Division of Comparative Physiology and Biochemistry, Society for Integrative and
Comparative Biology
The Origin and Evolution of the Surfactant System in Fish: Insights into the Evolution of
Lungs and Swim Bladders
Author(s): Christopher B. Daniels, Sandra Orgeig, Lucy C. Sullivan, Nicholas Ling,
Michael B. Bennett, Samuel Schürch, Adalberto Luis Val, and Colin J. Brauner
Source: Physiological and Biochemical Zoology, Vol. 77, No. 5 (September/October 2004), pp.
732-749
Published by: The University of Chicago Press. Sponsored by the Division of Comparative
Physiology and Biochemistry, Society for Integrative and Comparative Biology
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732
The Origin and Evolution of the Surfactant System in Fish: Insights
into the Evolution of Lungs and Swim Bladders*
Christopher B. Daniels1,†
Sandra Orgeig1
Lucy C. Sullivan1,‡
Nicholas Ling2,§
Michael B. Bennett3
Samuel Schürch4
Adalberto Luis Val5
Colin J. Brauner6
1
School of Earth and Environmental Sciences, University of
Adelaide, Adelaide, South Australia 5005, Australia;
2
Department of Zoology, University of Auckland, Auckland,
New Zealand; 3School of Biomedical Sciences, Department of
Anatomy and Developmental Biology, University of
Queensland, St. Lucia, Queensland 4072, Australia;
4
Department of Physiology and Biophysics, University of
Calgary, Calgary, Alberta T2N 4N1, Canada; 5Instituto
Nacional de Pesquisas da Amazônia (INPA), Manaus,
Amazonas 69083, Brazil; 6Department of Zoology, University
of British Columbia, 6270 University Boulevard, Vancouver,
British Columbia V6T 1Z4, Canada
Accepted 11/19/03
Online enhancement: color figure.
ABSTRACT
Several times throughout their radiation fish have evolved either
lungs or swim bladders as gas-holding structures. Lungs and
swim bladders have different ontogenetic origins and can be
used either for buoyancy or as an accessory respiratory organ.
Therefore, the presence of air-filled bladders or lungs in different groups of fishes is an example of convergent evolution.
We propose that air breathing could not occur without the
* This article was presented at the symposium “How to Live Successfully on
Land If One Is a Fish: The Functional Morphology and Physiology of the
Vertebrate Invasion of the Land,” Sixth International Congress of Comparative
Physiology and Biochemistry, Mount Buller, Victoria, Australia, 2003.
†
Corresponding author; e-mail: chris.daniels@adelaide.edu.au.
‡
Present address: Department of Microbiology and Immunology, University of
Melbourne, Victoria 3010, Australia.
§
Present address: Department of Biological Sciences, University of Waikato,
Private Bag 3105, Hamilton, New Zealand.
Physiological and Biochemical Zoology 77(5):732–749. 2004. 䉷 2004 by The
University of Chicago. All rights reserved. 1522-2152/2004/7705-3061$15.00
presence of a surfactant system and suggest that this system
may have originated in epithelial cells lining the pharynx. Here
we present new data on the surfactant system in swim bladders
of three teleost fish (the air-breathing pirarucu Arapaima gigas
and tarpon Megalops cyprinoides and the non-air-breathing
New Zealand snapper Pagrus auratus). We determined the presence of surfactant using biochemical, biophysical, and morphological analyses and determined homology using immunohistochemical analysis of the surfactant proteins (SPs). We
relate the presence and structure of the surfactant system to
those previously described in the swim bladders of another
teleost, the goldfish, and those of the air-breathing organs of
the other members of the Osteichthyes, the more primitive airbreathing Actinopterygii and the Sarcopterygii. Snapper and
tarpon swim bladders are lined with squamous and cuboidal
epithelial cells, respectively, containing membrane-bound lamellar bodies. Phosphatidylcholine dominates the phospholipid
(PL) profile of lavage material from all fish analyzed to date.
The presence of the characteristic surfactant lipids in pirarucu
and tarpon, lamellar bodies in tarpon and snapper, SP-B in
tarpon and pirarucu lavage, and SPs (A, B, and D) in swim
bladder tissue of the tarpon provide strong evidence that the
surfactant system of teleosts is homologous with that of other
fish and of tetrapods. This study is the first demonstration of
the presence of SP-D in the air-breathing organs of nonmammalian species and SP-B in actinopterygian fishes. The extremely high cholesterol/disaturated PL and cholesterol/PL ratios of surfactant extracted from tarpon and pirarucu bladders
and the poor surface activity of tarpon surfactant are characteristics of the surfactant system in other fishes. Despite the
paraphyletic phylogeny of the Osteichthyes, their surfactant is
uniform in composition and may represent the vertebrate
protosurfactant.
Introduction
Many species of fishes can breathe air. Fish utilize a number
of different structures for aerial gas exchange, including the
skin, gills, mouth and buccal cavity, intestine, and other specifically evolved chambers. In particular, lungs appeared independently several times. Lung structure can vary from the
simple, transparent, baglike structures of rope fish and bichirs
(Polypteriformes, Cladista) to more complex compartmentalized structures in lungfish (Dipnoi). Lungs are likely to have
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Surfactant in Air-Breathing Organs of Fish
first appeared in the placoderms. Moreover, the possible presence of lungs in placoderm fossils indicates that these animals
must also have had a breathing mechanism and possibly a
respiratory oscillator for responding to changes in blood and
lung gases. Recent evidence suggests that such mechanisms
must have been in place before the lungs themselves evolved
(Perry et al. 2001). However, placoderm lungs are not homologous with the original Osteichthyian lungs, because placoderm lungs were most likely derived from the anterior pharynx (Denison 1941; Perry et al. 2001). Moreover, placoderm
lungs may have served a different function. They may have
been important in promoting neutral buoyancy for these
heavily armored fish. With the extinction of the placoderms,
lungs disappeared in the stem group and are lacking in all
cartilaginous fishes. Chondrichthyians are less dense than placoderms, because they lack armor plates, and they use squalene
in their liver for buoyancy (Hickman et al. 2001). However, it
is likely that lungs reappeared in the stem ancestor of the Osteichthyes. In both branches of the Osteichthyes (Sarcopterygii,
i.e., lungfish and the most primitive Actinopterygii, i.e.,
Polypteriformes), the lungs appear as paired ventral structures
derived from the posterior pharynx and posterior to the gills
(reviewed in Perry et al. 2001). Primitive Devonian osteichthyian fish may have breathed air in response to low environmental O2 and used the neutral buoyancy that an air-filled
lung provides to rest at the surface (Dehadrai and Tripathi 1976;
Fange 1983).
However, it is now accepted that while lungs occur in the
most primitive fishes and tetrapods, their ontogenetic origin is
different from that of swim bladders (Perry et al. 2001). The
swim bladder is an unpaired air-holding structure arising dorsally from the posterior pharynx (Perry et al. 2001). Swim
bladders are usually regarded as the primary buoyancy aid and
also support hearing by amplifying sound in the Ostariophysi
(Johansen 1968). They are, however, employed in breathing in
gars (Ginglymodi) and bowfin (Halecomorphi), where they are
termed “pulmonoid” because of the arterial supply from the
sixth branchial arch. In some teleosts, including a basal teleost,
the pirarucu (Arapaima gigas), and a derived teleost, the tarpon
or oxeye herring (Megalops cyprinoides), the swim bladder,
which is supplied by a swim bladder artery, is respiratory (Perry
et al. 2001).
