This paper not to be cited without prior reference to the author.

. International Council for theExploration of the Sea

C.t-I. 1980/L:36Biological Oceanography Cttee.

Ultrastructural Studies on the Developing Cranial Cartilagein several species of larval fish

by

Joel E. Bodammer1

U.S. Department of CommerceNational Harine Fisheries Service

Northeast Fisheries CenterOxford Laboratory

Oxford. t-laryl~nd 21654U.S.A•.

Olondrocytes observed in procartilage found under the dcveloping forebräinof striped bass (Horone saxatilis). winter flounder (Pseudopleuronectes americanus) •. ,and.·surnrner flounder· (Paralichthys dentatus) larvae had two distinctly different .appearances. Some cells appeared'to be actively elaborating and secrcting an

. amorphous glycoprotein-like material (presumably chondromucoprotein) \'lhich res·embl'ed.,the slightly more condensed substance found in the matrix that surrounded thcm •

. Others appeared to be degenerate or effete·in.that thcir cytoplasmwas disruptedwith large vesicles, their nuclei were condensed and heterochromatic, and in someinstances their cell membranes were no longer intact; Interstitial growth of thecartilage appeared to be minimal for neither cell division nor isogenie groups

·of chondrocyteswere readily observed. The cartilage was delimited by a weIldeveloped perichondrium with growth presurnably occurring by apposition. Therelevance of chondrogenesis to feeding in larval fishes and the possible effectsof contaminants on normal development are considered.

\RESilllE

Les chondrocytes (les cellules chondroides) observes dans le,pro-cartilagequi se trouve sous l'avant-cerveau larvaire des perches Tayees (t-forone saxatilis),des flets d'6t6s et d'hivers (Paralichthys dentatus et Pseudopleuronectes americanus)

'avaient deux aspects qui sont distinctement diff6rents. L'on de la deux morphologiesavait des cellules qui activement 6laboraient et s6cr6taient une mati~re amorpheet ressemb16e au glycoprot6ide (par prcsomption. un chondromucoprot6idc) qui .ressembltiit la substance un peu plus concentrcc qui se trouvait dans la matriceentourante. L'autre conformation de ces cellules se montrait degenerec ouepuiseeparce que lecytoplasma etait Tompu par des grosses v6sicules. les noyaux 6taientconcentres et h6terochromatiques et, de tcmps en temps, les membranes cellulairesn'etaient plus intactes. Le d~veloppem~nt l'interstitiel se montraitminimurn

Ipaper to be presented by Dr. Kenneth Sherrnan, Chairman, Biologi~al OceanographyCornrnittee. . .. ,; .

parce que souvent on n'a pas observe la division des cellules ou les groupes des·cellules isogenes. Le cartilage etait delimite par un perichondre et on suppose ~

qu'un cartilage nouveau se formera par suite du developpement appositiona1. Onexaminait 1e rapport de la chondrogenese a la nourriture par des poissons larvairesaussi bien que les effets possibles des contaminations sur 1e d6veloppement normaldes poissons.

INTRODUCTION

Upon depletion of their embryonic reserves, larval fish that cannot feedexogenously or obtain adequate numbers of prey starve und usua11y die (O'Conne11,1976). Frequently,.laporatory studies .of fe~ding and grO\'/th in fish larvae havefocused their attention on numerical relationships between predator and prey·(predator-prey density) and its importance to survival (see Werner and Blaxter,1980 for references). Studies of this type are often based on the premise thatlaboratory~reared larvae develop normally and are as capable of fee~ing as those ~from the natural environment. Larval fish reared in captivity, however, may

. demonstrate skeletal (Komada. 1980) or alimentary tract abnormalities (T\'lOngoand HacCrinunon, 1977) ",hich may affect or preclude their ability to capturc oringest prey.

This report presents results from aseries of introductory studies (Bodammer,1979) on scveral species (~brone saxatilis, Pseudopleuronectes americunus,' andParalichthys dentatus) of fish larvae which are being cultured in thc laboratory.The objective of this research is to examine.developing cells and tissues (e.g.,sensory, digestive, skeletal) that playa role in feeding. Hopefully, the resultsof this research \'1i11 be useful in assessing the effects of larval fish culturctechniques (Blaxter, 1969) and eventually those of environmental contaminants onlarval development and survival.

The larvae examined in this study exhibitcd no deformations in skeletalstructure of the llead (or.elsewhere), and the data presented are intcnded.toprovide insights on the status of crunial cartilage development in normal animals.

HATERIALS /\ND HETlIODS

Fish Larvae

l~inter flounder und summer flounder larvae were obtained from the NationalNarine Fisheries Service Laboratory, Narragunsett, Rhode Island, where they ,..erehatched and reared according to techniques developed by Smigielski und Arnold(1972) and Smigielski '(1975). Thc winter flounder 1arvae Here sacrificed 6 daysafter hatching, \'1hile the summer flounder Here 28 days old at thc time they werepreserved. Food was observed in the digestive tract of all specimens examined.

