sexual maturation and spawning of cottus reinii (pisces

Biogeography 23.

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Sexual Maturation and Spawning of Cottus reinii (Pisces: Cottidae) in the Chinai River around Lake Biwa, Central Japan

Yasuhiro Fujioka1*, Yuko Kido 1 and Makoto Uenishi 2

1 Lake Biwa Museum, Oroshimo 1091, Kusatsu, Shiga, 525-0001 Japan2 Faculty of Science and Technology, Ryukoku University, Seta, Otsu, Shiga, 520-2194 Japan

Abstract: The sexual maturation and spawning characteristics of Cottus reinii, a sculpin endemic to the Lake Biwa area, central Japan, were investigated in the Chinai River. Fish larger than 3 cm SL inhabit mainly the lower reaches of the river throughout the year. The breeding season was winter and spring (February-April) in the river, mostly coinciding with that of the fish inhabiting Lake Biwa. A large difference in the body size at 50% sexual maturation between male (6.80 cm SL) and female (5.32 cm SL) was inferred from the logistic curves. Egg masses laid on the top of a boulder (nest) were found from early February to April in riffles along the lower course of the river, and each mass, consisting of multiple clutches of eggs, was guarded by a single male. These results suggested that C. reinii larger than 3 cm SL in the Chinai River migrate upstream from the lake and breed at a body size larger than ca. 6 cm SL for males and ca. 5 cm SL for females.

Key words: Cottus reinii, Utsusemi-kazika, Freshwater sculpin, Spawning, Maturation, River

Introduction

Throughout the freshwater drainages of the Japanese Archi-pelago, Cottus amblystomopsis, C. hangiongensis and C. reinii (small egg- and middle egg-type) display amphidromous life style (Goto, 2001; Yokoyama & Goto, 2011). C. reinii, which is endemic to Japan, distributes mainly in the middle and/or lower reaches of rivers from Hokkaido to Kyushu Island (Na-kabo, 2013). This species descends to the sea after hatching and migrates upstream at sizes larger than 1.8 cm SL (Komada & Yamada, 1996; Shinagawa et al., 2002). Fukui et al. (2007) reported that the spawning season of this fish is winter (January and February) and the ripe females lay 1-3 times. The lacus-trine-type of C. reinii also lives in Lake Biwa and is known to spawn the smallest eggs in C. reinii (Kurawaka, 1976, 1992; Goto, 2001).

In Lake Biwa the previous report on C. reinii has revealed that the fish begins its benthic life at a size of 1 cm SL at depths of 20-30 m and dwells in the bottom from lake shore to offshore waters throughout the year (Fujioka et al., 2015). The spawning season begins in late February and end in May (Fu-jioka & Kido, 2020). There is no large difference in the size of sexual maturation between male and female (Fujioka & Kido, 2020). In the rivers around the lake, this species inhabits in the lower reaches, where its range of distribution does not overlap with that of C. pollux which lives in the upper reaches (Fujioka et al., 2014).

In the lake, rapidly growing fish can mature at one year af-ter hatching and all of the fish breed more than two years old.

In the ovary of mature females, the oocytes separate into two layers of ripe and immature oocytes just before spawning, sub-sequently ripe eggs were laid one or two more times (Fujioka & Kido, 2020).

Hitherto, there are no reports on the life of this species in rivers around the lake. Especially, there is no data related to the sexual maturation and spawning of the fish in rivers. This study aims to elucidate the size at sexual maturation and the season of spawning compared with those of the fish dwelling in the lake.

