Suisan Gakkaishi Nippon 55(6), 1001-1006 (1989)

Hybridization Experiment Between Female Crucian Carp and Male Grass Carp*1

Midoriko Kasama*2 and Hiromu Kobayasi*2 (Received December 7, 1988)

Intergeneric cross was preformed with the crucian carp Carassius carassius •Š•~ the grass carp Ctenopharyngodon idellus •‰. The hybrids were analyzed morphologically and karyologically , and were compared with the parental species . These hybrids possessed a mosaic of character expres sions that showed overall intermediacy though superficial appearances were similar to the crucian carp parent, and had the karyotype that incorporated two genomes of the crucian carp parent and

one of the grass carp parent.

Hybridization experiments have been carried the uppermost base of the ventral fin. "Pectoral out by many investigators with salmonid, cyp fin origin to ventral fin origin" is measured from rinid, cobitidid and gasterosteid fishes. With cyp the point at the uppermost base of the pectoral rinid fishes, Suzuki1-11) has performed exper fin to the uppermost base of the ventral fin. "Ventral fin origin to anal fin origin" is measured imental hybridizations among several species.

However, these studies have dealt almost exclusive from the uppermost point of base of the ventral

ly with survival analysis, viability and morpho fin to the anterior base of the anal fin. All mer

logical analysis, and with only a few species as the istic and morphometric characters analyzed were

parents. They have also paid few interest to chosen on the basis of marked and consistent chromosome morphology. differences between the parental species. The

As part of investigation on morphological and hybrid index computed following Hubbs and karyological analyses of various artificial hybrids Kuronuma15) and Hubbs et al.16) was used to com among the Cyprinida, this study described in pare hybrid character states to the parental spe

tergeneric hybrids between the crucian carp, cies. Index values between 30 and 70 were con

Carassius carassius •Š and the grass carp, Cte sidered intermediate. Scales at the region between

nopharyngodon idellus •‰. the anterior base of the dorsal fin and the lateral line were plucked out, and examined after staining with alizarin red S. Pharyngeal, jaw and opecular Materials and Methods bones were cleared in 1% KOH and stained with

Fishes used in this study were Ca. carassius alizarin red S to examine their morphological

and Ct. idellus, abbreviated to Ca. c. in the Table•@ 1, features. The terminology for the scales and the

had been collected from Holland and bred in our pharyngeal bones was that of Chu,17) and the

laboratory, and Ct. idellus, Ct. i were raised in terms for the other bones were those of Weisel. 18)

our laboratory. Artificial fertilization was per Chromosome study was carried out using cells

formed as in the foregoing experiment. 12) Hy form the primary culture of the scale epithelium.

brids used for morphological and karyological The cells were fixed in Carnoy's fluid, flame-dried

analysis were about one and a half years old. on slides and stained with Giemsa.

Methods for meristic counts and morphometric measurements were followed Matsubara13) and Results Hubbs and Lagler.14) Measurements were read from a dial caliper to 0.1mm. We added three About 41% of 532 eggs produced from this measures not typically taken on cyprinid fishes. cross developed into embryos up to the gastrular "Dorsal fin origin to ventral fin origin" is the dis stage, and mortality during this stage was high. tance from the anterior base of the dorsal fin to Embryos hatched at 92 to 139h after fertilization,

*1 Hybridization experiments in Cyprinida-II. *2 Japan Women's University, Mejirodai, Bunkyo, Tokyo 112, Japan (笠間 緑 子, 小 林 弘:日 本 女 子 大 学). 1002 Kasama and Kobayas i

Table 1. Comparison of Meristic and Morphometric characters of Ca. carassius •Š•~Ct. idellus •‰ with parental species. X is mean, r range and N samle size

