215
The Distribution of Mitochondria-Rich Cells in the Gills of
Air-Breathing Fishes
Hui-Chen Lin*
Wen-Ting Sung
Department of Biology, Tunghai University, Taichung 407,
Taiwan, Republic of China
Accepted 10/21/02
ABSTRACT
Respiration and ion regulation are the two principal functions
of teleostean gills. Mainly found in the gill filaments of fish,
mitochondria-rich cells (MRCs) proliferate to increase the
ionoregulatory capacity of the gill in response to osmotic challenges. Gill lamellae consist mostly of pavement cells, which
are the major site of gas exchange. Although lamellar MRCs
have been reported in some fish species, there has been little
discussion of which fish species are likely to have lamellar
MRCs. In this study, we first compared the number of filament
and lamellar MRCs in air-breathing and non-air-breathing fish
species acclimated to freshwater and 5 g NaCl L⫺1 conditions.
An increase in filament MRCs was found in both air-breathing
and non-air-breathing fish acclimated to freshwater. Lamellar
MRCs were found only in air-breathing species, but the number
of lamellar MRCs did not change significantly with water conditions, except in Periophthalmus cantonensis. Next, we surveyed the distribution of MRCs in the gills of 66 fish species
(including 29 species from the previous literature) from 12
orders, 28 families, and 56 genera. Our hypothesis that lamellar
MRCs are more likely to be found in air-breathing fishes was
supported by a significant association between the presence of
lamellar MRCs and the mode of breathing at three levels of
systematic categories (species, genus, and family). Based on this
integrative view of the multiple functions of fish gills, we should
reexamine the role of MRCs in freshwater fish.
Introduction
Fish gills are organs with multiple functions, including gas
exchange, ion regulation, and acid-base balance (Laurent and
* Corresponding author; e-mail: [email protected].
Physiological and Biochemical Zoology 76(2):215–228. 2003. 䉷 2003 by The
University of Chicago. All rights reserved. 1522-2152/2003/7602-2084$15.00
Perry 1991; Jurss and Bastrop 1995; Marshall and Bryson 1998;
Randall and Brauner 1998; Evans et al. 1999). The four pairs
of branchial arches in the teleost consist of filaments and lamellae, both covered with epithelial cells, which can be further
characterized into pavement cells, mitochondria-rich cells
(MRCs, formerly chloride cells), mucous cells, and undifferentiated cells (Laurent 1984; Perry 1998; Evans et al. 1999).
Pavement cells account for more than 90% of the gill surface
(mostly in the lamellae) and are considered to be the site of
gas exchange (Laurent and Dunel 1980; Perry 1998), although
recent evidence suggests that pavement cells may also play an
important role in ion and acid-base regulation (Perry 1998;
Evans et al. 1999). MRCs are mainly found in the gill filaments
of teleosts. In general, MRCs are believed to be the site of ion
extrusion by fish in seawater and ion uptake by fish in freshwater (Laurent and Perry 1991; Perry 1998; Evans et al. 1999;
Piermarini and Evans 2000).
Nevertheless, several studies have reported the presence of
lamellar MRCs in various teleost species, and lamellar MRCs
are inducible in certain species acclimated in fresh or deionized
water. For example, proliferation of lamellar MRCs was found
in Anguilla anguilla after 1 wk acclimation in deionized water
(Laurent and Dunel 1980) and also was found in the freshwateracclimated Atlantic stingray (Dasyatis sabina; Piermarini and
Evans 2000). Most of the conclusions on the role of lamellar
MRCs have been drawn on a species-specific basis, and thus
far there has been no systematic investigation of their functions.
Because of their rather large and oval shape, lamellar MRCs
increase diffusion distance and therefore impede gas exchange
(Greco et al. 1995; Perry 1998; Piermarini and Evans 2000). If
a fish can exchange gases from other parts of the body, the gills
may play a more important role in alternative physiological
functions such as osmoregulation and acid-base balance. This
directed our attention to air-breathing fishes, which have the
ability to exchange gases directly with the aerial environment
(Graham 1997). Depending on their behavioral and habitat
conditions, air-breathing fishes are categorized into two modes,
amphibious and aquatic (Graham 1997). The air-breathing organs include the skin, the lungs and respiratory gas bladders
of the more primitive teleosts, and structures derived from
buccal, pharyngeal, and branchial cavities and also from different parts of digestive tracts of the more advanced teleosts
(Graham 1997).
Since air breathing has evolved independently many times
among the fishes (Graham 1997) and this trait can be found
among species from various habitats, it is necessary to consider
216 H.-C. Lin and W.-T. Sung
the divisions of fish according to their life history and habitats.