Materials and Methods

The study was done in the Chinai River (R) which flows into the North Basin of Lake Biwa, central Japan from January ’97 to July ’98 (Fig. 1). We already revealed the distributions of C. reinii and C. pollux (large-egg type) in the river and the former was observed in the lower reaches (as far as 2.5 km from the river mouth) (Fujioka et al. 2014). In the river, repeated observations and sample collections were usually conducted by three persons using a hand-net and glass-bottom viewing box for one hour along a 100m stretch of the river at two survey points, traversing the route one or two times, between a course of 0.5 km and 2.4 km upstream (Fig. 1). The collected sculpins were identified as C. reinii or C. pollux (large-egg type) by the external appearance and number of pectoral fin rays following Nakabo (1993). Total number of C. reinii collected in the river was 355 (male; 201 and female; 154). Standard length (SL) of male and female ranged from 3.01 to 11.25 cm SL in male and 3.13 to 9.51 cm SL in fe-male. The fish were preserved in 10% formalin solution at the survey sites and body weight (BW; 0.001g), standard length

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−*Corresponding author: yasuhirofujioka@outlook. jp

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Yasuhiro Fujioka, Yuko Kido and Makoto Uenishi

(SL; 0.01cm), and gonad weight (GW; 0.001g) were measured in the laboratory. The gonadosomatic index (GSI=100×GW/BW) was calculated based on these measurements. Gonads removed from the abdomen were observed closely. To deter-mine the body size at 50% sexual maturation for male and fe-male, the fish collected during the development and spawning months (between December and April) were assigned to body size classes (SL) of 10 mm, and the ratios of mature males and females in each size class (Px; 0.00-1.00) were calculated. A logistic curve (Px=eax+b/1+eax+b, or ln (Px/1-Px=ax+b) was fit-ted against SL (x) classes to the ratios of mature fish (Px). The size at 50% sexual maturation was estimated from the equa-tions for both male and female. Each ripe ovum in ovary from 23 females from January to March ’97 and ’98 was separated from the ovarian membrane and directly enumerated in order to elucidate the fecundity of the fish. To estimate the number of eggs cared for by a male, we collected egg masses spawned in the nests on 5 February ’97, and 11 March, and 3 April ’98. The different clutches in each egg mass were separated based on differences in egg colors and development stages (e.g., eyed egg stage), and the number of eggs per clutch was count-ed. To know hatching size of the fish, some eggs were incubat-ed in the laboratory at room temperature (5-8℃) and the newly hatched fry were stored in 5% formalin solution and measured for total length (TL).

The samples in the study have been deposited at the Lake Biwa Museum (LBM), Kusatsu, Shiga, Japan (No. 1210055406-1210055429).

Results

Single or bimodal frequency distributions for male and female were detected except in June ’97 (Fig. 2). The lower mode consisting of 3-5 cm SL in January ’97 could be fol-lowed through the monthly frequency histograms to the mode of 5-6 cm in October ’97. A new lower mode consisting of 3-4 cm SL was detected in December ’97 and March ’98. Mean

Fig. 1. Location of the study river and survey points about 0.5 (St. 1), and 2.4 km (St. 2) upstream from the river mouth, respectively. There is about 1 m difference between the upper and lower reach in the river at the weir set near St.2.

Fig. 2. Changes in standard length (SL) frequency distributions of C. reinni collected in the Chinai R from January 1997 to July 1998. Black and white columns indicate males and females, respective-ly.

Fig. 3. Water temperatures in the Chinai R during the study.

TL of fry hatched from eggs in the laboratory on 11 March ’97 was 0.59±0.014 cm (mean±SD) (range: 0.55-0.61 cm). During the study in the river, the water temperature varied between 5.1℃ (February ’98) and 28.1℃ (July ’98) (Fig. 3).

