Japanese Journal of Ichthyology 魚 類学 雜誌 Vol. 39, No. 3 1992 39巻3号1992年

Fertility of Triploid Backcross Progeny, (Gengoroubuna Carassius auratus

cuvieri •Š •~ Carp Cyprinus carpio •‰)F1 •Š •~ Carp or Gengoroubuna •‰

Feng Zhang,1'2 Takashi Oshiro1 and Fumio Takashima1

Department of Aquatic Biosciences, Tokyo University1 of Fisheries , 4-5-7 Konan, Minato-ku, Tokyo 108, Japan 2Yangtze Ri ver Fisheries Research Institute, The Chinese Academy of Fishery Sciences, Shashi, Hubei Province, China

Abstract The fertility of triploid backcross progeny , (gengoroubuna Carassius auratus cuvieri •Š•~carp Cyprinus carpio •‰)F1 •Š•~gengoroubuna or carp •‰ is reported . In females, spawning could be induced by the injection of pituitary extracts. The fertilization rates were from 52 .4 to 88.6%, and free swimming rates from 2.6 to 34.4%. Many normal larvae were obtained after crossing with the sperm of gengorou - buna. However, after crossing with sperm of the loach Misgurnus anguillicaudatus , most of the embryos died before hatching and no normal larvae were obtained . Sperm could not be stripped from the progeny of either type of triploid backcross after the injection of pituitary extracts. Under light microscope, each seminal lobule in the testis was mainly occupied by

spermatocytes in various stages. Some spermatids were found in part of the lobules, but no spermatozoa were observed. These facts suggest that the laboratory-reared , triploid backcross progeny may represent an interme- diate step in the evolution of a gynogenetic triploid form.

Several teleost species produce all-female offspring

by gynogenesis (Hubbs and Hubbs, 1932; Cherfas, Materials and Methods 1966; Schultz, 1967; Kobayasi, 1971; Cimino, 1972; Echelle et al., 1988; Fan and Shen, 1990; Dawley et Experimental fish. Common carp, Cyprinus al., 1987). Most of them may have originated from carpio, and goldfish, Carassius auratus auratus, from hybrids of related species (Schultz, 1967, 1971; Ab- Yoshida Research and Training Station, Tokyo Uni- ramoff et al., 1968). versity of Fisheries and gengoroubuna, C. auratus Gynogenetic triploid ginbuna, Carassius auratus cuvieri, from the Fisheries Experimental Station, Sai- langsdorfii, in Japan are also considered to have tama Prefecture were used in this study. Loach, originated from hybrids of different species or sub- Misgurnus anguillicaudatus, were purchased from species (Sekiya and Honma, 1973, 1975; Shimizu et the fish market in Tokyo. Hybrids (F1) between al., 1991.). Ojima et al. (1975) reported that triploid female gengoroubuna and male carp were produced larvae, all females and morphologically similar to in 1985. In 1988, fresh egg of a F1 female were

ginbuna, were produced by backcrossing of (funa fertilized with fresh sperm of gengoroubuna or carp, carp •‰)F1 •Š•~carp •‰. Furthermore, Cherfas•Š•~ and and two types of triploid backcross progeny were Ilyasova (1980) reported that the backcross of produced. These were reared in indoor tanks until

(silver crucian carp •Š•~carp •‰)F1 •Š•~carp •‰ res- maturation and used for this study. ulted in triploids. In these studies, however, the Ploidy determination and sex distinction. The fertility of triploid backcross progeny of crucian carp ploidy levels were determined by DNA content and and carp was not reported. In the present study, we chromosome number of somatic cells. DNA content examined the fertility of triploid backcross progeny of red blood cells (RBC) was measured by flow of gengoroubuna, Carassius auratus cuvieri, and carp cytometry after propidium iodide (PI)-staining to clarify the evolutionary history of gynogenetic (Allen, 1983). Chromosome preparations were made triploid ginbuna. from leucocyte cultures (Ojima et al., 1970).

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Fig. I. Mitotic metaphase chromosomes from backcross progeny (gengoroubuna •Š•~carp •‰) F1 •Š•~

gengoroubuna•‰. Fig. 2. Eggs from backcross progeny (gengoroubuna •Š•~carp •‰) F1 •Š•~gengoroubuna •‰. Small size eggs (se)

and large size eggs (le) were observed. Bar indicates 1mm.

Fig. 3. Cytological observation of testis in triploid backcross progeny (gengoroubuna •Š•~carp •‰)F1

gengoroubuna •‰. The primary spermatocyte (psc), secondary spermatocyte•Š•~ (ssc), and spermatid (spt) are delimited. Bar indicates 30ƒÊm.