Striped bass 1arvae were obtained from the Edenton National Fish Hatchery,Edenton, North Carolina, where they were cultured using techniques deve10ped atthat faci1ity (Atstupenas, pers. commun.). The larvae were raised in continuous­flo\'1 tanks at water temperatures bet\.,reen 17-19°C and fed brine shrimp severa1times daily. The specimens used were sacrificed 9 days after hatching; however,the gut contents of thesQ fish Here not examined.

Light and E1ectron Nicroscopy

The 1arvae were fixed for light and electron microscopy by immersing them inice cold (0-4°C). 290 glutaraldehyde adjusted to pli 7.4 with 0.1 1'1 phosphate buffer.After rinsing in buffer solution. they were allowcd to come to room temperatureand postfixed for 1 hr in osmium tetroxide in 0.1 1'1 phosphate buffer at pli 7.4.They were then·stained en bloc with aqueous urany1 acetate according to the methodof Terzakis (1968), dehydrated in a graded series of ethanols and propylene oxide.infiltrated. and embedded in Spurr (1969) low viscosity medium. Thick sections(1.0-2.0 ~m) were made withglass knives. stained with toluidine blue. ·and examinedwith the light microscope. Thin sections were obtained with a diamond knife andstained with alcoholic uranyl acetate and lead citrate before being examined witha Zeiss BI 9 52 electron microscope.

RESULTS AND DISeUSSION

In winter flounder larvae (Fig. I). the cartilagenous plate lying dorsal to·the oral chamber (Oe. Fig. 1) contained a number of oval-shaped cllondrocytes lyingin close proximity to each other as is typical for procartilage. The nuc1ei ofthe cells were round and of variable staining intensity. The cytoplasm of thechondrocytes varied from a uniform light blue to a darkly stained. condensedcondition accompanied by large vacuoles. The chondrocyteswere not in pairs orclusters (isogenic groups). nor was the capsu1e and lacuna normally surroundingthese cells in hyaline type cartilage observed. Except for its width. ~lich waslarger in striped bass and summer flounder larvae. the cartilagenous plate occupiedthe same anatomical position in all three species. In summer flounder larvae.however, more chondrocytes appeared to have the condensed and vacuolated cytoplasmthan \.;as observed for the otherspecies.

U1 trastructurally, the chondrocytes in \'/inter flounder larvae (Figs. 2. 3)procartilage demonstrated several different morphological types which are presumedto be related to their age and secretory activity. In Figure 2. a putative effetecell (EC) is shO\m near the periphery of the cartilage. Cells of this typeroutinely had a disrupted cytoplasm containing many large empty spaces--hencetheir vacuolar appearance with the light microscope. A mature secretory cell(SC. Fig. 2) is present next to the effete cel1. and its cytoplasm is fil1edwith vesicles which contained a moderately dense material that resembled thenonfibril1ar, amorphous substance seen in the adjacent matrix (1'~. Fig. 2).50mewhat younger appearing secretory cells (Fig. 3) also contained numerousvesicles; however, the stainable material within them was less condensed thanin the vesic1es of the mature chondrocytes. Their nuclei demonstrated a euchromaticappearance suggesting ongoing genetic·activity (note contrast \'Iith effete cellshmm in Fig. 2). The mitochondria of these cells were quite large and had tubularcristae (l-f. Fig. 3). The Go1gi comp1ex. hO\'/ever, \'/as not particularly noticeableand consisted of on1y a fcw flattened saccules and very smal1 vesicles.

The electron microscopic appcarance of the chondrocytes was similar in allthe larval fish spccies cxamined. Perhaps owing to their age. greater numbersof degenerate cells appeared to be present in the procartilage plate of stripedbass and summer flounder larvac (Fig. 4). These older larvac, howcvcr, provcd

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to be more suitable specimens for possible observations of cartilage growth byapposition in which nel" chondrocytes are added along the periphery of the peri- ­chondrium (PC, Fig. 5). This type of growth al10ws for the distribution of materialin a manner such that the appropriate form and size of tllC carti1agc is attained

,(Be1anger, 1977).

The causes of ske1etal abnorma1ities invo1ving the head and jaws of larvalfishes are incomplete1y understood (Rosentha1 and A1derdice, 1976). As Komada(1980) has ShOlvn, these anomalies may be much more preva1ent in cu1tured fishcompared to those captured from their natural environment. Because 1aboratory­raised larval fish are often used in various studies on gro\vth and surviva1 (e.g.,feeding energetics, physiology, pollution effects), we are beginning our researchby studying the normal morpho10gy of ee11s and tissues that are invo1ved in feedingand l.;hich may be affected by either laboratory conditions or environmental pollutants(Hehr! e and Huyer, 1977; Couch et al., 1979; Ozoh, 1980).

