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The Origin and Evolution of the Surfactant System in Fish: Insights into the Evolution ofLungs and Swim BladdersAuthor(s): Christopher B. Daniels, Sandra Orgeig, Lucy C. Sullivan, Nicholas Ling,Michael B. Bennett, Samuel Schürch, Adalberto Luis Val, and Colin J. BraunerSource: Physiological and Biochemical Zoology, Vol. 77, No. 5 (September/October 2004), pp.732-749Published by: The University of Chicago Press. Sponsored by the Division of ComparativePhysiology and Biochemistry, Society for Integrative and Comparative BiologyStable URL: http://www.jstor.org/stable/10.1086/422058 .

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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 Schurch4

Adalberto Luis Val5

Colin J. Brauner6

1School of Earth and Environmental Sciences, University ofAdelaide, Adelaide, South Australia 5005, Australia;2Department of Zoology, University of Auckland, Auckland,New Zealand; 3School of Biomedical Sciences, Department ofAnatomy and Developmental Biology, University ofQueensland, St. Lucia, Queensland 4072, Australia;4Department of Physiology and Biophysics, University ofCalgary, Calgary, Alberta T2N 4N1, Canada; 5InstitutoNacional de Pesquisas da Amazonia (INPA), Manaus,Amazonas 69083, Brazil; 6Department of Zoology, Universityof 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 eitherlungs or swim bladders as gas-holding structures. Lungs andswim bladders have different ontogenetic origins and can beused either for buoyancy or as an accessory respiratory organ.Therefore, the presence of air-filled bladders or lungs in dif-ferent 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: [email protected].‡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. � 2004 by TheUniversity of Chicago. All rights reserved. 1522-2152/2004/7705-3061$15.00

presence of a surfactant system and suggest that this systemmay have originated in epithelial cells lining the pharynx. Herewe present new data on the surfactant system in swim bladdersof three teleost fish (the air-breathing pirarucu Arapaima gigasand tarpon Megalops cyprinoides and the non-air-breathingNew Zealand snapper Pagrus auratus). We determined the pres-ence of surfactant using biochemical, biophysical, and mor-phological analyses and determined homology using immu-nohistochemical analysis of the surfactant proteins (SPs). Werelate the presence and structure of the surfactant system tothose previously described in the swim bladders of anotherteleost, the goldfish, and those of the air-breathing organs ofthe other members of the Osteichthyes, the more primitive air-breathing Actinopterygii and the Sarcopterygii. Snapper andtarpon swim bladders are lined with squamous and cuboidalepithelial cells, respectively, containing membrane-bound la-mellar bodies. Phosphatidylcholine dominates the phospholipid(PL) profile of lavage material from all fish analyzed to date.The presence of the characteristic surfactant lipids in pirarucuand tarpon, lamellar bodies in tarpon and snapper, SP-B intarpon and pirarucu lavage, and SPs (A, B, and D) in swimbladder tissue of the tarpon provide strong evidence that thesurfactant system of teleosts is homologous with that of otherfish and of tetrapods. This study is the first demonstration ofthe presence of SP-D in the air-breathing organs of nonmam-malian species and SP-B in actinopterygian fishes. The ex-tremely high cholesterol/disaturated PL and cholesterol/PL ra-tios of surfactant extracted from tarpon and pirarucu bladdersand the poor surface activity of tarpon surfactant are charac-teristics of the surfactant system in other fishes. Despite theparaphyletic phylogeny of the Osteichthyes, their surfactant isuniform in composition and may represent the vertebrateprotosurfactant.

Introduction

Many species of fishes can breathe air. Fish utilize a numberof different structures for aerial gas exchange, including theskin, gills, mouth and buccal cavity, intestine, and other spe-cifically evolved chambers. In particular, lungs appeared in-dependently several times. Lung structure can vary from thesimple, transparent, baglike structures of rope fish and bichirs(Polypteriformes, Cladista) to more complex compartmental-ized structures in lungfish (Dipnoi). Lungs are likely to have



Surfactant in Air-Breathing Organs of Fish 733

first appeared in the placoderms. Moreover, the possible pres-ence of lungs in placoderm fossils indicates that these animalsmust also have had a breathing mechanism and possibly arespiratory oscillator for responding to changes in blood andlung gases. Recent evidence suggests that such mechanismsmust have been in place before the lungs themselves evolved(Perry et al. 2001). However, placoderm lungs are not ho-mologous with the original Osteichthyian lungs, because pla-coderm lungs were most likely derived from the anterior phar-ynx (Denison 1941; Perry et al. 2001). Moreover, placodermlungs may have served a different function. They may havebeen important in promoting neutral buoyancy for theseheavily armored fish. With the extinction of the placoderms,lungs disappeared in the stem group and are lacking in allcartilaginous fishes. Chondrichthyians are less dense than pla-coderms, because they lack armor plates, and they use squalenein their liver for buoyancy (Hickman et al. 2001). However, itis likely that lungs reappeared in the stem ancestor of the Os-teichthyes. In both branches of the Osteichthyes (Sarcopterygii,i.e., lungfish and the most primitive Actinopterygii, i.e.,Polypteriformes), the lungs appear as paired ventral structuresderived from the posterior pharynx and posterior to the gills(reviewed in Perry et al. 2001). Primitive Devonian ostei-chthyian fish may have breathed air in response to low envi-ronmental O2 and used the neutral buoyancy that an air-filledlung provides to rest at the surface (Dehadrai and Tripathi 1976;Fange 1983).

However, it is now accepted that while lungs occur in themost primitive fishes and tetrapods, their ontogenetic origin isdifferent from that of swim bladders (Perry et al. 2001). Theswim bladder is an unpaired air-holding structure arising dor-sally from the posterior pharynx (Perry et al. 2001). Swimbladders are usually regarded as the primary buoyancy aid andalso support hearing by amplifying sound in the Ostariophysi(Johansen 1968). They are, however, employed in breathing ingars (Ginglymodi) and bowfin (Halecomorphi), where they aretermed “pulmonoid” because of the arterial supply from thesixth branchial arch. In some teleosts, including a basal teleost,the pirarucu (Arapaima gigas), and a derived teleost, the tarponor oxeye herring (Megalops cyprinoides), the swim bladder,which is supplied by a swim bladder artery, is respiratory (Perryet al. 2001).