Fig. 4. Cytological observation of testis in a diploid goldfish. The sperm (spin) and secondary spermatocyte

(ssc) are shown. Scale bar indicates 30ƒÊm.

The sex of backcross progeny was determined by testes of loach were each diluted to four times their observation of secondary sexual characters, such as original volume with Ringer's solution. Eggs were the pearl organ or genital pore, and by inspecting ova divided into two groups and inseminated with dil- in the ovary by means of cannulation. uted sperm of gengoroubuna and loach, respectively. Fertility check. Females. In 1991, eggs could Fertilization rate, eyed egg rate, hatching rate and be stripped from some of the female triploid back- free swimming (A free swimming eleutheroembryo cross progeny after intraperitoneal injection of pitu- is defined as an individual that has developed beyond itary extracts from the silver carp, Hypophthalmicht- the period of attachment to the substrate) rate were hys molitrix. Sperm of gengoroubuna and squashed examined in each group (50-600 eggs) used in the

•\ 230•\ Zhang et al.: Fertility of Cyprinid Triploids

crossing experiments. 6.5 (n=6) and 143.0•}1.2 (n=6), respectively.

Males. The bellies of 20 male triploid backcross The sex ratio is shown in Table 1. In the case of the backcross (gengoroubuna •Š•~carp •‰ ) F1 •Š•~carp •‰ progeny were squeezed whether or not sperm could be stripped out. For histological observations, testes , 3 females and 6 males resulted. On the other were removed and fixed with Bouin's solution for 12 hand, the backcross (gengoroubuna •Š•~carp •‰)F1

hrs. After dehydration with an ascending ethanol gengoroubuna •‰ •Š•~resulted in 11 females and 26

series and finally with toluene, the testes tissues were males.

embedded in paraplast. Sections 7m in thickness Fertility of female backcross progeny. Spawning

were stained with Mayer's haematoxylin and eosin. was induced in 4 out of 11 progeny from the back-

Testes of diploid goldfish were also investigated for cross (gengoroubuna •Š•~carp •‰) F1 •Š•~gengoro-

comparison. ubuna •‰ after injection of pituitary gland extracts.

Backcross progeny. The ploidy levels of the off- Two egg sizes were distinguished, being about 1.25

spring of backcross progeny were also determined by mm and 1.10mm in diameter (Fig. 2). As shown in

flow cytometry, as mentioned above. Table 2, after insemination with sperm of gengorou- buna, normal fry were obtained. When two back-

cross progeny (nos. 1 and 2) were inseminated with Results

Ploidy level and sex ratio of backcross progeny. Table 1. Sex ratios of backcross progeny Chromosome spreads of backcross progeny are

shown in Fig. 1, the chromosome number being 150.

When the DNA content of diploid goldfish measured

by flow cytometry was adjusted to a channel number

of 100, the relative values of the triploid backcross *F1 was a gengoroubuna •Š•~carp•‰ cross . progeny (carp and gengoroubuna •‰) were 136.3•}

Table 2. Crossing experiments with females of backcross progeny [(gengoroubuna •Š•~carp •‰) F1 •Š•~

gengoroubuna •‰]

Table 3. Crossing experiments of females with backcross progeny [(gengoroubuna •Š•~carp •‰)F1 •Š•~carp •‰]

•\ 231•\ 魚類学雑誌 Japan. J. Ichthyol. 39(3),1992 sperm of the loach, some free swimming, actively backcross progeny are shown in Table 4. When the feeding larvae were obtained, but all of them showed DNA content of diploid carp measured by flow cyto- extreme deformities and died within 3 months after metry was adjusted to a channel number of 200, the hatching. At no time were normal fry obtained from relative DNA contents of 4 individuals (nos. 1-4) crossing with loach sperm. showed more than 350, the others showing about The results of the crossing experiments using back- 230. It is suggested that the first 4 individuals were cross progeny [(gengoroubuna •Š•~carp•‰)F1•Š•~ tetraploids, and the other 2, hyperdiploids (approxi- carp•‰] are shown in Table 3. Two out of 3 individ- mately 2.5n). uals spawned after the injection of pituitary gland extracts. In a cross with gengoroubuna •‰, a fertiliza- Discussion tion rate of 77.9-88.6% and free swimming rate of

