Japan. J. Ichthyol. 魚 類 学 雑 誌 41 (3): 338-342, 19 94 41 (3): 338-342, 1 9 94

mation pertaining to this subject was available. Embryonic Growth during Gestation of the n the present study, the changes inI total length, Viviparous Eelpout, Zoarces elongatus body weight and dry weight were investigated as

criteria for growth during gestation in Z. elongatus. Yasunori Koya,1 Toshitaka Ikeuchi,2 In addition, tracer experiments on the mechanism Takahiro Matsubara,1 Shinji Adachi2 and site of nutrient absorption by the embryo were and Kohei Yamauchi2 performed. Hokkaido National Fisheries1 Research Institute, 116 Katsurakoi, Kushiro, Hokkaido 085, Japan Materials and Methods 2Department of Biology, Faculty of Fisheries, Female Zoarces elongatus were caught by angling Hokkaido University,3-1-1 Minato-cho, Hakodate, Hokkaido041, Japan in Akkeshi Bay, eastern Hokkaido, Japan, during May to October 1992. Thirty-four fish were subse-

(ReceivedJuly 20, 1994;in revisedform September13, 1994; quently transferred to Usujiri Fisheries Laboratories, acceptedOctober 14, 1994) Hokkaido University, and kept in an indoor 1000

liter circular tank with flowing sea water under nat-

ural photoperiod conditions. At monthly intervals

The viviparous mode of reproduction in teleosts during the gestation period (September to February, was categorized as lecithotrophy and matrotrophy Koya et al., 1993), four to ten females were anesthe- on the basis of maternal-fetal trophic relationships tized with ethyl 4-aminobenzoate and the ovaries (Wourms, 1981). Lecithotrophic embryos derive removed. Embryos were removed from the ovaries their nutrition solely from yolk reserves, whereas and the total lengths and wet weights measured, matrotrophic embryos depend on a supply of mater- before being dried for 48 hr at 80•Ž. The dry weight nal nutrients during gestation (Wourms, 1981, was then determined. 1991). Designation of a particular species as lecitho- An in vitro tracer experiment was carried out in trophic or matrotrophic has been based on a compar- order to determine the site of embryonic nutrient ison between the dry weight or total organic weight absorption. Isolated from the ovaries, embryos were

of the egg and that of the full-term embryo (Wourms placed into L-15 medium (pH7.5, Sigma Chemical et al., 1988). Co.) containing 10mM HEPES, 100mg/l strepto- Within the Zoarcidae, viviparity appears to have mycin sulfate and 75mg/l penicillin G potassium. evolved only in three species of the genus Zoarces Bovine serum albumin (BSA, fraction V, Sigma Che- (Nelson, 1993). Heretofore, almost all the informa- mical Co.) at a concentration of 1% w/v was used as tion available on zoi.rcid viviparity has been derived a tracer. Embryos were incubated for 8-12 hr at from studies of the European species, Z. viviparus 12•Ž and then fixed with Bouin's solution for 12 hr (e.g. Korsgaard, 1986), with little being known at 4•Ž. After washing three times with 0.1M about the other species. Z. elongatus is a viviparous sodium phosphate buffer (PBS, pH 7.4) containing teleost, which inhabits the Pacific coast of northern 10% sucrose for 6 hr at 4•Ž, they were embedded in