Ca. c. Hybrid Ct. i. Index Character value X r N X r N X r N Body depth/standard length 0.404 0.401-0.413 20 0.375 0.361-0.395 41 0.255 0.249-0.261 20 19.4 Caudal peduncle depth/body depth 0.381 0.371-0.386 20 0.419 0.408-0.429 41 0.529 0.508-0.545 20 25.6 Body width/body depth 0.544 0.467-0.592 20 0.538 0.502-0.561 41 0.748 0.727-0.769 20 6.3 Head width/head depth at occiput 0.561 0.532-0.590 20 0.616 0.602-0.630 41 0.815 0.795-0.834 20 21.6 Orbital length/head depth at occiput 0.176 0.157-0.190 20 0.221 0.206-0.238 41 0.246 0.213-0.290 20 64.2* Interorbital width/head depth at pupil 0.613 0.553-0.668 20 0.712 0.686-0.735 41 0.967 0.909-1.017 20 27.9 Dorsal fin origin to ventral fin origin/ pectoral fin origin to ventral fin origin 1,501 1.446-1.597 20 1.579 1.461-1.667 41 0.876 0.822-0.930 20 0.0 Pectoral fin origin to ventral fin origin/ ventral fin origin to anal fin origin 1.009 0.938-1.055 20 0.899 0.836-0.957 41 1.132 1.071-1.208 20 0.0 Longest pectoral fin ray length/ pectoral fin origin to ventral fin origin 0.575 0.564-0.586 20 0.914 0.820-0.992 41 0.658 0.608-0.762 20 100.0 Dorsal fin base length/ longest dorsal fin ray length 2.344 2.243-2.446 20 1.137 1.047-1.151 41 0.560 0.535-0.591 20 67.6* Anal fin base length/ longest anal fin ray length 0.899 0.804-0.956 20 0.575 0.534-0.611 41 0.664 0.641-0.685 20 100.0 Dorsal fin rays 19 18-21 20 14 14-15 41 8 8 45,4* Pectoral fin rays 14 13-16 20 16 16-17 41 20 19-20 20 33.3* Ventral fin rays 8 8 20 9 9 41 9 9 100.0 Lateral line scales 32 31-33 20 30 27-32 41 39 37-42 20 0.0 Gill-rakers 28 28-30 20 24 21-24 41 16 14-18 20 33,3* Pharyngeal teeth formula- 18 32 17 0.0 outer row on left side 0 0 0 0 2 2 inner row on left side 4 4 4 4 5 4-5 inner row on right side 4 4 4 4 4 4-5 outer row on right side 0 0 0 0 2 2 Vertebrae 31 29-33 20 32 31-33 - 41 42 40-43 20 9.0 * Consideredintermediate. Hybrid index=36.3 Female Crucian Carp •~ Male Grass Carp Hybridizati on 1003

Fig. 1. Hybrid and parental species. Carassius carassius (a), carassius carassius •Š•~ •@ Cteno pharyngodon idellus •‰(b), an d Ctenopharyngodon idellus (c). Bars equal 10mm.

