Transition in organ function during the evolution of air-breathing; insights from Arapaima gigas, an obligate air-breathing teleost from the Amazon
1 Department of Zoology, University of British Columbia, 6270 University
Blvd, Vancouver, BC, Canada, V6T 1Z4,
2 Department of Biology, San Diego State University, 5500 Campanile Drive,
San Diego, CA 92182, USA,
3 Centro Interdisciplinar de Investigação Marinha e Ambiental
(CIIMAR), Universidade do Porto, Rua dos Bragas 177, 4050-123 Porto,
Portugal,
4 Department of Zoology, University of Guelph, Guelph, ON, Canada, N1G
2W1
5 National Institute for Research in the Amazon (INPA), Laboratory of
Ecophysiology and Molecular Evolution, Ave André Araújo 2936,
CEP 69083-000, Manaus, AM, Brazil
* Author for correspondence (e-mail: brauner{at}zoology.ubc.ca)
Accepted 19 January 2004
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Summary |
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Key words: air-breathing, gills, kidney, Arapaima gigas, Osteoglossum bicirrhosum, gas exchange, ionoregulation, acidbase balance, nitrogenous waste excretion
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Introduction |
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One of the most impressive air-breathing fishes is Arapaima gigas,
which is endemic to the Amazon Basin. It is one of the largest freshwater
fishes in the world, reaching a length of 3 m and weighing as much as 250 kg
(Salvo-Souza and Val, 1990;
Graham, 1997
). Arapaima
gigas is an osteoglossid teleost that has a modified swim-bladder, which
is used as an air-breathing organ, and is among the most aerially dependent of
fishes. It drowns in
10 min without access to air, despite the presence
of gills (Val and Almeida-Val,
1995
). The males carry the fertilized embryos and newly hatched
larvae in their mouths (Salvo-Souza and
Val, 1990
) and, early in development, A. gigas is a water
breather up to
9 days post-hatch (see
Graham, 1997
). Thus, during
development, A. gigas undergoes a transition from an exclusive
water-breather to an air-breather, representing an impressive model system to
investigate how the transition from aquatic to aerial respiration affects gill
design. Furthermore, it represents a model system to determine whether
air-breathing is associated with the transition of physiological processes
from the gills to the kidney; an area ripe for investigation in the evolution
of air-breathing (Graham,
1997
).
The first portion of this commentary will focus on how environment and development influence gill design in water-breathing fishes. This will be used to facilitate interpretation of the observed changes in the gills during development in A. gigas. The remainder of the commentary will focus on the evidence that exists for the relocation of physiological processes from the gills to the kidney in A. gigas.
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Physiological constraints influencing gill design |
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The majority of nitrogenous waste excretion in freshwater fishes appears to
occur across the gills as passive NH3 diffusion
(Wood, 1993;
Wilkie, 1997
); therefore,
conditions that facilitate respiratory gas exchange will, for the most part,
facilitate NH3 diffusion. The same is not true for ionoregulation
or acidbase balance, both of which require specific cell types with
specialized ion exchangers or pumps appropriately placed in the gill
epithelium. In freshwater fishes, the mitochondria-rich (MR) cells (chloride
cells), which are responsible for Cl and Ca2+
uptake, are generally localized to the trailing edge of the filamental
epithelium at the base of the lamellae and within the inter-lamellar regions
(Perry, 1997
). This design
permits relatively efficient ion and acidbase regulatory exchanges
across the non-respiratory portion of the gill, while optimizing conditions
for gas diffusion across the lamellar epithelium. This prioritization in gill
design can be superseded during ionic or acidbase regulatory challenges
(Goss et al., 1995
), which are
particularly pronounced when fish are acclimated to soft water
(Greco et al., 1996
). Because
of the need to maintain active ion uptake under sub-optimal conditions, MR
cells proliferate on both the filamental and lamellar epithelium of the gill.
This acts to increase the capacity of the gill to actively take up
Ca2+ and Cl from the water but results in an
increase in the lamellar blood-to-water diffusion distance, which directly
compromises gas exchange in the absence of compensatory measures such as
hyperventilation or changes in whole-blood oxygen affinity (Perry,
1997
,
1998
). Thus, changes to the
general pattern of gill design can be induced by environmental alterations;
however, this is associated with a cost.
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Physiological constraints influencing gill design in larval fishes |
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Based upon morphological analyses, gills of larval fishes may develop first for ion regulation or acidbase balance and secondarily for gas exchange. As the cutaneous surface becomes incapable of satisfying the respiratory needs of the animal, due to the reduction in surface area-to-volume ratio that accompanies growth, a shift from ion or acidbase regulation to gas exchange as the principal factor influencing gill design may occur. Thus, the physiological process that is most limited under a given condition may have the greatest influence on gill design, but this may vary with developmental stage or environmental conditions. This may also be the case for the gills of air-breathing fishes, such as A. gigas, where different selective pressures from those observed in water-breathing fishes have a marked influence on gill design.
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Changes in gill structure during development in Arapaima gigas |
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Three different sizes of A. gigas (10 g, 100 g and 1 kg) were
obtained to investigate changes in gill morphology, ultrastructure and
immunohistochemistry during development. The smallest available size (10 g) is
larger than that at which the transition from water- to air-breathing occurs,
but fish of this size are still less dependent upon aerial respiration
relative to 1 kg A. gigas and can survive for twice as long without
access to air (20 min vs 10 min, respectively; C. J. Brauner and A.
L. Val, unpublished). Scanning electron microscopy (SEM) of gills from 10 g
fish revealed that they possess well-developed lamellae, typical of
water-breathing fish gills (Fig.
1A). While the lamellae are compact relative to those of other
water-breathers such as trout (O. mykiss), they are more similar to
those of O. bicirrhosum (Fig.
