Scale Growth and Squamation Chronology for the LaboratoryReared Hermaphroditic Fish Rivulus marmoratus (Cyprinodontidae)

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1 Japanese Journal of Ichthyology Vol.34, No Scale Growth Squamation Chronology for the LaboraryReared Hermaphroditic Fish Rivulus marmoratus (Cyprinodontidae) EunHo Park SeungHwi Lee (Received February 2, 1987) Abstract Scale morphology, growth the squamation chronology are described for the her maphroditic fish Rivulus marmoratus reared in the laborary. The s are round or oval shaped cycloid type, their sizes are about mm in diameter. The number of ridges increases more rapidly relative the body growth of the fish in early stages, but this increase is proportionate growth subsequently. Three loci of development have been identified. The s first appeared on the center of the parietal region at 8 days after hatching. The second locus of formation was on the lateral line of the posterior end of the caudal peduncle. A third locus was later observed on the lower right corner of the operculum. The final squamation was completed at 6 weeks after hatching. The oviparous cyprinodontid fish Rivulus marmo ratus Poey inhabiting the brackish waters through out the Caribbean is the only known vertebrate that naturally exhibits functional hermaphroditism with internal selffertilization (Harringn, 1963). This species has received special attention not only because of interest in reproductive biology (Har ringn, 1967, 1968, 1971, 1975) but also because of its potential as a laborary animal for the study of experimental carcinogenesis (Park Kim, 1984a). R. marmoratus has several desirable attributes as an experimental animal for educational re search purposes in fish biology: A given popula tion of this species is genetically homozygous due functional hermaphroditism (Kallman Harringn, 1964). The generation time is only 46 months; every day a mature individual lays several eggs which are large enough (1.8mm, diameter) hle, they develop normally at room temperature outside the body (Harringn Crossman, 1976) in 14 days thus enabling us examine detailed developmental sequences through their transparent chorion. In addition, the fish are small (35cm), hardy easy cultivate in the laborary. The early life hisry including sex determina tion of this species has been meticulously docu mented (Harringn, 1961, 1963, 1967, 1968, 1971, 1975; Harringn Kallman, 1968). Little study, however, has been conducted on the detailed anamy of this remarkable species. As part of an investigation of the comparative anamy of this species, the morphology, growth development studied accumulate biological information on this species. This information may serve as a baseline in normal morphogenesis of s during larval juvenile stages of R. marmoratus. Materials methods Laborary culture. The parental sck of R. marmoratus was obtained from the Zoology In stitute Museum, University of Hamburg, Federal Republic of Germany in Adult fish kept singly in 20l glass aquarium filled with 15l of tap water. The salinity of water was adjusted 15 ñ with crude salts. All fish kept in a room where the temperature was adjusted at 25 }1 Ž which was illuminated by fluores cent lamps for 14 hours daily. Larvae fish younger than 1 month fed brine shrimp (Artemia salina) nauplii (eggs from Metaframe, Calif.), adult fish nourished with chicken liver, Drosophila earthworm. Sampling analysis. Fertilized eggs separated from parent fish incubated in 1l glass chambers. The hatched larvae ex amined under a lowpower microscope any which appeared grossly abnormal removed. The normal larvae cultivated in homogeneous age groups for analysis. The fish density was

2 Park Lee: Fig. 1. Phomicrographs Bar for fish=1cm. maintained of s from different bars for s =0.2mm. identically groups avoid Rivulus marmoratus (1fish/l) density in locations growth of the skin chronology. Scale from ten 3monthold experimental dependent Squamation inhi in Rivulus marmoratus morphology was adult specimens. adult. examined bition. Fish analyze hatching. fixed remove tures. cleared analysis 10 ñ body starved empty neutral which stained fish described fish of in pattern prior The fixed H2O2 alizarin S method 1984b). age struc red the Kim, each Scale morphology. The s of adult R. marmoratus the typical cycloid type ranging from round oval shape, their sizes about mm in diameter (Fig. 1). The small est s oval distributed on the ventral side of the trunk. The largest round has an anterior field medially, with concave anterolateral margins found only on the parietal region. The remaining body surface was covered with medium sized round s (Fig. 1). canal, alkaline obscure with (Park day Results after alimentary according examined growth 1 weeks formalin. with pigments whole intervals 12 for the bleached Scales previously Ten in on appropriate until sacrificed specimens at development They being sampled group for squamation 477

