Richardson in light and dar

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Th e eyes o f t eleo st fish ad ap t t o d ifferen t ligh t levels t h ro u gh ch an ges in ret in al st ru ct u re. So m e o f t h ese ret in al ch an ges in resp on se to ligh t cycles in clu d e: m ovem en ts of th e m yoid (AREY 1975), m igration of m elan in gran u les (ES-SOUNNI & ALI 1986, BURNSIDE 2001) an d flu ctu ation s in th e p re-syn ap tic m em bran e an d th e term in al bu tton s (BRAEKEVELTet al. 1998b, BURN SID E 2001), am o n g o t h ers. ES-SO UN N I & ALI (1986) an d BRAEKEVELTet al. (1998a) rep orted several ch an ges wh en com -p arin g th e retin a of teleost fish ad a-p ted to ligh t an d to d ark-n ess. Th ese iark-n clu d ed a ch aark-n ge iark-n th e sh ap e of lip id s d rop lets,

d etach m en t of ou ter segm en ts of p h otorecep tors, form ation of m u ltilobu lar bod ies, alteration s in th e n u m ber of m acrop h -ages an d m elan olysosom es, an d oth er organ elles.

Retin om otor or p h otom ech an ical m ovem en ts are ch ar-acterized by th e m igration of con es, rod s an d p igm en t ep ith e-liu m in resp on se to ch an ges in en viron m en tal ligh t con d ition s (ALI 1971 , EASTM AN 1988, MACDO N ALD & MO N TG O M ERY 1991, BURNSIDE 2001).

Cells of th e retin al p igm en t h ave been sh own to h ave a m ech an ism to tran sp ort m elan in gran u les n ot on ly in resp on se

Notohenia

coriiceps

Richar

Richardson in light and dar

dson in light and dar

dson in light and dark-conditions

k-conditions

Lucélia Donatti & Edith Fanta

Departamento de Biologia Celular, Setor de Ciências Biológicas, Universidade Federal do Paraná. Caixa Postal 19031, 81531-970 Curitiba, Paraná, Brasil. E-mail: [email protected]

ABSTRACT. The Antarctic fish Nototheniacoriiceps Richardson, 1844 lives in an environment of daily and annual photic variation and retina cells have to adjust morphologically to environmental luminosity. After seven day dark or seven day light acclimation of two groups of fish, retinas were extracted and processed for light and transmission electron microscopy. In seven day dark adapted, retina pigment epithelium melanin granules were aggregated at the basal region of cells, and macrophages were seen adjacent to the apical microvilli, between the photoreceptors. In seven day light adapted epithelium, melanin granules were inside the apical microvilli of epithelial cells and macrophages were absent. The supranuclear region of cones adapted to seven day light had less electron dense cytoplasm, and an endoplasmic reticulum with broad tubules. The mitochondria in the internal segment of cones adapted to seven day light were larger, and less electron dense. The differences in the morphology of cones and pigment epithelial cells indicate that N. coriiceps has retinal structural adjustments presumably optimizing vision in different light conditions.

KEY WORDS. Photic variation; photoreceptor; retinomotor movements.

RESUMO. UltrUltraestrUltrUltrUltraestraestraestraestrutrutrutrutrutraaaaa dodo epitéliodododoepitélioepitélioepitélioepitélio pigmentarpigmentarpigmentarpigmentarpigmentar dadadadada rrrrretinaetinaetina eetinaetinaeeee dosdosdosdosdos conesconesconesconescones dododododo peixpeixpeixpeixpeixeeeee AntárticoAntárticoAntárticoAntártico AntárticoNototheniaNototheniaNototheniaNototheniaNototheniacoriicepscoriicepscoriicepscoriicepscoriiceps Richar