2). Arapaima gigas grows very quickly, and the next
largest group (100 g) is only 45 days older than the 10 g fish; however,
changes in gill structure have taken place, and the lamellae have become less
discernible (Fig. 1B). Within
45 months, fish have reached 1 kg, and the changes in gill morphology
are striking. The lamellae are no longer visible by SEM, and the gills consist
of what appear to be smooth, column-shaped filaments
(Fig. 1C). The filaments of 1
kg A. gigas appear qualitatively similar to the recent SEM images
obtained for the crucian carp (Carassius auratus) in normoxia
(Sollid et al., 2003
).
However, in carp, several days exposure to hypoxia results in pronounced
lamellar protrusion from the filaments that is associated with the
disappearance of the interlamellar cell mass, indicating that environmental
conditions exert reversible effects on gross gill morphology in carp. The
filling of the interlamellar space during development in A. gigas is
likely to be non-reversible; however, this remains to be investigated.
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|
Light microscopy provides insight into the developmental changes of the
gills. Again, 10 g fish possess gills similar to those of other
water-breathers, with MR cells localized to the interlamellar region of the
filament (Fig. 3A). In 100 g
fish, the interlamellar region of the gill becomes partially filled with
developing cells, including MR cells. Consequently, the lamellae expand
laterally and become stubby in appearance, consistent with that observed under
SEM. MR cells at this stage are even observed on the tips of the lamellae
(Fig. 3B). In gills from 1 kg
fish, interlamellar regions become completely filled due to cellular
proliferation, predominantly with MR cells, again consistent with gross
anatomical changes observed under SEM. The MR cells are large and extensive on
the outer layer of the filament. Immunohistochemistry reveals strong
immunofluorescence for Na+/K+-ATPase in the outer
epithelium of the filament of the 1 kg fish, consistent with the location of
the MR cells in the light micrographs. Immunolabelling of the
Cl/HCO3 anion exchanger 1 (AE1)
is restricted to the red blood cells (Fig.
4), and preliminary experiments indicate that
Na+/H+ exchanger 2 (NHE2) may be co-localized in
Na+/K+-ATPase-immunoreactive cells in the apical region.
Further studies are required to verify the presence of NHE2, which is rare
among freshwater fishes; however, in acid-tolerant dace (Tribolodon
hakonensis), an apical NHE has been found associated with MR cells
(Hirata et al., 2003).
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In both the light micrographs and immunohistochemistry, it is clear that
the diffusion distance between the blood and the water in the 1 kg fish is
very large relative to the 10 g A. gigas and other water-breathers.
It is not surprising that so little oxygen uptake occurs across the gills at
this stage. Despite the large diffusion gradient, the majority of
CO2 is excreted into the water
(Randall et al., 1978), with
up to 79% presumably excreted across the gills
(Brauner and Val, 1996
). Such
significant CO2 excretion across the gills is accomplished in part
by the large bloodwater CO2 partial pressure
(PCO2) gradient. Blood PCO2 values are
2630 mmHg (1 mmHg= 133.3 Pa) in similarly sized A. gigas
(Randall et al., 1978
), which
is far greater than the 23 mmHg measured in most water-breathing
fishes. The high PCO2 values in A. gigas result
from the reduced total gill surface area (characteristic of obligate
air-breathers in general; see Graham,
1997
) and the increased diffusion distance (Figs
3,
4), both of which limit
CO2 excretion across the gills. It is intriguing to think that
A. gigas lives in a continuous state of compensated respiratory
acidosis, which is supported by the large difference between plasma
Na+ and Cl concentrations (150 mmol
l1 and 60 mmol l1, respectively; R.
Gonzalez and C. J. Brauner, unpublished data). Given the large dependence of
the gills for CO2 removal, it is clear that conditions for
CO2 excretion do not impose large selection pressures to optimize
gill design for gas exchange at this stage, and the gills appear to be more
designed for ionoregulation or acidbase balance.
The gills in 1 kg fish possess a twofold-higher density of MR cells for a
given distance along the filament relative to the 10 g animals (V. Matey and
C. J. Brauner, unpublished data). Interestingly, the unidirectional
Na+ uptake rate in resting undisturbed A. gigas (1 kg) is
quite low (70 nmol g1 h1; R. Gonzalez,
personal communication) relative to other freshwater fishes, seemingly
paradoxical given the high density of MR cells. An overall reduction in gill
surface area and an increase in bloodwater diffusion distance of the
gill of A. gigas will greatly reduce diffusive ion loss across the
gills, which in water-breathing fishes is the primary surface for ion efflux.
In fish that possess low ion efflux, unidirectional uptake of ions is also low
(McDonald et al., 1991) and,
thus, the low unidirectional Na+ uptake rate in A. gigas
may be a reflection of low gill ion permeability more than anything else. The
role of the gills in vivo will be best evaluated by analyzing ion
transport during an ionoregulatory or acidbase regulatory challenge,
where the full potential of the gills may be revealed.
The architecture of the gill appears to vary dramatically with development
in A. gigas. Shortly after becoming an air-breather (i.e. 10 g),
secondary lamellae are evident and the appearance of the gills is similar to
that of a closely related water-breather where conditions for efficient gas
transfer have a large influence on gill design. With development, the gills of
A. gigas appear to become better designed for ionoregulation or
acidbase balance, particularly by the time A. gigas reaches 1
kg. This is similar to that observed in larval fishes (but chronologically
reversed), where pressures related to ionoregulation and acidbase
balance appear to have a greater influence on gill design than those for gas
exchange early in development. The role of the gills in whole-body
ionoregulation and acidbase balance in 100 g A. gigas
remains to be investigated.
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Transition of branchial physiological function to the kidney in Arapaima gigas |
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Conclusions |
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Acknowledgments |
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