Freshwater (FW) fishes are generally classified into primary,
secondary, and peripheral FW fishes (Myers 1938 [cited in
Helfman et al. 1997]). While primary FW fishes spend their
entire life cycles in FW, secondary FW fishes are generally restricted to FW but may occasionally enter saltwater (Helfman
et al. 1997). Peripheral FW fish species have taken up more or
less permanent residence in FW or spend part of their life cycle
in FW and another part in marine habitats (Helfman et al.
1997).
In this study, we conducted two independent experiments
to elucidate whether the likelihood of possessing lamellar MRCs
is correlated with the life history and air-breathing capability
of the fish. First, we compared the relative changes in the numbers of filament and lamellar MRCs in air-breathing and nonair-breathing fishes when they were maintained in FW and
brackish water (5 g NaCl L⫺1; hereafter, this artificial seawater
will be referred to only as 5 g L⫺1). Second, the distribution of
both filament and lamellar MRCs in 12 orders, 28 families, 56
genera, and 66 species (including 29 species from the literature)
was examined by scanning electron microscopy, zinc-iodide
osmium staining (ZIO), or immunofluorescent staining. We
proposed that air-breathing fish are more likely to have lamellar
MRCs while non-air-breathing fish are less likely to have them.
The association between air-breathing capability and the presence of lamellar MRCs was tested. We also examined the association between the distribution of lamellar MRCs and the
divisions of FW fishes.
Material and Methods
Fish-Rearing and Acclimation Conditions
Most FW fishes cannot tolerate either full seawater or deionized
water (Helfman et al. 1997). For FW species, fish were first
acclimated in FW or 2 g L⫺1 artificial saltwater for at least 1
wk (but not longer than 1 mo) and then transferred to FW
and 5 g L⫺1, respectively, for 1 wk. For peripheral FW species,
fish were first acclimated in 7 g L⫺1 artificial saltwater for at
least 1 wk (but not longer than 1 mo) and then further transferred to FW and 5 g L⫺1, respectively, for 1 wk. Water compositions for FW and 5 g L⫺1 were as follows (in mM): Na⫹
1.30, 85.22; K⫹ 0.17, 4.10; Ca2⫹ 0.30, 2.95; Mg2⫹ 0.21, 4.17;
Cl⫺1 1.09, 76.25; and pH 7.39, 7.20, respectively.
Fish were kept in aerated water at 28⬚ Ⳳ 1⬚C on a photoperiod of 14 L : 10 D and were fed daily with commercial pellets.
Artificial saltwater was prepared by adding commercial salt
(Coralife Scientific Grade Marine Salt, Carson, Calif.) to local
tap water and aerating the mixture for at least 1 mo. About
one-half of the tank water was changed every 3 d.
Sample Preparation
Gill filaments from one side of the second branchial arch were
removed and prefixed in phosphate-buffered, 4% paraformaldehyde (Sigma) and 5% glutaldehyde (Wako) for 8–12 h at
4⬚C, pH 7.4. They were rinsed three times with 0.1 M phosphate
buffer (PB; [0.2 M NaH2 PO4 7 2H2O ⫹ 0.2 M Na2HPO4 7
2H2O] : deionized water p 1 : 1, pH p 7.2) before being postfixed with phosphate-buffered 1% osmium tetroxide for 1–2
h. The tissues were dehydrated in seven ascending concentrations of ethanol (30%–100%) and in 100% acetone and were
then dried using a Hitachi HCP-2 critical-point drier. After
sputter-coating for 3 min with a gold-palladium complex using
an Eiko iB-2 vacuum evaporator, the filaments were examined
using a scanning electron microscope (SEM; Hitachi, S-2300).
Gill filaments from the other side of the second branchial
arches were collected for either ZIO or immunofluorescent
staining. For ZIO fixation, we followed the procedures described by Watrin and Mayer-Gostan (1996). In particular, the
zinc iodide (ZnI2) solution must be prepared immediately before use (0.24 g ZnI2 powder [Aldrich] in redeionized water to
8 mL total volume). Tissue samples were fixed in the dark for
24–30 h, depending on the size of the gills, and rinsed with
deionized water for 4–6 h before serial alcohol/xylene dehydration and paraffin embedding. Six-micrometer sections
(Leica, RM2025) were glued on glass slides coated with polyl-lysine (Sigma). After paraffin removal (with 100% xylene, 2
min # 3 times), sections were mounted with Permount
mounting medium. According to Greco et al. (1995) and Watrin and Mayer-Gostan (1996), MRCs with numerous plasma
membrane infoldings will give a black color with ZIO staining.