ACKNOWLEDGr.fENT

I wish to thank Dr. G.C. Laurence and the rest of the staff members of theLarval Fish Physiology Investigation at the National Marine Fisheries ServiceNarragansett Laboratory, Narragansett, Rhode Is1and, for providing the larvalfish.

LITERATURE CITED

Belanger, L.F. 1977. The skeletal tissues, pp. 205-249. In L. Weiss and R.O.Greep (eds.), lIistology. McGrm.;-Hill Inc., New York.

Blaxter, J.H.S. 1969. Deve1opment: eggs and 1arvae, pp. 177-252. In W.S. Hoarand D.J. Rundall (eds.), Fish Physiology. Vol. 3. Academic Press, New York.

Bodammer, J.E. '1979., An introductory light and eleetron microscopic study of theolfuetory epithelium in striped bass (t-Iorone saxatilis) und '\"inter flounder ...(Pseudopleuroncctes americanus) larvae. Int. Counc. Exp1or. Sea C.r-I. 1979/~

G:38, 7 pp.

Couch, J.A., J.T. Winstead, D.J. Hansen, and L.R. Goodman. 1979.dysp1asia in young fish cxposed to the hcrbicide trif1uralin.2: 35-42.

VertebralJ. Fish. Dis.

Komuda, N. 1980. Incidcnce of gross mal formations and vertebral anoma1ies ofnatural and hatchcry Plccog1ossus a1tivc1is. Copeia 1980, pp. 29-35.

t-Iehrle, P.t-I. and F.L. ~Iaycr. 1977. Bone devclopmcnt and grmvth of fish as affectedby toxaphene, pp. 301-314. In I.H. Suffet (cd.), Fatc'of Po11utunts in Airand Water Environments. Parr-2. Wilcy Intcrscicnce Publishers, Nmv York.

O'Conncll, C.P. 1976. lIistologica1 criteria for diagnosing thc starving conditionin carly post yolk sae larvae of thc northcrn anehovy, Engraulis mordax Girard.J. Exp. Nar. Biol. Beol. 25: 285-312.

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Ozoh, P.T.E. 1980. Effeets of reversible incubations of zebrafish eggs in copperand lead ions with or without shell membranes. Bul1. Environ. Contam. Toxicol.24: 270-275.

Rosenthai, H. and D.F. Alderdiee. 1976. Sublethai effects of environmentalstressors, natural and pollutional, on marine fish eggs and larvae. J. Fish.Res. Board Can. 33: 2047-2065.

Smigie1ski, A.S. 1975. Hormone-induced spawnings of the summerflounder andrearing of the larvae in the laboratory. Prog. Fish-Cult. 37: 3-8.

Smigielski, A.S. and C.R. Arnold. 1972. Separating and incubating winter floundereggs. Prog. Fish-Cult. 34: 113.

Spurr, A.R. 1969. A low-viscosity epoxy resin embedding medium for e1eetronmieroscopy. J. Ultrastruct. Res. 26: 31-43.

Terzakis, J.A. 1968. Uranyl acetate, a stain and a fixative. J. U1trastruct.Res. 22: 168-184.

Twongo, T.K. and H.R. r.lacCrimmon. 1977. Histogenesis of the oropharyngeal andoesophagea1 mucosa as re1ated to early feeding in rainbow trout, Sa1mogairdneri Richardson. Can. J. Zool. 55: 116-128.

Wemer, R.G. and J.H.S. B1axter. 1980. Growth and survival of larval herring(Clupea harengus) in relation to prey density. Can. J. Fish. Aquat. Sei.37: 1063-1069. ~

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FIGURE LEGENDS

Fig. 1. Light micrograph of a winter flounder larvae in cross-sectionillustrating chondrocytes within the procartilage plate (CP) near the dorsalaspect of the oral cavity (aC). X693.

Fig. 2. Electron micrograph of chondrocytes from winter flounder larvaefeaturing an effete cell (EC) adjacent to an active secretory cell (SC). (O~I =oral mucosa; MA = cartilage matrix; N = cell nucleus). X7,220.

Fig. 3.procartilage.

Electron micrograph of developing chondrocytes in winter flounder(N = cell nucleus; M = mitochondrion). X7,220.

Fig. 4. Electron micrograph of two effete cells (EC) observed in theprocartilage of a striped bass larvae. X7,220.

Fig. 5. Electron micrograph of the procartilage from a striped bass larvaeillustrating a putative developing cell (DC) located near the perichondrial celllayer (PC). X7,220:

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