Fisheries Science 61(2), 203-208 (1995)

Annual Reproductive Cycle and Spawning Characteristics of the Female Kichiji Rockfish Sebastolobus macrochir

Yasunori Koya, Tomonori Hamatsu, and Takahiro Matsubara

Hokkaido National Fisheries Research Institute, Fisheries Agency of Japan , Katsurakoi, Kushiro, Hokkaido 085, Japan

(Received June 24, 1994)

The ovarian morphology, annual reproductive cycle and spawning characteristics of the kichiji rockfish Sebastolobus macrochir collected from the Pacific coast of southeastern Hokkaido , was examined on the basis of histological observation. The ovaries of this fish are categorized as cystovarian

type. The ovarian parenchyma, stroma, and blood vessels run longitudinally through the center of each ovary, suggesting a specialized structure for formation of gelatinous egg masses.

The female kichiji rockfish begins vitellogenesis from August to October , and continues yolk accumulation until March. Post-ovulatory follicles or oocytes at final maturation stage were observed in March and April, suggesting that spawning was carried out mainly in this period. During the spawning season, there were two clutches of developing oocyte, or post-ovulatory follicle and one clutch of oocyte in the ovary. Therefore, females seem to spawn twice within a single spawning season. Fish which finished spawning rapidly form yolk vesicles for the next reproduction, and maintain a resting condition for several months.

Gonad somatic index (GSI) corresponds clearly to ovarian maturity, and could be divided into immature (GSI < 2), spent (2 < GSI < 3), and vitellogenic or maturation (GSI > 3) during the spawning season. This indicates that ovarian maturity can be evaluated on the basis of GSI. By this method, the minimum maturation size of the female kichiji rockfish was estimated to be 151 to 175 mm in standard length.

Key words: rockfish, reproductive cycle, oogenesis, ovary, maturity, spawning

The kichiji rockfish Sebastolobus macrochir is distributed in the Pacific off northern Japan south to Suruga Bay, Okhotsk Sea, and Bering Sea, and is an important commercial fish caught by trawl net or bottom gill net on the Pacific side of northern Japan. The catch quantity of kichiji rockfish has decreased remarkably in recent years, from 1292 tons in 1983 to 274 tons in 1992. Appropriate resource management of this fish is needed to restore previous catch levels.

For managing kichiji rockfish resources, clarification of the reproductive biology, particularly the process of gonadal development and spawning characteristics of this species is urgently needed. The spawning season of the kichiji rockfish has been estimated by the pelagic egg balloons of this species sampled in the field,1) by exterior observation of oocytes and monthly changes in the gonad somatic index,2) and by the process of male testicular development.3) However, there have been no studies which clarify correctly the process of ovarian development based on histological observations.

In the present study, four phases of ovarian maturity of the kichiji rockfish based on histological observation were defined, and the annual reproductive cycle, features of ovarian development, and number of spawning times were clarified. Furthermore, the mechanism of formation of the

gelatinous egg masses spawned by this species is also discussed in terms of the ovarian structure.

Materials and Methods

Fish

The kichiji rockfish used in the present study were caught by bottom trawl net on the Pacific coast of eastern Hokkaido between December 1992 and October 1993. A total of 364 females were measured for standard length (length between the tip of the upper jaw and base of the caudal fin), body weight (BW) and gonadal weight (GW). The gonad somatic index (GSI) was calculated by the expression GSI = 100 x GW / BW.

Histology

For histological observation, the ovaries of 60 females

were removed, fixed in Bouin's solution, embedded in

paraffin, sectioned at 5ƒÊm, and stained with Derafield's

haematoxylin and eosin. To identify yolk vesicles, some of

the sections were treated with periodic acid Schiff (PAS)

reagent.

Measurement of Oocyte DiameterFor measurement of oocyte diameter, part of the ovari

an tissue was fixed with 10% formalin. Then, each oocyte was isolated and the diameter was measured by a profile projector. Since the oocytes prior to or during final maturation had a long globular shape, the long axis was measured. The diameter of fresh oocytes was also measured to

204 Koya et al,

indicate the range of live oocyte size at each development stage.

