doi 10.1098/rstb.2000.0661

Thecornealsurface of aquaticvertebrates: microstructureswithoptical andnutritional function?

H.BarryCollin 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

surfacearea of 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 of salt water( n 1.340, microscopyto examine the cornealsurfaces of35 verte- ˆ ˆ Jerlov1 976)or freshwater ( n 1.333,Horva ¨th &Varju¨ brates fromthe 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 centralcornea, 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 marine species (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 2 126 713 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 published results 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 mucousgranules can be expelled.( b)Micrographof § thecorneal surface of theblack shark Dalatiaslicha ,showingthe dense pattern of microvilliwith somemicroplicae. ( c) The cornealsurface of theblow¢ sh Torquigenerp leurogramma ,showingthe long, sometimes continuous microridges that lie parallel to eachother and to thecell border. An unusualfeature of thisbony ¢ sh,compared with allothers so fardescribed, is thepresence oftheextensions or noduleson theside of themicroridges adjacent to thecell borders. ( d)Numerousmicrovilli on thecorneal surfaceof theAustralian lung¢ sh Neoceratodusforsteri .At thecell borders are larger microprojectio ns,some of which havehollowed centres.The functionof theselatter microprojection sisunknown. Scale bars, 1 m m. salts andwater from the corneato maintain an Di¡erences betweenthe cell densities ofcentral and appropriatelevel of dehydrationof the stroma toensure a peripheralcorneas are known to occur in some species. clear,transparent cornea.Similarly ,the lowerdensities in Inboth the rabbit(Doughty & Fong1 992)and the non-aquaticvertebrates assist inthe evaporationof water sandlance Limnichthyes fasciatus (Collin& Collin1 997),the fromthe corneaand therefore aiddeturgescence. cornealepithelial cells arelarger in the peripherythan in

Phil. Trans.R. Soc.Lond. B (2000) 1174H. B.Collin and S. P .Collin The corneal surface in aquatic vertebrates

Figure 2. (a)The cornealsurface of theloggerhead turtle Carettacaretta ,showingthe dense carpet of microvilli,some of which appearto be aggregated into clumps. ( b)Micrographof the corneal surface of theornate lizard Ctenophorusornatus , with its patternof shortmicroridges that appear to berandomlyarranged. ( c)While mostother birds have microvilli, the fairy penguin Eudyptulaminor hasa patternof microridges that are shorter than those of bony¢ shesand randomly arranged. ( d ) The dense patternof microvillion thecorneal surface of theemu Dromaiusnovaehollandiae .This patternappears to betypicalof non-aquatic birds.Scale bars, 1 m m. the centre. However,these di¡erences werenot investi- mammals, birds, reptiles andamphibians are covered gatedin this study. withsmall membrane-boundprojections in the formof ¢nger-likemicrovilli (80^1 20nm indiameter ,Collin& Collin20 00)and microplicae. The corneal surface ofthe 3.SURFACE MICROPROJECTIONS cartilaginous¢ shes studied alsoshow the presence of Scanningelectron micrographsshowing the appear- microvilliand microplicae. The microvilli on the surface anceof the cornealsurface ofvarious vertebrate species ofthe corneaof the blackshark Dalatias licha are shown areshown in ¢ gures 1and2. The corneal surfaces ofmost in ¢gure 1b.

Phil.Trans.R. Soc.Lond. B (2000) The corneal surface in aquatic vertebrates H. B.Collin and S. P.Collin1 175