Richar Richar Richar

Richardsondsondsondsondson submetidosubmetidosubmetidosubmetidosubmetido ààààà luzluzluzluzluz eee aoeeaoaoao escuraoescurescurescurescuro.o.o.o. O peixe Antártico o. Nototheniacoriiceps Richardson, 1844 habita meios com variações fóticas diária e anual e as células da retina se adaptam morfologicamente a esta luminosidade ambiental. Dois grupos de peixes foram aclimatados durante sete dias à luz constante ou ao escuro constante. Após secção medular, as retinas foram extraídas e processadas para microscopia de luz e microscopia eletrônica de transmissão. No epitélio pigmentar da retina adaptado sete dias ao escuro, os pigmentos de melanina agregam-se na base coroidal das células epiteliais pigmentares e macrófagos são encontrados no interior do processos apicais entre as células fotorreceptoras. No epitélio adaptado sete dias à luz os pigmentos de melanina se dispõem ao longo das projeções apicais das células epiteliais pigmentares e os macrófagos são ausentes. A região supranuclear dos cones adaptados sete dias à luz possui um citoplasma menos elétron denso devido a presença de canais do retículo endoplasmático que se dilatam aumentando o seu lúmen. As mitocôndrias existentes no segmento interno dos cones adaptados à luz são maiores, e menos elétron densas. As diferenças na morfologia dos cones e nas células epiteliais pigmentares indicam que N.coriiceps possui mecanismos celulares de ajustes na retina provavelmente otimizando a capacidade visual em diferentes situações de luz.

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34 L. Donatti & E. Fanta

to ch an ges in ligh t levels (ALI 1975) bu t also th rou gh regu la-tion by en d ogen ou s circad ian rh yth m s (ALI 1975). However if th e p igm en t ep ith elial cells are isolated from th e retin a, th ey d o n ot resp on d to ch an ges in ligh t (BRUENNER & BURNSIDE 1986). In ligh t ad ap t ed ret in as, t h e p ro st aglan d in E1 can in d u ce retin om otor m ovem en t in con es an d p igm en t ep ith elial cells (CAVALLARO & BURNSIDE 1988).

Th e cytoskeleton an d th e en d op lasm ic reticu lu m are two cell elem en ts in volved in p h otom ech an ical ad ap tation to d ark-n ess aark-n d ligh t (FERREROetal. 1979). Th ese p h otom ech an ical re-sp on ses in volve elon gation of th e m yoid in p h otorecep tors, an d a red istribu tion of m icrotu bu les th at slid e alon g th e len gth of m yoid . Th is slid in g is gen erated by th e in teraction of m icro-tu bu les d istribu ted as strip es in sp ecific m yoid region s (WARREN & BURNSIDE 1978). Th e d istribu tion an d ap p earan ce of th e en -d op lasm ic reticu lu m in th e in tern al segm en t of th e p h otorecep tors ch an ges with ad ap tation to ligh t. As th e in ferior ellip -soid of con es an d rod elon gates th e d en sity of th e en d op lasm ic reticu lu m is red u ced (FERREROetal. 1979).

Fish h abitats an d retin al resp on ses are related . Dep en d -in g on sp ecies an d life stage, fish live -in a variety of h abitats with d ifferen t ligh t regim es, from su rface water to great d ep th s, an d th ey m ay m ake exten sive d iu rn al vertical m igration s. Th e in terspecific differen ces in retin al respon ses u n der differen t ligh t con d ition s m ay be a con seq u en ce of ecological ad ap tation s. Sp ecies m ay be ad ap ted to a certain ligh t regim e an d ad ap ta-tion to ligh t is often faster th an ad ap tata-tion to d ark (ALI & HANYU 1963, ALI & ANCTIL 1968, ALI 1971). Retin om otor resp on ses m ay b e relat ed t o b eh avio r in sch o o lin g fish (KEEN LEYSIDE 1955, LOUKASHKIN & GRANT 1959), or to feedin g an d m igration (ALI 1971, FANTAetal. 1994, DONATTI & FANTA 2002), am on g oth er factors. N otothenia coriiceps Rich ard son , 1844 (Nototh en iid ae) lives in relatively sh allow coastal An tarctic waters, wh ere th e p h otop eriod an d ligh t q u ality in term s of wavelen gth can vary sign ifican t on a d aily an d / or season al. Ackn owled gin g th e oc-cu rren ce of th is variation ou r objectives in clu d ed a q u alitative d escrip tion of u ltrastru ctu ral m orp h ological ch an ges an d fu n c-tion al strategies of th e retin a th at en able th e visu al system of N ototheniacoriiceps to fu n ction in ligh t an d d arkn ess.