Although males with GSI of less than 0.2 were seen every month, the GSI values of the remaining males were over 0.8 (Fig. 4). Fish with developed testes were seen between Oc-tober and April, with a peak in December ’97 (GSI; 1.8-3.2, mean=2.5). The GSI of mature males in June ’98 declined to less than 0.2 indicating the end of spawning activity. The GSI of some females were always less than 0.2. GSI values of the remaining fish increased between October to April, with the values ranging from 3.3 to 20.4 in March ’98. Ovaries with ripe eggs were observed from January to April, afterward GSI

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of females declined to less than 1.0 in June ’98.Since the gonadal development was coincident with that of

the fi sh collected from Lake Biwa reported by Fujioka & Kido (2020), sexual maturation was classifi ed into four stages from the external shape, color, and GSI of gonads after Fujioka & Kido (2020). In male (Fig. 5): “Immature”; threadlike and under 0.2 GSI, “Developing”; swelling and white or yellowish in color, and 0.3-1.8 GSI, “Maturing”; thick and triangle, and over 1.9 GSI, “Spent”; thin and grayish, and under 1.0 GSI. In female (Fig. 6): “Immature”; threadlike or slightly swelling and pale pink under 2 GSI, “Developing”; visible oocytes as small drops and orange in color, and 3-20 GSI, “Spawning”; oocytes separate into two layers of large ripe eggs and im-mature oocytes within the ovary and the number of ripe eggs decreases leaving the immature eggs in the ovary, “Spent”; ripe eggs disappear, and irregular sized immature oocytes remain in the ovary. Fig. 7 shows the monthly changes in percentages of sexual maturation. In males, the “Developing” gonadal stage was seen from October to January, subsequently “Maturing” stage appeared from January to April. In females, “Spawning” fish were seen in two months of February and March. There were “Immature” fish throughout the year in males and females.

Fig. 4. Seasonal changes in gonadosomatic index (GSI) of male (up-per) and female (lower) C. reinii collected in the Chinai R.

Fig. 7. Seasonal changes in sexual maturation stages of C. reinii of male (upper) and female (lower) collected in the Chinai R. Each sexual maturation stage of C. reinii in the river is described in the text.

Fig. 5. The development of testes in Cottus reinii. a: “Immature” tes-tis, b: “Developing” testes, c: “Maturing” testis.

Fig. 6. The mature ovary in Cottus reinii. a: appearance of ripe eggs, b: sectional appearance of “Spawning” ovary (The black and white allows indicate separated mature eggs and small sized im-mature eggs, respectively.).

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Judging from the specimens caught in the reproductive sea-son between December ’97 and April ’98, the smallest mature male and female were 6.73 cm SL and 5.36 cm SL, respec-tively. The maximum sizes of immature fi sh were 7.45 cm SL for male and 5.92 cm SL for female. The logistic equations adapted for the relationships of mature fi sh ratios to standard length both male and female were represented Px= e11.201x-

76.169/1+e11.201x-76.169 for male and Px= e18.421x-97.981/1+e18.421x-97.981

for female (Fig. 8). The body size at 50% sexual maturation for male and female calculated from the equations were 6.80 cm for male and 5.32 cm for female, respectively.

Among 23 mature females, the number of mature ova (Y) increased with standard length (X) from 745 to 2378 (Fig. 9), and the best fi tting equation to represent this relationship was: Y=97.47e0.378X (r=0.93). Egg masses laid on the top of a boulder (nest) were found from early February to April in rapids along the lower course of the Chinai R, and each mass consisted of several clutches of eggs with different developing stages and colors ranging from yellowish to brownish (Fig. 10, Table 1). A nest was composed of single boulder half-buried in gravel or sand, and one mature male guarded the eggs in each nest. In fi ve nests observed, male SL ranged from 7.36 cm to 10.39 cm, and the number of clutches ranged from 2 to 10. The number of eggs per clutch ranged from 618 to 2968, for a total number of eggs per nest of 2332-20449.

Discussion

In this study, the GSI of mature males of C. reinii attained a peak from October to December and relatively high values were seen until April in the Chinai R. Temporal changes in the GSI of mature females differed slightly from those of the males, with a peak two months later and a marked decrease in April. These changes in the gonad condition of both males and females and the occurrence of spawning females indicate that the breeding season is from February to April in the Chinai R, mostly coinciding to that of the fi sh inhabiting in Lake Biwa (Fujioka & Kido, 2020).