Results

General Anatomy of the OvaryThe paired ovaries of the kichiji rockfish are nearly

equal in size and are located in the posterio-dorsal part of the abdominal cavity (Fig. IA). Each ovarian lobe fuses caudally following a united oviduct. Figure 1B shows a cross section of the mid-ovarian lobe during the spawning season. Ovarian stroma and blood vessels run longitudinally through the center of each ovary (Fig. 1B). Each ovarian follicle projects radially from the central stroma. The ovarian stroma are connected to the ovarian wall only at the rostral part of the ovarian lobe. Therefore, the ovarian cavity situated between the surrounding ovarian wall and ovarian follicles radiates in all directions (Fig. 1B, 6A). The inner surface of the ovarian wall facing the ovarian cavity is covered with a simple columnar epithelial layer

(Fig. 1C). There is a gelatinous substance in the ovarian cavity during the spawning season. The gelatinous substance was divided into two distinct layers based on its stainability with haematoxylin. The outer layer consisted of fibrous material which was stained faintly with haematoxylin, and the inner layer was a homogeneous material which was stained deeply with haematoxylin (Fig. 1C). Both layers were showed PAS-positive reaction (Fig. 1D), suggesting that these substances contained polysaccharide.

Histology of Developing OocytesThe morphological changes of the developing oocytes of

kichiji rockfish were divided into eight stages following the

classification of Yamamoto.4)1) Early peri-nucleolus stage (Fig. 2A): Oocytes are 40

to 140ƒÊm in diameter and the ooplasm is stained deeply by

haematoxylin. Chromonemata are scattered throughout

the nucleus, and several nucleoli are distributed along the

nuclear membrane.

2) Late peri-nucleolus stage (Fig. 2B): Oocytes are 110

to 200 ƒÊm in diameter. The ooplasm becomes slightly less

stainable by haematoxylin compared to the early peri

nucleolus stage. Yolk nucleus is often visible in ooplasm.

Oil droplet-like vacuoles appear in the ooplasm.

3) Yolk vesicle stage (Fig. 2C, D): Oocytes are 140 to

300,um in diameter. The yolk vesicles are stained by eosin

and PAS-positive reactions appear in the ooplasm.

4) Early yolk globule stage (Fig. 2E, F): Oocytes are 240

to 360 ƒÊm in diameter. Yolk globules stained by eosin ap

pear and are gradually accumulated in ooplasm. The yolk

vesicles are scattered throughout the ooplasm.

5) Late yolk globule stage (Fig. 2G, H): Oocytes are 360

to 500ƒÊm in diameter (550 to 700ƒÊm when fresh), easily

visible to the naked eye. The yolk globules fill the entire

ooplasm, and the yolk vesicles line the cortical part of the

ooplasm. The oil droplet-like vacuoles merge with each

other to form several larger vacuoles.

6) Migratory nucleus stage (Fig. 2I): Oocytes become

long and globular in shape, and have a long axis of 400 to

750 ƒÊm (650 to 950ƒÊm when fresh). The germinal vesicle

migrates to the cortical part of the ooplasm. The yolk glob

ules begin to fuse to each other. A large vacuole which

seems to be an oil droplet is visible in the center of the

ooplasm.

7) Maturation stage (Fig. 2J): Oocytes have a long axis

of 640 to 760ƒÊm (800 to 1100 ƒÊm when fresh). The germi

Fig. 1. Ovarv of the kichiii rockfish.

bv, blood vessel; g, gelatinous substance; oc, ovarian cavity; ow, ovarian wall. A: Exterior view of a paired ovary. B: Cross-section of ovary.

H-E staining. Scale bar= 1 mm. C: Cross-section of ovary at spawning season. H-E staining. Gelatinous substance fills ovarian cavity. Arrow indi

cates the ovarian wall epithelium. Scale bar =100ƒÊm. D: Cross-section of ovary at the spawning season. PAS staining. Scale bar= 100 ƒÊm.

Reproductive Cycle of Kichiji Rockfish 205

Fig. 2. Oocyte of the kichiji rockfish.n, nucleus; od, oil droplet-like vesicle; yv, yolk vesicle. A, B, C, E, G, I and J, H-E staining; D, F and H, PAS staining. A: Early peri-nucleolus

stage. Scale bar =50ƒÊm. B: Late peri-nucleolus stage. Scale bar =50ƒÊm. C: Yolk vesicle stage. Scale bar =50 ƒÊm. D: Oocyte at same stage as C.