魚類学雑誌 Fig. 2. Japan. J. Ichthyol. Phomicrograph of Bar=100ƒÊm. 34(4), 1988 longitudinal e, epidermis; Culture d, Temp.; Phoperiod; Density; Fig. 3. Diagram tal length showing section dermis; Rivulus marmoratus hypodermis; m, muscle; skin showing s, ; sp, imbrication.

3 魚類学雑誌 Fig. 2. Japan. J. Ichthyol. Phomicrograph of Bar=100ƒÊm. 34(4), 1988 longitudinal e, epidermis; Culture d, Temp.; Phoperiod; Density; Fig. 3. Diagram tal length showing section dermis; Rivulus marmoratus hypodermis; m, muscle; skin showing s, ; sp, imbrication. pocket. region against 25 }1 Ž 14hrs./day 1fish/liter the increase of Rivulus of h, marmoratus of circuli during number on s from the parietal 12 weeks after for 10 specimens. 478 hatching. Each point represents age the mean

4 Park Lee: Rivulus marmoratus Squamation 8 days 28 days 10 days 14 days 32 days 21 days 23 days 35 days 25 days 42 days Fig. 4. Diagrammatic representation of sequence of squamation in Rivulus marmoratus after hatching. 8 days, x=7.09 }0.28mm; 10 days, x=7.77 }0.18mm; 14 days, x=8.01 }0.31mm; 21 days, x= mm; 23 days, x=8.74 }0.36mm; 25 days, x=8.78 }0.27mm; 28 days, x=9.71 }0.57mm; 32 days, x=10.21 }0.56mm; 35 days, x=11.73 }0.94mm, 42 days, x=16.1 }1.28mm. x=average tal length }S.D. The s imbricated with the posterior field exposed; the anterior lateral fields embedded in the dermis. Hislogically, they lay obliquely in a pocket surrounded by dermal tissue, which in turn covered by an epidermal layer a heavy layer of mucus (Fig. 2). Clear ridges (circuli) visible in every, they discontinuous at the radii on the anterior field (Fig. 1). Scale growth. The s from a restricted area on the parietal region where the fisrt s appear ed carefully examined the number of ridges counted against the age size (tal length) of the fish. Fig. 3 shows the results ob