In our previous study of C. reinii in Lake Biwa, the size

Fig. 8. Changes in the ratio of mature fi sh in relation to body size (SL) for male (upper) and female (lower) and size at 50% sexual maturation (black square) in the river.

The logistic equations adapted for the relationships of mature fi sh ratios to standard length for both males and females.

Fig. 9. Relationship between fecundity (Y) and body size (X) of C. reinii in the river.

Table 1. Male size and number of eggs in the nest .

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of 50 % maturation was similar between male (5.23 cm SL) and female (5.41 cm SL) and there was a small difference be-tween male (4.78 cm SL) and female (4.50 cm SL) in the size of smallest maturation (Fujioka & Kido, 2020). In this study, however, there was a large difference of more than 1.4 cm in the size between the sexes in the Chinai R. Such a sexual size dimorphism in mature C. reinii in the river, as shown in other congeners (Goto 1987a, 1989a; Natsumeda et al., 1997), is generally caused by different growth rates and/or age of matu-rity between the sexes (Goto 1984b, 1987b, 1989c; Natsumeda et al., 1997). Since sexual size dimorphism of Cottus species has been shown to be due to some environmental differences (Natsumeda, 2011), the sexual size difference of the fi sh in-habited in the Chinai R is presumed to be derived from some environmental differences between the river and the lake such as the density of the fish and/or condition for the resources related to breeding like the availability of a boulders for nests. Furthermore, we observed several clutches of eggs at different developmental stages in single nest. This fact indicates that C. reinii are polygamous similar to other sculpins (Goto 1989b). Polygamy may be strongly related to sexual size dimorphism (Goto 1984b, 1989b), but how the growth process differs in detail between male and female was not clarifi ed in this study.

Since the mean TL of hatched larvae in C. reinii was 0.59 cm in this study, this fish is thought to descend river to the lake at this size, and begin its benthic life at 1.0 cm SL in June soon after the planktonic existence. Afterwards the fry grows up to 3-5 cm SL by November and 3-6 cm SL by March in the lake (Fujioka et al., 2015). From these reports, each single mode of 3-6 cm between January and October ’97 in the fre-quency distributions of SL in the river (Fig. 2) is assumed to be under-yearling (before April) and yearling (after May) fi sh. As the smallest mature male and female were 6.73 cm SL and 5.36 cm SL in the study, the age at sexual maturation in the river is inferred to be 2 years old or more in both sexes.

The previous study reported that various body sizes larger than 1.0 cm SL of C. reinii were caught in the lake (Fujioka &

Kido, 2020). On the other hand, we could not fi nd fi sh smaller than 3.0 cm SL throughout the year in the river. This result suggests that the size at the upstream migration from the lake begins is larger than 3.0 cm SL. This size of C. reinii was larger than the size of upstream migration from the sea for C. kazika (13-15 mm TL) (Goto, 1989c; Kinoshita et al., 1999), C. amblystomopsis (11-14 mm TL) (Goto, 1984a), C. hangion-gensis (13-16 mm TL) (Goto, 1984a), and C. reinii (small-egg type) (18-25 mm TL) (Komada & Yamada, 1996). Since we could not show the season and range of size at the upstream migration of the fi sh in this study, further studies related to the upstream migration from the lake in C. reinii are needed.

In the present study, spawning of C. reinii took place in nests using boulders in rapids of the river. Egg masses guarded by single males were actually detected. If suitable boulders for nests and sites for spawning are not abundant in the lake, males may prefer to inhabit rivers rather than the lake. C. extensus, endemic to Bear Lake, USA, has been studied in connection with its spawning habits and the pelagic behavior of its larvae in the lake; suitable spawning sites are reported to be limited in the lake (Ruzycki et al., 1998). The distribution of boulders for spawning is confi ned to a part of shore bottom in Lake Biwa (Kotani, 1971), but suitable boulders for nests are considered to be most abundant in the rivers fl owing into the lake. From this difference in the availability of resources for spawning between the lake and rivers, C. reinii may show preference to inhabit in rivers.