Scale bar =50 ƒÊm. E: Early yolk globule stage. Scale bar =100ƒÊm. F: Oocyte at same stage as E. Scale bar =100ƒÊm. G: Late yolk globule stage.

Scale bar= 100ƒÊm. H: Oocyte at same stage as G. Scale bar= 100ƒÊm. I: Migratory nucleus stage. Scale bar= 100ƒÊm. J: Ripe egg stage. Scale

bar= 100ƒÊm.

nal vesicle breaks down. The yolk globules fuse to each

other. and oocvtes become transparent.

8) Ripe egg stage: The ovulated eggs are colorless and

transparent. The long and short axis of the eggs fixed with

10% formalin are about 1300ƒÊm and 850ƒÊm, respec

tively. The ovulated eggs are maintained in the ovarian cav

ity, embedded in the gelatinous substance.

Maturity of the OvaryOvarian maturity of kichiji rockfish was divided into

four phases as follows on the basis of the frequency of each oocyte stage.

I) Immature phase: Ovarian follicles consist of oocytes at the stage prior to the yolk vesicle stage. Distinct postovulatorv follicles are not observed in the ovary.

‡U) Vitellogenic phase: Clutches of developing oocytes are in the early or late yolk globule stage. Distinct post

ovulatory follicles are not observed in the ovary.

‡V) Maturation phase: The most developed clutch of

oocytes is in the migratory nucleus stage, maturation

stage, or ripe egg stage. Post-ovulatory follicles are often

observed.

‡W) Spent phase: Post-ovulatory follicles exist in the

ovary. Ovarian follicles consist of oocytes at the pre-vitel

logenic stage. Oocytes at the late yolk globule stage or

migratory nucleus stage, which seem to be the residue of

spawned clutches, are infrequently observed.

Table 1. Appearance of each ovarian maturity in the female kichiji

rockfish sampled each month

Numerals indicate the numbers of fish.

Seasonal Changes in Ovarian Maturity

Ovarian maturity in each month is shown in Table 1. In December and February, the ovarian condition was immature phase or vitellogenic phase. In March, in addition to the immature and vitellogenic phase, females in the maturation phase and spent phase appeared. Therefore, some fish have already finished spawning by late March. In April, all fish observed were maturation phase, suggesting this to be the spawning season. In May, six of eight were spent phase (migratory nucleus or late yolk globule stage oocytes in Table 2 were judged reserve fund oocytes), indicating the termination of spawning. Fish entering the vitellogenic phase appeared in August and October.

From August to February, the atresia of oocytes were often observed. These atresia occurred mostly on a part of vitellogenic oocytes.

206 Koya et al.

Table 2. Changes in stage composition of oocytes in the female kichiji rockfish

Symbols indicate the occurrence of oocytes, categorized as few (no mark), some (-), and many (+).

Spawning Season and the Number of Spawning TimesThe composition of oocyte stages in each month is

shown in Table 2. The females having post-ovulatory folli

cles appeared from March to May, indicating that this

period is the spawning season of this species. In April, several specimens had post-ovulatory follicles in spite of

having maturing oocytes. This indicates that this species is

a multiple spawner which spawns more than twice during a

single spawning season. Furthermore, except for one speci

men, all specimens had two clutches of developing oocytes

with no post-ovulatory follicles, or one clutch of maturing

Fig. 3. Frequency of diameter of the developing oocyte fixed with 10%

formalin in three individuals (A, B, and C) during the spawning season.

Two clutches of oocytes at the stages of late yolk globule and

migratory mucleus are divided at 750ƒÊm in diameter.

oocytes with post-ovulatory follicles. This suggests that this species spawns twice during a spawmng season.

In order to confirm the above, the composition of the di

ameter of the developing oocyte was examined. The two

distinct clutches of oocytes diameter were shown in three

of thirteen individuals which had two clutches of develop

ing oocytes. In the three specimens, two distinct clutches

were divided at 750ƒÊm diameter (Fig. 3). The numbers of

larger oocytes and smaller oocytes were 167 and 172 (Fig.

3A), 116 and 95 (Fig. 3B), and 75 and 53 (Fig. 3C), at a ra

tio of about 1:1. This result strongly suggests that this spe

cies ovulates and spawns twice during a single spawning

season.