and about 33% of the eggs produced larvae. But but actually were intermediate in only 5 of the 18 40% of the larvae suffered from edema, bent characters analyzed (Table 1). In general ap bodies and circulatory disorder, and died at l to pearance, thier body coloration and body outline 5 days after hatching. The survivors were seemed made them appear more similar to crucian carps to develop normally, but many of them could not (Fig. 1). The Body side was light and a golden feed and died within 10 days after hatching. Many copper-red, and each fin was dark red. But the larvae that did feed died with the lapse of time. dark spot at the base of the caudal fin characteristic Finally 56 hybrids were reared untill the the adult of the crucian carp was absent from all hybrids at -like stage, and 41 subjected to analyses. The the adult-like stage. The body was laterally com others are subsequently reared for further study pressed. The ratios of body depth/standard of fertility. length and caudal peduncle depth/body depth The hybrids exhibited general intermediacy, were closer to those of the crucian carp. But the 1004 Kasama and Kobayasi lateral view outline from breast to belly was some were more similar to those of the crucian carp what straight, similar to the grass carp. The ratio than the grass carp (Fig. 2a). The basal margin of dorsal fin origin to ventral fin origin/pectoral fin was wavy, and the radial grooves were all round. origin to ventral fin origin was closer to the crucian But pattern of pigmentation, situation of the carp. The ratios of body width/body depth, forcus and density of the apical circuli.were more interorbital width/head depth at pupil and head or less similar to those of the grass carp. The width/head depth at occiput were closer to the number of gill-rakers was intermediate. The crucian carp. The average of orbital length/head length of the gill-rakers in relation to the gill depth at occiput in all hybrids fell between the filaments was longer than that of the grass carp parental averages, but the ranges overlapped those and closer to that of the crucian carp (Fig. 2b). for grass carps. The mouth was oblique, giving The corner of the gill arch was relatively distinct the front of the snout a profile resembling that of compared with crucian carps. the crucian carp. The outline of each fin was The pharyngeal arches were similar to those of similar to that of the crucian carp, but not as the crucian carp (Fig. 2c, d). In all hybrids, the round. The ratios of dorsal fin base length/ pharyngeal bones did not have so broad width of longest dorsal fin ray length and the number of anterior and posterior process as crucian carps, dorsal fin rays were intermediate. The ratio of and the processes were somewhat arched com anal fin base length/longest anal fin ray length pared with those of crucian carps. The tooth was much closer to the grass carp. The ratio of platform was as elevated as in crucian carps. The longest pectoral fin ray length/pectoral fin origin situation of the pitted surface was intermediate to ventral fin origin was much closer to the grass between the two species, and it reached the middle carp, with the posterior tip of the pectoral fins region of the anterior limbs. The characteristics being near or at the ventral fin origin. The ratio of the jaw bones and the opecular bone series of pectoral fin origin to ventral fin origin/ventral tended to be similar to those of the crucian carp. fin origin to anal fin origin was much closer to The anterior tip of the dentary of the hybrids the crucian carp. The number of pectoral fin with a distinct symphyseal knob had as narrow rays was relatively similar to the crucian carp. width as that of the crucian carp, giving the The number of ventral fin rays was nine, this num mandible a thin profile. The opercle of the ber was typical for the grass carp. The caudal fin opecular bone series was distinctly different forked stronger than that of crucian carps. between the parental species, had a narrower The number of lateral line scales was more width and narrower opecular process than the similar to that of the crucian carp. The scales grass carp, this resembling that of the crucian carp. The number of vertebrae was much closer to that of the crucian carp. The gas-bladder was intermediate in its shape (Fig. 2e). The outward form of the anterior and posterior chambers was similar to the crucian carp, but the posterior chamber was longer in length than that of the crucian carp, thus being closer to that of the grass carp. The intestine was much closer to that of the crucian carp in its coiling form (Fig. 2f). The gonads of the hy brids were slender and semitransparent in the spawning season, and adhered to the lateral sides of the gas-bladder. The chromosome number of the hybrids was 124 (Fig. 3), while it was 2n=100 for the crucian Fig. 2. Some morphological characters of Ca. carp and 2n=48 for the grass carp. The karyo carassius •Š•~ Ct. idellus •‰. Lateral view of type of the crucian carp consisted of 20 met scale (a) and first right gill arch (b); dorsal (c) and ventral (d) views of right pharyngeal arch acentrics, 40 submetacentrics and 40 acrocentrics,

and teeth; and lateral view of gas-bladder (e) and that of the grass carp involved 16 metacentrics, and intestine (f). Bars equal 2mm for a, c, d 20 submetacentrics and 12 acrocentrics. These and 5mm for b, e, f. results for the parental species agreed with those Female Crucian Carp •~ Male Grass Carp Hybridization 1005

Fig. 3. Karyotype of Ca. carassius •Š•~ Ct. idellus •‰. Arrows show chromosomes with satellites.

reported by Kobayasi et a1.19)and Ojima et al..20) crucian carp in most of the characters resulted Chromosomes of the hybrids were distinguished from the unbalanced inheritance from the parental into 28 metacentrics, 50 submetacentrics and 46 species. acrocentrics, of which 24 chromosomes: 8 met Suzuki5) reported that intergeneric hybrids be tacentrics, 10 submetacentrics and 6 acrocentrics tween Carassius auratus auratus •Š and Gnathopogon were chosen out as a genome belonging to the elongatus elongatus •‰ were similar to Ca. auratus grarss carp on the basis of its karyotype. The auratus. He stated that this may have resulted other 100 chromosomes: 20 metacentrics, 40 sub from the relatively greater gap of the phylogeneti metacentrics and 40 acrocentrics consisted of 50 cal relationship between Ca. auratus auratus •@ and pairs of homologous chromosomes: 10 pairs of G. elongatus elongatus compared with the other metacentrics, 20 pairs of submetacentrics and 20 intergeneric Gnathopogon hybrids, which were pairs of acrocentrics. The 5th chromosome pair typically intermediate. The present data suggest in the acrocentric group showed a satellite on the that his hybrids between Ca. auratus auratus •Š short arm. These 100 chromosomes were identical and G. elongatus elongatus •‰ have karyotypes to the karyotype of the crucian carp in both incorporating two genomes of Ca. auratus auratus number and morphology and could belong to two and one genome of G. elongatus elongatus. Our genomes of the crucian carp. findings support caution against discussing the •@interrelationship among parental species of various