MATERIAL AND METHODS

N ototheniacoriiceps was collected by tram m el n et in Ad -m iralt y Bay (62º09’S, 58º26’W ), Kin g Geo rge Islan d , So u t h Sh etlan d s, An tarctica, at abou t 20m d ep th , d u rin g th e su m m er (Decem ber an d Jan u ary) of 1998/ 1999. In d ivid u als of a total len gth of 36.5 ± 2 cm (m ean ± S.D.) (n = 28) were m ain tain ed in ligh t p roof tan ks, u n d er con trolled con d ition s at th e Brazil-ian An tarctic Station Com an d an te Ferraz.

Th e well-aerated water was kep t at a tem p eratu re of 0 ± 0.5ºC (m ean ± S.D.), p H 7.0 ± 0.5 (m ean ± S.D.), an d salin ity 34.5 ± 1.0 p p t (m ean ± S.D.). Grou p s of 14 N. corriceps were m ain tain ed for seven d ays in con tin u ou s ligh t, or con tin u ou s d arkn ess. Darkn ess tan ks were covered with a black ligh t p roof

sh eet. For con tin u ou s ligh t con d ition s, a d ayligh t flu orescen t lam p was on above th e tan k an d rem ain ed on all d u rin g th e exp erim en t.

Fish were eu th an ized by m ed u llar section an d after d e-cerebration th e retin as of d ark ad ap ted fish were extracted an d fixed u n d er a low level red ligh t. Ligh t ad ap ted fish retin as were extracted u n d er d ayligh t flu orescen t lam p .

For op tical m icroscop y, retin as were fixed in Bou in ’s flu id for eigh t h ou rs (CULLING et al. 1985) an d p rocessed rou tin ely for trad ition al h istology accord in g to CLARK (1981). Sam p les were em bed d ed in Parap last Plu s® (Sigm a), section ed at 2-4 µ m , stain ed with Haem atoxylin -Eosin (H.E.) an d p h otom icro-grap h s were taken with a p h otom icroscop e (m od el PM 10AD, Olym p u s, Jap an ).

For tran sm ission electron m icroscop y, th e retin as were cu t in to sm all p ieces an d fixed in m od ified Karn ovsky at 4ºC (CULLING et al. 1985). Du rin g p ost fixation a 2% solu tion of osm iu m tetroxid e bu ffered in 0.2M cacod ylate (p H. 7.2) was u sed , an d sam p les were con trasted with 2% u ran yl acetate. Th e sam p les were d eh yd rated in an eth an ol series, em bed d ed in Ep on -812 in a balan ced m ixtu re (LUFT 1961). Th e con trast of section s was en h an ced with 2% u ran yl acetate (WATSON 1958), an d lead n itrate (REYNOLDS 1963). Th ey were exam in ed u n d er a electron m icroscop e (JEM 1200 EXII, JEOL, Jap an ).

RESULTS

N ototh en ia coriiceps h as a ret in a wit h t en cells layers. However, fish ad ap ted to 7-d ay ligh t or seven d ay d ark con d i-tion s d iffered in som e retin al p rop erties. Th e p osii-tion of th e m elan in p igm en t in th e p igm en t ep ith eliu m d iffered in both ligh t regim es (Figs 1 an d 2). After seven d ay in th e d ark th e m elan in gran u les were aggregated in th e coroid al base of th e p igm en ted ep ith elial cells, exp osin g th e tip s of th e extern al segm en ts of th e p h otorecep tors (Fig. 3). After seven d ays u n -d er ligh t th e m elan in p igm en t was locate-d in si-d e th e ap ical p ro ject io n s o f t h e p igm en t ep it h eliu m cells, p ro t ect in g t h e extern al segm en ts of th e p h otorecep tors (Fig. 4).