Relation between Ovarian Maturity and Gonad Somatic

Index

Gonad somatic indices (GSI) of each maturity stage are compared in March (Table 3). The results show that the GSI range of immature phase (0.54-0.76%), vitellogenic and maturation phase (3.33-5.44%), and the spent phase (2.21-2.97%) were clearly separated at between 2% and 3%. This indicates that it is possible to determine ovarian maturity from the GSI during the spawning season.

Female Maturation SizeFigure 4 shows the relationship between standard length

Table 3. Relation between gonad somatic index and ovarian

maturity in the samples obtained in March

Reproductive Cycle of Kichiji Rockfish 207

Fig. 4. Standard length and gonad somatic index (GSI) of females in

the spawning season.GSI was divided into three groups; immature (less than 2), spent

(more than 2 to less than 3), and mature (more than 3). Closed symbols indicate the specimens whose maturity was confirmed histologi

cally. Samples were obtained in March (A), and in April (B).

Fig. 5. Female maturation rate for different standard lengths in the

spawning season.

Samples were obtained in March (A), and in April (B). Numerals in parentheses indicate the number of fish examined.

and GSI during the spawning season (March and April). The individuals of vitellogenic and maturation phase or spent phase were more than 150 mm in standard length. The proportional frequency of immature fish (immature phase) and mature fish (vitellogenic, maturation, and spent phase) for each class of standard length is shown in Fig. 5. The ratio of mature fish rose as standard length increased. The minimum maturation size was from 151 to 175 mm standard length.

Discussion

Structure of Ovary and Formation of Gelatinous Egg Mass

The ovaries of teleosts are generally divided into gymnovarian and cystovarian types on the basis of their anatomical relationship to the place of ovulation.') Moreover, the cystovarian type is divided into four types based on histological characteristics.) The ovary of the kichiji rockfish, which is completely covered by the ovarian wall and has stroma and blood vessels in the center of the ovarian lobes, belongs to the cystovarian type 11-3, following the classification of Takano.o This type of ovary has been reported in Dendrochirus brachyptelus,7) Helicolenus hilgendorfi,6) and Sebastolobus alascanus.8) All three species belong to scorpaenidae. There are several species which spawn gelatinous egg masses in scorpaenidae, including these three species. 1,9-14)

In the egg mass of the kichiji rockfish, the eggs are embedded in a gelatinous matrix that forms a hollow balloon.') In the present study, it was confirmed that the gelatinous substance is secreted into the ovarian cavity prior to ovulation (Fig. 6A). Therefore, eggs seem to be embedded into the gelatinous substance when ovulation occurs (Fig. 6B). The egg masses form a paired hollow balloon after spawning (Fig. 6C, D). Thus, it seems that the cystovarian type II-36) is a specialized structure for forming the egg mass.

Reproductive Cycle

The annual reproductive cycle of kichiji rockfish has been investigated by exterior observation of oocytes and gonad somatic index, and the spawning season of this species has been broadly estimated. 1,2) In the present study, we made histological observations of the ovary of kichiji rock

Fig. 6. Schematic illustration of the formation of gelatinous egg masses in the kichiji rockfish.

Ovary at the pre-ovulatory period (A), post-ovulatory period (B) , spawning (C), and gelatinous egg mass spawned (D) .

208 Koya et al.

fish in order to clarify the process of ovarian development, which we determined was as follows.

Female kichiji rockfish seem to begin vitellogenesis from

August to October and continue accumulation of yolk

globules for five to seven months. During this period, oo

cyte diameter increases from 240ƒÊm to 500ƒÊm. From

March to April, oocytes at final maturation, ovulated eggs

or post-ovulatory follicles were often observed. In May ,

post-ovulatory follicles were obseved, but not vitellogenic

oocytes, and the post-spawning females appeared in large

numbers. Therefore, the spawning season of this species

seems to be mainly March and April. Fish which finished

spawning seemed to rapidly form yolkk vesicles for the next

reproductive phase, and to maintain a resting condition

for several months until the beginning of vitellogenesis.

The spawning season of kichiji rockfish has been estimated as being from March to June based on field sampling data of egg masses,1) from January to April based on morphological changes in the ovaries and the oocytes,2) or from February to May based on histological examination of the testes.3) The present results agree approximately with all these previous results.

