doi 10.1098/rstb.2000.0661 Thecornealsurface of aquaticvertebrates: microstructureswithoptical andnutritional function? H.Barry Collin 1 and ShaunP.Collin 2* 1Department of Optometry and Vision Sciences, University of Melbourne,Parkville, 3052,Victoria, Australia 2Department of Zoology,University of Western Australia, Nedlands,6907,Western Australia, Australia Theanterior surface ofthe mammaliancornea plays an important role in maintaining a smoothoptical interface andconsequently a sharpretinal image. The smooth surface is producedby a tear ¢lm, which adheresto a varietyof microprojections, which increase the cell surface area,improve the absorbanceof oxygenand nutrients andaid in the movementof metabolic products across the outercell membrane. However,little is knownof the structural adaptationsand tear ¢lm supportprovided in other vertebrates fromdi¡ erent environments.Using ¢ eldemission scanningelectron microscopy,this study examinesthe densityand surface structure ofcorneal epithelial cells inrepresentative species ofthe classes Cephalaspidomorphi,Chondrichthyes, Osteichthyes, Amphibia,R eptilia,A vesand Mammalia, includingsome Marsupialia.V ariationsin cell densityand the structure andoccurrence ofmicroholes, microridges,microplicae and microvilli are described withrespect tothe demandsplaced upon the corneain di¡ erent aquaticenvironments such asmarine and freshwater .Aprogressivedecrease in epithelialcell densityoccurs frommarine (e.g. 29 348cells mm 72 inthe Doversole Microstomius paci¢cus ) toestuarine orfreshwater (e.g.5999 cells mm 72 inthe blackbream Acanthopagrus butcheri )toterrestrial (e.g.2 126cells mm 72 inthe Australiankoala Phascolarctos cinereus )vertebrates, indicatingthe reduction inosmotic stress across the cornealsurface. The microholes found in the SouthernHemisphere lampreys,namely the pouchedlamprey ( Geotria australis )andthe shortheadlamprey ( Mordacia mordax ) represent openingsfor the release ofmucus, whichmay protect the corneafrom abrasion during their burrowingphase. Characteristic ofmarine teleosts, ¢ngerprint-likepatterns ofcorneal microridges are aubiquitousfeature, covering many types ofsensory epithelia (including the olfactoryepithelium and the oralmucosa) .Likemicroplicae and microvilli, microridges stabilize the tear ¢lm tomaintain a smoothoptical surface andincrease the surface areaof the epithelium, assisting indi¡ usion and active transport. Theclear interspeci¢ c di¡erences incorneal surface structure suggestan adaptive plasticity inthe compositionand stabilization of the cornealtear ¢lm invarious aquatic environments. Keywords: cornea;corneal surface ;epithelium;microvilli;microprojections surface areaof the super¢cial corneal epithelial cells in 1.INTRODUCTION most terrestrial vertebrates, is thoughtto improve the Inair ,the majorrefracting component of the vertebrate stabilityof the tear ¢lm. However,the surface ofmost, if eyeis the curvedair ^tear interface.The curvature of the notall, aquatic corneas is coveredwith a mucouscoating. tear ¢lm re£ects the shapeof the anteriorcorneal surface, Inbony ¢ shes, this isstabilizedby a series ofmembranous wherethe mucus layerof the tear ¢lm is adsorbedonto microridges,which are relatively straight cornealsurface the epithelialcell membranes. Inwater ,the anterior microprojections,have few branches, may be up to about cornealsurface contributes little tothe totalrefractive 20 m minlength and generally run parallel to each other powerof the eye,due to the small di¡erence betweenthe andto the cell margins. refractiveindexes of the tear ¢lm andthe epithelialcells Inthis study,weused ¢eldemission scanningelectron (n 1.365,Sivak 1982) and that ofsalt water( n 1.340, microscopyto examine the cornealsurfaces of35 verte- ˆ ˆ Jerlov1 976)or freshwater ( n 1.333,Horva ¨th &Varju¨ brates from the followingclasses, Cephalaspidomorphi ˆ 1995). (twospecies) ,Chondrichthyes(four) ,Osteichthyes (13), Thepresence of¢nger-likemicroprojections (microvilli) Amphibia(two) ,Reptilia(three) ,Aves(seven) and andrandomly directed curvedand branched membranous Mammalia(four, including two Marsupialia) (table 1 ). folds up to 2 m mlong(microplicae) ,whichincrease the Tissues wereprepared using glutaraldehyde ¢ xation, osmium tetroxidepost-¢ xation and dehydration with alcohol.F ollowingthese procedures,there waslittle *Author andaddress for correspondence: The Departmentof Anatomical Sciences,University of Queensland, Brisbane, Queensland4072, evidenceof a layerof mucus. From electron micrographs, Australia([email protected]) . whichwere mostly of the central cornea,we analysed the Phil. Trans.R. Soc.Lond. B (2000) 355, 1171^1176 1171 © 2000The RoyalSociety 1172H. B.Collin and S. P .Collin The corneal surface in aquatic vertebrates Table 1. Species