FisheriesScience 64(4), 517-521 (1998)

Application of Otolith Microchemistry for Distinguishing between Amphidromous and Non-amphidromous Stocked Ayu, Plecoglossus ƒ¿ltivelis

Tsuguo Otake*1 and Kazuo Uchida*2 *1Faculty of Bioresources , Mie University, Kamihama, Tsu 514-8507, Japan *2Ueda Station , National Research Institute of Fisheries Science, Komaki, Ueda 386-0031, Japan

(Received November 9, 1997)

Ontogenic change patterns in otolith Sr:Ca ratios were examined in amphidromous, landlocked (Lake Biwa population) and hatchery-reared ayu, so as to determine the usefulness of otolith microchem istry for discrimination between amphidromous and stocked non-amphidromous individuals. Otolith Sr:Ca ratios of amphidromous ayu fluctuated strongly along the life history transect in accordance with the migration pattern from sea to freshwater, via brackish water in the river mouth. On the contrary, the Sr:Ca ratios of Lake Biwa and hatchery-reared ayu remained at consistently low levels throughout the otolith. The higher ratios in amphidromous ayu, in the otolith region from the center to 220-400 ,ƒÊm equivalent to the first 90-110 days from hatching, corresponded to the initial sea-going period, sug gesting that otolith Sr:Ca ratios are affected by ambient water salinity or the seawater-freshwater gradient in Sr concentration. No relationship between changes in the Sr:Ca ratio and water temperature was found. The findings clearly indicated that otolith Sr:Ca ratios reflected individual life histories, ena bling amphidromous ayu to be distinguished from stocked non-amphidromous ayu.

Key words: otolith Sr:Ca ratios, ayu, amphidromous form, stocked non-amphidromous form

The ayu, Plecoglossus altivelis, is an amphidromous os -ing, which may result in genetic changes in the amphidro merid fish with a one-year life span. Newly-hatched larvae mous population, resulting in reduced survival of (6 mm long) drift downstream to the sea in autumn, sea-going larvae and consequent recruitment. However, remaining there over the winter months. Juveniles of the finding of evidence for such interbreeding has necessi about 60 mm total length migrate upstream in spring, tated a means of distinguishing between amphidromous growing to 150-300 mm by the end of the following sum and Lake Biwa-sourced reproductive individuals at a mer.1)In addition to such amphidromous ayu, landlocked spawning site. Established methods, such as otolith micro populations are known from Lake Biwa2) and several other structure ,7) and isozyme3) and mitochondria DNA,4) have lakes in Japan. not resulted in clear separation of the two juvenile forms. The ayu is one of the most important species for Recently, concentration ratios of strontium (Sr) to calci freshwater fisheries and recreational fishing in Japan, um (Ca) in the otoliths of some fishes have been shown to where juveniles have been extensively stocked in many be correlated with ambient water salinity (or Sr concentra rivers. The majority of stocked juveniles (70%) are taken tion) at the time of otolith precipitation,8,9) enabling the from the landlocked population in Lake Biwa, the remain reconstruction of seasonal and ontogenic migration pat der comprising amphidromous wild juveniles (10%) and terns of individual anadromous fishes.10,12)The objectives hatchery-reared juveniles (20%). Despite the extensive of the present study are to examine the otolith Sr:Ca ratios stocking of juveniles, such has not resulted in an increase in amphidromous and non-amphidromous landlocked in the amphidromous ayu populations, the number of up ayu, and to ascertain the appropriateness of otolith Sr:Ca stream-migrating wild juveniles having decreased in most ratios for discriminating between multiple-sourced ayu in rivers. river populations. The establishment of a sound discrimi Seki et al.3) and Pastene et al.,4) who examined isozymes nation method would accelerate studies not only on the and mitochondrial DNA as genetic markers, suggested evaluation of stocking on recruitment, but also on the eco that heavy stocking of Lake Biwa-sourced juveniles had logical and behavioral effects of stocking on native river not significantly altered the genetic composition of river populations. populations, indicating a low contribution of the Lake Biwa stock population to recruitment. Azuma5) and Taba Materials and methods ta and Azuma6) suggested that the Lake Biwa population matures earlier than river populations (i .e. in late summer), A total of six amphidromous (body length: 89-103 mm), even after transplantation , the low tolerance of the larvae seven landlocked (body length: 67-95 mm) and five hatch to sea water at high temperatures resulting in a low larval ery-reared ayu juveniles (body length: 74-92 mm) were survival rate . Pastene et al.4) pointed out that the native analyzed for otolith microchemistry. The amphidromous and stocked populations occurred together in the spawn juveniles were collected in Okouzubunsui, an artificial ing ground, suggesting the possibility of their interbreed branch of the Shinano river, 500 m from the river mouth 518 Otake and Uchida

on 17 May, 1996, and the landlocked juveniles in Lake otolith growth increments were not well-defined in the Biwa in March, 1996 and reared in a pond near the lake till hatchery-reared fish and one of the Lake Biwa specimens, 10 May, 1996. The collection sites in Lake Biwa were not the examination of daily growth increments and assign specified. The hatchery-reared juveniles were obtained ment of content data to them were not carried out in those from the hatchery of Gunma Prefectural Fisheries Ex fishes.