Discussion hybrids only on the basis of morphological •@ analysis.

Although the hybrid index for this cross was Kobayasi et a1. 21, 22) noted that some back-cross within the range considered intermediate, 13 of hybrids of F1 fishes between Carassius auratus the 18 characters were not, and 10 of them closer langsdorfii •Š and Cyprinus carpio •‰ or between to the crucian carp. The phenotype of the hy Carassius auratus subsp. •Š and Ca. carassius •‰ brids was similar to the crucian carp. The karyo had karyotypes consisting of two genomes of the type of the hybrids incorporated two genomes of maternal species and one genome of the paternal the crucian carp and one of the grass carp. These species. Fishes belonging to the genus Carassius data confirm that the tendency to resemble the seem to produce offspring by a specific fertiliza- 1006 Kasama and Kobayasi

tion mechanism in intergeneric or interspecific Univ. J., 34, 107-112 (1987). crosses. Further study of this is needed . 13) K. Matsubara: Fish Morphology and Hier archy, 1st ed., Ishizaki-shoten press, Tokyo, 1955, References pp. 60-69. (in Japanese). 14) C. L. Hubbs and K. F. Lagler: Fishes of the 1) R. Suzuki: Nippon Suisan Gakkaishi , 28, 992- Great Lake Region (Ann. Arbor), Univ. 997 (1962). Michigan Press, 1970, pp. 19-26. 2) R. Suzuki: Jap. J. Genet., 38, 89-96 (1963). 15) C. L. Hubbs and K. Kuronuma: Pap . Mich. 3) R. Suzuki: Nippon Suisan Gakkaishi , 29, 421- Acad. Sci., Arts and Lett. 27, 267-306 (1942). 423 (1963). 16) C. L. Hubbs, L. C. Hubbs, and R . E. Johnson: 4) R. Suzuki: Nippon Suisan Gakkaishi , 29, 655- Contr. Lab. Vert. Biol. Univ. Mich., 22, 1-7 657 (1963). (1943). 5) R. Suzuki: Annot. Zool. Jap ., 36, 203-207 17) Y. T. Chu: Biol. Bull. St . John's Univ., Shang (1963). hai, 2, 1-225 (1935). 6) R. Suzuki: Jap. J. Ichthyol ., 10, 39-42 (1963). 18) G. F. Weisel: J. Morphol ., 96, 333-358 (1955). 7) R. Suzuki: Jap. J. Ichthyol., 12, 18-22 (1964). 19) H. Kobayasi, Y. Kawashima , and N. Takeuchi: 8) R. Suzuki: Jap. J. Ichthyol., 13, 64-68 (1965). Jap. J. Ichthyol., 17, 153-159 (1970) . (in Japanese). 9) R. Suzuki: Bull. Freshwater Fish . Res. Lab., 14, 20) Y. Ojima, M. Hayashi, and K . Ueno: Jap. J. 91-95 (1965). Genet., 47, 431-440 (1972). 10) R. Suzuki: Bull. Freshwater Fish. Res. Lab ., 21) H. Kobayasi and K . Nakano: Jap. Women's 16, 65-69 (1966). Univ. J., 23, 113-122 (1976). (in Japanese) . 11) R. Suzuki: Bull. Freshwater Fish. Res. Lab., 22) H. Kobayasi and M. Hashida: Jap . Women's 18, 113-155 (1968). Univ. J., 24, 121-133 (1977) . (in Japanese). 12) M. Kasama and H. Kobayasi: Jap . Women's

Nippon Suisan Gakkaishi: Formerly Bull . Japan. Soc. Sci. Fish .