For immunofluorescent staining, fish gills were fixed with a
2% paraformaldehyde (Sigma) and 0.5% glutaldehyde (Wako)
mixture at 4⬚C for 10 min and rinsed three times with 0.1 M
PB. Samples were placed in 30% sucrose/deionized water for
1 h before embedding with tissue-freezing medium (Shandon).
Sections 8–10 mm thick (Leica, Jung frigocut 2800N) were
glued on glass slides coated with poly-l-lysine (Sigma) and were
rinsed three times with 0.1 M PB. Sections were placed in 10%
normal goat serum at room temperature for 1 h and then
incubated at 4⬚C for 14–16 h with a 1 : 100 dilution of a-5
monoclonal antibody against the a-subunit of chicken Na⫹,K⫹ATPase (Developmental Studies Hybridoma Bank, Iowa City,
Iowa). After washing (three times) in 0.1 M PB, the sections
were incubated for 1 h at room temperature in a 1 : 200 dilution
of fluorescein isothiocyanate–conjugated goat anti-mouse IgG
(Jackson).
The Relative Numbers of MRCs in Filaments and Lamellae
Our goal was to compare the numbers of filament and lamellar
MRCs in both air-breathing and non-air-breathing fishes with
salinity change. Six fish species were included in this experi-
Mitochondria-Rich Cells in the Gills of Air-Breathing Fishes
ment: Colisa lalia, Trichigaster trichopterus, Periophthalmus cantonensis, Oreochromis mossambicus, Monodactylus argenteus,
and Toxotes jaculator. The first three species are air breathers
while the latter three are not. They were acclimated and transferred, respectively, to FW and 5 g L⫺1 following the procedure
described previously. For every fish species, at least four individuals from each water condition were collected for MRC
determination. Within the same individual, at least four different regions were examined and averaged for filament MRCs.
Ten lamellae per fish gill were randomly chosen for quantification.
The afferent side of a gill filament from the second branchial
arch was examined using SEM (Hitachi, S-2300). Because the
size of the filament varied among species, the samples were
examined under different magnifications. Therefore, the actual
areas for counting MRCs on the SEM screen were not the same,
ranging from 400 to 1,080 mm2. For comparison, we later standardized the results to become number of MRCs per 1,000
mm2.
Lamellar MRCs were examined from the sagittal sections
after ZIO fixation preparation. To avoid MRCs located on the
base of a lamella, only those cells that were at least 5 mm away
from the base were included. The lengths of the lamellae were
determined by the use of Image-Pro Plus 4.0 data-analysis software (Media Cybernetics, Silversprings, Md.), and the numbers
of lamellar MRCs were later standardized as number of MRCs
per 100 mm of lamella in length. The number of MRCs in FW
divided by the number of those in 5 g L⫺1 gave an indication
of the degree of variation with salinity change.
The Association between Lamellar MRCs and Air-Breathing
Capability in 66 Fish Species
To examine the likelihood of having lamellar MRCs in airbreathing fish, we examined lamellar MRCs of the FW and
peripheral species under FW and 5 g L⫺1 conditions and of
marine species under seawater conditions (35 g L⫺1). At least
five individuals per species were examined for both filament
and lamellar MRCs. We followed Nelson (1994) for classification and nomenclature of the fish. For analysis purposes, fish
were classified as (1) air-breathing fish, (2) fish species that
have never been described as air breathers but belong to families
with known air-breathing species, and (3) non-air-breathing
fish according to Graham (1997). Air-breathing fish are generally further classified as those that have specific air-breathing
organs (amphibious mode) and those that have the ability of
aquatic surface respiration (aquatic mode) species (Graham
1997). We pooled these two groups of air-breathing species in
this analysis. The three species (Epiplatys fasciolatus, Fundulus
heteroclitus, and Glossogobius olivaceus) that have never been
described as air breathers but belong to the families with known
air-breathing species were not included in the genus- and
species-level statistical analyses. There were four categories in
217
the analysis of number of lamellar MRCs (Tables 1, 2): (1) a
definite presence of lamellar MRCs, (2) no lamellar MRC
found, (3) few if any lamellar MRCs, and (4) medium-dependent lamellar MRCs. When more than one species was examined in the same genus, we determined which category the
genus belonged to depending on whether these species were
unanimous in terms of the presence of lamellar MRCs. The
same criteria were applied to determine the category at the
family level, except that those genera that contain species with
no known air breathers but that belong to air-breathing families
were pooled into the air-breathing families. Species in the category of few lamellar MRCs had less than three incidences of
a single MRC among the 10 or more lamellae examined under
SEM. We assumed that these few isolated MRCs should not
have too much of an effect on either impeding gas exchange
or enhancing ion regulation, and, therefore, those species were
pooled into the category of no lamellar MRCs for further statistical analysis.