Pulmonary surfactant plays a crucial role in the physicalforces acting at the air-liquid interface in the lungs during thedynamic changes in surface area and volume that occur duringinspiration and expiration. Surfactant consists of disaturatedphospholipids (DSP), unsaturated phospholipids (USP), neu-tral lipids (predominately cholesterol), and surfactant proteins(SPs). Four SPs have been described in mammals: SP-A, SP-B, SP-C, and SP-D. Surfactants have also been located anddescribed in fish swim bladders, including those that are usedfor buoyancy (e.g., in goldfish, eels, and carp; Daniels andSkinner 1994; Orgeig and Daniels 1995; Rubio et al. 1996;

Sullivan et al. 1998; Prem et al. 2000; Bourbon and Chailley-Heu 2001) and gas exchange (gar; Smits et al. 1994), and alsoin lunged fishes (rope fish, bichirs, and the three species ofsarcopterygian lungfishes; Smits et al. 1994; Orgeig and Daniels1995; Sullivan et al. 1998).

In this article, we use previously published biochemical andmorphological analyses of the surfactant system of primitivefish and add new information on this system in teleosts (twofreshwater-inhabiting air breathers, the pirarucu A. gigas andthe tarpon M. cyprinoides, and one marine fish, the snapperPagrus auratus). This review (with new data) demonstrates thatthe surfactant system of fishes is most likely homologous withthat of the tetrapods, despite the different ontogenetic originsand functional aspects of the air-breathing organs (Rubio et al.1996; Sullivan et al. 1998; Prem et al. 2000). In addition, thehigh-cholesterol, low–saturated phospholipid composition ofsurfactant found in fishes and in the ancient sarcopterygian,the Australian lungfish (Neoceratodus forsteri), represents a veryprimitive, poorly surface-active mixture, which we term a “pro-tosurfactant” (Daniels and Skinner 1994; Daniels et al. 1995a;Orgeig and Daniels 1995; Daniels and Orgeig 2001). A prim-itive, if not the original, function of fish surfactant is likely tobe acting as an antiadhesive (Daniels and Skinner 1994), butsurfactant may also prevent fluid from entering the bladder orlung, prevent oxidative damage to the epithelial lining, and actas an antiseptic/antibiotic (Daniels and Orgeig 2001). We alsooutline the evolution of the surfactant system in the fish, withparticular emphasis on postulating a mechanism whereby ahomologous system could appear in structures derived inde-pendently several times.

Material and Methods

Fish

The tarpon Megalops cyprinoides occurs in a wide range ofmarine and freshwater habitats, including East Africa, SoutheastAsia, Japan, Tahiti, Australia’s tropical seas, and the freshwatersof far northern Australia. The fish can tolerate a wide range ofhabitats, including landlocked lagoons and oxbow lakes(termed billabongs in Australia), with waters ranging from pH5.2 to 9.1 and temperatures between 23� and 34�C (Merrickand Schmida 1984). It has been reported that M. cyprinoideswill die in aquaria if prevented from reaching the surface (Mer-rick and Schmida 1984), and the closely related Atlantic tarpon(Megalops atlanticus) will regularly ventilate the swim bladderby a characteristic surface roll, even in normoxic waters (Gra-ham 1976). Therefore, air breathing may be essential for thisspecies, particularly in water with low oxygen levels (Graham1997).

The pirarucu Arapaima gigas is one of the largest freshwaterfishes in the world, reaching up to 4.5 m and 250 kg (Graham1997). It is an obligate air-breathing teleost from the Amazonthat will drown in 10–20 min without access to normoxic air.



734 C. B. Daniels, S. Orgeig, L. C. Sullivan, N. Ling, M. B. Bennett, S. Schurch, A. L. Val, and C. J. Brauner

The degree of aerial dependence is related to size. After hatch-ing, A. gigas is a water breather but quickly becomes an airbreather by the time it is about 18 mm long, 8–9 d posthatch(Graham 1997). To determine whether there is an effect of sizeon surfactant composition of the air-breathing organ, we col-lected two size classes, 30–70 g and 550–1,000 g. Unfortunately,these were the only sizes that could be obtained. Both sizes arecapable of surviving anoxic water by securing all O2 uptakefrom the air, and both groups secure approximately 80% oftheir metabolic O2 requirement from air in normoxic water (C.J. Brauner and A. L. Val, personal observations; Stevens andHoleton 1978). Clearly, air breathing is essential for this species.

The snapper Pagrus auratus is a demersal marine species inthe family Sparidae and is found in New Zealand, Australia,and Japan. Species of Sparid snappers comprise major com-mercial aquaculture and wild fisheries in many areas of theworld. Sparids are Perciform fishes possessing a euphysoclistousswim bladder, which is solely a hydrostatic organ and does notperform any respiratory function. The swim bladder developsas an extrusion of the gut wall and is first filled by swallowingair via the pneumatic duct at around 5 d after hatching. Byaround 30 d after hatching, the pneumatic duct has closed andthe 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 Ter-ritory, Australia) or the Brisbane River (southern Queensland,Australia). We killed the fish by bathing them in a solution of1% MS-222 and removed the tissues overlying the swim blad-der. A tube 2 mm in diameter was passed into the swim bladdervia the pharyngeal opening, and the air was then sucked outwith a 20 mL syringe. The bladder was lavaged with 5–6 mLof ice-cold 0.15 M NaCl. The saline was withdrawn, and thelavaging process was repeated twice. The lavage was centrifugedat 150 g for 10 min to remove cellular debris and lyophilized.Material for biochemical analysis, transmission electron mi-croscopy (TEM), and immunohistochemistry was collectedfrom this species.

Pirarucu (Arapaima gigas). Six small fish (33–66 g) and threelarger fish (550, 655, and 1,006 g) were obtained from Boutiquedo Peixe Vivo and Amazonas Ecopeixe S.A. and held at theNational Institute for Research of the Amazon on a naturallight cycle for 3 wk before sampling. During this time, fish werefed goldfish at a ration of 1% body weight per day. Fish werekilled in a solution of 1% MS-222, a tube was inserted intothe pharyngeal opening of the swim bladder, and lavage wasobtained as described above for tarpon. The entire ventral lowerhalf of the swim bladder was removed and weighed before andafter lyophilization. Only lavage material was collected fromthis species.