Pulmonary surfactant plays a crucial role in the physical
forces acting at the air-liquid interface in the lungs during the
dynamic changes in surface area and volume that occur during
inspiration and expiration. Surfactant consists of disaturated
phospholipids (DSP), unsaturated phospholipids (USP), neutral lipids (predominately cholesterol), and surfactant proteins
(SPs). Four SPs have been described in mammals: SP-A, SPB, SP-C, and SP-D. Surfactants have also been located and
described in fish swim bladders, including those that are used
for buoyancy (e.g., in goldfish, eels, and carp; Daniels and
Skinner 1994; Orgeig and Daniels 1995; Rubio et al. 1996;
733
Sullivan et al. 1998; Prem et al. 2000; Bourbon and ChailleyHeu 2001) and gas exchange (gar; Smits et al. 1994), and also
in lunged fishes (rope fish, bichirs, and the three species of
sarcopterygian lungfishes; Smits et al. 1994; Orgeig and Daniels
1995; Sullivan et al. 1998).
In this article, we use previously published biochemical and
morphological analyses of the surfactant system of primitive
fish and add new information on this system in teleosts (two
freshwater-inhabiting air breathers, the pirarucu A. gigas and
the tarpon M. cyprinoides, and one marine fish, the snapper
Pagrus auratus). This review (with new data) demonstrates that
the surfactant system of fishes is most likely homologous with
that of the tetrapods, despite the different ontogenetic origins
and functional aspects of the air-breathing organs (Rubio et al.
1996; Sullivan et al. 1998; Prem et al. 2000). In addition, the
high-cholesterol, low–saturated phospholipid composition of
surfactant found in fishes and in the ancient sarcopterygian,
the Australian lungfish (Neoceratodus forsteri), represents a very
primitive, poorly surface-active mixture, which we term a “protosurfactant” (Daniels and Skinner 1994; Daniels et al. 1995a;
Orgeig and Daniels 1995; Daniels and Orgeig 2001). A primitive, if not the original, function of fish surfactant is likely to
be acting as an antiadhesive (Daniels and Skinner 1994), but
surfactant may also prevent fluid from entering the bladder or
lung, prevent oxidative damage to the epithelial lining, and act
as an antiseptic/antibiotic (Daniels and Orgeig 2001). We also
outline the evolution of the surfactant system in the fish, with
particular emphasis on postulating a mechanism whereby a
homologous system could appear in structures derived independently several times.
Material and Methods
Fish
The tarpon Megalops cyprinoides occurs in a wide range of
marine and freshwater habitats, including East Africa, Southeast
Asia, Japan, Tahiti, Australia’s tropical seas, and the freshwaters
of far northern Australia. The fish can tolerate a wide range of
habitats, including landlocked lagoons and oxbow lakes
(termed billabongs in Australia), with waters ranging from pH
5.2 to 9.1 and temperatures between 23⬚ and 34⬚C (Merrick
and Schmida 1984). It has been reported that M. cyprinoides
will die in aquaria if prevented from reaching the surface (Merrick and Schmida 1984), and the closely related Atlantic tarpon
(Megalops atlanticus) will regularly ventilate the swim bladder
by a characteristic surface roll, even in normoxic waters (Graham 1976). Therefore, air breathing may be essential for this
species, particularly in water with low oxygen levels (Graham
1997).
The pirarucu Arapaima gigas is one of the largest freshwater
fishes in the world, reaching up to 4.5 m and 250 kg (Graham
1997). It is an obligate air-breathing teleost from the Amazon
that will drown in 10–20 min without access to normoxic air.
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734 C. B. Daniels, S. Orgeig, L. C. Sullivan, N. Ling, M. B. Bennett, S. Schürch, A. L. Val, and C. J. Brauner
The degree of aerial dependence is related to size. After hatching, A. gigas is a water breather but quickly becomes an air
breather by the time it is about 18 mm long, 8–9 d posthatch
(Graham 1997). To determine whether there is an effect of size
on surfactant composition of the air-breathing organ, we collected two size classes, 30–70 g and 550–1,000 g. Unfortunately,
these were the only sizes that could be obtained. Both sizes are
capable of surviving anoxic water by securing all O2 uptake
from the air, and both groups secure approximately 80% of
their metabolic O2 requirement from air in normoxic water (C.
J. Brauner and A. L. Val, personal observations; Stevens and
Holeton 1978). Clearly, air breathing is essential for this species.
The snapper Pagrus auratus is a demersal marine species in
the family Sparidae and is found in New Zealand, Australia,
and Japan. Species of Sparid snappers comprise major commercial aquaculture and wild fisheries in many areas of the
world. Sparids are Perciform fishes possessing a euphysoclistous
swim bladder, which is solely a hydrostatic organ and does not
perform any respiratory function. The swim bladder develops
as an extrusion of the gut wall and is first filled by swallowing
air via the pneumatic duct at around 5 d after hatching. By
around 30 d after hatching, the pneumatic duct has closed and
the swim bladder loses its connection to the gut.
Lavage Procedure
Tarpon (Megalops cyprinoides). Four fish (body mass range:
236–540 g) were captured from the Mary River (Northern Territory, Australia) or the Brisbane River (southern Queensland,
Australia). We killed the fish by bathing them in a solution of
1% MS-222 and removed the tissues overlying the swim bladder. A tube 2 mm in diameter was passed into the swim bladder
via the pharyngeal opening, and the air was then sucked out
with a 20 mL syringe. The bladder was lavaged with 5–6 mL
of ice-cold 0.15 M NaCl. The saline was withdrawn, and the
lavaging process was repeated twice. The lavage was centrifuged
at 150 g for 10 min to remove cellular debris and lyophilized.
Material for biochemical analysis, transmission electron microscopy (TEM), and immunohistochemistry was collected
from this species.
Pirarucu (Arapaima gigas). Six small fish (33–66 g) and three
larger fish (550, 655, and 1,006 g) were obtained from Boutique
do Peixe Vivo and Amazonas Ecopeixe S.A. and held at the
National Institute for Research of the Amazon on a natural
light cycle for 3 wk before sampling. During this time, fish were
fed goldfish at a ration of 1% body weight per day. Fish were
killed in a solution of 1% MS-222, a tube was inserted into
the pharyngeal opening of the swim bladder, and lavage was
obtained as described above for tarpon. The entire ventral lower
half of the swim bladder was removed and weighed before and
after lyophilization. Only lavage material was collected from
this species.
Snapper (Pagrus auratus). Snapper (body mass p 500 g) were
captured by line fishing in the Hauraki Gulf, New Zealand.
Fish were killed by stunning and pithing, and a midventral
incision was made from the pelvic girdle to the vent to expose
the gut cavity. Both the internal and external surfaces of the
swim bladder were fixed before excision to optimize tissue fixation and maintain the in situ arrangement of the delicate
mucosal surface. We ligated the esophagus and removed all
abdominal organs, taking care not to damage the swim bladder.