Incontrast, the cornealsurfaces ofmost bony¢ shes the ubiquitousneed for either structural supportor nutri- possess nomicrovilli and are covered with microridges tionalexchange in an aquatic environment, microridges (120^227nm inwidth, Collin & Collin20 00),whichare havealso been described inepithelia associated with oftencontinuous, frequently orientated parallel to each sensorystructures. Theseinclude the conjunctivaland otherand to the cell border,andnever cross fromone cell nasalepithelia of the sandlance(Collin & Collin1 997) toanother (Collin & Collin20 00)(¢ gure 1 c).Following andthe olfactoryepithelium, oralmucosa, neuromasts of comparisonsof a number ofbony ¢ shes, the patterns of the lateralline receptors, electroreceptors, chemosensory microridges,although similar ,appearto be di¡ erent and cells andtaste budsin a rangeof teleosts (reviewedin possiblyspecies speci¢c. Asomewhatunusual feature of Collin& Collin200 0). the microridgepattern is foundin the blow¢sh Torquigener pleurogramma (¢gure 1c),wherethere aresmall extensions 5.SURFACE MICROHOLES ornoduleson the outerside ofthe microridgesadjacent to the cell border.Todate,the onlybony ¢ sh foundto possess Intwo species ofSouthern Hemisphere lampreys, microvilli,instead of microridges, is the Australianlung- microholesare present inthe cornealsurface (¢gure 1 a). ¢sh Neoceratodus forsteri (¢gure 1d ). Theseholes are quite large (395 15nm inthe pouched § Althoughmicroridges are a characteristic ofthe lamprey Geotria australis and 245 177nm inthe short- § cornealsurface ofbony ¢ shes, there aresome species of headlamprey Mordacia mordax )andrepresent openings reptiles that alsopossess microridges.The ornate lizard forthe release ofmucous granules, which may form a Ctenophorus ornatus hasa pattern ofmicroridges(¢ gure 2 b) coatingover the corneato protect it againstabrasion that areboth narrower (96 13nm) andshorter than duringburrowing. Microholes (324 76nm) similar to § § those foundin bony ¢ shes. Inaddition, the microridges those observedin the lampreysare also present inthe pre- arearranged in an apparently random pattern, rather metamorphicsalamander (axolotl) Ambystoma mexicanum . thanaligned parallel to each other andto the cell border. Theseholes also permit the release ofmucous granules Amongother members ofthe Reptilia,the corneasof the ontothe cornealsurface (H. B.Collin and S. P .Collin, crocodile Crocodilus porosus andthe loggerheadturtle unpublisheddata) . Caretta caretta arecovered with microvilli (¢ gure 2 a). Goblet cells arepresent inthe cornealepithelium ofthe Thecorneal surfaces ofmost mammals andbirds, salamander¢sh L.salamandroides (Collin& Collin1996) . includingthe emu (¢gure 2 d),arecovered with microvilli Astheir habitatof small pondsdries outin the summer and/ormicroplicae. However, there arealso numerous months, L.salamandroides burrowsinto the mudand aesti- microridgesin the centralcorneal surface ofthe vates,inducing the release ofmucus fromthe gobletcells, Australiankoala Phascolarctos cinereus andin the partially whichcovers the surface ofthe cornea.Holes inthe marineaquatic fairy penguin Eudyptula minor (¢gure 2c), cornealsurface havealso been found in the bar-tailed whichclosely resemble those ofthe ornatelizard C. ornatus £athead Platycephalus endrachtensis (Collin& Collin20 00) (¢gure 2b). andthe sandlance Limnichthyes fasciatus (Collin& Collin 1997),twospecies whichare partially buried when lying inwait for prey with eyes protruding.However ,these are 4.THE ROLE OFSURFACE MICROPROJECTIONS ca. 2 m mindiameter ,arepresent onlyin the peripheral Inhumans and other mammals, severalfunctions have corneaand are situated atthe junctionsof three and beensuggested for the microvilliand microplicae. They sometimes fourcells. increase the surfacearea of the super¢cial epithelial cells, whichaids the absorptionof oxygen (Beuerman & The authorswish tothank Michael Archer of the Pedrosa1996) and other nutrients intothe cells and Departmentof Zoology ,The Universityof W esternAustralia assists the movementof metabolicproducts from the cells. andBrendon Gri¤ n ofthe Centre for Microscopy and Microanalysis,The Universityof New South W ales.S.P .C. Inaddition, they stabilize the tear ¢lm, whichis essential wasan Australian Research Council (AR C)QueenElizabeth forclear vision in an aerial or terrestrial environment IIResearch F ellow.This workwas funded by AR Cand (P¢ster 1973). NationalHealth and Medical Research Council Grants to Inaquatic vertebrates, the roleof the microvilliand S.P.C. microridgesis less clear.Astable tear ¢lmisnotessential inthe productionof a clearretinal image due to the minimalrefractive index di¡ erence betweenthe cornea REFERENCES andthe surroundingwater .However,stabilizingthe Beuerman,R. W.&Pedrosa,L. 1996Ultrastructure of the mucouslayer on the cornealsurface may enable it toact humancornea. MicroscopyRes.Tech. 33, 320^335. asaprotectivecoating, which is particularlyimportant in Christensen,P .1982The distributionof Lepidogalaxias those species, such asthe salamander¢sh Lepidogalaxias salamandroides andother small freshwater ¢ shesin thelower salamandroides ,that burrowinto the sand.Although the southwestof W esternAustralia. J.R.Soc.W est.Aust. 65, cell surfacearea of microridges is less thanthat ofmicro- 131^141. villi,microridges will still result ina signi¢cant increase Collin,H. B. &Collin,S. 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Phil.Trans.R. Soc.Lond. B (2000)