W h en ad ap ted to d ark con d ition s, th e p igm en t ep ith e-liu m con tain ed m ore m em bran es d etach ed from th e extern al segm en ts of th e p h otorecep tors, d isp ersed in its cytop lasm , m ore p h agosom es, an d m ore m yeloid bod ies associated with lip id d rop lets (Figs 5, 6 an d 8). Macrop h ages were fou n d close to th e Bru ch ’s m em bran e an d alon gsid e th e ap ical p rocesses, between th e p h otorecep tors (Fig. 7). Th ere were n o m acrop h -ages in th e p igm en t ep ith eliu m of an im als ad ap ted to ligh t.

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pran uclear region an d n eck (Fig. 11). Th e m itoch on dria in th e ellipsoids were m ore electron -den se an d sm aller in dark con di-tion s wh en com pared to ligh t con didi-tion s (Figs 10 an d 12).

Th e su p ran u clear region was less electron -d en se in con es ad ap ted to d ayligh t th an in th ose ad ap ted to d arkn ess, becau se th e tu bu les in th e en d op lasm ic reticu lu m exp an d , en largin g th e lu m en of th e en d op lasm ic reticu lu m (Figs 13 an d 15). In seven d ay d ark ad ap ted con es, th e cytop lasm is m ore electron d en se an d th e en d op lasm ic reticu lu m h as n o en larged tu bu les (Fig. 14).

DISCUSSION

Th e retin a of N ototheniacoriiceps is com p osed of ten h is-t o lo gic a l la y e r s, b e in g sim ila r is-t o is-t h a is-t o f se v e r a l o is-t h e r n otoh en ioid s an d typ ical teleosts (EASTMAN 1988, GRÖTZNER & FANTA 1998, DONATTI & FANTA 2001, FANTAet al. 2001). Th ey exh ibit fou r typ es of con es, rod s, p igm en ted ep itexh eliu m an d n eu -ron al layers (GRÖTZER & FANTA 1998, DONATTI & FANTA 2001).

Som e au th ors h ave described th e retin al organ ization an d visu al fu n ction of Nototh en ioid fish (FANTAet al. 1994, 1999, 2001, GRÖTZER & FANTA 1998, MACDONALD & MONTGOMERY 1998, DONATTI & FANTA 2001, 2002). However, th ere are several ch an ges in th e retin al u ltrastru ctu ral ch aracteristics th at occu r as a con -seq u en ce of ad ap tation to con tin u ou s ligh t or d ark con d ition s th at were n ot yet q u alitatively d escribed . Th is stu d y d etected st ru ct u ral d ifferen ces in ret in as o f ligh t an d d ark ad ap t ed N ototheniacoriiceps related to retin om otor m ovem en ts, tu bu les of en d op lasm atic reticu lu m , an d to th e d en sity of cytop lasm an d m itoch on d ria.

Th e exten t of retin om otor m ovem en ts in retin al p h oto-recep tors, an d p igm en t ep ith eliu m , are in d icators of th e activ-ity of th e retin a. Usu ally, isovolu m etric ch an ges, sim ilar to th ose of a m u scle con traction , are attribu ted to a system of m icrotu -bu les in th e p h otorecep tors (BURNSIDE 1978, WARREN & BURNSIDE 1978). Retin om otor m ovem en ts rep osition p h otorecep tor ou ter segm en ts an d screen in g p igm en t in th e retin al p igm en t ep ith eliu m so ith at p h otorecep tors are op tim ally exp osed to in -com in g ligh t in th e d ark in th e case of rod s an d u n d er ligh t con d ition s in th e case of con es (ALI 1971, 1975). Th is cap acity m igh t be im p ortan t for N.coriiceps to en able th e fish to fin d t h eir p rey u n d er ligh t an d d ark co n d it io n s, o r even wh en weath er ch an ges, resu ltin g in su d d en ch an ges in ligh t con d i-tion s. (DONATTI & FANTA 2002)