The gut cavity was then filled with fixative (2% paraformaldehyde, 2% gluteraldehyde, 0.1 M sodium cacodylate, 375
mOsm, pH 7.8). In order to fix the inside of the swim bladder
without altering its volume, the swim bladder gases were gradually replaced with fixative. A 20-mL syringe was half filled
with fixative and the syringe needle introduced to the swim
bladder lumen through the epaxial muscle. One milliliter of
fixative was introduced, followed by the withdrawal of the same
volume of swim bladder gas. This reciprocal replacement was
repeated until the lumen was entirely filled. Only material for
TEM was collected for this species.
Electron Microscopy and Immunohistochemistry
After in situ fixation of the snapper swim bladder (see above)
for 1 h, the ventral swim bladder wall was carefully excised
from the body and cut into small strips of approximately
2 # 5 mm and continued to fix for 24 h in 2% paraformaldehyde, 2% gluteraldehyde, 0.1 M sodium cacodylate, 375
mOsm, pH 7.8. In the case of the tarpon, sections of the bladder
wall and associated respiratory tissue bands forming the accessory breathing organ were cut into small pieces and fixed
for TEM, as previously described (Sullivan et al. 2001).
In the tarpon, sections for immunohistochemistry were prepared by freeze substitution and immunolabelling, as previously
described (Sullivan et al. 1998). The primary antibodies to human SP-A and SP-D and bovine SP-B were purchased (Chemicon Australia, Boronia, Australia).
Surfactant Composition
Methods for measuring total phospholipids, disaturated phospholipids, and phospholipid headgroups have been published
previously (Daniels and Skinner 1994; Orgeig and Daniels 1995;
Daniels et al. 1999). We measured cholesterol in the neutral
lipid fraction (reconstituted with heptane) with a high-performance liquid chromatography (HPLC) system (LC1500 HPLC
pump, LC1610 autosampler, DP800 data interface; GBC Instruments, Melbourne, Australia). We injected 30 mL onto a
silica column (Alltech 4.6 # 250 mm with 5 mm spherical silica)
equilibrated with a hexane : isopropanol (99 : 1 v/v) mobile
phase (HPLC grade; APS Chemicals, Greenfields, Australia).
Samples were eluted (1 mL/min) and measured at 206 nm
(Model 2151 LKB Bromma variable-wavelength UV detector).
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Surfactant in Air-Breathing Organs of Fish
735
Figure 1. Appearance of the respiratory portion of the swim bladder of the tarpon Megalops cyprinoides. The surface consists of a thin layer
of capillaries (C) and an overlying layer of epithelial cells (EC). AS p airspace. N p nucleus. Scale bar p 10 mm.
A backpressure regulator (100 psi) was fitted to the detector
outlet to minimize the formation of bubbles in the flow cell.
Retention time for cholesterol was 10.2 min, and total run time
per sample was 25 min.
SP-B in lavage samples from tarpon and pirarucu was quantified in a competitive ELISA based on a previously published
method (Lesur et al. 1993). The primary antibody, rabbit antibovine SP-B polyclonal antibody (Chemicon Australia), and
the secondary antibody, antirabbit IgG-HRP conjugate (Sigma,
Sydney, Australia), were both used at a dilution of 1 : 1,000.
The reaction was developed in Sigma Fast tablets (Sigma), and
the absorbance at 492 nm was determined with a Titertek MCC
Multiskan (Titertek, Huntsville, Ala.). The amount of SP-B in
the lavage samples was compared to the amount of total protein, which was determined by the method of Lowry et al.
(1951).
Surface Activity
Surface activity for an aliquot of lyophilized lavage from one
tarpon was determined after the lavage was reconstituted in
water and centrifuged at 40,000 g (Beckman Optima TLX Ultracentrifuge; Beckman, Sydney, Australia) for 30 min to pellet
large aggregate material. A phosphorus assay of the pelleted
material and the supernatant revealed that 190% of the phospholipids remained in the supernatant. The supernatant was
lyophilized again and reconstituted in a buffered salt solution
(140 mM NaCl, 10 mM HEPES, 2.5 mM CaCl2, pH 6.9) to a
concentration of 20 mg/mL of phospholipid.
We used a leakproof system, the captive bubble surfactometer
(CBS), which enables the determination of surface tension, area,
and volume of a bubble with a surfactant film at the air-water
interface (Schürch et al. 1992). We used a previously published
method for the preparation of small sample volumes (Codd et
al. 2002). We measured the rate of adsorption for the first 5
min and then performed quasi-static compressions (Codd et
al. 2002). From these we determined the minimum surface
tension upon final compression (STmin), the maximum surface
tension before compression (STmax), and the percent surface
area of compression (%SAcomp), which is a measure of the extent
of compression required to achieve STmin. Measurements were
performed both at room temperature (22⬚C) and at 37⬚C. We
recorded the bubble continuously and calculated bubble volume, area, and surface tension from digitized bubble images,
using height and diameter (Schoel et al. 1994). We compared
these results to values obtained for other fish species using this
and other techniques.
Results
Electron Microscopy and Immunohistochemistry
Tarpon. The respiratory surface of the swim bladder accessory
breathing organ consisted of a thin layer of blood capillaries
and an overlying layer of epithelial cells (Fig. 1). There were a
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736 C. B. Daniels, S. Orgeig, L. C. Sullivan, N. Ling, M. B. Bennett, S. Schürch, A. L. Val, and C. J. Brauner
Figure 2. Cuboidal epithelial cells in the lining of the swim bladder of the tarpon Megalops cyprinoides. The cells have a large nucleus and
contain either numerous electron-lucent structures (A, arrows, scale bar p 2 mm) or electron-dense structures (B, arrows, scale bar p 1 mm).
number of cuboidal epithelial cells containing a dark nucleus
and numerous electron-lucent structures (Fig. 2A) that appear
to represent one form of lamellar body (Table 1). Other lamellar
bodies appeared as granular, electron-dense structures (Figs.
2B, 3A), and still others consisted of concentric rings of lamellae
in a membrane-bound vesicle (Fig. 3B). Different structures
for the lamellar bodies are also reported for the snapper (see
below) and may represent a developmental sequence. At a
higher magnification, SP-A, SP-B, and SP-D were located
within the tarpon swim bladder. SP-A and SP-B were located
both within the cells and in the airspaces. The location of SPA appeared to be quite diffuse (Fig. 3A), whereas SP-B was
found to be associated with lamellar bodies (Fig. 3B). A small
amount of SP-D staining was found in the air spaces only (Fig.
3C).
Snapper. The lining of the swim bladder lumen was composed
of squamous epithelial cells. These cells were generally in the
region of 200 nm thick, except for a central thickening due to
the presence of the nucleus and adjacent mitochondria. Cells
were joined peripherally by tight junctions at the lumenal surface and formed extensive interdigitations with adjoining cells
that extended for 1–2 mm from the position of cellular abutment. The apical surface of the epithelium was interrupted by
occasional microvilli that appeared more common toward the
cell periphery. This cell type was characterized by the presence
of numerous lamellar bodies or cytosomes. Cytosomes appeared to be more common in the region of central thickening
close to the nucleus (Fig. 4A, 4B). The cytosomes of the lumenal
epithelium were of the form described by Creasey et al. (1974)
as simian and common in human Type II alveolar cells, although their description of this structure as having concentric
lamellae may be misleading and appears to depend on the plane
of sectioning and possibly the developmental stage of the cytosome (Table 1). For example, a lamellar body with lamellae
fused at the poles may appear concentric if sectioned equatorially. As with the tarpon, several different morphs of cytosome were recognized and may represent a developmental sequence based on the assumption that all eruptant cytosomes
appeared to be of the concentric lamellate form. The proposed
developmental sequence for these cytosomes is described in
Figure 4C.