Some features of the distribution, growth, maturation, and breeding of C. reinii in the Chinai R were revealed in the present study. The egg sizes of the fi sh Plecoglossus altivelis altivelis and the shrimp Palaemon paucidens inhabiting Lake Biwa are signifi cantly smaller, and the brood size greater, than the usual latitudinal trends for these species (Azuma, 1973; Iguchi, 2001; Nishino, 1980). C. reinii has the smallest egg and fry sizes, and the higher fecundity than other freshwater species of Cottus. This fi sh is presumed to have acquired these life history characteristics through evolution within Lake Biwa basin system (Goto, 2001). Further ecological studies of C. reinii in the lake proper are needed to elucidate the evolu-tionary pressure leading to the origin of this species with the smallest egg size among Japanese freshwater sculpins.

Acknowledgements

We thank Dr. M. J. Grygier (Lake Biwa Museum) for his critical reading of the manuscript.

References

Azuma, M., 1973. Studies on the variability of the landlocked ayu-fi sh, Plecoglossus altivelis T. et S., in Lake Biwa VI. Considerations on the grouping and features of variability.

Fig. 10. Egg masses of Cottus reinii laid on the top of a boulder. The different clutches in each mass can be separated based on differ-ences in egg colors and developmental stages.

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Jpn. J. Ecol., 23: 255-265. (In Japanese with English ab-stract)

Fujioka, Y., Kido, Y., Uenishi, M., Yoshioka, M. & Kashiwagi, M., 2014. Distribution of Cottus reinii and Cottus pollux in rivers around Lake Biwa, central Japan. Biogeography, 16: 31-37.

Fujioka, Y., Sakai, A. & Ide, A., 2015. Horizontal and verti-cal distribution of Cottus reinii (Pisces: Cottidae) in Lake Biwa, central Japan. Biogeography, 17: 13-20.

Fujioka, Y. & Kido, Y. 2020. Spawning season and size at maturity of Cottus reinii (Pisces: Cottidae) in Lake Biwa, central Japan. Bull. Shiga Pref. Fish. Exp. St., 56: 11-20. (In Japanese with English abstract)

Fukui, K., Fujii, R., Tahara, D., Hayakawa, Y. & Koya, Y., 2007. Annual changes in gonadal histology and serum profiles of sex steroids in Kajika, Cottus sp. SE (small egg type), under rearing conditions. Jpn. J. Ichthyol., 54: 173-186.

Goto, A., 1984a. Comparative ecology of young-of-the year between two amphidromous species of Cottus in Hokkaido 1. Upstream migration and growth. Bull. Fac. Fish. Hokkai-do Univ., 35: 133-143.

Goto, A., 1984b. Sexual dimorphism in a river sculpin, Cottus hangiongensis. Ichthyol. Res., 31: 161-166.

Goto, A., 1987a. Life-history variations in males of the river sculpin. Cottus hangiongensis, along the course of a river. Env. Biol. Fish., 19: 82-92.

Goto, A., 1987b. The distribution and speciation in freshwa-ter Cottidae, In: Mizuno, N. & Goto, A. (eds), Freshwater fishes in Japan: 156-166. Tokai University Press, Tokyo. (In Japanese)

Goto, A., 1989a. Growth patterns, and age and size at maturity in female Cottus hangiongensis, with special reference to their life-history variation. Jpn. J. Ichthyol., 36: 90-99.