A h igh er n u m ber of d etach ed m em bran es of th e extern al segm eextern t of p h otorecep tors, aextern d th e p reseextern ce of m acrop h -ages was observed in th e p igm en t ep ith eliu m of N . coriiceps ad ap ted to d arkn ess. Th is loss of th e ou ter segm en ts of p h oto-recep tors followed by p h agocytosis by th e p igm en t ep ith eliu m cells is n orm al in som e retin as (BALKEMA & BUNT-MILAM 1982, BRAEKEVELTetal. 1998a, DONATTI & FANTA 1999), d u e to rh yth m ic t u rn o ver o f it s cellu la r o rga n elles (O ’DAY & YO U N G 1 9 7 8 , BRAEKEVELT 1980), an d is in ten sified in con seq u en ce of h igh er p h otorecep tors activity. Th e balan ce between m em bran e syn

-th esis an d d egrad ation m ain tain s -th e extern al segm en t, m ain ly of rod s, in a relatively con stan t len gth . Th e rem oval of th ese extern al segm en ts can be d on e m acrop h ages (BRAEKEVELT 1980), wh ich wou ld exp lain th eir p resen ce in th e p igm en t ep ith eliu m in th is stu d y. In con trast, evid en ce of d isk sh ed d in g by con es is very rare, an d h as n ot been sh ow to be a regu larly recu rrin g p rocess. (O’DAY & YOUNG 1978). Th ey d o n ot exist given in th e literatu re th at exp lain s th e occu rren ce of a h igh er n u m ber of d etach ed m em bran es of th e extern al segm en t of p h otorecep -tors in th e an im als ad ap ted to d arkn ess

Lip id drop lets, associated with th e m yeloid body, were foun d in greater quan tity in th e darkadapted retin a. Th eir n um -ber can in crease due to th e degradation of ph agosom es, an d th is m ay explain th e lipid droplets in dark-adapted N.corriceps.

In N.coriiceps ad ap ted to d arkn ess th e p resen ce of electron d en se cytop lasm , an d an en d op lasm ic reticu lu m with tu -bu les th at h ave a sm all d iam eter, in d icates a red u ction in

syn th etic activity an d in th e release of m etabolites to th e extern al segm en t of p h otorecep tors.

Th e q u alitative an alysis of th e retin a of N.coriiceps dem -on strates th at th is sp ecies p resen ts th e typ ical cellu lar an d su b-cellu lar m ech an ism s for retin al stru ctu ral ad ju stm en ts th at can op tim ize th e visu al fu n ction of th e retin a.

ACKNOWLEDGEMENTS

Th e au t h o rs t h an k: t h e C N Pq fo r fin an cial su p p o rt (Projects n u m ber 490.103/ 99-5 an d 480.265/ 00-3), an d for a Prod u ctivity in Research Stip en d to E. Fan ta (300.831/ 93-5); th e CAPES for a PICDT-Doctorate Stip en d to L. Don atti; th e SECIRM (Brazilian Navy) an d th e staff of th e Brazilian An tarc-tic Station Com an d an te Ferraz for all logistarc-tical su p p ort an d I. Everson (BAS, UK) for valu able su ggestion s to th e m an u scrip t.

REFERENCES

ALI, M.A. 1971. Les résp o n ses ret in o m o t rices: caract ères et m ecan ism es. Visio n Research 1 1: 1225-1228.

ALI, M.A. 1975. Retin om otor resp on ses, p . 315-355. In: M.A. ALI (Ed .). Visio n inFish es. New York, Plen u m Press, 836p . ALI, M.A. & I. HANYU. 1963. A co m p arat ive st u d y o f ret in al stru ctu re in som e fish es from m od erately d eep waters of th e Western North Atlan tic. Can adianJo urn alo fZo o lo gy 4 1: 225-241.

ALI, M.A. & M. ANCTIL. 1968. Co rrelat io n en t re la st ru ct u re rétin ien n e et l’h abitat ch ez Stiz oz teidonvitreum vitreum et S. Can aden se. Jo u rn al o f th e Fish e re s Re se arc h Bo ard o f Can ada 2 5: 2001-2003.