Cytosomes appear to form from membrane-bound, electrondense aggregations of an amorphous material. Brooks (1970)
reports formation of epithelial cytosomes in trout from similar
electron-dense bodies and states that this material appeared to
be composed of very tightly packed membranes. The lamellate
structure of these bodies in the snapper could only be distinguished close to the site at which lamellae were separating from
the electron-dense material (Fig. 4C). These osmiophilic lamellae begin to form and separate centrally but remain attached
peripherally in an equatorial, or possibly polar, adhesion plaque
(Fig. 4D). Total separation of the individual lamellae to form
a truly concentric structure seems to occur just before eruption
from the apical surface of the cell.
The gas gland epithelium contains multilamellar bodies, or
cytosomes, of the form previously described for the toadfish
gas gland (Morris and Albright 1975, 1977) and classified as
subsimian by Creasey et al. (1974). This cytosome has lamellae
that traverse from one side of the structure to the other (Fig.
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Table 1: Morphological characteristics of surfactant from fishes (Osteichthyes)
Type II Cells
(and Analogues)a
Lamellar
Bodies
Tubular
Myelin
?
⫹
⫹
⫹
?
?
⫹(*)
⫹
⫹(*)
⫹
⫹
⫹
⫹
⫹
?
⫺
?
?
⫹
⫹
⫺
?
⫹
⫹
⫹
?
?
Acerina cernua
Coregonus lavaretus
Fundulus heteroclitus
Gadus callarias
Opsanus tau
Amphipnous cuchia
Anabas tetudineus
Channa punctatus
?
?
?
?
?
⫹(*)
⫺
⫹(*)
⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
?
?
?
?
?
⫺
⫺
⫺
Channa striatus
⫹(*)
⫺
⫺
Clarias batrachius
Heteropneustes fossilis
Misgurnus fossilis
⫹(*)
⫹(*)
⫹(*)
⫺
⫺
⫹
⫺
⫺
⫺
Pagrus auratus
⫹
⫹
⫹
Megalops cyprinoides
⫹
⫹
?
Species
Dipnoi:
Protopterus annectens
Protopterus sp.
Lepidosiren paradoxa
L. paradoxa
Neoceratodus forsteri
N. forsteri
Teleostei:
Salmo gairdneri
Anguilla vulgaris
Lota lota
Halecomorphi:
Amia calva
Polypteriformes:
Polypterus senegalensis
?
⫹
?
⫺
⫺
⫺
Polypterus ornatipinnis
⫹
⫹
⫺
Other Forms and Comments
Osmiophilic inclusion bodies in cells and
in alveolar space
(*)Only one cell type
Extracellular material also present
(*)Only one cell type
Type II cells can be isolated; secrete LP
Nonciliated (columnar or cuboidal?) cells
that secrete mucus-like material into
lumen of swim bladder
Very isolated LBs in cells of the gas
gland
(*)Cuboidal cells; air sac instead of lung
Labyrinthine organ
(*)Cuboidal cells; suprabranchial
chamber
(*)Cuboidal cells; suprabranchial
chamber
(*)Cuboidal cells with vesicles; air sac
(*)Cuboidal cells; air sac
(*)Goblet epithelial cell of respiratory
intestine
Different forms of LBs; they possibly
represent a developmental sequence,
i.e., only eruptant LBs have
concentric lamellae
Different forms of LBs (electron-lucent,
electron-dense, or with concentric
lamellae); SPs A, B, D present in Type
II cells and airspace
References
1
2
3
4
3
5
6
7
8
8
9
10
10
10
11, 12
11, 12
11, 12
11, 12
11, 12
12, 13
14
15
15
3
Electron-opaque granules in rare cells in
secretory crypts
“Classical-appearing” Type II cells
16
13
Note. A plus sign indicates presence, a minus sign absence, and a question mark unresolved or not attempted. LB p lamellar body. References: (1) Klika and
Janout 1967; (2) DeGroodt et al. 1960; (3) Hughes 1973; (4) Hughes and Weibel 1976; (5) Wood et al. 2000; (6) Brooks 1970; (7) Dorn 1961; (8) Jasinski and
Kilarski 1964; (9) Fahlen 1967; (10) Copeland 1969; (11) Munshi 1976; (12) Hughes and Munshi 1973; (13) Marquet et al. 1974; (14) Jasinski 1973; (15) this
study; (16) Klika and Lelek 1967.
a
Asterisks are defined in the first entry in the “Other Forms and Comments” column.
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738 C. B. Daniels, S. Orgeig, L. C. Sullivan, N. Ling, M. B. Bennett, S. Schürch, A. L. Val, and C. J. Brauner
4E). Eruption of this form of cytosome through the apical cell
surface was not observed. Simian or concentrically lamellate
cytosomes of the form common to the general lumenal epithelium were also observed and seen to occur in eruption from
the epithelium. It was notable that eruption of this form of
cytosome could occur either from the apical surface onto the
surface of the lumen or through the basal surface into underlying circulatory sinuses. Extracellular material was occasionally
observed on the lumenal surface of the gas gland, trapped between apical microvilli (Fig. 4F). The transverse structure of
this material (Fig. 4G) resembles the cytosomal tubular myelin
of the vertebrate lung. As the crosshatched structure of tubular
myelin is highly characteristic of surfactant, it is likely that the
material in the snapper gas gland is surfactant.
Surfactant Composition
Tarpon. The disaturated phospholipid (DSP) content of the
lavage material was very low and was measurable in only one
of the four tarpon fish samples (%DSP/PL p 4.04). The cholesterol (Chol) to DSP ratio (mg/mg) in this one sample was
5.65. The cholesterol to phospholipid (PL) ratio (mg/mg) in the
lavage was 0.258 Ⳳ 0.069 (mean Ⳳ SE, n p 4; Table 2). Thinlayer chromatography (TLC) revealed that phosphatidylcholine
(PC) was the predominant PL in lavage, accounting for 71%
of total PL. The only other significant lipids were sphingomyelin
(18.4%) and a combination of phosphatidylserine and phosphatidylinositol (7.7%). Lysophosphatidylcholine (1%), phosphatidylglyerol (1%), and phosphatidylethanolamine (0.3%)
were present only in trace amounts (Table 2). The ELISA demonstrated the presence of SP-B in lavage from two of the tarpon.
There were 2 pg of SP-B per mg of total protein in one animal
and 17.6 pg of SP-B per mg of total protein in the other.
Pirarucu. The %DSP/PL in the lavage of the small fish was
26.85 Ⳳ 3.35 (mean Ⳳ SE, n p 6). The Chol/PL and Chol/DSP
ratios (mg/mg) were 0.266 Ⳳ 0.02 and 1.03 Ⳳ 0.08 (mean Ⳳ
SE, n p 6), respectively (Table 2). The Chol/PL ratio of the
lavage from the large fish was 0.34 Ⳳ 0.114 (mean Ⳳ SE, n p
3) and did not differ from that of the small fish. We did not
measure DSP in the large pirarucu. SP-B was measurable by
ELISA in the lavage of one of the large pirarucu. The lavage
of this fish contained 44.6 pg SP-B per mg of total protein.