examined and corneal epithelial cell densities measured celldensity class species common name (cells mm7 2) Cephalaspidomorphi Geotriaaustralis pouchedlamprey b 5511 2025 § Cephalaspidomorphi Mordaciamordax shortheadlamprey b 11 041 2752 § Chondrichthyes Galeocerdocuvier tiger sharkb 8286 3212 § Chondrichthyes Squalisacanthias spinydog¢ sh a 4176 2470 § Chondrichthyes Dalatiaslicha black sharka 14 492 4056 § Chondrichthyes Hydrolaguscolliei rat¢sha notmeasured Osteichthyes Acanthopagrusbutcheri black breamb 5999 1976 § Osteichthyes Neoceratodusforsteri Australianlung¢ sh b 7843 2214 § Osteichthyes Hippocampusangustus WestAustralian seahorse a 14 186 2385 § Osteichthyes Hippoglossoideselassodon £atheadsole a 28 860 9214 § Osteichthyes Lepidogalaxiassalamandroides salamander¢sh d 21 880 4200 § Osteichthyes Torquigenerp leurogramma blow¢shb notmeasured Osteichthyes Aldrichettaforsteri yellow-eyemullet b 10 943 4538 § Osteichthyes Platycephalusendrachtensis bar-tailed£ athead b 11 171 5740 § Osteichthyes Amniabatacaudavittatus yellow-tailtrumpeter b 13 453 1385 § Osteichthyes Nematalosavlaminghi Perthherring b 19 639 4010 § Osteichthyes Myoxocephalusp olyacanthocephalus greatsculpin a 22 615 4800 § Osteichthyes Polyperagreenii lobe¢n snail¢sh a 28 229 9334 § Osteichthyes Microstomiusp aci¢cus Dover solea 29 348 12 917 § Amphibia Ambystomamexicanum salamander(axolotl) c 2918 1753 § Amphibia Xenopuslaevis Africanclawed toad c 7095 3470 § Reptilia Crocodilusp orosus crocodile c 2283 824 § Reptilia Ctenophorusornatus ornatelizard c 5095 1746 § Reptilia Carettacaretta loggerheadturtle c 4191 1672 § Aves Phoenicopteruschilensis Chilean£ amingo 5944 2042 § Aves Eolophusroseicapillus Australiangalah 11 561 6175 § Aves Struthiocamelus SouthAfrican ostrich 7658 2397 § Aves Phoenicopterusruber Caribbean£ amingo 8674 3034 § Aves Dromaiusnovaehollandiae emu 5250 2076 § Aves Eudyptalaminor fairypenguin a notmeasured Aves Bubo strix barred owl 5351 1325 § Mammalia Pseudorcacrassidens falsekiller whale c 2211 794 § Mammalia Ovis aries sheep notmeasured Mammalia Sminthopsiscrassicaudata fat-taileddunnart 9785 9900 § Mammalia Phascolarctoscinereus Australiankoala 2126 713 § a Marinespecies. b Estuarineor freshwaterspecies. c Groupedwith terrestrialand aerial species for statistical analysis. d Classi¢ed with themarine species because it lives in highosmolality freshwater ponds ( Christensen1982) . size andshape of the surface epithelialcells andthe showsa signi¢cant di¡ erence ( p 0.000018)between the ˆ natureof the surface epithelialmicroprojections in the cornealepithelial cell densities ofthe aquaticspecies contextof their functionin di¡ erent (predominantly (17 602 9604cells mm 72)comparedwith the aerialand § aquatic)environments. terrestrial vertebrates (3755 2067cells mm 7 2), where § the latter groupincluded two species ofamphibians and the falsekiller whale Pseudorca crassidens . 2.EPITHELIAL CELL DENSITIES Anexaminationof the environmentsof the aquaticspecies Thecorneal surfaces inall of the species studied is indicatesthat marinespecies (22553 8878cells mm 72) § formedof essentially straight-sided pentagonaland hexa- havea signi¢cantly ( p 0.0034)higher epithelial cell ˆ gonalepithelial cells. Twoexceptions are the pouched densitythan species that livein either freshwater orin lamprey Geotria australis ,whichpossess some ovalcells bothsalt waterand freshwater (estuarine) environments amongthe predominantlypolygonal cells (¢gure 1 a) and (10 529 5341cells mm 72).Thus,there appearsto be a § the Australiankoala Phascolarctos cinereus ,inwhich the progressivelowering of the cornealepithelial cell density polygonalcells sometimes appearrounded. frommarine vertebrates throughthe estuarine andfresh- Therewas great variation in the cornealsurface waterspecies tothe aerialand terrestrial vertebrates. The epithelialcell densities measured inthis study(table 1 ). lowestdensities occuramong the mammals, includingthe Thecell densities rangefrom 2126 713 cell mm72 in the falsekiller whale P.crassidens . § Australiankoala P. cinereus (amarsupial) to 29 348 Inaddition to giving more strength tothe corneal 12917cells mm 72 inthe Doversole Microstomius paci¢cus surface,the greaterepithelial cell densities inthe salt § (abony ¢ sh).Analysisof these andother publishedresults watervertebrates maypermit more activetransport of Phil.Trans.R. Soc.Lond. B (2000) The corneal surface in aquatic vertebrates H. B.Collin and S. P .Collin1 173 Figure 1. (a)The cornealsurface of thepouched lamprey Geotriaaustralis ,showingthe presence of aroundto ovalcell in the midstof otherwisepentagonal and hexagonal cells. There are numerous small microplicae but the predominant feature is the presenceof manyrelatively large (395 15nm)microholes, through which