AREY, L.B. 1 9 7 5 . Th e o ccu rren ce a n d t h e sign ifica n ce o f ph otom ech an ical ch an ges in th e vertebrate retin a – an h is-torical survey. Jo urn alCo m parativeNeuro lo gy25: 535-554. BALKEMA, G.W.JR & A.H. BUNT-MILAM. 1982. Con e ou ter segm en t sh ed d in g in th e gold fish retin a ch aracterized with 3_{H-fu cose}

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BRAEKEVELT, C.R. 1980. Wan d erin g p h ago cyt es at t h e ret in al ep it h eliu m -p h o t o recep t o r in t erface in t h e t eleo st ret in a. Visio n Research2 0: 495-499.

BRAEKEVELT, C.R.; S.A. SMITH & B.B. SMITH. 1998a. Fin e stru ctu re of th e retin al p igm en t ep ith eliu m of Oreochrom isniloticus L. (Cich lid ae; Teleo st ei) in ligh t - an d d ark-ad ap t at io n . Th e An ato m icalReco rd, Oaklan d , 2 5 2: 444-452.

BRAEKEVELT, C.R.; S.A. SMITH & B.B. SMITH. 1998b Ph otorecep tor fin e stru ctu re in Oreochrom isniloticus L. (Cich lidae; Teleostei) in ligh t- an d d ark-ad ap tation . Th eAn ato m icalReco rd2 5 2: 453-461.

BRUENNER, U. & B. BURNSIDE. 1986. Pigm en t gran u le m igration in iso lat ed cells o f t h e t eleo st ret in al p igm en t ep it h eliu m . In v estigativ eOph th alm o lo gy&VisualScien ce2 7: 16-34. BURNSIDE, B. 1978. Th in (actin ) an d th ick (m yosin like) filam en ts in th e con e con traction of teleost retin a. Jo urn al o f Cell Bio lo gy7 8: 227-246.

BURNSIDE, B. 2001. Lih t an d circad ian regu lation of retin om otor m ovem en t. Pro gressin BrainResearch 1 3 1: 477-485. CAVALLARO, B. & B. BURNSIDE. 1988. Prostaglan d in s E1, E2 an d D2

in d u ce d ark-ad ap t ive ret in o m o t o r m o vem en t s in t eleo st retin al con es an d RPE. In v estigativ e Op h th alm o lo gy & VisualScien ce2 9: 882-891.

CLARK, G. 1981. Stain in g p ro ced u res. Baltim ore, Willian s & Wilkin s, 512p .

CU LLIN G, C .F.A.; R.T. ALLISO N & W.T. BARR. 1 9 8 5 . Ce l l u l a r Path o lo gyTech n ique. Lon d on , Bu tterworth , 4th_{ed ., 642p .}

DONATTI, L. & E. FANTA. 1999. Morph ology of th e retin a in th e fr e sw a t e r fish M et y n n is roosev et i Eige n m a n n , 1 9 1 5 (Ch aracidae, Serrasalm in ae) an d th e effects of m on och rom atic red ligh t. RevistaBrasileiradeZo o lo gia16 (1): 151-173. DONATTI, L. & E. FANTA. 2001. Ultrastru ctu re of p h otosen sory

cells an d p igm en t ep ith eliu m in th e retin a of th e An tarctic fish N otothenianeglecta Nybelin (Nototh en iid ae). An tarctic Reco rd4 5 (3): 297-310.

DONATTI, L. & E. FANTA. 2002. In flu en ce of p h oto p eriod on visu -al p rey d etection in th e an tarctic fish N otothenia neglecta Nybelin . An tarcticScien ce1 4 (2): 146-150.

EAST M AN, J. T. 1 9 8 8 . O c u l a r m o r p h o l o g y i n An t a r c t i c n ototh en ioid fish es. Jo urn al Mo rph o lo gy1 9 6: 283-306. ES-SO UNNI, A. & M.A. ALI. 1986. Ult rast ru ct u re o f t h e ret in al

p igm en t ep it h eliu m o f ligh t -a n d d a rk-a d a p t ed Yo u n g p igm en ted , an d m atu re silver eels, Anguillaanguilla (p isces, Teleostei). Zo o m o rph o lo gy1 0 6: 179-184.