Surface Activity
The adsorption of surfactant from the tarpon swim bladder
was extremely slow. At 22⬚C the surfactant adsorbed slightly
Figure 3. Immunogold labeling of SP-A (A; scale bar p 0.2 mm), SPB (B; scale bar p 0.5 mm), and SP-D (C; scale bar p 0.5 mm) in the
respiratory swim bladder of the tarpon Megalops cyprinoides. SP-A was
localized in the air spaces (filled arrows) and in the epithelial cells (open
arrows), whereas SP-B appeared to be primarily associated with lamellar bodies (arrows). The presence of SP-D was restricted only to
the airspaces (arrows).
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Figure 4. Multilamellar bodies (cytosomes) and tubular myelin in the snapper (Pagrus auratus) swim bladder. A, Cytosome erupting from
lumenal epithelium (scale bar p 1 mm). B, Semilamellate cytosome (scale bar p 1 mm). C, Progressive development of cytosomes from electrondense amorphous granules to semilamellate form (scale bar p 1 mm). D, Semilamellate cytosome, lamellae fused equatorially (scale bar p 2
mm). E, Subsimian cytosome (scale bar p 250 nm). F, Lumenal surface material, tubular myelin (scale bar p 250 nm). G, Transverse structure
of the material in F (scale bar p 100 nm).
739
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Table 2: Surfactant composition in fishes (Osteichthyes)
Species
740
Dipnoi:
Neoceratodus forsteri a
Lepidosiren paradoxab
L. paradoxaa
Protopterus annectensa
Teleostei:
Hoplias malabaricusb
Hoplerythrinus unitaeniatusb
Erythrinus erythrinusb
Arapaima gigasb
A. gigasc
Megalops cyprinoidesc
Carassius auratus (posteriord
swim bladder)
Ginglymodi:
Lepisosteus osseuse
Polypteriformes:
Calamoichthys calabaricuse
Polypterus senegalensise
n
Percent of PLs
TA/B PL
(⬚C) (mg/gWL) PC PG PE SM PI
Chol
DSP
DSP/PL
PS LPC/Unk (mg/gWL) Chol/PL (mg/gWL) Chol/DSP (%)
3
23
1–3 RT
3
23
4
23
.084
ND
.22
.61
59.6 .5 14.4 15.3
46.0 8.0 19.0 11.0
73.9 1.8 6.9 7.3
80.2 3.0 3.0 5.6
10.2
0
0
9.3
7.9
0
0
.8
.4
.022
ND
.010
.028
.242
ND
.044
.052
.006
ND
.059
.126
3.66
1–3
1–3
1–3
1–3
6
4
RT
RT
RT
RT
RT
RT
ND
ND
ND
ND
ND
ND
70
69
93
39
ND
71
0
7
5
0 25
2
0
3
2
6
7 24
ND ND ND
1
.3 18.4
0 12
0
2
2
0
8
8
ND
7.7
5
0
0
8
ND
1
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
.266
.258
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1.03
5.65
ND
ND
ND
ND
26.85
!4
9
23
.14
75
1.2
5–8
23
.50
76.4 1.4
2–9
2–8
23
23
.23
.097
.169
.222
8.9
ND
27.8
20.4
4.7 15.9
5.1
0
.051
.328
.036
1.41
20.8
4.6
5.9
7.9
3.0
.091
.175
.069
1.30
14.1
ND ND ND ND
ND ND ND ND
ND
ND
ND
ND
.044
.021
.182
.208
.022
.008
2.05
1.72
9.54
11.6
Note. TA/B p ambient or body temperature; PL p phospholipid; gWL p grams of wet lung mass; PC p phosphatidylcholine; PG p phosphatidylglycerol; PE p phosphatidyl-ethanolamine;
SM p sphingomyelin; PI p phosphatidylinositol; PS p phosphatidylserine (a single value for PI and PS indicates that the bands could not be resolved); LPC p lysophosphatidylcholine;
unkwn p unknown PL; Chol p cholesterol; DSP p disaturated PL. RT p roomtemperature (22⬚–25⬚C); ND p not determined.
a
Orgeig and Daniels 1995.
b
Phleger and Saunders 1978.
c
This study.
d
Daniels and Skinner 1994.
e
Smits et al. 1994.
Surfactant in Air-Breathing Organs of Fish
741
Surfactant Proteins
The presence of the surfactant lipids and proteins (SP-A, SPB, and SP-D) provides strong evidence that the surfactant system of all fishes is homologous with that of the tetrapods.
Furthermore, the surfactant system located in the lungs or swim
bladders of fish (lungfish, bichirs, gar, carp, eels, goldfish, snapper, pirarucu, and tarpon) is likely to be homologous (Daniels
and Skinner 1994; Rubio et al. 1996; Sullivan et al. 1998; Prem
Figure 5. Rate of adsorption of tarpon (Megalops cyprinoides) surfactant
(10 mg/mL PL) to the air-liquid interface of a bubble in the captive
bubble surfactometer. The surface tension at the end of adsorption is
the equilibrium surface tension (STeq).
faster than at 37⬚C (Fig. 5), and it reached a slightly lower STeq
(22⬚C: 38 mN/m; 37⬚C: 41 mN/m). Upon the first quasi-static
compression cycle, tarpon surfactant demonstrated relatively
high values for STmax (22⬚C: 41.0 mN/m; 37⬚C: 41.1 mN/m),
STmin (22⬚C: 19.5 mN/m; 37⬚C: 21.7 mN/m), and %SAcomp
(22⬚C: 75.3%; 37⬚C: 75.8%; Fig. 6). Surface tension–lowering
properties, including STmin and %SAcomp, were marginally better
at 22⬚ than at 37⬚C (Fig. 6). Film stability was poor, as seen
from the slight progressive increase in STmin following successive
quasi-static cycles (Fig. 6; Table 3).
Discussion
Morphology
Cells containing lamellar bodies and surfactant lipids and proteins are easily observable in the swim bladder of the tarpon
and the snapper. It is also clear that the function of the gasholding structure (respiration or buoyancy) is irrelevant to the
requirement for a surfactant system in fish (Table 1). The surfactant-secreting cells in the tarpon and the snapper do not
differ structurally from those of other fishes and possess the
same characteristics as the alveolar Type II epithelial cells found
in tetrapod lungs (Daniels and Orgeig 2001). They demonstrate
microvilli on the apical surface, and the lamellar bodies are
membrane-bound and of similar size and appearance to those
observed in all other vertebrate groups, including fishes (Table
1; Hughes 1970, 1973; Hughes and Weibel 1978; Prem et al.
2000; Wood et al. 2000; Daniels and Orgeig 2001).
Figure 6. Quasi-static (QS) surface tension–area relations for tarpon
(Megalops cyprinoides) surfactant on the captive bubble surfactometer
at (A) 22⬚C and (B) 37⬚C; the first, second, and fourth QS cycles (qs1,
qs2, and qs4, respectively) are given for comparison. The lower limb
of each curve represents compression and the upper expansion. The
lowest point at the end of compression represents STmin. The change
in surface area compression (%SAcomp) required to reach STmin is an
indication of the surface tension–lowering quality of the sample. Sample concentration was 10 mg/mL PL. A color version of this figure is
available in the online edition of the journal.