Japan. Embryos of Z. elongatus are retained in the paraffin (m.p. 52-54•Ž) and sectioned at 5 gm thick- ovarian cavity for more than five months after fertil- ness. Immunohistochemistry procedures were car- ization (Koya et al., 1993). Ripe eggs of Z. elong- ried out using the second antibody procedure and the

atus are very large, about 4.4 mm in diameter (Koya peroxidase-antiperoxidase complex (PAP) technique et al., 1993), indicating that the embryos have access of Sternberger et al. (1970). Deparaffinized sections to abundant yolk reserves during early development. were treated with 1% H202 in methanol to inhibit In addition, the embryos of this species grow from endogenous peroxidase activity and then incubated 29.1mm at hatching to 61.0mm by late gestation in 10% normal goat serum to avoid non-specific (Koya et al., 1993). Hence, it is thought that the binding of antibodies. After rinsing with distilled embryos received some maternal nutrients after yolk water followed by PBS, the sections were subjected resorption. In order to determine whether Z. elong- to a solution of rabbit anti-BSA antiserum (Wako atus is lecithotrophic or matrotrophic, it is necessary Pure Chemical Industries, LTD.) diluted 1:5000. to examine embryonic growth, especially the change After washing with PBS, a solution of goat anti- in dry weight during gestation. However, no infor- rabbit IgG antiserum (Wako Pure Chemical Indust-

― 3 3 8 ― Embryonic Growth of Viviparous Eelpout

A

B

Fig. 1. Growth of embryo in Zoarces elongatus during gestation . A) Changes in total length (mean•}SE). Inset photographs show yolk-sac embryo in October (a) and juvenile in February (b); B) changes in body weight (•›) and dry weight (•œ) (mean•}SE). ries, LTD.) was applied. The sections were then Results incubated with PAP (Jackson Immuno Research Laboratories, Inc.) and any end product on the Embryo growth. -Figure 1 shows the changes in total length (TL), wet weight and dry weight of sections indicative of BSA localization made visible with 3,3'-diaminobenzidine. The specificity of im- embryos during gestation. The ripe eggs of Zoarces munohistochemical staining was examined in the elongatus were 4.4mm in diameter, their wet and dry weights being 45mg and 7mg, respectively. Fertili- control sections which were not subjected to rabbit anti-BSA antiserum. For routine histological obser- zation occurred in September after which the em- bryos developed before hatching in October. Within vations, sections were stained with haematoxylin and eosin (H-E). one month after hatching (Fig. 1A, inset a), the

― 3 3 9 ― Y. Koya et al.

Fig. 2. Photomicrographs of the sections of Zoarces elongatus embryos in October. H-hind-gut; M-mid-gut; n-nucleus. A) Sagittal section. Haematoxylin-eosin (H-E) staining. Scale bar=1mm; B) sagittal section. Immunostaining of the PAP method using anti serum against BSA. Reaction products were detected in the mid- and hind-gut. Scale bar=1mm; C) H-E staining section of the mid-gut epithelium. Scale bar=10ƒÊm ; D) H-E staining section of the hind-gut epithelium. Scale bar=10ƒÊm; E) immunostaining section of the mid-gut epithelium. Scale bar=10ƒÊm; F) immunostaining section of the hind-gut epithelium. Scale bar=10ƒÊm. embryos were 39 mm TL, 170mg and 20mg in wet chemical observations of BSA uptake was performed and dry weights, respectively, and had a yolk-sac. on the sagittal sections that extended from the head Embryos completed yolk absorption in late Novem- to the anus. Figure 2A shows a photomicrograph of ber, having attained 50mm TL, and 300mg and 35 a H-E stained section of a yolk-sac embryo, 39mm in mg in wet and dry weights, respectively. In Febru- total length, in October. The abdominal cavity of the ary, the embryos grew further (Fig. 1A, inset b), embryo was occupied by a well-developed intestine, reaching 57 mm TL, and 540 mg in wet weight (65 recognizable as two parts owing to their morpho- mg dry weight). logical differences. The front part of the intestine Absorption of external protein. Immunohisto- (mid-gut) was a simple winding tube, whereas the