29.7-34.4% were achieved. Although more than half Hubbs (1955) gave evidence that Poecilia formosa, of the embryos died before hatching, a large number a well-known gynogenetic, all-female species, was a of normal cross progeny were obtained. On the other hybrid, by crossing P. sphenops with P. latipinna. hand, the hatching rate using loach sperm was lower Reciprocal crosses of the latter species under labora- than that with gengoroubuna sperm. All of the larvae tory conditions produced bisexual hybrids, which in showed extreme deformities and died in early stages turn produced intermediate offspring following back- before free swimming and feeding. crossing. Fertility of male backcross progeny. The males Schultz (1969) reported a hybrid complex from the of both types of triploid backcross progeny had many Rio Fuerte of northwestern Mexico. The complex pearl organs on the pectoral fins, but did not produce consisted of two bisexual species, Poeciliopsis mona- sperm on stripping after the injection of pituitary cha and P. lucida, and 3 unisexual derivatives, 2 of gland extracts. However, it was found that the testes which were triploid (P. 2 monacha-lucida and P. had developed to a certain extent and that the monacha-2lucida) and the other, diploid (P. mona- gonado-somatic index (GSI) attained 1.39•}0.71% cha-lucida). The karyotype of the unisexual diploid

(n=20) in the breeding season. Under light micros- approximated that of the hybrid of P. monacha and copy, each seminal lobule of the testes was occupied P. lucida. During oogenesis the paternally derived P. mainly by primary spermatocytes, secondary sper- lucida chromosomes were lost, the P. monacha set matocytes and spermatids, but no spermatozoom being transmitted to the egg. Since the all-female were observed (Fig. 3). On the other hand, the form mated mainly with males of P. lucida, the seminal lobules of the testes of diploid goldfish were hybrid nature of the fish was maintained. Schultz occupied by spermatozoa (Fig. 4). coined the term "hybridogenesis" for this mode of Ploidy level of the offspring of backcross progeny. reproduction. Subsequently, Schultz (1973) bred The relative DNA content of 6 offspring of triploid unisexual hybrids of P. monacha and P. lucida in the laboratory, these being all-female diploids that were

Table 4. Relative DNA contents of the offspring of indistinguishable from wild P. monacha-lucida. triploid backcross progeny Cherfas (1980) obtained triploid hybrids by back- crossing carp and diploid silver crucian carp, Caras- sius auratus gibelio, and suggested that the evolution- ary development of gynogenetic, triploid populations of the latter species might have originated from hybridization, as in Poeciliopsis. In the present study, we also obtained triploid, backcross progeny of gengoroubuna and carp, which were bisexual. The females were fertile, but the males could not produce sperm. Populations of triploid silver crucian carp, Caras- sius auratus gibelio, in northeast China include only 5-25% males (Shen et al., 1983), which cannot *1F , was a gengoroubuna carp cross.*2 produce functional sperm (Jiang et al. 1982). Like- Funa was a gengoroubuna. wise, males constitute about 40% of a family of

•\ 232•\ Zhang et al.: Fertility of Cyprinid Triploids gynogenetic, triploid ginbuna, kept in our laborato- Yang and S.E. Lin. 1982. Artificial and natural gynogen- ry, but nevertheless cannot produce sperm (un- esis in crucian carp. Acta Hybrobiol. Sinica, 7: 471-477. Kobayasi, H. 1971. A cytological study on gynogenesis of published data). It is very interesting that triploid the triploid ginbuna (Carassius auratus langsdorfii). backcross females are fertile, while the males are not, Zool. Mag., 80: 316-322. similar properties to those of the triploid ginbuna Ojima, Y., M. Hayashi and K. Ueno, 1975. Triploidy population mentioned above. appeared in the backcross offspring from funa-carp cross- On the other hand, the offspring of backcross ing. Proc. Japan Acad., 51: 702-706. progeny might be composed of tetraploid and hyper- Ojima, Y., S. Hitotsumachi and M. Hayashi, 1970. A blood diploid (approximately 2.5n) individuals. Triplaid culture method for fish chromosomes. Japan. J. Genet. backcross females may produce two types of ova; 3n 45, 161-162. ova, which combine with 1n sperm to make 4n Schultz, R.J. 1967. Gynogenesis and triploidy in the vivi- larvae, and 1.5n ova, which combine with 1n sperm parous fish Poeciliopsis. Science, 157: 1564-1567. to make 2.5n larvae. Apparently, the triploid back- Schultz, R.J. 1969. Hybridization, unisexuality, and poly- cross progeny did not reproduce by gynogenesis, ploidy in the teleost Poecliopsis (Poeciliidae) and other vertebrates. Am. Nat., 103(934): 605-619. because perfect fertilization occurred. However, Schultz, R.J. 1971. Special adaptive problems associated some triploid backcross progeny produced 3n ova, with unisexual fishes. Am. Zool., 11: 351-360. indicating that meiotic division did not occur during Schultz, R.J. 1973. Unisexual fish: Laboratory synthesis of oogenesis as in natural triploid ginbuna. It is possible a "species." Science, 179: 180-181. that the triploid backcross progeny may represent an Sekiya, S. and Y. Honma. 1973. Racial variation and local intermediate step in the evolution of a gynogenetic distribution of the funa (genus Carassius) in Niigata and triploid form. Sado Island (a preliminary report). Proc. Japan. Soc. Syst. Zool., 9: 63-70. Sekiya, S. and Y. Honma. 1975. Morphological and cyto- Literature Cited logical studies on the ironfish occurring in Niigata and Sado Island. Proc. Japan. Soc. Syst. Zool., 11: 53-60. Abramoff, P., R.M. Darnell and J.S. Balsano. 1968. Electrophoreticdemonstration of the hybridorigin of the Shen, J.B., G.R. Wang and Z.T. Fan. 1983. The ploidy of crucian carp and its geographical distribution in Heilong- gynogeneticteleost Poecilia formosa. Am. Nat., 102:555- 558. jiang main river. J. Fish. China, 7 (2): 87-94. Allen,S.K. 1983.Flow cytometry:Assaying Experimental Shimizu, Y., T. Oshiro, F. Takashima and M. Sakaizumi. 1991. Origin of the triploid "ginbuna" Carassius auratus polyploidfish and shellfish.Aquacul., 33: 317-328. Cherfas, N.B. 1966. Natural triploid in female of the langsdorfii. Zool. Sci., 7 (6): 1056. unisexualform of the goldfishCarassius auratus gibelio (Received March 13, 1992; accepted May 11, 1992) Bloch. Genetika, 12: 16-24. Cimino, M.C. 1972. Meiosis in triploid all-female fish (Poeciliopsis,Poeciliidae). Science, 175: 1484-1486. フ ナ ・コ イ3倍 体 雑 種 の 生 殖 能 力 Dawley, R.M., R.J. Schultz and K.A. Goddard. 1987. 張 峰 ・尾 城 隆 ・隆 島 史 夫