Snapper (Pagrus auratus). Snapper (body g) weremass p 500captured by line fishing in the Hauraki Gulf, New Zealand.Fish were killed by stunning and pithing, and a midventralincision was made from the pelvic girdle to the vent to exposethe gut cavity. Both the internal and external surfaces of theswim bladder were fixed before excision to optimize tissue fix-ation and maintain the in situ arrangement of the delicatemucosal surface. We ligated the esophagus and removed allabdominal organs, taking care not to damage the swim bladder.The gut cavity was then filled with fixative (2% paraformal-dehyde, 2% gluteraldehyde, 0.1 M sodium cacodylate, 375mOsm, pH 7.8). In order to fix the inside of the swim bladderwithout altering its volume, the swim bladder gases were grad-ually replaced with fixative. A 20-mL syringe was half filledwith fixative and the syringe needle introduced to the swimbladder lumen through the epaxial muscle. One milliliter offixative was introduced, followed by the withdrawal of the samevolume of swim bladder gas. This reciprocal replacement wasrepeated until the lumen was entirely filled. Only material forTEM 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 excisedfrom the body and cut into small strips of approximately

mm and continued to fix for 24 h in 2% paraformal-2 # 5dehyde, 2% gluteraldehyde, 0.1 M sodium cacodylate, 375mOsm, pH 7.8. In the case of the tarpon, sections of the bladderwall and associated respiratory tissue bands forming the ac-cessory breathing organ were cut into small pieces and fixedfor TEM, as previously described (Sullivan et al. 2001).

In the tarpon, sections for immunohistochemistry were pre-pared by freeze substitution and immunolabelling, as previouslydescribed (Sullivan et al. 1998). The primary antibodies to hu-man SP-A and SP-D and bovine SP-B were purchased (Chem-icon Australia, Boronia, Australia).

Surfactant Composition

Methods for measuring total phospholipids, disaturated phos-pholipids, and phospholipid headgroups have been publishedpreviously (Daniels and Skinner 1994; Orgeig and Daniels 1995;Daniels et al. 1999). We measured cholesterol in the neutrallipid fraction (reconstituted with heptane) with a high-perfor-mance liquid chromatography (HPLC) system (LC1500 HPLCpump, LC1610 autosampler, DP800 data interface; GBC In-struments, Melbourne, Australia). We injected 30 mL onto asilica column (Alltech with 5 mm spherical silica)4.6 # 250 mmequilibrated with a hexane : isopropanol (99 : 1 v/v) mobilephase (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).




Figure 1. Appearance of the respiratory portion of the swim bladder of the tarpon Megalops cyprinoides. The surface consists of a thin layerof capillaries (C) and an overlying layer of epithelial cells (EC). . . Scale mm.AS p airspace N p nucleus bar p 10

A backpressure regulator (100 psi) was fitted to the detectoroutlet to minimize the formation of bubbles in the flow cell.Retention time for cholesterol was 10.2 min, and total run timeper sample was 25 min.

SP-B in lavage samples from tarpon and pirarucu was quan-tified in a competitive ELISA based on a previously publishedmethod (Lesur et al. 1993). The primary antibody, rabbit an-tibovine SP-B polyclonal antibody (Chemicon Australia), andthe 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), andthe absorbance at 492 nm was determined with a Titertek MCCMultiskan (Titertek, Huntsville, Ala.). The amount of SP-B inthe lavage samples was compared to the amount of total pro-tein, which was determined by the method of Lowry et al.(1951).

Surface Activity

Surface activity for an aliquot of lyophilized lavage from onetarpon was determined after the lavage was reconstituted inwater and centrifuged at 40,000 g (Beckman Optima TLX Ul-tracentrifuge; Beckman, Sydney, Australia) for 30 min to pelletlarge aggregate material. A phosphorus assay of the pelletedmaterial and the supernatant revealed that 190% of the phos-pholipids remained in the supernatant. The supernatant waslyophilized again and reconstituted in a buffered salt solution

(140 mM NaCl, 10 mM HEPES, 2.5 mM CaCl2, pH 6.9) to aconcentration 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-waterinterface (Schurch et al. 1992). We used a previously publishedmethod for the preparation of small sample volumes (Codd etal. 2002). We measured the rate of adsorption for the first 5min and then performed quasi-static compressions (Codd etal. 2002). From these we determined the minimum surfacetension upon final compression (STmin), the maximum surfacetension before compression (STmax), and the percent surfacearea of compression (%SAcomp), which is a measure of the extentof compression required to achieve STmin. Measurements wereperformed both at room temperature (22�C) and at 37�C. Werecorded the bubble continuously and calculated bubble vol-ume, area, and surface tension from digitized bubble images,using height and diameter (Schoel et al. 1994). We comparedthese results to values obtained for other fish species using thisand other techniques.

Results

Electron Microscopy and Immunohistochemistry

Tarpon. The respiratory surface of the swim bladder accessorybreathing organ consisted of a thin layer of blood capillariesand an overlying layer of epithelial cells (Fig. 1). There were a




Figure 2. Cuboidal epithelial cells in the lining of the swim bladder of the tarpon Megalops cyprinoides. The cells have a large nucleus andcontain either numerous electron-lucent structures (A, arrows, scale mm) or electron-dense structures (B, arrows, scale mm).bar p 2 bar p 1

number of cuboidal epithelial cells containing a dark nucleusand numerous electron-lucent structures (Fig. 2A) that appearto represent one form of lamellar body (Table 1). Other lamellarbodies appeared as granular, electron-dense structures (Figs.2B, 3A), and still others consisted of concentric rings of lamellaein a membrane-bound vesicle (Fig. 3B). Different structuresfor the lamellar bodies are also reported for the snapper (seebelow) and may represent a developmental sequence. At ahigher magnification, SP-A, SP-B, and SP-D were locatedwithin the tarpon swim bladder. SP-A and SP-B were locatedboth within the cells and in the airspaces. The location of SP-A appeared to be quite diffuse (Fig. 3A), whereas SP-B wasfound to be associated with lamellar bodies (Fig. 3B). A smallamount of SP-D staining was found in the air spaces only (Fig.3C).

Snapper. The lining of the swim bladder lumen was composedof squamous epithelial cells. These cells were generally in theregion of 200 nm thick, except for a central thickening due tothe presence of the nucleus and adjacent mitochondria. Cellswere joined peripherally by tight junctions at the lumenal sur-face and formed extensive interdigitations with adjoining cellsthat extended for 1–2 mm from the position of cellular abut-ment. The apical surface of the epithelium was interrupted byoccasional microvilli that appeared more common toward thecell periphery. This cell type was characterized by the presenceof numerous lamellar bodies or cytosomes. Cytosomes ap-peared to be more common in the region of central thickeningclose to the nucleus (Fig. 4A, 4B). The cytosomes of the lumenalepithelium were of the form described by Creasey et al. (1974)

as simian and common in human Type II alveolar cells, al-though their description of this structure as having concentriclamellae may be misleading and appears to depend on the planeof sectioning and possibly the developmental stage of the cy-tosome (Table 1). For example, a lamellar body with lamellaefused at the poles may appear concentric if sectioned equa-torially. As with the tarpon, several different morphs of cyto-some were recognized and may represent a developmental se-quence based on the assumption that all eruptant cytosomesappeared to be of the concentric lamellate form. The proposeddevelopmental sequence for these cytosomes is described inFigure 4C.