perimental Station on 11 May, 1996. They had been reared in artificial sea water (4 ppt salinity) from September 27, Results 1995 (hatching date) to January 1, 1996, and subsequently in spring-sourced freshwater until the sampling. The Ca Daily ages of the amphidromous and landlocked Lake and Sr concentrations of the artificial seawater were 51.45 Biwa ayu ranged from 189-243 d, averaging 223.3 d

ppm and 0.165 ppm, respectively, determined using induc (•} 16.6 SD), and 192-230 d, averaging 208.4 d (•} 13.7 SD), tively coupled plasma atomic emission spectrometry (ICP respectively. The average back-calculated birthdate of the AES), as described by Otake et al.13) Such concentrations Shinano river specimens was November 4, 1995, which con were one-eighth and one-fiftieth of natural seawater, re formed with the usual hatching period in the area (early spectively. The water temperature of the hatchery pond September to late November).15) On the other hand, the fluctuated between 13.0 and 22.0•Ž during the first month average back-calculated birthdate of Lake Biwa ayu was and between 13.0 and 18.0•Ž throughout the remaining November 19, 1995, which was outside the usual hatching period. The three types of ayu juveniles were thereafter period, i.e. late August to late October.7) This may have transported to ponds at Ueda Station of National resulted from undercounting of the daily increments of the Research Institute of Fisheries Science and maintained Lake Biwa ayu, because their outer otolith regions were there until they were sacrificed and the sagittal otoliths re distorted, possibly due to the long term rearing in the moved, on 14-16 June, 1996. Water temperatures of the pond, and outer daily increments were consequently un cast adjacent to the Shinano river (surface and 30 m clear in the ground otoliths. deep) and the river mouth (surface and 8 m deep) were X-ray intensity maps (Fig. 1) showed that otolith Sr con provided by Niigata Prefectural Inland Water Fisheries Ex tent changed dramatically along the life history transect in perimental Station. Water temperature data for Lake Biwa amphidromous ayu, contrasting with the Sr content in (surface and 30 m deep) were provided by Shiga Prefectur Lake Biwa ayu, which were at much lower levels and un al Fisheries Experimental Station. Water temperatures to derwent only small changes. Ca content was relatively con which the three types of ayu juveniles were subject just be stant throughout the otoliths of both groups. Sr:Ca ratios fore transportation to the ponds at Ueda Station were in the otoliths of the amphidromous ayu increased just out 11.0•Ž, 13.6•Ž and 15.0•Ž, respectively. The water tem side the core and remained high until the portion 220-400 peratures of the later ponds fluctuated between 14.4 and ,ƒÊm from the otolith center (Fig. 2), which corresponded 17.3•Ž during the final rearing period (mid May-mid to age 90-110 d (Fig. 3). The ratios subsequently decreased June). outward to the edge, rapidly outer the last 120-190 ƒÊm. After measuring the body length, sagittal otoliths were The timing of the rapid decrease in the ratios correspond removed from each fish and stored in 99% ethanol until ed to age 160-200 d. The ratio averaged 8.18 •~ 10-3 their preparation for electron microprobe analyses of the (•}0.45 •~ 10-3 SD) in the high "plateau" and 2.19 •~ 10-3 Sr:Ca ratios using a wavelength dispersive X-ray electron (•}0.60 •~ 10-3 SD) on the edge of the otolith. On the con microprobe (JEOL JXA-733), as described by Otake et trary, otolith Sr:Ca ratios of Lake Biwa and hatchery al 13,14) Each otolith was embedded in epoxy resin (Struers, reared ayu were very low and did not show distinct Epofix), mounted on a glass slide and then ground to re changes along the life-history transect (Figs. 2, 3), as veal a sagittal section through the core of the otolith. The shown in the X-ray intensity map. In otoliths of the Lake surface was polished further with 1 ,ƒÊm diamond paste, Biwa ayu, the ratios averaged 1.75 •~10-3 (•}0.56 •~ 10-3 cleaned in an ultrasonic bath and rinsed with deionized SD) overall, with a slight increase between 200 and 400 ƒÊm water. The surface of the polished otolith was coated with from the core, which corresponded to age 70-150 d. The carbon by high vacuum evaporation. All otoliths were ratios of the inner region from the center to 200-400, ƒÊm used for "life-history transect" analyses of Ca and Sr con averaged 1.29 •~ 10-3 (•}0.53 •~ 10-3 SD) and increased to tent, executed at 5 ,ƒÊm intervals across the longest axis of 2.02 •~ 10-3 (•}0.60 •~ 10-3 SD) in the outer region. The ra the otolith from the center to the edge. The accelerating tios of hatchery-reared ayu averaged 0.69•~ 10-3 voltage and beam current were 15 kV and 7 nA, respec (•}0.33 •~ 10-3 SD). These findings suggested that otolith tively. The electron beam was focused on a point ca. 5 ƒÊm Sr:Ca ratios in ayu fluctuated according to ambient water in diameter. Each value presented was the average of three salinity or a large seawater-freshwater gradient in Sr con counts (each counting time: 4.0 s). centration.12) Following microprobe analyses for Sr and Ca content, The ratios of the otolith core region, reflecting the daily growth increments of each otolith were observed un spawning ground environment, averaged 7.37 •~ 10-3 der a light microscope with a camera lucida at •~ 500 mag (•}2.57 •~ 10-3 SD) in amphidromous ayu, which was nification. Microprobe measurement points were assigned much higher than those of the Lake Biwa to every group of ten otolith daily increments from the (1.56 •~ 10-3•}0.42 •~ 10-3 SD) and hatchery-reared ayu hatch check. The averages of content data included in the (1.26 •~ 10-3•}0.44 •~ 10-3 SD) (Fig. 3). The ratio in the am distance covered by each pool of ten increments were re phidromous ayu should be similar to those in the other garded as representative of each ten growth increments two types of ayu, since all ayu spawn in freshwater. The and nsed for the "life-history trancect" analyses.Since difference seems to have been caused by the otolith grind Otolith Sr:Ca Ratios in Ayu 519