FANTA, E.; A.A. MEYER; S.R. GRÖ TZNER & M.F. LUVIZO TTO. 1994. Co m p arat ive st u d y o f t h e feed in g st rat egy an d act ivit y p attern s of two An tarctic fish Trem atom usnewnesi Bou len ger, 1902 an d Gobionotothengibberifrons (Lön n berg, 1905) (Pisces, Nototh en iid ae) u n d er d ifferen t ligh t con d ition s. An tarctic

Reco rd3 8: 13-29.

FANTA, E.; F.S. RIOS; A.A. MEYER & S.R. GRÖTZNER. 2001. Ch em ical an d visu al system s in feed in g beh aviou r of th e An tarctic fish Oph t h alm olycu s am beren sis (Teleo st ei, Zo a rcid a e). An tarcticReco rd3 8: 13-29.

FANTA, E.; L. DONATTI & S. FRAIBERGUER. 1999. Visu al su fficien cy in food an d in itiation of feed in g beh aviou r in th e an tarctic fish trem atom usnewnesi Bou len ger, 1902. An tarcticReco rd 4 3 (2): 221-236.

FERRERO, E.; M. ANCTIL & M.A. ALI. 1979. Ultrastructural correlates of retin om otor resp on ses in in n er segm en ts of vertebrate ph otoreceptors. Révue Can adien n e de Bio lo gie 38: 249-264. GRÖTZER, S.R. & E. FANTA. 1998. Com p arative m orp h ology of th e ret in a o f t h e An t a rct ic fish Gobion ot ot h en gibberif ron s (Lö n n b erg, 1905), Trem atom us n ew n esi Bo u len ger, 1902, Lepidonotothen nudifrons (Lön n berg, 1905) an d N otothenia neglecta Nybelin , 1951. PesquisaAn tárticaBrasileira3: 31-47.

KEEN LEYSID E, M .H .A. 1 9 5 5 . So m e a sp ect s o f t h e sch o o lin g beh aviou r of fish . Beh av io ur8: 183-248.

LOUKASHKIN, A.S. & N. GRANT. 1959. Beh aviou r an d reaction s of t h e Pa cif sa rd in e, Sardin ops caeru la (gira d ), u n d er t h e in flu e n ce o f w h it e a n d co lo r e d ligh t s a n d d a r k n e ss. Pro ceedin gs o f th e Califo rn ia Academ ic o f Scien ces2 9 (15): 509-548.

LUFT, J.H . 1961. Im p ro vem en t s in ep o xy resin em b ed d in g m eth od s. Th e Jo u rn al o f Bio p h y sical an d Bio ch em ical Cy to lo gy9: 409-414.

MACDONALD, J.A. & J.C. MONTGOMERY. 1991. Th e sen sory biology of n ototh en ioid fish , p . 145-162. In: G. DI PRISCO; B. MARESCA & B. TOTA (Ed s). Bio lo gyo fAn tarcticFish . Berlim , Sp rin ger Verlag, 292p .

MACDONALD, J.A. & J.C. MONTGOMERY. 1998. Evolu tion of sen soty system s: a com p arison of An tarctic an d d eep -sea ich th yo-fau n a, p . 329-338. In: G. DI PRISCO; E. PISANO & A. CLARKE (Ed s).

Fish es o f An tarctica: a bio lo gical o verview. Milan , Sprin ger Verlag, 363p .

O’DAY, W.T. & R.W. YOUNG. 1978. Rh yth m ic d aily sh ed d in g of ou ter-segm en t m em bran es by visu al cells in th e gold fish . Jo urn alo fCellBio lo gy7 6: 593-604.

REYNOLDS, E.S. 1963. Th e u se of lead citrate at h igh p H as an electron -op aq u e stain in electron m icroscop y. Jo urn al o f CellBio lo gy 1 7: 208-212.

WARREN, R.R. & B. BURNSIDE. 1978. m icrotu bu les in con e m yoid elon gation in th e teleost retin a. Jo urn al o f Cell Bio lo gy 7 8: 247-259.

WATSO N, M.L. 1958. St ain in g o f t issu e sect io n s fo r elect ro n m icroscop y with h eavy m etals. Th eJo urn alo fBio ph y sical an d Bio ch em icalCy to lo gy4 (4): 475-478.