Number of Spawning TimesThe kichiji rockfish has been thought to be a single

spawner based on the results of naked eye observation of the mature ovary.1) However, the present study strongly suggests that this species is a multiple spawner. The vitellogenic oocytes developed almost synchronously until the late yolk globule stage. When the leading clutch oocytes began final maturation, two clutches which consisted of oocytes at the late yolk globule stage and the maturation stage were clearly distinguishable. Furthermore, the ratio of both clutches was about 1:1 in three specimens. These results strongly suggest that this species spawns twice during a single spawning season.

The spawning interval could not be clarified in the

present study. However, it is estimated that the interval between spawning was less than one month, because spent females appeared late in March, and the pre-spawning females and those which had completed the first spawning in April already became spent in May.

Fecundity is an important reproductive parameter for resource estimation. For measurement of total fecundity of a multiple spawner, it is necessary to measure it before the first spawning. Measurement of the fecundity of kichiji rockfish has been previously attempted, 2,15) but whether the samples were individuals before or after the first spawning was not confirmed. Therefore, it is not clear whether the results indicated total fecundity or batch fecundity. In the case of measurement of fecundity of the kichiji rockfish, it is necessary to count the numbers of eggs at final maturation for batch fecundity, and to count the develop-

ing oocytes in the pre-spawning ovary for total fecundity .

Acknowledgements The authors wish to thank Dr. K. Watanabe, Dr. T. Wada, and Mr. K. Yabuki, Hokkaido National Fisheries Research In.

stitute, for their useful suggestions. For technical help, we are greatly in. debted to the staff of the Demersal Fish Resource Laboratory and Pelagic Fish Resource Laboratory, Hokkaido National Fisheries Research In

stitute.

References

1) H. Fukataki: Pelagic egg balloons from the western north Pacific referrable to Sebastolobus macrochir (Gunther). Bull. Jap. Reg. Fish. Res. Lab., (11), 91-100 (1963).

2) M. Mikawa: Maturation and spawning of kichiji rockfish. Report of the Japan Scientific Council on the Fisheries Resources, North. ern Groundfish Section, (16), 42-52 (1981) (in Japanese).

3) Y. Koya, T. Hamatsu, and T. Matsubara: Testicular development of male Sebastolobus macrochir in the Pacific coast of southeastern Hokkaido. Bull. Hokkaido Natl. Fish. Res. Inst., (58), 1-8 (1994) (in Japanese).

4) K. Yamamoto: Studies on the formation of fish eggs I. Annual cycle in the development of ovarian eggs in the flounder, Liopsetta ob

scura. J. Fac. Sci. Hokkaido Univ., Ser. VI, Zool., 12, 362-378 (1956).

5) W. S. Hoar: Reproduction, in "Fish Physiology" (ed. by W. S. oar and D. J. Randall), Vol. 3, Academic Press, New York, 1957,

pp. 1-72.6) K. Takano: Ovarian structure and gametgenesis, in "Reproductive

Biology of Fish and Shellfish" (ed. by F. Takashima and I. Hanyu), Midori-Shobo, Tokyo, 1989, pp. 3-34 (in Japanese).

7) L. Fishelson: Ultrastructure of the epithelium of the ovary wall of Dendrochirus brachypterus (Pteroidae: Teleostei). Cell Tiss. Res., 77, 375-381 (1977).

8) D. L. Erickson and E. K. Pikitch: A histological description of shortspine thornyhead, Sebastolobus alascanus, ovaries: structures associated with the production of gelatinous egg masses. Environ,

Biol. Fishes, 36, 273-282 (1993).9) L. Fishelson: Ethology and reproduction of pteroid fishes found in

the Gulf of Aqaba (Red Sea), especially Dendrochirus brachypterus (Cuvier) (Pteroidae: Teleostei). Pubblicazioni delta Stazione

Zoologica di Napoli, 39 (Suppl.), 635-656 (1975).10) D. H. Graham: A treasury of New Zealand fishes, A. H. & A. W.

Reed, Wellington, 1956, p. 424.11) G. Krefft: A contribution to the reproductive biology of Helicole

nus dactylopterus (De la Roche, 1809) with remarks on the evolution of Sebastinae. Rapp. P.-v. Ran . Cons. perm. int. Explor.