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742 C. B. Daniels, S. Orgeig, L. C. Sullivan, N. Ling, M. B. Bennett, S. Schürch, A. L. Val, and C. J. Brauner
Table 3: Surface activity of surfactant from fishes (Osteichthyes)
Species and TA (⬚C)
Dipnoi:
Protopterus annectens:
RT
Lepidosiren paradoxa:
RT
37
24
Neoceratodus forsteri:
RT
Teleostei:
Carassius auratus:
RT
22
Megalops cyprinoides:
22h
37h
Hoplias malobaricus:
37
Hoplerythrinus unitaeniatus:
37
Arapaima gigas:
37
Halecomorphi:
Amia calva:
RT
Ginglymodi:
Lepisosteus osseus:
RT
24
Device
Calculated from bubble shapeb
WBd
WBe
Min ST
(mN/m)
%SA Stability
Comp Ratios
!.1
22
22
Bubble
Surpellic Property/
Clickinga Surface Activity
Stableb
⫹c
Fully surpellicb
Stableb
⫹d
Fully surpellicb
Very lowe
70
⫹f
Partially surpellicb
⫺c
Partially surpellicb
Very lowe
Calculated from bubble shapeb
PBSg
20
27.4
CBS
19.5
21.7
75.3
75.8
Very low
Very low
WBd
22
70e
Very lowe
WBd
22
80e
Very lowe
WBd
22
75e
Very lowe
Calculated from bubble shapeb
4
2–6
Calculated from bubble shapeb
WBe
17.0 Ⳳ 1.0
70
⫺c
Partially surpellicb
⫺c
Partially surpellicb
Very lowe
Note. TA p ambient temperature; Min ST p minimum surface tension; %SA Comp p percent surface area compression required to achieve min ST;
RT p room temperature (22⬚–25⬚C). WB p Wilhelmy balance; PBS p pulsating bubble surfactometer; CBS p captive bubble surfactometer. The number of
measurements performed regards whole lung extracts, not lavage. “Surpellic” is a term coined by Pattle to describe the ability of lavage material to exist as stable
bubbles and demonstrate bubble clicking. “Stability ratio” is a measure of a bubble’s stability as determined by the change in surface area over a standard length
of time after stabilisation. “Bubble clicking” is a phenomenon involving rapid changes in bubble shape and surface tension immediately after formation in water.
It indicates that the fluid involved is capable of attaining very low surface tension.
a
A plus sign indicates presence and a minus sign absence.
b
Pattle 1976.
c
Hughes 1967.
d
Phleger and Saunders 1978.
e
Daniels et al. 1998b.
f
Hughes 1973.
g
Daniels and Skinner 1994.
h
This study.
et al. 2000). Given that the lung and swim bladder have separate
ontogenetic origins and arise from different pharyngeal regions
(Perry et al. 2001), we can conclude that the surfactant system
must predate the appearance of both lungs and swim bladders.
Significant recent evidence has demonstrated surfactantcontaining lamellar bodies secreted from epithelial cells in the
mammalian gut (Engle and Alpers 2001). SP-A and SP-D co-
localize with these structures (Rubio et al. 1995; Engle and
Alpers 2001). Furthermore, SP-A antibodies also cross-react
with material from both the intestine and swim bladder of the
carp (Rubio et al. 1996). We therefore postulate that the surfactant system originated in the epithelial cells lining the pharynx. Here the SPs may have played an important role in the
innate immune system of the organism.
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Surfactant in Air-Breathing Organs of Fish
The hydrophilic SPs, SP-A and SP-D, belong to the family
of proteins known as collectins that are characterized by Cterminal carbohydrate recognition domains. These proteins are
thought to function as molecules of the innate immune system
by recognizing a broad spectrum of pathogens, including viruses, bacteria, and fungi, as well as allergens, such as pollen
grains and mite allergens (Haagsman and Diemel 2001). In
addition to their presence in gut epithelium, SPs have also been
described in mucosal surfaces of other tissues, for example, the
middle ear duct, esophagus, stomach, and peritoneal cavity
(Van Rozendaal et al. 2001), as well as in the genitourinary
tract (Bourbon and Chailley-Heu 2001). Hence, these proteins
may perform a generalized function in mucosal immunity, as
these sites are directly exposed to the environment. Mucosal
immunity involves both infectious defense and tolerance
against environmental and dietary antigens (Haagsman and
Diemel 2001). Furthermore, given that the epithelial lining of
these structures arises from different regions of the pharynx,
mucosal immunity may represent the ancient function of SPs
in the pharynx.
However, it is not only the SPs that are expressed in gut
mucosa. Surfactant-like lipid material enriched in phosphatidylcholine and demonstrating surface tension–lowering capabilities has also been reported as lining the gut epithelium
(Engle and Alpers 2001). Although the function of these lipids
is not clear, they are likely to have a role in protecting the
underlying epithelial cells from both mechanical and chemical
injury and may be involved in the binding of molecules for
biochemical processing (Engle and Alpers 2001). It is also possible that the lipids may have a role in dealing with interfacial
tension of liquids of different viscosities in the gastrointestinal
tract and other mucosal surfaces (Sullivan et al. 1998).
Hence, it is possible that the surfactant system could have
migrated with the epithelial cells during the out-pouching
events associated with the generation of either lungs or swim
bladders from different regions of the pharynx. To our knowledge, no analysis has been performed on the presence of SPs
in other gas-exchange organs of fish, such as the gills, buccal
cavity, or specialized respiratory regions of intestine. However,
given that these structures also originate from the pharynx, it
is likely that SPs are present in these structures.
This study is the first demonstration of the presence of SPD in the lungs of nonmammalian species and the first to demonstrate SP-B in actinopterygian fishes. SP-B has been previously detected in chicken and salamander lungs (Zeng et al.
1998; Miller et al. 2001), and SP-A is well documented in fish
(Rubio et al. 1996; Sullivan et al. 1998; Prem et al. 2000). The
functions of the SPs in mammals are not yet fully understood.
SP-A and SP-D are thought to function predominantly in immune defense by promoting the uptake of numerous pathogens
by macrophages (Haagsman and Diemel 2001). SP-B, on the
other hand, is intricately involved with the surfactant PLs and
is important in regulating the biophysical activity of the sur-
743
factant film (Haagsman and Diemel 2001). It is likely that both
the biophysical and immune functions of the SPs are important
in the lungs and swim bladders of fish. Moreover, the crossreactivity between the mammalian antibodies and the native,
in situ fish proteins implies that the tertiary structures of all
three proteins are highly conserved. This is remarkable, because
in spite of major differences in the structure and function of
gas-holding structures, both within a lineage and between distantly related animals, the structure of these proteins and their
retention as part of the surfactant system have not been affected.
We conclude that the proteins must be essential to both the
function of the surfactant system and the operation of lungs
and swim bladders. The genes for these proteins, therefore, may
have extremely low mutation rates, or it may be that any mutation is extremely deleterious and is selected out. This evidence
powerfully supports the hypothesis that a functional surfactant
system is a crucial prerequisite for the evolution of aerial gas
exchange using a lung or swim bladder.
Surfactant Lipids
The predominant lipid in all vertebrates examined is phosphatidylcholine (PC; Daniels and Orgeig 2001), and the disaturated form of this lipid, dipalmitoylphosphatidylcholine
(DPPC), is usually regarded as responsible for the reduction in
surface tension (Veldhuizen et al. 1993). However, the relative
abundance of DSP, USP, and cholesterol varies greatly between
different species (Daniels et al. 1995a). The surfactant of mammals, birds, and reptiles is rich in DSP. However, the surfactant
in the lungs of the air-breathing, ray-finned fishes (Polypteriformes and Ginglymodi) and the teleost swim bladders (Fig.