Clonal reproductionand polyploidyin unisexualhybrids 天 然3倍 体 雌 性 発 生 ギ ン ブ ナの 雑 種 起 源説 を検 討 す る ため,3 of Phoxinuseos and Phoxinusneogaeus (Pisces; Cyprini- 倍 体 交 雑 種(ゲ ンゴ ロ ウ ブ ナ ♀ × コ イ♂)F1♀ ×ゲ ンゴ ロ ウ ブ ナ dae). Copeia, 1987:275-283. ♂ ま た は コイ ♂ の 生 殖 能 力 につ い て検 討 した.雌 は 脳 下 垂 体 ホ Echelle, A.A., A.F. Echelle, L.E. Debault and D.W. ル モ ン注 射 に よ って 人 工 採 卵 で き,得 られ た卵 を ゲ ン ゴ ロ ウ ブ Durham. 1988.Ploidy levelsin silversidefishes (Atherin- ナ ま た は ドジ ョ ウの精 子 で再 交 雑 した.そ の結 果,ゲ ンゴ ロ ウ ブ idae,Menidia) on the Texascoast: Flow-cytometricanal- ナ の精 子 で媒 精 した場 合 の受 精 率 は52.4-88.6%,自 由遊 泳 率 は ysis of the occurrence of allotriploidy.J. Fish Biol., 32: 2.6-34.4%に 達 し,正 常 な稚 魚 が多 数 得 られ た.一 方,ド ジ ョウ 835-844. 精 子 で 媒 精 した 場 合,孵 化 率 は3-15%に 達 した もの の,ほ とん Fan, Z.T. and J.B. Shen. 1990.Studies on the evolutionof ど の 個 体 は奇 形 の ま ま死 亡 し,正 常 稚 魚 は得 られ な か った.雄 は,ホ ル モ ン注 射 を行 な って も採 精 で きな か った.精 巣 の 光 顕観 bisexualreproduction in crucian carp (Carassiusauratus 察 の 結 果,精 小 葉 に 精 母 細 胞 と少 数 の 精 細 胞 が 観 察 さ れ た もの gibelio Bloch). Aquacul., 84: 235-244. の,精 子 は認 め られ な か った.こ れ らの結 果 か ら,こ こで 作 った Hubbs, C.L. 1955.Hybridization between fish species in 人 為3倍 体 交 雑 種 は 自然 雌 性 発 生 集 団 へ 進 化 す る 中 間 ス テ ッ プ nature. Syst. Zool., 4: 1-20. に相 当 す る も の と考 え られ た. Hubbs, C.L. and L.C. Hubbs. 1932.Apparent partheno- genesis in nature, in a form of fish of hybrid origin. (張 ・尾 城 ・隆 島:108東 京 都 港 区 港 南4-5-7東 京 水 産 Science,76: 628-630. 大 学 資 源 育 成 学 科;張:中 国 湖 北 省 沙 市 市 中 国 水 産 科 学 Jiang, Y.G., H.X. Yu, B.D. Chen, S.C. Liang, D.L. 研 究 院 長 江 水 産 研 究 所)

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