Cytosomes appear to form from membrane-bound, electron-dense aggregations of an amorphous material. Brooks (1970)reports formation of epithelial cytosomes in trout from similarelectron-dense bodies and states that this material appeared tobe composed of very tightly packed membranes. The lamellatestructure of these bodies in the snapper could only be distin-guished close to the site at which lamellae were separating fromthe electron-dense material (Fig. 4C). These osmiophilic la-mellae begin to form and separate centrally but remain attachedperipherally in an equatorial, or possibly polar, adhesion plaque(Fig. 4D). Total separation of the individual lamellae to forma truly concentric structure seems to occur just before eruptionfrom the apical surface of the cell.

The gas gland epithelium contains multilamellar bodies, orcytosomes, of the form previously described for the toadfishgas gland (Morris and Albright 1975, 1977) and classified assubsimian by Creasey et al. (1974). This cytosome has lamellaethat traverse from one side of the structure to the other (Fig.



Table 1: Morphological characteristics of surfactant from fishes (Osteichthyes)

SpeciesType II Cells(and Analogues)a

LamellarBodies

TubularMyelin Other Forms and Comments References

Dipnoi:Protopterus annectens ? � ? 1Protopterus sp. � � ? Osmiophilic inclusion bodies in cells and

in alveolar space2

Lepidosiren paradoxa �(*) � ? (*)Only one cell type 3L. paradoxa � � � Extracellular material also present 4Neoceratodus forsteri �(*) � ? (*)Only one cell type 3N. forsteri � � ? Type II cells can be isolated; secrete LP 5

Teleostei:Salmo gairdneri � � � Nonciliated (columnar or cuboidal?) cells

that secrete mucus-like material intolumen of swim bladder

6

Anguilla vulgaris ? � ? 7Lota lota � � ? Very isolated LBs in cells of the gas

gland8

Acerina cernua ? � ? 8Coregonus lavaretus ? � ? 9Fundulus heteroclitus ? � ? 10Gadus callarias ? � ? 10Opsanus tau ? � ? 10Amphipnous cuchia �(*) � � (*)Cuboidal cells; air sac instead of lung 11, 12Anabas tetudineus � � � Labyrinthine organ 11, 12Channa punctatus �(*) � � (*)Cuboidal cells; suprabranchial

chamber11, 12

Channa striatus �(*) � � (*)Cuboidal cells; suprabranchialchamber

11, 12

Clarias batrachius �(*) � � (*)Cuboidal cells with vesicles; air sac 11, 12Heteropneustes fossilis �(*) � � (*)Cuboidal cells; air sac 12, 13Misgurnus fossilis �(*) � � (*)Goblet epithelial cell of respiratory

intestine14

Pagrus auratus � � � Different forms of LBs; they possiblyrepresent a developmental sequence,i.e., only eruptant LBs haveconcentric lamellae

15

Megalops cyprinoides � � ? Different forms of LBs (electron-lucent,electron-dense, or with concentriclamellae); SPs A, B, D present in TypeII cells and airspace

15

Halecomorphi:Amia calva ? � ? 3

Polypteriformes:Polypterus senegalensis � � � Electron-opaque granules in rare cells in

secretory crypts16

Polypterus ornatipinnis � � � “Classical-appearing” Type II cells 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.




Figure 3. Immunogold labeling of SP-A (A; scale mm), SP-bar p 0.2B (B; scale mm), and SP-D (C; scale mm) in thebar p 0.5 bar p 0.5respiratory swim bladder of the tarpon Megalops cyprinoides. SP-A waslocalized in the air spaces (filled arrows) and in the epithelial cells (open

arrows), whereas SP-B appeared to be primarily associated with la-mellar bodies (arrows). The presence of SP-D was restricted only tothe airspaces (arrows).

4E). Eruption of this form of cytosome through the apical cellsurface was not observed. Simian or concentrically lamellatecytosomes of the form common to the general lumenal epi-thelium were also observed and seen to occur in eruption fromthe epithelium. It was notable that eruption of this form ofcytosome could occur either from the apical surface onto thesurface of the lumen or through the basal surface into under-lying circulatory sinuses. Extracellular material was occasionallyobserved on the lumenal surface of the gas gland, trapped be-tween apical microvilli (Fig. 4F). The transverse structure ofthis material (Fig. 4G) resembles the cytosomal tubular myelinof the vertebrate lung. As the crosshatched structure of tubularmyelin is highly characteristic of surfactant, it is likely that thematerial in the snapper gas gland is surfactant.

Surfactant Composition

Tarpon. The disaturated phospholipid (DSP) content of thelavage material was very low and was measurable in only oneof the four tarpon fish samples ( ). The cho-%DSP/PL p 4.04lesterol (Chol) to DSP ratio (mg/mg) in this one sample was5.65. The cholesterol to phospholipid (PL) ratio (mg/mg) in thelavage was ( , ; Table 2). Thin-0.258 � 0.069 mean � SE n p 4layer 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 phos-phatidylinositol (7.7%). Lysophosphatidylcholine (1%), phos-phatidylglyerol (1%), and phosphatidylethanolamine (0.3%)were present only in trace amounts (Table 2). The ELISA dem-onstrated 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 animaland 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( , ). The Chol/PL and Chol/DSP26.85 � 3.35 mean � SE n p 6

ratios (mg/mg) were and (0.266 � 0.02 1.03 � 0.08 mean �

, ), respectively (Table 2). The Chol/PL ratio of theSE n p 6lavage from the large fish was ( ,0.34 � 0.114 mean � SE n p

) and did not differ from that of the small fish. We did not3measure DSP in the large pirarucu. SP-B was measurable byELISA in the lavage of one of the large pirarucu. The lavageof this fish contained 44.6 pg SP-B per mg of total protein.