5 Japan. J. Ichthyol. 34(4), 1988 tained. Each point represents the average num ber of ridges found for at least ten s from each age group of fish. There was a relatively high rate of growth (ridge increase) during the early stage of fish growth, then a steady decline when the number of ridges increased pro portionately the growth of the fish. No annuli formed even in eight fish older than 2 years in the laborary. Squamation. The origin of s the squa mation for R. marmoratus are illustrated in Fig. 4. The initial site of formation was near the center of the parietal region. The first appeared on the center of the junction of the frontal parietal bones at 8 days after hatching (average tal length; 7.09 }0.28mm). No s observed on fish younger than 8 days. Scale development progressed concentrically cover the entire head surface. The second locus of squamation was midlaterally at the posterior end of the caudal peduncle. Scale formation on this site proceeded anteriorly along a midlateral line, met with the s from the first locus on the dorsal region of the trunk. At 32 days after hatching, the s of a third locus developed on the lower right corner of the operculum. Squa mation here progressed dorsally anteriorly, finally connecting with those from the first locus. At this time (5 weeks post hatching), the squa mation was nearly completed except in the an terior region of the operculum the central region of the belly. Scale formation was com pleted at 6 weeks post hatching. Nearly the en tire body surface was completely d. The average size of specimens at this stage was mm, the number of s along } their lateral line was counted be 4151(x }S.D.= 46.1 }2.3, n=102). The degree of squamation in fish within the same age groups was somewhat variable among individuals. The larger fish observed show more developed squamation than smaller ones. Discussion The present study is the first description of the morphology, growth developmental sequence of the s in R. marmoratus. The gross mor phology hislogical arrangement of s are similar other teleosts (Harder, 1975). Scale growth has been extensively studied determine the age, life hisry rate of growth of the fishes. The pattern of growth versus the growth of R. marmoratus is also similar that of other fishes investigated (Wallin, 1957). Most studies on squamation chronology have been made with species of the families Carangidae (Berry, 1960), Casmidae (White, 1977), Cen trarchidae (Siefert, 1965; Conley Witt, 1966; Cooper, 1971), Clupeidae (Chapn, 1967), Coregonidae (Hoagman, 1970), Cyprinidae (Ward Leonard, 1954; McCrimmon Swee, 1967; Andrews, 1970; Armstrong, 1973), Esocidae (Franklin Smith, 1960), Hexagrammidae (Fukuhara Fushimi, 1984), Megalopidae (Harringn, 1958), Pomamidae (Silverman, 1975), Salmonidae. (Elson, 1939; Neave, 1943; Brown Bailey, 1952; Warner Havey, 1961) Sciaenidae (Priegel, 1966). No report has been, however, published for any member of the family Cyprinodontidae. The squamation pattern of R. marmoratus dif fers from that of other species. Although the location number of loci where the squamation initiates vary among species, the single locus on the caudal peduncle has been reported in most species may be the most commonly occurring pattern (Conley Witt, 1966; Priegel, 1966; Andrews, 1970; White, 1977). Two loci have been found in the zebrafish, Brachydanio rerio (Armstrong, 1973) in the greenling, Hexagram mos otakii (Fukuhara Fushimi, 1984), one on the anterior portion of the trunk the other on the caudal peduncle. In the black crappie (Pomoxis nigromaculatus), four loci of de velopment have been identified; squamation first appears at the posterior of the anal fin anterior of the pelvic fins. A fourth locus is the most anterior region of the lateral line (Cooper, 1971). Even though the loci may vary, squamation usually progresses along the lateral line then on the dorsum venter. This pattern is fairly consistent among the many species investigated. R. marmoratus is unusual in the number of loci (3), the squamation pattern, the parietal region as the initial site of development. Individual variation in the degree of de velopment within the same age groups of R. marmoratus suggests that development is correlated with both size age of fish. Scale development in the zebrafish is related length