Goto, A., 1989b. Reproductive style and tactics in freshwater sculpins, In: Goto, A. & Maekawa, K. (eds), Reproductive behavior in fish: 73-84. Tokai University Press, Tokyo. (In Japanese)

Goto, A., 1989c. Ayukake. In: Kawanabe, H. & Mizuno, N. (eds), Freshwater Fishes of Japan: 655. Yama-kei Publish-ers, Tokyo. (In Japanese)

Goto, A., 2001. Patterns in the segregation of life-style in diadromy: Sculpins. In: Goto, A., & Iguchi, K. (eds), Evo-lutionary Biology of Egg Size in Aquatic Animals: 171-190, Kaiyu-sha, Tokyo. (In Japanese)

Iguchi, K., 2001. Adaptations from individuals to populations: Ayu fish. In: Goto, A., & Iguchi, K. (eds), Evolutionary Biology of Egg Size in Aquatic Animals, 43-64, Kaiyu-sha, Tokyo (In Japanese)

Kinoshita, I., Azuma, K., Fujita, S., Takahashi I., Niimi K. &

Harada, S. 1999. Early life history of a catadromous sculpin in western Japan. Env. Biol. Fish., 54: 135-149.

Komada, N., & Yamada, K., 1996. Activity and growth rate of Japanese sculpin, Cottus pollux, during in its upstream migration in the Nagara River. J. Growth, 35: 37-44. (In Japanese)

Kotani, A., 1971. The bottom lay and its environment. In: The research group of the Lake Biwa Quasi-National Park (ed.), The research reports in the Lake Biwa Quasi- national park: 125-175. The research of the Lake Biwa Quasi-Na-tional Park, Otsu. (In Japanese)

Kurawaka, K., 1976. Study in speciation in fish. Ph.D. disser-tation, Kyoto University, pp 1-55. (In Japanese with English abstract)

Kurawaka, K., 1992. Imi-ron. Doubutsu-sha, Tokyo, 142 pp. (in Japanese).

Nakabo, T., 1993. Cottidae. In: Nakabo, T. (ed.), Fishes of Ja-pan with Pictorial Key to the Species: 557. Tokai University Press, Tokyo. (In Japanese)

Nakabo, T., 2013. Cottidae. In: Nakabo, T. (ed.), Fishes of Ja-pan with Pictorial Key to the Species, Third edition: 1160-1175, Tokai University Press, Tokyo. (In Japanese)

Natsumeda, T., Kimura, S. & Nagata, Y., 1997. Sexual size dimorphism, growth and maturity of the Japanese fluvial sculpin, Cottus pollux (large egg type), in the Inabe River, Mie Prefecture, central Japan. Ichthyol. Res., 44: 43-50.

Natsumeda, T., 2011. Diversity in reproduction and ecology, and conservation of a fluvial sculpin. In: Munehara, H., Goto, A. & Yabe, M., (eds), Diversity of Cottoid fishes – Adaptation and Evolution: 158-175. Tokai University Press, Hadano. (In Japanese)

Nishino, M., 1980. Geographical variations in body size, brood size and egg size of a freshwater shrimp, Palaemon paucidens De Haan, with some discussion on brood habit. Jpn. J. Limno., 41: 185-202.

Ruzycki, J. R., Wurtsbaugh, W.A., & Lay, C., 1998. Reproduc-tive ecology and early life history of a lacustrine sculpin, Cottus extensus (Teleostei, Cottidae). Env. Biol. Fish., 53: 117-127.

Shinagawa, J., Kaji, K., Kohno, H., & Fujita, K., 2002. Onto-genetic intervals based on the development of swimming- and feeding-functions in the amphidromous Cottus pollux larvae and juveniles. J Tokyo Univ. Fish., 88: 25-32. (In Japanese with English abstract)

Yokoyama, R. & Goto, A., 2011. Molecular phylogeny and life-history evolution in freshwater sculpins (Pisces: Cotti-dae). In: Munehara, H., Goto, A., & Yabe, M. (eds), Diver-sity of Cottoid fishes – Adaptation and Evolution: 43-65. Tokai University Press, Hadano. (In Japanese)

(Received November 17, 2020; Accepted February 12, 2021)

sexual maturation and spawning of cottus reinii (pisces

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