The association between air-breathing capability and the distribution of lamellar MRCs was tested using a 2 # 2 Fisher exact
statistical analysis. The experiments and handling of the animals
comply with the current laws of Taiwan, Republic of China.
Results
An increase in filament MRCs was found in both air-breathing
and non-air-breathing fish acclimated to FW, except Monodactylus argenteus. Lamellar MRCs were only found in airbreathing species, but the numbers of lamellar MRCs did not
change significantly with water conditions, except in Periophthalmus cantonensis (Fig. 1). Both Colisa lalia and Trichigaster
trichopterus are primary FW fish with labyrinths in their gill
chambers to enhance air breathing. For both species, a significantly higher number of filament MRCs were found in those
from FW than from 5 g L⫺1 (Fig. 2). However, no difference
was found in the number of lamellar MRCs between the two
media in either species.
Periophthalmus cantonensis is an amphibious, peripheral FW
fish. The number of MRCs in filaments was significantly higher
in FW. On the contrary, lamellar MRCs were significantly higher
in 5 g L⫺1 than in FW (Fig. 2).
The secondary FW fish Oreochromis mossambicus had no
MRCs in the lamellae, and the number of filament MRCs was
significantly higher in FW than in 5 g L⫺1. In the two peripheral
FW fish M. argenteus and Toxotes jaculator, very few lamellar
MRCs could be found at the base of the lamellae. A significantly
higher number of filament MRCs were found in FW for T.
jaculator, while there was no significant difference in the number of filament MRCs between the two water conditions for
M. argenteus (Fig. 2).
We examined 37 fish species for the presence of lamellar
MRCs. Among them, 19 species were examined in 5 g L⫺1 and
FW conditions; two in both saltwater and FW; 13 in only FW;
Table 1: Examination of the presence of lamellar mitochondria-rich cells (MRCs) in the gills of 66 fish species, including 29 species reported in the previous
literature
Medium
Order, Family, and Species
218
Air-breathing fish with specific air-breathing organs:
Perciformes:
Belontiidae:
Betta splendens
Colisa lalia
Macropodus opercularis
Trichogaster leeri
Trichogaster trichopterus
Gobiidae:
Periophthalmus cantonensis
Periophthalmodon schlosseri
Boleophthalmus pectinirostris
Scartelaos virides
Cypriniformes:
Cobitididae:
Cobitis taenia
Misgurnus anguillicaudatus
Siluriformes:
Loricariidae:
Hypostomus plecostomus
Callichthyidae:
Corydoras aeneus
Acipenseriformes:
Lepisosteidae:
Lepisosteus osseus
Anguilliformes:
Anguillidae:
Anguilla anguilla
Anguilla japonica
Anguilla rostrata
Anguilla marmorata
Cyprinodontiformes:
Aplocheilidae:
Division SW 5 g L⫺1 FW Methodology
1⬚
1⬚
1⬚
1⬚
1⬚
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
p
p
p
p
⫹
⫹a
⫺
⫹
⫺
Na⫹,K⫹-ATPase immunohistochemistry
References
Wilson et al. 2000
f
1⬚
1⬚
⫹ LM and TEM
⫹ LM and TEM
Pisam et al. 1990
Kikuchi 1977
1⬚
⫹ LM, TEM, and SEM
Fernandes and Perna-Martins 2001
1⬚
⫹
2⬚
⫹
p
p
p
p
⫺
⫺
⫹
⫺ LM and TEM
⫺ LM and TEM
⫹ Na⫹,K⫹-ATPase immunohistochemistry
and quantified
f SEM and TEM
f
Laurent and Dunel 1980
Shirai and Utida 1970
Sasai et al. 1998
Perry et al. 1992a, 1992b
Rivulus marmoratus
Air-breathing fish with aquatic surface respiration
capability:
Cypriniformes:
Cyprinidae:
Balantiocheilus melanopterus
Barbus tertrazona
Labeo erythrurus
Carassius auratus
p
⫺
⫺ LM and TEM
1⬚
1⬚
1⬚
1⬚
⫹
Carassius carassius
Cyprinus carpio
1⬚
1⬚
⫹
219
Gobio gobio
Paracheilognathus himantegus
Fish not described as air breathers in Graham
(1997) but belonging to families with
known air-breathing species:
Cyprinodontiformes:
Cyprinodontidae:
Epiplatys fasciolatus
Fundulus heteroclitus
Perciformes:
Gobiidae:
Glossogobius olivaceus
Non-air-breathing fish:
Characiformes:
Characidae:
Pristella maxillaries
Prionobrama filigera
Colossoma bidens
Serrasalmus nattereri
Siluriformes:
Ictaluridae:
Ictalurus nebulosus
Perciformes:
Cichlidae:
Astrenotus ocellatus
Haplochromis ahli
1⬚
1⬚
2⬚
2⬚
p
⫺
⫺
⫺
f
⫹
f
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺ SEM and quantified
Na⫹,K⫹-ATPase immunohistochemistry
Kikuchi 1977
Lee et al. 1996a
Kikuchi 1977
Kikuchi 1977
Hwang 1988
Lee et al. 1996a
Pisam et al. 1990
Hossler et al. 1985
Katoh et al. 2000
⫹
1⬚
1⬚
1⬚
1⬚
⫺
⫺
⫺
⫺
1⬚
f
2⬚
2⬚
LM and TEM
SEM and quantified
LM and TEM
LM and TEM
LM and TEM
SEM and quantified
LM and TEM
King et al. 1989
⫺
⫺
⫺
⫺
SEM and TEM
Perry et al. 1992a, 1992b
Table 1 (Continued)
Medium
Order, Family, and Species
Melanochromis auratus
Oreochromis aureus
Oreochromis mossambicus
Oreochromis niloticus
220
Pelvicachromis pulcher
Pterophyllum scalare
Symphysodon aequifasciatus
Tropheus duboisi
Cichlasoma biocellatum
Cyprinodontiformes:
Oryziidae:
Oryzias latipes
Poecilidae:
Lebistes reticulatus
Poecilia reticulata
Poecilia latipinna
Perciformes:
Lobotidae:
Datnioides quadrifasciatus
Monodactylidae:
Monodactylus sebae
Monodactylus argenteus
Percichthyidae:
Lateolabrax japonicus
Toxotidae:
Toxotes jaculator
Salmoniformes:
Salmonidae:
Oncorhynchus mykiss
(also known as Salmo gairdneri)
Oncorhynchus keta
Division SW 5 g L⫺1 FW Methodology
2⬚
2⬚
2⬚
2⬚
2⬚
2⬚
2⬚
2⬚
2⬚
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
References
LM and TEM
Avella et al. 1993
LM and TEM
LM and TEM
Cioni et al. 1991
Avella et al. 1993
LM
Mattheli and Stroband 1971
⫺
⫺ SEM and quantified
Lee et al. 1996b
⫺
⫺
⫺ SEM
⫺ LM
⫺
Pisam et al. 1987
Shikano et al. 1998
p
⫺
⫺
p
p
⫺
f
f
f
2⬚
2⬚
2⬚
2⬚
p
⫺
⫺
p
f
p
p
⫹ Na⫹,K⫹-ATPase immunohistochemistry
⫺
⫹
Hirai et al. 1999
f
f
⫹
⫹
⫹
SEM and quantified
Treated with cortisol; SEM
SEM
Chum salmon fry Na⫹,K⫹-ATPase
immunohistochemistry
⫹ Mature chum salmon Na⫹,K⫹-ATPase
immunohistochemistry
Laurent and Hebibi 1989
Perry et al. 1992a, 1992b
Laurent and Perry 1990
Uchida and Kaneko 1996;
Uchida et al. 1996
Uchida et al. 1997
Salmo trutta
221
Osmeriformes:
Plecoglossidae:
Plecoglossus altivelis
Perciformes:
Centropomidae:
Ambassis gymnocephalus
Leiognathidae:
Leiognathus nuchalis
Percichthyidae:
Morone saxatilis
Teraponidae:
Terapon jarbua
Mugiliformes:
Mugilidae:
Mugil cephalus
Pleuronectiformes:
Soleidae:
Solea solea
Scophthalmidae:
Scophthalmus maximus
p
⫺
p
⫹ SEM and quantified
⫹ Na⫹,K⫹-ATPase immunohistochemistry
Brown 1992
Seidelin et al. 2000
⫹ LM and TEM
Hwang 1988
m
⫺
⫺
m
⫺
⫺
m
⫺
⫺ LM, TEM, and SEM
⫺
m
King and Hossler 1991
⫺
m
⫺
SEM
Hossler et al. 1979
m
⫺
LM and TEM
Dunel-Erb and Laurent 1980
m
⫺
⫺
⫺ LM and TEM
ZIO
Pisam et al. 1990
Watrin and Mayer-Gostan 1996
Note. Fish were classified into three groups: (1) air-breathing fish, (2) non-air-breathing fish, and (3) fish species that have never been described as air breathers but belong to the families with known
air-breathing species, according to Graham (1997). The divisions of the fish families used are primary freshwater (FW) fish (1⬚), secondary FW fish (2⬚), peripheral FW fish (p), and marine fish (m),
following Helfman et al. (1997). The 37 species we examined were acclimated in both 5 g L⫺1 artificial saltwater and FW. In the “Medium” columns, we present the results as a definite presence of lamellar
MRCs (⫹), no lamellar MRCs (⫺), or few if any lamellar MRCs (f). SW p saltwater.
a
Acclimated at 15 g L⫺1.