―3 4 0 ― Embryonic Growth of Viviparous Eelpout

posterior part (hind-gut) was thickened having nu- as "trophotaeniae" or "follicular pseudoplacenta," in merous, intricately-arrayed folds (Fig. 2A). Positive the embryos of viviparous teleosts are known (see immune localization of BSA was detected in both the Wourms, 1981). In Z. viviparus, Kristoffersson et al. mid- and hind-guts (Fig. 2B). In the mid-gut, the (1973) suggested that an enlarged hind-gut with a cytoplasm of the epithelial cell was stainable by H-E, hypertrophied intestinal epithelium was the main site with the nucleus being localized in the center of the of nutrient absorption. The present study indicated cell (Fig. 2C). On the other hand, the epithelial cells that the embryos of Z. elongatus absorb maternal of the hind-gut were occupied by a vacuole-like nutrients both from the simple mid-gut as well as structure which was not stained H-E, and had the from the enlarged hind-gut. Based on apparent nucleus localized in the basal part of the cell (Fig. 2 differences in the absorptive area, the hind-gut sur- D). Positive immuno-reaction in the mid-gut occur- passing the mid-gut, it was concluded that the hind- red in the cytoplasm of most of the epithelial cells gut is the main site of nutrient absorption in Z. (Fig. 2E), although that in the hind-gut epithelium elongatus. The difference in the morphology of mid- was restricted to the cortical and thin lateral parts of and hid-gut epithelial cells may reflect differences in the cytoplasm (Fig. 2F). In the control sections, no function. positive reactions were observed. Similar results were obtained from embryos that had completed yolk absorption by late November. Acknowledgments The authors wish to thank the staff of Usujiri Discussion Fisheries Laboratory, Faculty of Fisheries, Hok- kaido University for rearing the specimens. The present study demonstrated that embryos of Zoarces elongatus increased from 7 mg to 65 mg in dry weight during gestation. Such an increase indi- Literature Cited cates that Z. elongatus is a matrotrophic species in Bretschneider,L.H. and J.J.D. DeWit. 1947. Sexual which embryos depend on a continual supply of endocrinologyof non-mammalianvertebrates. Elsevier, maternal nutrients. Various grades of maternal de- Amsterdam. 146 pp. pendence occur in matrotrophy, involving both the Korsgaard, B. 1986. Trophic adaptations during early initial yolk reserves as well as the amount of nutri- intraovarian developmentof embryos of Zoarces vivi- ents provided during gestation. For example, yolk parus. J. Exp. Mar. Biol. Ecol., 98: 141-152. reserves are lacking in the eggs of Embiotoca lateralis Koya, Y., S. Ohara, T. Ikeuchi, S. Adachi, T. Matsubara (Wourms, 1981), whereas abundant yolk reserves and K. Yamauchi. 1993. The reproductive cycle of occur in the eggs of Zoarces viviparus (Bretschneider female Zoarces elongatus, a viviparous teleost. Bull. and DeWit, 1947; Korsgaard, 1986). Z. elongatus HokkaidoNatl. Fish. Res. Inst., (57): 9-20. (In Japanes forms very large eggs (4.4 mm in diameter) which with Englishabstract.) Kristoffersson,R., S. Broberg and M. Pekkarinen. 1973. have abundant reserves of yolk (Koya et al., 1993). Histology and physiologyof embryotrophe formation, Thus, they are similar to Z. viviparus in terms of their embryonicnutrition and growth in the eel-pout,Zoarces initial provision of nutrients. viviparus(L.). Ann. Zool. Fennici., 10: 467-477. In Z. elongatus, the dry weight increased from 7 Nelson, J.S. 1993. Fishesof the world, 3rd ed. John Wiley mg ripe eggs to 20mg for embryos that still had a and Sons, New York. 614 pp. yolk-sac one month after hatching. The results of Sternberger,L.A., P.H. Hardy, Jr., J.J. Cuculisand H.G. tracer experiments demonstrated that the intestine of Meyer. 1970. The unlabeled antibody-enzymemethod of embryos having a yolk-sac had the ability to absorb immunohistochemistry.Preparation and properties of protein. The embryos of Z. elongatus thus begin to soluble antigen-antibody complex (horseradish perox- receive maternal nutrients from their intestine idase-antihorseradishperoxidase) and its use in iden- during the period of yolk absorption. Therefore, the tification of spirochetes. J. Histochem.Cytochem., 18: 315-333. embryos of this species utilize their own yolk and Wourms, J.P. 1981. Viviparity:The maternal-fetalrela- maternal nutrients during early gestation, thereafter tionshipin fishes. Am. Zool., 21: 473-515. depending solely on additional maternal nutrients for Wourms, J.P. 1991. Reproduction and developmentof more than three months. Sebastes in the context of the evolution of piscine vivi- Several types of external trophic adaptations, such parity. Env. Biol. Fish., 30: 111-126.