Surface Activity

The adsorption of surfactant from the tarpon swim bladderwas extremely slow. At 22�C the surfactant adsorbed slightly



739

Figure 4. Multilamellar bodies (cytosomes) and tubular myelin in the snapper (Pagrus auratus) swim bladder. A, Cytosome erupting fromlumenal epithelium (scale mm). B, Semilamellate cytosome (scale mm). C, Progressive development of cytosomes from electron-bar p 1 bar p 1dense amorphous granules to semilamellate form (scale mm). D, Semilamellate cytosome, lamellae fused equatorially (scalebar p 1 bar p 2mm). E, Subsimian cytosome (scale nm). F, Lumenal surface material, tubular myelin (scale nm). G, Transverse structurebar p 250 bar p 250of the material in F (scale nm).bar p 100



740

Table 2: Surfactant composition in fishes (Osteichthyes)

Species nTA/B

(�C)PL(mg/gWL)

Percent of PLs Chol(mg/gWL) Chol/PL

DSP(mg/gWL) Chol/DSP

DSP/PL(%)PC PG PE SM PI PS LPC/Unk

Dipnoi:Neoceratodus forsteria 3 23 .084 59.6 .5 14.4 15.3 10.2 0 .022 .242 .006 3.66 8.9Lepidosiren paradoxab 1–3 RT ND 46.0 8.0 19.0 11.0 0 0 0 ND ND ND NDL. paradoxaa 3 23 .22 73.9 1.8 6.9 7.3 9.3 .8 .010 .044 .059 .169 27.8Protopterus annectensa 4 23 .61 80.2 3.0 3.0 5.6 7.9 .4 .028 .052 .126 .222 20.4

Teleostei:Hoplias malabaricusb 1–3 RT ND 70 0 7 5 0 12 5 ND ND ND ND NDHoplerythrinus unitaeniatusb 1–3 RT ND 69 0 25 2 0 2 0 ND ND ND ND NDErythrinus erythrinusb 1–3 RT ND 93 0 3 2 2 0 0 ND ND ND ND NDArapaima gigasb 1–3 RT ND 39 6 7 24 8 8 8 ND ND ND ND NDA. gigasc 6 RT ND ND ND ND ND ND ND ND .266 ND 1.03 26.85Megalops cyprinoidesc 4 RT ND 71 1 .3 18.4 7.7 1 ND .258 ND 5.65 !4Carassius auratus (posteriord

swim bladder) 9 23 .14 75 1.2 4.7 15.9 5.1 0 .051 .328 .036 1.41 20.8Ginglymodi:

Lepisosteus osseuse 5–8 23 .50 76.4 1.4 4.6 5.9 7.9 3.0 .091 .175 .069 1.30 14.1Polypteriformes:

Calamoichthys calabaricuse 2–9 23 .23 ND ND ND ND ND ND .044 .182 .022 2.05 9.54Polypterus senegalensise 2–8 23 .097 ND ND ND ND ND ND .021 .208 .008 1.72 11.6

Note. or body temperature; ; of wet lung mass; ; ; ;T p ambient PL p phospholipid gWL p grams PC p phosphatidylcholine PG p phosphatidylglycerol PE p phosphatidyl-ethanolamineA/B

; ; (a single value for PI and PS indicates that the bands could not be resolved); ;SM p sphingomyelin PI p phosphatidylinositol PS p phosphatidylserine LPC p lysophosphatidylcholine

; ; PL. (22�–25�C); determined.unkwn p unknown PL Chol p cholesterol DSP p disaturated RT p roomtemperature ND p nota Orgeig and Daniels 1995.b Phleger and Saunders 1978.c This study.d Daniels and Skinner 1994.e Smits et al. 1994.




Figure 5. Rate of adsorption of tarpon (Megalops cyprinoides) surfactant(10 mg/mL PL) to the air-liquid interface of a bubble in the captivebubble surfactometer. The surface tension at the end of adsorption isthe equilibrium surface tension (STeq).

Figure 6. Quasi-static (QS) surface tension–area relations for tarpon(Megalops cyprinoides) surfactant on the captive bubble surfactometerat (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 limbof each curve represents compression and the upper expansion. Thelowest point at the end of compression represents STmin. The changein surface area compression (%SAcomp) required to reach STmin is anindication of the surface tension–lowering quality of the sample. Sam-ple concentration was 10 mg/mL PL. A color version of this figure isavailable in the online edition of the journal.

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-staticcompression cycle, tarpon surfactant demonstrated relativelyhigh 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–loweringproperties, including STmin and %SAcomp, were marginally betterat 22� than at 37�C (Fig. 6). Film stability was poor, as seenfrom the slight progressive increase in STmin following successivequasi-static cycles (Fig. 6; Table 3).

Discussion

Morphology

Cells containing lamellar bodies and surfactant lipids and pro-teins are easily observable in the swim bladder of the tarponand the snapper. It is also clear that the function of the gas-holding structure (respiration or buoyancy) is irrelevant to therequirement for a surfactant system in fish (Table 1). The sur-factant-secreting cells in the tarpon and the snapper do notdiffer structurally from those of other fishes and possess thesame characteristics as the alveolar Type II epithelial cells foundin tetrapod lungs (Daniels and Orgeig 2001). They demonstratemicrovilli on the apical surface, and the lamellar bodies aremembrane-bound and of similar size and appearance to thoseobserved in all other vertebrate groups, including fishes (Table1; Hughes 1970, 1973; Hughes and Weibel 1978; Prem et al.2000; Wood et al. 2000; Daniels and Orgeig 2001).

Surfactant Proteins

The presence of the surfactant lipids and proteins (SP-A, SP-B, and SP-D) provides strong evidence that the surfactant sys-tem of all fishes is homologous with that of the tetrapods.Furthermore, the surfactant system located in the lungs or swimbladders of fish (lungfish, bichirs, gar, carp, eels, goldfish, snap-per, pirarucu, and tarpon) is likely to be homologous (Danielsand Skinner 1994; Rubio et al. 1996; Sullivan et al. 1998; Prem




Table 3: Surface activity of surfactant from fishes (Osteichthyes)

Species and TA (�C) DeviceMin ST(mN/m)

%SAComp

StabilityRatios

BubbleClickinga

Surpellic Property/Surface Activity

Dipnoi:Protopterus annectens:

RT Stableb �c Fully surpellicb

Lepidosiren paradoxa:RT Calculated from bubble shapeb !.1 Stableb �d Fully surpellicb

37 WBd 2224 WBe 22 70 Very lowe

Neoceratodus forsteri:RT �f Partially surpellicb

Teleostei:Carassius auratus:

RT Calculated from bubble shapeb 20 �c Partially surpellicb

22 PBSg 27.4 Very lowe

Megalops cyprinoides:22h CBS 19.5 75.3 Very low37h 21.7 75.8 Very low

Hoplias malobaricus:37 WBd 22 70e Very lowe

Hoplerythrinus unitaeniatus:37 WBd 22 80e Very lowe

Arapaima gigas:37 WBd 22 75e Very lowe

Halecomorphi:Amia calva:

RT Calculated from bubble shapeb 4 �c Partially surpellicb

Ginglymodi:Lepisosteus osseus:

RT Calculated from bubble shapeb 2–6 �c Partially surpellicb

24 WBe 17.0 � 1.0 70 Very lowe

Note. temperature; Min surface tension; %SA surface area compression required to achieve min ST;T p ambient ST p minimum Comp p percentA

temperature (22�–25�C). balance; bubble surfactometer; bubble surfactometer. The number ofRT p room WB p Wilhelmy PBS p pulsating CBS p captive

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 separateontogenetic origins and arise from different pharyngeal regions(Perry et al. 2001), we can conclude that the surfactant systemmust predate the appearance of both lungs and swim bladders.Significant recent evidence has demonstrated surfactant-containing lamellar bodies secreted from epithelial cells in themammalian gut (Engle and Alpers 2001). SP-A and SP-D co-

localize with these structures (Rubio et al. 1995; Engle andAlpers 2001). Furthermore, SP-A antibodies also cross-reactwith material from both the intestine and swim bladder of thecarp (Rubio et al. 1996). We therefore postulate that the sur-factant system originated in the epithelial cells lining the phar-ynx. Here the SPs may have played an important role in theinnate immune system of the organism.




The hydrophilic SPs, SP-A and SP-D, belong to the familyof proteins known as collectins that are characterized by C-terminal carbohydrate recognition domains. These proteins arethought to function as molecules of the innate immune systemby recognizing a broad spectrum of pathogens, including vi-ruses, bacteria, and fungi, as well as allergens, such as pollengrains and mite allergens (Haagsman and Diemel 2001). Inaddition to their presence in gut epithelium, SPs have also beendescribed in mucosal surfaces of other tissues, for example, themiddle ear duct, esophagus, stomach, and peritoneal cavity(Van Rozendaal et al. 2001), as well as in the genitourinarytract (Bourbon and Chailley-Heu 2001). Hence, these proteinsmay perform a generalized function in mucosal immunity, asthese sites are directly exposed to the environment. Mucosalimmunity involves both infectious defense and toleranceagainst environmental and dietary antigens (Haagsman andDiemel 2001). Furthermore, given that the epithelial lining ofthese structures arises from different regions of the pharynx,mucosal immunity may represent the ancient function of SPsin the pharynx.

However, it is not only the SPs that are expressed in gutmucosa. Surfactant-like lipid material enriched in phosphati-dylcholine and demonstrating surface tension–lowering capa-bilities has also been reported as lining the gut epithelium(Engle and Alpers 2001). Although the function of these lipidsis not clear, they are likely to have a role in protecting theunderlying epithelial cells from both mechanical and chemicalinjury and may be involved in the binding of molecules forbiochemical processing (Engle and Alpers 2001). It is also pos-sible that the lipids may have a role in dealing with interfacialtension of liquids of different viscosities in the gastrointestinaltract and other mucosal surfaces (Sullivan et al. 1998).

Hence, it is possible that the surfactant system could havemigrated with the epithelial cells during the out-pouchingevents associated with the generation of either lungs or swimbladders from different regions of the pharynx. To our knowl-edge, no analysis has been performed on the presence of SPsin other gas-exchange organs of fish, such as the gills, buccalcavity, or specialized respiratory regions of intestine. However,given that these structures also originate from the pharynx, itis likely that SPs are present in these structures.

This study is the first demonstration of the presence of SP-D in the lungs of nonmammalian species and the first to dem-onstrate SP-B in actinopterygian fishes. SP-B has been previ-ously 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). Thefunctions of the SPs in mammals are not yet fully understood.SP-A and SP-D are thought to function predominantly in im-mune defense by promoting the uptake of numerous pathogensby macrophages (Haagsman and Diemel 2001). SP-B, on theother hand, is intricately involved with the surfactant PLs andis important in regulating the biophysical activity of the sur-

factant film (Haagsman and Diemel 2001). It is likely that boththe biophysical and immune functions of the SPs are importantin the lungs and swim bladders of fish. Moreover, the cross-reactivity between the mammalian antibodies and the native,in situ fish proteins implies that the tertiary structures of allthree proteins are highly conserved. This is remarkable, becausein spite of major differences in the structure and function ofgas-holding structures, both within a lineage and between dis-tantly related animals, the structure of these proteins and theirretention as part of the surfactant system have not been affected.We conclude that the proteins must be essential to both thefunction of the surfactant system and the operation of lungsand swim bladders. The genes for these proteins, therefore, mayhave extremely low mutation rates, or it may be that any mu-tation is extremely deleterious and is selected out. This evidencepowerfully supports the hypothesis that a functional surfactantsystem is a crucial prerequisite for the evolution of aerial gasexchange using a lung or swim bladder.

Surfactant Lipids

The predominant lipid in all vertebrates examined is phos-phatidylcholine (PC; Daniels and Orgeig 2001), and the disat-urated form of this lipid, dipalmitoylphosphatidylcholine(DPPC), is usually regarded as responsible for the reduction insurface tension (Veldhuizen et al. 1993). However, the relativeabundance of DSP, USP, and cholesterol varies greatly betweendifferent species (Daniels et al. 1995a). The surfactant of mam-mals, birds, and reptiles is rich in DSP. However, the surfactantin the lungs of the air-breathing, ray-finned fishes (Polypteri-formes and Ginglymodi) and the teleost swim bladders (Fig.7; Table 2) have a Chol/DSP ratio that is up to 15 times higherthan that of the tetrapods (Smits et al. 1994). Similarly, themost primitive of the lobe-finned fishes, the Australian lungfishNeoceratodus forsteri has DSP/PL, Chol/DSP, and Chol/PL ratiosof the same order as those of the ray-finned, air-breathing fishes(Daniels et al. 1995a; Orgeig and Daniels 1995; Table 2). How-ever, two derived species of lobe-finned fishes, the South Amer-ican and African lungfishes Lepidosiren paradoxa and Protop-terus annectens, have a surfactant that is between two and threetimes richer in DSP than N. forsteri or the primitive, air-breathing, ray-finned fishes (Polypteriformes and Ginglymodi;Orgeig and Daniels 1995; Table 2). The derived lungfish alsohave Chol/PL ratios that are between one-third and one-fifththose of N. forsteri, the Polypteriformes, and the Ginglymodi.Furthermore, on the basis of their Chol/PL and Chol/DSP ra-tios, the surfactants of the teleost fishes (pirarucu, tarpon, andgoldfish) group more closely with those of the primitive,lunged, air-breathing, ray-finned fishes and N. forsteri. Collec-tively, these species and groups (N. forsteri, Teleosts, Polypter-iformes, Ginglymodi) have Chol/PL ratios that are betweenthree- and sevenfold greater and Chol/DSP ratios that are be-tween four- and 25-fold greater than those in the derived lung-




Figure 7. Schematic diagram of the evolutionary sequence of air-breathing organs among the fishes. The ontogenetic origin (i.e., ventral ordorsal) and the evolution of lungs, swim bladders, and their blood supply and the loss of respiratory function are indicated in italics.