7; Table 2) have a Chol/DSP ratio that is up to 15 times higher
than that of the tetrapods (Smits et al. 1994). Similarly, the
most primitive of the lobe-finned fishes, the Australian lungfish
Neoceratodus forsteri has DSP/PL, Chol/DSP, and Chol/PL ratios
of the same order as those of the ray-finned, air-breathing fishes
(Daniels et al. 1995a; Orgeig and Daniels 1995; Table 2). However, two derived species of lobe-finned fishes, the South American and African lungfishes Lepidosiren paradoxa and Protopterus annectens, have a surfactant that is between two and three
times richer in DSP than N. forsteri or the primitive, airbreathing, ray-finned fishes (Polypteriformes and Ginglymodi;
Orgeig and Daniels 1995; Table 2). The derived lungfish also
have Chol/PL ratios that are between one-third and one-fifth
those of N. forsteri, the Polypteriformes, and the Ginglymodi.
Furthermore, on the basis of their Chol/PL and Chol/DSP ratios, the surfactants of the teleost fishes (pirarucu, tarpon, and
goldfish) group more closely with those of the primitive,
lunged, air-breathing, ray-finned fishes and N. forsteri. Collectively, these species and groups (N. forsteri, Teleosts, Polypteriformes, Ginglymodi) have Chol/PL ratios that are between
three- and sevenfold greater and Chol/DSP ratios that are between four- and 25-fold greater than those in the derived lung-
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744 C. B. Daniels, S. Orgeig, L. C. Sullivan, N. Ling, M. B. Bennett, S. Schürch, A. L. Val, and C. J. Brauner
Figure 7. Schematic diagram of the evolutionary sequence of air-breathing organs among the fishes. The ontogenetic origin (i.e., ventral or
dorsal) and the evolution of lungs, swim bladders, and their blood supply and the loss of respiratory function are indicated in italics.
Resp. p respiratory; fn p function. Figure modified from Perry et al. (2001).
fish (L. paradoxa and P. annectens; Table 2). Therefore, on a
holistic scale, it appears that the surfactant lipid composition
of all the ray-finned and primitive lobe-finned fishes is relatively
similar, in that they have a high-cholesterol, low-PL, low-DSP
mixture. On the other hand, the derived lobe-finned fishes (P.
annectens and L. paradoxa) have a surfactant composition much
more similar to that of the tetrapods. However, upon closer
examination it is apparent that among the ray-finned and primitive lobe-finned fishes there are some subtle differences in
surfactant composition that may be related to the function of
the gas-filled organ. For example, the goldfish (Table 2) is not
an air breather, and it has the highest Chol/PL ratio of all the
fish. Hence, it appears that the less the air-breathing organ is
used for gas exchange, the more cholesterol its surfactant contains. This high-cholesterol, low-PL, low-DSP mixture may represent the protosurfactant (Daniels et al. 1995a).
The functions of this mixture will differ significantly from
those of the surfactants of tetrapods, because it is poorly surface
active and highly fluid (see below). We have previously argued
that given the extremely smooth, nonseptated lungs of the polypterid fishes, the smooth swim bladder walls of the goldfish,
and the comparatively large respiratory units of the gar, lungfish, and other teleosts, a highly surface-active mixture may not
be required. It is possible that the larger size of the respiratory
units themselves may confer stability by substantially reducing
the collapse pressures that are present in the much smaller
alveoli of mammals (Daniels et al. 1998a). In these lungs or
swim bladders, a highly fluid (i.e., high-Chol, high-USP, lowDSP) mixture may assist in the easy spreading of the surfactant
from (presumably isolated) clumps of secretory cells to cover
the entire surface (Daniels et al. 1995b). Furthermore, such a
mixture is adequate to perform the antiadhesive function that
we have demonstrated for goldfish swim bladders, ray-finned,
air-breathing fish (Ginglymodi and Polypteriformes), and many
reptiles and amphibians (Daniels et al. 1995a). It is also possible
that the unusual combination of lipids, in particular the large
amount of cholesterol, in fish swim bladder surfactant may
serve as an antioxidant (Szebeni and Toth 1986), as the swim
bladders of most physoclist teleosts are exposed to hyperbaric
oxygen (Pelster 2001).
There are also differences in the relative abundance of the
PL head groups between the fishes and the tetrapods. In all
cases, PC is the dominant head group, but there is significant
variation in the presence and abundance of the minor PLs.
Surfactant isolated from mammals contains about 10% phosphatidylglycerol (PG) and only trace amounts of phosphatidylethanolamine (PE) and sphingomyelin (Daniels and Orgeig
2001). PG is relatively uncommon in vertebrate surfactants
(restricted only to mammals, a few amphibians, the lungfishes,
and one of three snake species that have been examined; Daniels
and Orgeig 2001). The surfactant isolated from N. forsteri contains large amounts of sphingomyelin and PE and only traces
of PG (Orgeig and Daniels 1995). The amount of sphingomyelin is even greater in the goldfish swim bladder than in the
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Surfactant in Air-Breathing Organs of Fish
Australian lungfish (Daniels and Skinner 1994). The most
abundant minor PL in fish surfactant is also sphingomyelin,
yet the surfactant essentially lacks PG. The significance of the
variation in the minor PLs, in particular PG, is unknown.
In terms of surfactant lipid composition, the strongest evidence that the surfactant system had a single evolutionary origin
comes from the similarity in composition between the Australian lungfish (N. forsteri), a primitive sarcopterygian closely
related to the stem ancestor of the tetrapods, and the primitive
actinopterygian air-breathing fish (Ginglymodi and Polypteriformes). Hence, the similarity in composition between primitive members of the two branches (Actinopterygii and Sarcopterygii) suggests that the surfactant system evolved once in
a common ancestor. Furthermore, the difference in the surfactant between these primitive representatives of the two
branches on the one hand and the more derived sarcopterygians
and tetrapods on the other hand supports our suggestion of a
protosurfactant.
Surface Activity
The surface activity of tarpon surfactant was marginally better
at 22⬚C than at 37⬚C, as reflected by a slightly lower STmin and
a reduced %SAcomp (Table 3). This presumably reflects an adaptation to function at temperatures that are more similar to
the body temperature experienced by the animal. It has previously been demonstrated that surfactant isolated from coldacclimated animals, such as marsupials and bats, which are
capable of undergoing torpor, functions more effectively at
room temperature (22⬚–24⬚C) than at 37⬚C. Conversely, if the
surfactant is isolated from the active animals, it is more active
at 37⬚C than at room temperature (Lopatko et al. 1998; Codd
et al. 2002). Whether fish are capable of adjusting their surfactant composition and function to match changes in body
temperatures is unknown. The activity of tarpon surfactant
certainly appears sensitive to temperature.
The surface activity of tarpon surfactant is extremely poor,
compared with that of mammalian surfactant measured under
the same conditions with the same device. Mammalian surfactant typically experiences an STmin of !1 mN/m, an STeq of
∼25 mN/m, and a %SAcomp of !20% (Schürch et al. 1992; Table
3). Moreover, the surface activity was also poor relative to that
measured in lizards on the Wilhelmy balance (Daniels et al.