222 H.-C. Lin and W.-T. Sung
Table 2: Summary of the number of species with lamellar mitochondria-rich
cells in the gills
Air-Breathing Fish
Division
⫹
⫺
1⬚
2⬚
p
m
15
1
2
0
0
0
3
0
⫹/⫺
f
2
0
2
0
0
0
2
0
Non-Air-Breathing Fish
Non–Air
Breather
but in AirBreathing
Family
⫹
⫹
⫺
0
0
1
0
0
2
0
0
0
0
1
0
⫺
⫹/⫺
f
4
15
1
7
1
0
2
0
0
0
5
0
Note. The notations are the same as in Table 1, and those that changed among the three media
(saltwater, 5 g L⫺1 artificial saltwater, and freshwater) are indicated as ⫹/⫺.
one in saltwater; one in 5 g L⫺1; and one in saltwater, 5 g L⫺1,
and FW conditions. For the 29 species reported in the previous
literature, there were two in saltwater; one in 5 g L⫺1; 10 in
FW; three in both 5 g L⫺1 and FW; 12 in both saltwater and
FW; and one in saltwater, 5 g L⫺1, and FW conditions.
There were 27 air-breathing fish, 36 non-air-breathing fish,
and three species that have never been described as air breathers
but belong to families with known air-breathing species according to Graham (1997). The results on the presence of lamellar MRCs of fish in different water conditions are presented
in Table 1. Lamellar MRCs were found in 20 species, among
which 18 were air breathers. There were 39 species that have
zero or few lamellar MRCs, and 32 of them were non–air
breathers. The number of species in each category is summarized in Table 2. There was a significant association between
the presence of lamellar MRCs and the mode of breathing on
all three levels of systematic categories (species, genus, and
family; Table 3). Seven species did not have a consistent observation among different media: Boleophthalmus pectinirostris,
Anguilla japonica, Monodactylus sebae, Lateolabrax japonicus,
Oncorhynchus mykiss (previously known as Salmo gairdneri),
Oncorhynchus keta, and Salmo trutta. These species were not
included in the statistical analysis but will be discussed separately later.
According to the division to which the fish family belongs
(Helfman et al. 1997), there were 22 primary FW species, 18
secondary FW species, 19 peripheral FW species, and seven
marine species included in this study (Table 2). Among them,
15 primary FW species had lamellar MRCs, and 15 secondary
FW species had no lamellar MRCs. There was a lot of variation
in the peripheral FW species, and no generalization can be
made currently. Most of the variation in the presence of lamellar
MRCs was found in peripheral FW species from the orders
Perciformes, Anguilliformes, Cyprinodontiformes, Salomoniformes, and Osmeriformes. None of the seven marine fish species had lamellar MRCs.
When the division of the fish and air-breathing capability
were considered simultaneously, we found that those primary
FW fish species that are also air breathers are more likely to
have lamellar MRCs. Because almost all secondary FW species
are non-air-breathing species (Graham 1997; Helfman et al.
1997), the confounded relationship was not resolved until we
examined Lepisosteus osseus, an air-breathing and secondary FW
species. However, it was not possible to conduct a statistical
analysis with both air-breathing capability and the division of
fish family considered at the same time due to the low number
of observations (less than five) in certain categories in Table 2.
Discussion
Although many authors have reported that lamellar MRCs can
be found in several fish species acclimated in FW or soft water
(see Table 1 for references) or can be induced by hormones
(Perry et al. 1992a, 1992b; Bindon et al. 1994; Perry 1998; Dang
et al. 2000), there has been little discussion as to which kinds
of fish are more likely to possess lamellar MRCs. Nevertheless,
it is suggested that lamellar MRCs may play a role in ion uptake
in FW species, such as FW trout Salmo gairdneri (Laurent and
Perry 1990), sea trout Salmo trutta (Brown 1992), rainbow trout
Oncorhynchus mykiss (Greco et al. 1995), chum salmon Oncorhynchus keta (Uchida et al. 1997), Japanese eel Anguilla japonica (Sasai et al. 1998), Japanese sea bass Lateolabrax japonicus (Hirai et al. 1999), and armored catfish Hypostomus
plecostomus (Fernandes and Perna-Martins 2001) and in ammonia elimination in the mudskipper Periophthalmodon schlosseri (Wilson et al. 2000). These additional roles played by gill
lamellae are not without a cost. According to a review by Perry
(1998), proliferation of lamellar MRCs would lead to an increase in blood-to-water diffusion distance and, therefore, an
impairment of gas transfer. In addition, several compensatory
physiological responses, such as hyperventilation and a higher
Mitochondria-Rich Cells in the Gills of Air-Breathing Fishes
223
Figure 1. Mitochondria-rich cells (MRCs) in gill lamellae. A, Immunofluorescent staining and (B) zinc-iodide osmium (ZIO) staining indicating
a definite presence of lamellar MRCs in Periophthalmus cantonensis. C, Immunofluorescent staining and (D) ZIO staining indicating filament
MRCs but no lamellar MRCs in Monodactylus argenteus. E, Negative control for immunofluorescent staining. F p gill filament; I p
interlamellar region; L p gill lamella. Arrowheads indicate the MRCs.