•\3 4 1 •\ Y. Koya et al.

Wourms, J.P., B.D. Grove and J. Lombardi. 1988. The は孵 化 して い た.孵 化 後1か 月 以 内 の 卵 黄 嚢 を 持 つ 胎 仔 は体 長 39mmで,乾 燥 重 量 は20mgに 増 加 して い た.そ の後 の妊 娠 期 を maternal-embryonic relationship in viviparous fishes. 通 じて 体 長,湿 重 量,お よ び乾 燥 重 量 と も直 線 的 に増 加 し,2月 Pages 1-134 in W.S. Hoar and D.J. Randall, eds. Fish に は体 長57mm,乾 燥 重 量65mgに 達 した.こ の よ うな乾 燥 重 量 physiology, 11B. Academic Press, San Diego. の増 加 は,本 種 が母 体 栄 養 依 存 型 の胎 生 魚 で あ る こ とを 示 して い る.ま た,孵 化 後 間 もな い胎 仔 の 乾 燥 重 量 が 未受 精 卵 の そ れ よ り も大 き か った こ と は,こ の 時 期 か らす で に 胎 仔 が 母 体 由来 の 胎 生 魚 ナ ガ ガ ジ の 妊 娠 期 に お け る 胎 仔 の 成 長 栄 養 を 摂 取 し て い る こ と を 示 唆 して い る.牛 血 清 ア ル ブ ミ ン (MW67,000)を トレー サ ー に用 い て胎 仔 の栄 養 吸 収 能 を 調 べ た 古 屋 康 則 ・池 内 俊 貴 ・松 原 孝 博 ・足 立 伸 次 ・山 内 晧 平 結 果,卵 黄 嚢 を持 つ 時 期 に は既 に腸 で 外 因 性 の 栄 養 を吸 収 で き る こ とが 示 され た.従 って,本 種 の胎 仔 は孵 化 後 自身 の 卵黄 嚢 を 吸 収 しつ つ 母 体 か ら も何 らか の栄 養 を 受 け,卵 黄 嚢 吸 収 後 は母 胎 生 魚 の ナ ガ ガ ジ(Zoarces elongatus)が,卵 黄 栄 養 依 存(lecit- 体 か らの栄 養 供 給 の み に依 存 して成 長 す る もの と思 わ れ る. hotrophy)お よ び母 体 栄 養 依 存(matrotrophy)の い ず れ の 胎生 型 に属 す る の か を 明 らか に す る た め に,1992年9月 か ら2月 ま で の 妊 娠 期 中 の 胎 仔 の 体 長,湿 重 量,お よ び乾 燥 重 量 の 変 化 を 調 べ (古 屋 ・松 原:〒085釧 路 市 桂 恋116北 海 道 区 水 産 研 究 た.本 種 の未 受 精 卵 は 直径4.4mm,乾 燥 重 量7mgで,胎 生 魚 の 所 資 源 管 理 部;池 内 ・足 立 ・山 内:〒041函 館 市 港 町3- 卵 と して は極 め て 大 きか っ た.9月 に受 精 した卵 は10月 下 旬 に 1-1北 海 道 大 学 水 産 学 部 淡 水 増 殖 学 講 座)

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