1998a). Lizard surfactant demonstrated intermediate surface
activity with an STmin of 13–14 mN/m, an STeq of 25 mN/m,
and a %SAcomp of 30%. However, the surface activity of tarpon
surfactant was very similar to that previously reported for other
ray-finned fishes (Teleostei and Ginglymodi) and lungfishes
(Daniels et al. 1998a; Table 3). The STmin values measured with
alternative instruments, that is, either the Wilhelmy balance or
the Enhörning Bubble Surfactometer, ranged from 17 to 26
mN/m (Daniels et al. 1998a). This study is the first to use the
modern, state-of-the-art CBS in combination with high PL
745
concentrations (10 mg/mL, compared with 1 mg/mL PL) to
measure the surface activity of fish surfactant. The CBS is currently regarded as the most sophisticated and accurate device
for measuring the biophysical behavior of pulmonary surfactant
mixtures (Veldhuizen et al. 1998; Schürch et al. 2001). The
other methodologies are less reliable, particularly for highly
unsaturated, highly fluid surfactant mixtures, as they tend to
suffer from leakage of PLs out of the surfactant film (Veldhuizen
et al. 1998; Schürch et al. 2001). Hence, the poor surface activity
previously reported for fish surfactant appears to be a true
reflection of its biophysical properties and is not an artifact of
low PL concentrations or inferior (leaky) measurement devices
(Table 3).
It is likely that the surface-active properties observed for fish
surfactant directly reflect the differences in lipid and possibly
protein composition that we have described. For example, the
concentration of DSP in the tarpon swim bladder is only 4%
or less of total PL, which is even lower than that of other airbreathing fishes (9%–14%; Table 2; Smits et al. 1994) and virtually nonexistent compared with that of reptiles, birds, and
mammals (30%–50%; Veldhuizen et al. 1998; Daniels and Orgeig 2001). Given that DSP, and in particular DPPC, is the
major PL responsible for achieving low surface tensions upon
compression (Veldhuizen et al. 1998), it is not surprising that
fish surfactant is unable to generate low surface tensions. A
further characteristic of fish surfactant that mitigates against
low surface tensions is the extremely high concentration of
cholesterol (Chol/PL: 0.2–0.3; Chol/DSP: 1.0–5.6), compared
with that in mammals (Chol/PL: ∼0.08; Chol/DSP: ∼0.12; Daniels et al. 1995a), reptiles (Chol/PL: ∼0.03–0.08; Chol/DSP:
∼0.1–0.3; Daniels et al. 1996; Daniels and Orgeig 2001), and
amphibians (Chol/PL: ∼0.03–0.1; Chol/DSP: ∼0.2–0.3; Daniels
et al. 1994; Daniels and Orgeig 2001). While cholesterol (of the
order of 5%–10% of total PL) is essential in surfactant to promote the spreading of the saturated PL film upon inspiration
(Notter et al. 1980; Fleming and Keough 1988), high concentrations of cholesterol tend to inhibit surfactant surface activity
(Notter et al. 1980). Yet another characteristic of surfactant
from the tarpon and pirarucu swim bladders is the very low
concentration of SP-B (2, 17, and 44 pg/mg protein in the three
fish tested), compared with 2 ng/mg protein in mice, determined
by the same method (Tokieda et al. 1999). As SP-B is closely
associated with the surfactant lipids and has a crucial role in
promoting PL adsorption to the surfactant film (Haagsman
and Diemel 2001), the up to 100-fold lower concentration in
lavage of this essential protein no doubt also contributes to the
poor surface-active properties of fish surfactant.
We have previously argued that nonmammals, due to their
larger respiratory units, less complex lungs, and greater natural
lung distensibility, do not require a surfactant mixture capable
of the extremely low surface tensions typical of mammalian
surfactants. A more detergent-like surfactant (demonstrating
higher STmin and less variation) would be perfectly adequate
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746 C. B. Daniels, S. Orgeig, L. C. Sullivan, N. Ling, M. B. Bennett, S. Schürch, A. L. Val, and C. J. Brauner
(Daniels et al. 1998a). Surfactant of nonmammals is not
primarily responsible for maintaining alveolar stability and increasing lung compliance but rather is required as an antiadhesive and an antiedema agent (Daniels et al. 1998a). Nevertheless, fish surfactant, due to its composition and function,
appears to be in a class of its own—the protosurfactant. The
extraordinary differences between fish surfactant and that of
mammals, reptiles, and amphibians suggest that fish surfactant
has functions that have not yet been elucidated. These may
include functioning as an innate immune system or as an antioxidant system or even simply the provision of a medium for
dissolving other antioxidant, antiviral, or antimicrobial agents.
Conclusion
It appears that the evolution of the surfactant system predated
the evolution of lungs and swim bladders. However, the functional evolution of the surfactant system is closely coupled with
that of lungs or swim bladders in the fish and lungs in the
tetrapods. It also appears that there are two different types of
surfactant, one in the actinopterygian fishes that is high in
cholesterol and USPs and one in the advanced sarcopterygian
fishes and tetrapods that is relatively low in Chol and USPs
and high in DSPs. Fish surfactant is likely to be highly spreadable but is not very surface active. The tetrapod surfactant is
much more surface active and may, therefore, allow the development of more complex lungs with smaller respiratory
units and a greater total respiratory surface area, paving the
way for the evolution of high-performance lungs in amniotes.
It is possible that fish surfactant is an archaic or “proto-” surfactant that evolved into tetrapod surfactants but is also important as a lipid lining for the swim bladders in the modern
fish.
Many reptilian, amphibian, and fish lungs are essentially baglike, with a large central airspace and lacking a bronchial tree,
and with few exceptions they can collapse completely (Bishop
and Foxon 1968; Guimond and Hutchison 1976; Hughes and
Vergara 1978; Martin and Hutchison 1979; Stark-Vancs et al.
1984; Brainerd et al. 1989; Frappell and Daniels 1991a, 1991b).
A very important function of surfactant in these lung types is
to prevent the epithelial surfaces from sticking together (Daniels
et al. 1995a). Nonmammalian surfactant does not significantly
influence inflation compliance. Given the highly derived nature
of the mammalian lung, it is likely that regulation of surface
tension during nonatelectatic breathing is a late development
in the evolution of surfactant. Based on our studies of reptilian,
amphibian, and piscine surfactants, we conclude that while
acting as an antiadhesive may have been the original function
of surfactant, it led naturally to the alveolar stability and compliance roles that dominate mammalian surfactant function.
The surfactant system has been highly conserved, morphologically and biochemically, throughout (and despite) the enormous radiation of air-breathing organs among the vertebrates.
The lipid composition is conserved, and homology of SP-A,
SP-B, and SP-D demonstrates a single evolutionary origin for
the system.
Acknowledgments
This study was supported by an Australian Research Council
(ARC) grant to C.B.D., an ARC research fellowship to S.O., a
postgraduate research scholarship to L.C.S., an ARC grant to
M.B.B., the Alberta Heritage Foundation for Medical Research,
the Canadian Institute of Health Research, the Silva Casa Foundation (S.S.), the Natural Sciences and Engineering Research
Council (Canada; C.J.B.), and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (Brazil; A.L.V.). We thank
Helen Blacker for assistance with lipid analyses, Stanley Cheng
for the surface activity measurements, and Mark Mano, who
assisted with the cholesterol analysis.
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