Mer., 150, 243-244 (1961).12) L. A. Lisovenko: Reproduction of rockfishes (family Scorpaenidae)

off the Pacific coast of South America. J. Ichthyol., 18, 262-268 (1979).

13) J. T. Moyer and M. J. Zaiser: Social organization and spawning be havior of the pteroine fish Dendrochirus zebra at Miyake-jima,

Japan. Jap. J. Ichthyol., 28, 52-69 (1981).14) W. G. Pearcy: Egg masses and early developmental stages of the

scorpaenid fish, Sebastolobus. J. Fish. Res. Bd Can., 19, 1169-1173 (1962).

15) T. Hamatsu: Maturity and Fecundity of kichiji rockfish on spring season in the Pacific coast of southeastern Hokkaido. Report of the

Japan Scientific Council on the Fisheries Resources, Northern Groundfish Section, (26), 33-39 (1993) (in Japanese).1) H. Fukataki: Pelagic egg balloons from the western north Pacific referrable to Sebastolobus macrochir (Gunther). Bull. Jap. Reg. Fish. Res. Lab., (11), 91-100 (1963).2) M. Mikawa: Maturation and spawning of kichiji rockfish. Report of the Japan Scientific Council on the Fisheries Resources, North ern Groundfish Section, (16), 42-52 (1981) (in Japanese).3) Y. Koya, T. Hamatsu, and T. Matsubara: Testicular development of male Sebastolobus macrochir in the Pacific coast of southeastern Hokkaido. Bull. Hokkaido Natl. Fish. Res. Inst., (58), 1-8 (1994) (in Japanese).4) K. Yamamoto: Studies on the formation of fish eggs I. Annual cycle in the development of ovarian eggs in the flounder, Liopsetta ob scura. J. Fac. Sci. Hokkaido Univ., Ser. VI, Zool., 12, 362-378 (1956).5) W. S. Hoar: Reproduction, in "Fish Physiology" (ed. by W. S. Hoar and D. J. Randall), Vol. 3, Academic Press, New York, 1957, pp. 1-72.6) K. Takano: Ovarian structure and gametgenesis, in "Reproductive Biology of Fish and Shellfish" (ed. by F. Takashima and I. Hanyu), Midori-Shobo, Tokyo, 1989, pp. 3-34 (in Japanese).7) L. Fishelson: Ultrastructure of the epithelium of the ovary wall of Dendrochirus brachypterus (Pteroidae: Teleostei). Cell Tiss. Res., 177, 375-381 (1977).8) D. L. Erickson and E. K. Pikitch: A histological description of shortspine thornyhead, Sebastolobus alascanus, ovaries: structures associated with the production of gelatinous egg masses. Environ, Biol. Fishes, 36, 273-282 (1993).9) L. Fishelson: Ethology and reproduction of pteroid fishes found in the Gulf of Aqaba (Red Sea), especially Dendrochirus brachypterus (Cuvier) (Pteroidae: Teleostei). Pubblicazioni delta Stazione Zoologica di Napoli, 39 (Suppl.), 635-656 (1975).10) D. H. Graham: A treasury of New Zealand fishes, A. H. & A. W. Reed, Wellington, 1956, p. 424.11) G. Krefft: A contribution to the reproductive biology of Helicolenus dactylopterus (De la Roche, 1809) with remarks on the evolution of Sebastinae. Rapp. P.-v. Ran. Cons. perm. int. Explor. Mer., 150, 243-244 (1961).12) L. A. Lisovenko: Reproduction of rockfishes (family Scorpaenidae) off the Pacific coast of South America. J. Ichthyol., 18, 262-268 (1979).13) J. T. Moyer and M. J. Zaiser: Social organization and spawning be havior of the pteroine fish Dendrochirus zebra at Miyake-jima, Japan. Jap. J. Ichthyol., 28, 52-69 (1981).14) W. G. Pearcy: Egg masses and early developmental stages of the scorpaenid fish, Sebastolobus. J. Fish. Res. Bd Can., 19, 1169-1173 (1962).15) T. Hamatsu: Maturity and Fecundity of kichiji rockfish on spring season in the Pacific coast of southeastern Hokkaido. Report of the Japan Scientific Council on the Fisheries Resources, Northern Groundfish Section, (26), 33-39 (1993) (in Japanese).