affinity of hemoglobin to oxygen, were observed following lamellar MRC proliferation (Perry 1998). Apparently, there is a
trade-off between gas exchange and ion regulation in gill
lamellae.
Whether there is a functional differentiation between gill
filaments and lamellae is constantly under debate. Because of
concurrent increases in MRCs on both filament and lamellar
epithelia of S. gairdneri in FW, Laurent and Perry (1990) suggested that the function of MRCs is the same in filaments and
lamellae. However, Uchida et al. (1996), in their study of anadromous chum salmon (O. keta) fry, first proposed the hypothesis that MRCs in the lamellae may be the site of ion uptake
in hypoosmotic conditions and those in the filament are for
salt secretion in hyperosmotic conditions. This hypothesis of a
functional differentiation in fish gills was later supported by
Sasai et al. (1998), examining the catadromous Japanese eel (A.
japonica), and by Hirai et al. (1999) in Japanese sea bass L.
japonicus, a euryhaline marine fish. Nevertheless, the hypothesis
proposed by Uchida et al. (1996) is limited to those species
that have lamellar MRCs and is not applicable to those that
have no lamellar MRCs.
In this study, it appeared that unequal changes in the numbers of filament and lamellar MRCs were found in the three
species of air-breathing fish acclimated to FW and 5 g L⫺1.
224 H.-C. Lin and W.-T. Sung
Figure 2. Comparison of filament and lamellar mitochondria-rich (MR) cells in six species of fish acclimated to freshwater and 5 g L⫺1 saltwater
conditions. A–C, Three species of air-breathing fish. D–F, Three species of non-air-breathing species. One asterisk and three asterisks indicate
significant difference according to Student’s t-test (P ! 0.05 and P ! 0.0001, respectively).
There were 1.99 to 3.86 times more filament MRCs but only
0.69 to 1.08 times more lamellar MRCs when they were in FW
as compared to when they were in 5 g L⫺1. However, when the
multicellular complex of filament MRCs found in saltwater was
taken into account (Hwang 1988; Pisam et al. 1990), we may
have underestimated the number of filament MRCs in the 5 g
L⫺1 condition, and the difference between the relative changes
of MRCs in filaments and lamellae could become insignificant.
Therefore, our results should support the discussion by Laurent
and Perry (1990) that filament and lamellar MRCs have similar
functions due to their concurrent change in number.
The reason to include aquatic surface respiration breathers
in the category of air breathers was that these species can obtain
oxygen through a gulp of air or a breath of oxygen-saturated
water. Because air contains about 30 to 40 times more oxygen
than water (depending on temperature, salinity, etc.), the physiological demand for oxygen should be relieved. Therefore, it
is not surprising to find lamellar MRCs in these species.
The comparative methods for explaining adaptations (Harvey and Pagel 1991; Harvey and Purvis 1991) address the importance of seeking independent evolutionary origins of characteristics rather than simply counting species as independent
data points. The two most popular phylogenetically based statistical methods are the independent contrast methods (Fel-
Mitochondria-Rich Cells in the Gills of Air-Breathing Fishes
225
Table 3: Association between the presence of lamellar MRCs and air-breathing capability
at species, genus, and family levels
Presence
of Lamellar
MRCs
Species:
Air-breathing species
Non-air-breathing species
Species in the families with known air-breathing species
Genera:
Air-breathing genus
Non-air-breathing genus
Genus in the families with known air-breathing species
Families:
Air-breathing family
Non-air-breathing family
Yes
No/Few
P
18
1
1
7
30
2
3.55 # 10⫺8
16
1
1
4
26
2
4.81 # 10⫺8
5
1
3
13
.011
Note. The three species that have never been described as air breathers but belong to families with known airbreathing species according to Graham (1997) were not included in the genus- and species-level statistical analyses.