; . Figure modified from Perry et al. (2001).Resp. p respiratory fn p function

fish (L. paradoxa and P. annectens; Table 2). Therefore, on aholistic scale, it appears that the surfactant lipid compositionof all the ray-finned and primitive lobe-finned fishes is relativelysimilar, in that they have a high-cholesterol, low-PL, low-DSPmixture. On the other hand, the derived lobe-finned fishes (P.annectens and L. paradoxa) have a surfactant composition muchmore similar to that of the tetrapods. However, upon closerexamination it is apparent that among the ray-finned and prim-itive lobe-finned fishes there are some subtle differences insurfactant composition that may be related to the function ofthe gas-filled organ. For example, the goldfish (Table 2) is notan air breather, and it has the highest Chol/PL ratio of all thefish. Hence, it appears that the less the air-breathing organ isused for gas exchange, the more cholesterol its surfactant con-tains. This high-cholesterol, low-PL, low-DSP mixture may rep-resent the protosurfactant (Daniels et al. 1995a).

The functions of this mixture will differ significantly fromthose of the surfactants of tetrapods, because it is poorly surfaceactive and highly fluid (see below). We have previously arguedthat given the extremely smooth, nonseptated lungs of the po-lypterid fishes, the smooth swim bladder walls of the goldfish,and the comparatively large respiratory units of the gar, lung-fish, and other teleosts, a highly surface-active mixture may notbe required. It is possible that the larger size of the respiratoryunits themselves may confer stability by substantially reducingthe collapse pressures that are present in the much smalleralveoli of mammals (Daniels et al. 1998a). In these lungs or

swim bladders, a highly fluid (i.e., high-Chol, high-USP, low-DSP) mixture may assist in the easy spreading of the surfactantfrom (presumably isolated) clumps of secretory cells to coverthe entire surface (Daniels et al. 1995b). Furthermore, such amixture is adequate to perform the antiadhesive function thatwe have demonstrated for goldfish swim bladders, ray-finned,air-breathing fish (Ginglymodi and Polypteriformes), and manyreptiles and amphibians (Daniels et al. 1995a). It is also possiblethat the unusual combination of lipids, in particular the largeamount of cholesterol, in fish swim bladder surfactant mayserve as an antioxidant (Szebeni and Toth 1986), as the swimbladders of most physoclist teleosts are exposed to hyperbaricoxygen (Pelster 2001).

There are also differences in the relative abundance of thePL head groups between the fishes and the tetrapods. In allcases, PC is the dominant head group, but there is significantvariation in the presence and abundance of the minor PLs.Surfactant isolated from mammals contains about 10% phos-phatidylglycerol (PG) and only trace amounts of phosphati-dylethanolamine (PE) and sphingomyelin (Daniels and Orgeig2001). 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; Danielsand Orgeig 2001). The surfactant isolated from N. forsteri con-tains large amounts of sphingomyelin and PE and only tracesof PG (Orgeig and Daniels 1995). The amount of sphingo-myelin is even greater in the goldfish swim bladder than in the




Australian lungfish (Daniels and Skinner 1994). The mostabundant minor PL in fish surfactant is also sphingomyelin,yet the surfactant essentially lacks PG. The significance of thevariation in the minor PLs, in particular PG, is unknown.

In terms of surfactant lipid composition, the strongest evi-dence that the surfactant system had a single evolutionary origincomes from the similarity in composition between the Austra-lian lungfish (N. forsteri), a primitive sarcopterygian closelyrelated to the stem ancestor of the tetrapods, and the primitiveactinopterygian air-breathing fish (Ginglymodi and Polypteri-formes). Hence, the similarity in composition between prim-itive members of the two branches (Actinopterygii and Sar-copterygii) suggests that the surfactant system evolved once ina common ancestor. Furthermore, the difference in the sur-factant between these primitive representatives of the twobranches on the one hand and the more derived sarcopterygiansand tetrapods on the other hand supports our suggestion of aprotosurfactant.

Surface Activity

The surface activity of tarpon surfactant was marginally betterat 22�C than at 37�C, as reflected by a slightly lower STmin anda reduced %SAcomp (Table 3). This presumably reflects an ad-aptation to function at temperatures that are more similar tothe body temperature experienced by the animal. It has pre-viously been demonstrated that surfactant isolated from cold-acclimated animals, such as marsupials and bats, which arecapable of undergoing torpor, functions more effectively atroom temperature (22�–24�C) than at 37�C. Conversely, if thesurfactant is isolated from the active animals, it is more activeat 37�C than at room temperature (Lopatko et al. 1998; Coddet al. 2002). Whether fish are capable of adjusting their sur-factant composition and function to match changes in bodytemperatures is unknown. The activity of tarpon surfactantcertainly appears sensitive to temperature.

