolar cells are the primary
the
vacuoles
so far
into the alimentary canal labral glands examined
in
ultrastructural
secretory
labral
gland opening cavity). The
in
secretory
Our the
discharged into the gut lumen after the maturation of the secretory cells.* In copepods, the
the vacuoles
should
vacuoles found present study.
be distinguished in the parastitic Ultrastructural
findings on the production of digestive enzymes will be reported in our forthcoming paper.*
ƒÀ-N-Acetylglucosaminidase are
known
to
lysaccharides. liurus koe,
all
inferred
be
contents
of
the
pepods an
it
tivities the
role
made by
difficulties
of
of
this
study.
of
of
of
food. proteaae
the
decisive
endocytotic of
of
to
well
in
coplay
ac-
method
Due no
the
these
glycosidases
visualize
only
further
In
the
cells
visualization
subject
the japon-
substrate-film
method
midgut
inhabiting
digestion
a
the
vacuolar
The the
this
localized
midgut
both
to
use
except
reaction
the
attempt
the
Neoergasilus
that
the
course
obtained
in
an
hosts,
hosts.30)
mucopolysacin
respectively.*1
likely
the
the
inhabiting
orientalis,
seems
of
acid
esophagus,
gill,
important We
of
ConchyOstrincola
by
yamagutii
Ergasilus
and
cavity
observed
Panaietis and
of and
secreted
frequently
cavity and
mantle
amount
was
mucopo-
food bifidus
mucus
considerable
skin
principal
the
to
charides
icus
decomposing
Modiolicola
inhabiting
mouth
in
The
quintus,
was A
and ƒÀ-glucuronidase
participate
results
were
defined
positive
vacuoles
Panaietis
protease
in
technical
of
the
yamagutii.*2 activities
will
be
study.
References 1)
A.
Steuer:
Arb.
zool.
Inst.
Univ.
Wien
ges
Hydrobiol
, 15,
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