6 Park Lee: Rivulus marmoratus Squamation rather than age (Armstrong, 1973). Age as a main facr in determining squamation has been, however, suggested in the black crappie (Ward Leonard, 1954; Cooper, 1971). Literature cited Andrews, A.K Squamation chronology of the fathead minnow, Pimephales promelas. Trans. Amer. Fish. Soc., 99: Armstrong, J.G Squamation chronology of the zebrafish (Cyprinidae), Brachydanio rerio. Copeia, 1973: Berry, F.H Scale scute development of the carangid fish, Caranx crysos (Mitchill). Quart. J. Florida Acad. Sci., 23: Brown, C.D.J. J.E. Bailey Time pattern of formation in Yellowsne cutthroat trout (Salmo clarkii lewisi). Trans. Amer. Microsc. Soc., 71: Chapn, R.B Scale development in the Gulf menhaden, Brevoortia patronus. Trans. Amer. Fish. Soc., 96: Conley, J.M. A. Witt. Jr The origin development of s in the flier, Centrarchus macropterus (Lacepede). Trans. Amer. Fish. Soc., 95: Cooper, J.A Scale development as related growth of juvenile black crappie, Pomoxis nigro maculatus Lesueur. Trans. Amer. Fish. Soc., 100: Elson, P.F Order of appearance of s in speckled trout. J. Fish. Res. Bd. Can., 4: Franklin, D.R. L.L. Smith. Jr Notes on development of patterns in northern pike, Esox lucius L. Trans. Amer. Fish. Soc., 89: 83. Fukuhara, O. T. Fushimi Squamation of larval greenling Hexagrammos otakii (Pisces Hexa grammidae) reared in the laborary. Bull. Japan. Soc. Sci. Fish., 50: Harder, W Anamy of fishes. E. Schweizer bartsche Verlag., Stuttgart, xii+612 pp. Harringn, R.W., Jr Morphometry ecology of small tarpon, Megalops atlantica Valenciennes, from transitional stage through onset of forma tion. Copeia, 1958: 110. Harringn, R.W., Jr Oviparous hermaphrodi tic fish with internal selffertilization. Science, 134: Harringn, R.W., Jr Twentyfourhour rhythms of internal selffertilization oviposition by hermaphrodites of Rivulus marmoratus. Physiol. Zool., 36: Harringn, R.W., Jr Environmentally con trolled induction of primary male gonochorists from eggs of the selffertilizing hermaphroditic fish, Rivulus marmoratus Poey. Biol. Bull., 132: Harringn, R.W., Jr Delimitation of the thermolabile phenocritical period of sex determina tion differentiation in the ongeny of the normally hermaphroditic fish, Rivulus marmoratus Poey. Physiol. Zool., 41: Harringn, R.W., Jr How ecological genetic facrs interact determine when self fertilizing hermaphrodites of Rivulus marmoratus change in functional secondary male, with a reappraisal of the modes of intersexuality among fishes. Copeia, 1971: Harringn, R.W., Jr Sex determination differentiation among uniparental homozygotes of the hermaphroditic fish Rivulus marmoratus (Cyprinodontidae: Atheriniformes). Pages in R. Reinboth, ed. Intersexuality in the animal kingdom. SpringerVerlag, Berlin. Harringn, R.W., Jr. R.A. Crossman, Jr Temperature induced meristic variation among three homozygous genotypes (clones) of the selffertilizing fish Rivulus marmoratus. Can. J. Zool., 54: Harringn, R.W., Jr. K.D. Kallman The homozygosity of clones of the selffertilizing herma phroditic fish, Rivulus marmoratus Poey (Cyprino dontidae, Atheriniformes). Amer. Nat., 102: Hoagman, W.J Early development on the Great Lakes coregonids, Coregonus artedii C. kivi. Pages in C.C. Lindsey C.S. Woods, eds. Biology of coregonid fishes. Univ. of Maniba Press, Winnipeg. Kallman, K.D. R.W. Harringn, Jr Evidence for the existence of homozygous clones in the selffertilizing hermaphroditic teleost Rivulus marmoratus Poey. Biol. Bull., 126: McCrimmon, H.R. U.B. Swee Scale formation as related growth development of young carp, Cyprinus carpio L.J. Can., 24: Fish. Res. Bd. Neave, F Scale pattern counting methods in relating certain trout other salmonids. Trans. Roy. Soc. Can., 30: Park, E.H. D.S. Kim a. Hepacarcino genicity of diethylnitrosamine the selffertilizing hermaphroditic fish Rivulus marmoratus (Teleosmi; Cyprinodontidae). J. Natn. Cancer Inst., 73: Park, E.H. D.S. Kim b. A procedure for staining cartilage bone of whole vertebrate larvae while rendering all other tissues transparent. Stain Technol., 59:

7 Japan. J. Ichthyol. 34(4), 1988 Priegel, G.R Early development in the freshwater drum, Aplodinitus grunniens Rafinesque. Trans. Amer. Fish. Soc., 95: Siefert, R.E Early development in the white crappie. Trans. Amer. Fish. Soc., 94: 182. Silverman, M.J Scale development in the bluefish, Pomamus saltatrix. Trans. Amer. Fish. Soc., 104: Wallin, O On the growth, structure developmental physiology of the of fishes. Rep. Inst. Fish. Res. Drottningholm, 38: Ward, H.C. E.M. Leonard Order of appearance of s in the black crappie, Pomoxis nigromaculatus. Proc. Oklahoma Acad. Sci., 33: Warner, K. K. Havey Body- relation ships in llocked salmon, Salmo salar. Trans. Amer. Fish. Soc., 90: White, D.S Early development pattern of formation in the spotted sucker, Minytrema melanops(casmidae). Copeia, 1977: (Department of Biology, College of Natural Sciences, Hanyang University, Seoul 133, Korea) Rivulus marmoratus Eun-Ho Park Seung-Hwi Lee

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