The surface activity of tarpon surfactant is extremely poor,compared with that of mammalian surfactant measured underthe same conditions with the same device. Mammalian sur-factant typically experiences an STmin of !1 mN/m, an STeq of∼25 mN/m, and a %SAcomp of !20% (Schurch et al. 1992; Table3). Moreover, the surface activity was also poor relative to thatmeasured in lizards on the Wilhelmy balance (Daniels et al.1998a). Lizard surfactant demonstrated intermediate surfaceactivity with an STmin of 13–14 mN/m, an STeq of 25 mN/m,and a %SAcomp of 30%. However, the surface activity of tarponsurfactant was very similar to that previously reported for otherray-finned fishes (Teleostei and Ginglymodi) and lungfishes(Daniels et al. 1998a; Table 3). The STmin values measured withalternative instruments, that is, either the Wilhelmy balance orthe Enhorning Bubble Surfactometer, ranged from 17 to 26mN/m (Daniels et al. 1998a). This study is the first to use themodern, state-of-the-art CBS in combination with high PL

concentrations (10 mg/mL, compared with 1 mg/mL PL) tomeasure the surface activity of fish surfactant. The CBS is cur-rently regarded as the most sophisticated and accurate devicefor measuring the biophysical behavior of pulmonary surfactantmixtures (Veldhuizen et al. 1998; Schurch et al. 2001). Theother methodologies are less reliable, particularly for highlyunsaturated, highly fluid surfactant mixtures, as they tend tosuffer from leakage of PLs out of the surfactant film (Veldhuizenet al. 1998; Schurch et al. 2001). Hence, the poor surface activitypreviously reported for fish surfactant appears to be a truereflection of its biophysical properties and is not an artifact oflow PL concentrations or inferior (leaky) measurement devices(Table 3).

It is likely that the surface-active properties observed for fishsurfactant directly reflect the differences in lipid and possiblyprotein composition that we have described. For example, theconcentration of DSP in the tarpon swim bladder is only 4%or less of total PL, which is even lower than that of other air-breathing fishes (9%–14%; Table 2; Smits et al. 1994) and vir-tually nonexistent compared with that of reptiles, birds, andmammals (30%–50%; Veldhuizen et al. 1998; Daniels and Or-geig 2001). Given that DSP, and in particular DPPC, is themajor PL responsible for achieving low surface tensions uponcompression (Veldhuizen et al. 1998), it is not surprising thatfish surfactant is unable to generate low surface tensions. Afurther characteristic of fish surfactant that mitigates againstlow surface tensions is the extremely high concentration ofcholesterol (Chol/PL: 0.2–0.3; Chol/DSP: 1.0–5.6), comparedwith that in mammals (Chol/PL: ∼0.08; Chol/DSP: ∼0.12; Dan-iels et al. 1995a), reptiles (Chol/PL: ∼0.03–0.08; Chol/DSP:∼0.1–0.3; Daniels et al. 1996; Daniels and Orgeig 2001), andamphibians (Chol/PL: ∼0.03–0.1; Chol/DSP: ∼0.2–0.3; Danielset al. 1994; Daniels and Orgeig 2001). While cholesterol (of theorder of 5%–10% of total PL) is essential in surfactant to pro-mote the spreading of the saturated PL film upon inspiration(Notter et al. 1980; Fleming and Keough 1988), high concen-trations of cholesterol tend to inhibit surfactant surface activity(Notter et al. 1980). Yet another characteristic of surfactantfrom the tarpon and pirarucu swim bladders is the very lowconcentration of SP-B (2, 17, and 44 pg/mg protein in the threefish tested), compared with 2 ng/mg protein in mice, determinedby the same method (Tokieda et al. 1999). As SP-B is closelyassociated with the surfactant lipids and has a crucial role inpromoting PL adsorption to the surfactant film (Haagsmanand Diemel 2001), the up to 100-fold lower concentration inlavage of this essential protein no doubt also contributes to thepoor surface-active properties of fish surfactant.

We have previously argued that nonmammals, due to theirlarger respiratory units, less complex lungs, and greater naturallung distensibility, do not require a surfactant mixture capableof the extremely low surface tensions typical of mammaliansurfactants. A more detergent-like surfactant (demonstratinghigher STmin and less variation) would be perfectly adequate




(Daniels et al. 1998a). Surfactant of nonmammals is notprimarily responsible for maintaining alveolar stability and in-creasing lung compliance but rather is required as an anti-adhesive and an antiedema agent (Daniels et al. 1998a). Nev-ertheless, fish surfactant, due to its composition and function,appears to be in a class of its own—the protosurfactant. Theextraordinary differences between fish surfactant and that ofmammals, reptiles, and amphibians suggest that fish surfactanthas functions that have not yet been elucidated. These mayinclude functioning as an innate immune system or as an an-tioxidant system or even simply the provision of a medium fordissolving other antioxidant, antiviral, or antimicrobial agents.

Conclusion

It appears that the evolution of the surfactant system predatedthe evolution of lungs and swim bladders. However, the func-tional evolution of the surfactant system is closely coupled withthat of lungs or swim bladders in the fish and lungs in thetetrapods. It also appears that there are two different types ofsurfactant, one in the actinopterygian fishes that is high incholesterol and USPs and one in the advanced sarcopterygianfishes and tetrapods that is relatively low in Chol and USPsand high in DSPs. Fish surfactant is likely to be highly spread-able but is not very surface active. The tetrapod surfactant ismuch more surface active and may, therefore, allow the de-velopment of more complex lungs with smaller respiratoryunits and a greater total respiratory surface area, paving theway for the evolution of high-performance lungs in amniotes.It is possible that fish surfactant is an archaic or “proto-” sur-factant that evolved into tetrapod surfactants but is also im-portant as a lipid lining for the swim bladders in the modernfish.

Many reptilian, amphibian, and fish lungs are essentially bag-like, with a large central airspace and lacking a bronchial tree,and with few exceptions they can collapse completely (Bishopand Foxon 1968; Guimond and Hutchison 1976; Hughes andVergara 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 isto prevent the epithelial surfaces from sticking together (Danielset al. 1995a). Nonmammalian surfactant does not significantlyinfluence inflation compliance. Given the highly derived natureof the mammalian lung, it is likely that regulation of surfacetension during nonatelectatic breathing is a late developmentin the evolution of surfactant. Based on our studies of reptilian,amphibian, and piscine surfactants, we conclude that whileacting as an antiadhesive may have been the original functionof surfactant, it led naturally to the alveolar stability and com-pliance roles that dominate mammalian surfactant function.

The surfactant system has been highly conserved, morpho-logically and biochemically, throughout (and despite) the enor-mous 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 forthe system.

Acknowledgments

This study was supported by an Australian Research Council(ARC) grant to C.B.D., an ARC research fellowship to S.O., apostgraduate research scholarship to L.C.S., an ARC grant toM.B.B., the Alberta Heritage Foundation for Medical Research,the Canadian Institute of Health Research, the Silva Casa Foun-dation (S.S.), the Natural Sciences and Engineering ResearchCouncil (Canada; C.J.B.), and Conselho Nacional de Desen-volvimento Cientifico e Tecnologico (Brazil; A.L.V.). We thankHelen Blacker for assistance with lipid analyses, Stanley Chengfor the surface activity measurements, and Mark Mano, whoassisted with the cholesterol analysis.

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