Species that fell into the category of few lamellar MRCs were pooled into the category of zero lamellar MRCs for
statistical analysis. Significance was calculated using 2 # 2 Fisher exact probability.
senstein 1985) and the generalized least squares models (Garland and Ives 2000). However, these two approaches are for
continuous variables rather than for discrete ones to which our
characters (i.e., presence of lamellar MRCs and air-breathing
ability) belonged. Instead, Ridley and Grafen (1996) and Grafen
and Ridley (1996) made an in-depth discussion on the discrete
comparative methods. In our analysis, we did not include any
phylogenetic tree due to the fact that the branch lengths of the
phylogeny or even the phylogeny itself are basically not known.
Instead, variation in the presence of lamellar MRCs was found
at the genus level in this study. Therefore, an intergeneric analysis should be considered as an independent comparison.
Nonetheless, a significant association between air-breathing capability and the presence of lamellar MRCs was found at the
species, genus, and family level (Table 3).
Primary FW fishes that are also air breathers usually have
lamellar MRCs, while those that are not air breathers (e.g., the
four species from Characiformes in Table 1) usually do not
have lamellar MRCs (Table 3). Most of the secondary FW species are not air breathers (Graham 1997), and no lamellar MRCs
were found. The only air-breathing secondary FW species examined in this study was Lepisosteus osseus, an ancient airbreathing fish according to Graham (1997), and it has MRCs
in the gill lamellae. However, until more air-breathing secondary species are examined, no suggestion can be made as to the
association between the presence of lamellar MRCs and airbreathing capability in the secondary FW fish species.
The situation became complicated in the peripheral FW
fishes, which primarily include the anadromous salmonids, catadromous anguillids, and certain Perciforme families (Table
1). Members from the two former groups have very complicated life histories and are susceptible to hormonal regulation
(such as cortisol and prolactin) during their salinity adaptation
(Laurent and Perry 1990; Perry et al. 1992a; McCormick 1994;
Uchida et al. 1998). Therefore, it should not be too surprising
to find inconsistent observations from different studies of A.
japonica (Shirai and Utida 1970; Sasai et al. 1998), O. mykiss
(Laurent and Hebibi 1989; Perry et al. 1992a, 1992b; Greco et
al. 1995), and O. keta (Uchida and Kaneko 1996; Uchida et al.
1996, 1997). The other two non-air breathers with lamellar
MRCs are the anadromous Plecoalossus altivelis (Hwang 1988)
and the amphydromous L. japonicus (Hirai et al. 1999).
The order Perciformes is the most advanced euteleostean
clade and contains at least 148 families and around 9,300 species
(Helfman et al. 1997). While most species in this order are
marine fish, there are eight families that are primary FW fish,
one family that is secondary FW fish, and nine families that
are peripheral FW. Together, these three divisions of FW fishes
account for about 2,100 species (Berra 1981). There are also
various types of air-breathing fishes within this order that deserve further discussion as soon as the phylogenetic systematics
becomes conclusive.
According to Graham (1997), air breathing is an ancient trait
that evolved independently in many fishes and there is great
diversity in this bimodal respiratory specialization. Of course,
the degree of air breathing should not be taken as a dichotomous trait but rather a continuous spectrum. But, in this study,
for the convenience of statistical analysis and sample size, we
only classified the species into air breathers and non-air breathers. Gills, as a multifunction organ, should constantly have
226 H.-C. Lin and W.-T. Sung
functional differentiation. That is, extrabranchial gas exchange
may lead to an increase in the role of ion regulation in gills.
Therefore, lamellar MRCs, which would greatly impede respiration, were found. However, since MRCs can also be found
in the gills of the hagfish Myxine glutinosa (Choe et al. 1999)
and in the gill lamellae of the FW stingray Dasyatis sabina
(Piermarini and Evans 2000), it is also considered as a rather
ancient trait. According to this rationale, the filament MRCs
are universally found in all fishes, and if air breathing should
evolve, distribution of lamellar MRCs may follow. Hirai et al.
(1999) observed the migration of MRCs from filaments to lamellae, which may explain the origin of lamellar MRCs.
In this study, only those species that have been documented
as air breathers were included in the analysis. For species that
have never been reported to be air breathers but belong to the
air-breathing families, a better description on their life histories
is needed. More studies on the physiology of the air-breathing
fishes from an integrative approach would allow a comparison
of the relative increase in both filament and lamellar MRCs
with salinity change in air-breathing and non-air-breathing
fishes.
Acknowledgments
We are grateful to Dr. Tsung-Han Lee and Dr. Pung-Pung
Hwang for their comments during the preparation of the manuscript. This study was supported by the National Science
Council (NSC 89-2311-B-029-006) and Academia Sinica (Major Group-Research Project), Taiwan, R.O.C. to H.C.L.
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