A. Amphidromous B. Lake Biwa

Fig. 1. X-ray intensity maps of Sr (upper) and Ca (lower) contents in otoliths from amphidromous (68 mm TL, A) and landlocked Lake Biwa ayu

(73 mmTL, B). The red region in the Sr content map of amphidromous ayu, indicating relatively high Sr content, occurs between hatching and 120 d. Scale bar=200 ƒÊm.

Fig. 3. Profiles of Sr:Ca ratios from the center to the edge of otoliths Fig. 2. Profiles of Sr:Ca ratios filtered with 8-point running mean from amphidromous and Lake Biwa ayu. values from the center to the edge of otoliths from three types of Each datum point represents the mean value of data for each 10 ayu. successive increments. 520 Otake and Uchida

migration pattern, indicating that the former reconstruct the migration history of individuals from seawater area to freshwater, via brackish water in the river mouth. Otolith Sr:Ca ratios of Lake Biwa and hatchery-reared ayu (non-amphidromous ayu) showed no marked fluctua tions in the life-history transect. The ratio levels in otoliths of hatchery-reared ayu were a little lower than those of Lake Biwa ayu, although the former were exposed to a higher salinity (4 ppt). This may reflect the considerably lower level of Sr in the artificial seawater (0.165 ppm: one fiftieth of natural seawater). A relationship between otolith Sr:Ca ratios and ambient salinity (or Sr concentration) has been ascertained in vari ous fishes, in particular anadromous fishes such as sea trout (Salmo trutta),10) striped bass (Morone saxatilis)9,11) and American shad (Alosa sapidissima),12) and has been used for understanding migration history or distinguishing between anadromous and non-anadromous populations. The present study ascertained that otolith Sr:Ca ratios of ayu closely reflected individual histories of amphidromy and clearly discriminated between amphidromous and non-amhidromous stocked fish including both landlocked and hatchery-reared forms. The ratio of the otolith region corresponding to the sea life period in amphidromous ayu, i. e. from the center to 220-400ƒÊm, representing the age from hatching to 90-110 d, is a key for distinguishing be tween amphidromous ayu and other, non-amphidromous Fig. 4. Profiles of Sr:Ca ratios from estimated birthdate to day of populations. This otolith microchemical technique should sacrifice and ambient water temperatures in Amphidromous (A) and make possible the plausible analysis of the contribution of Lake Biwa ayu (B). stocked fishes to overall spawning and fertilization and the Dotted lines indicate water temperature. possibility of interbreeding, which are important factors when assessing the impact of stocking on the ecology of natural amphidromous ayu populations. ing conditions, i.e. the area of the core exposed on the Acknowledgments We thank Dr. G. S. Hardy, Thames, New Zealand, ground surface was narrow, a 5,ƒÊm beam diameter for for his critical reading of the manuscript, and Dr. T. Ishii, National In EPMA analysis therefore being large enough to include stitute of Radiological Sciences, for his assistance in measuring the Sr con data from the adjacent region of the core corresponding to centration of water samples by ICP-AES. We are also grateful to Niigata the sea life period. The large standard deviation for the ra Prefectural Inland Water Fisheries Experimental Station, Shiga Prefec tios of the core region in the amphidromous ayu otoliths tural Fisheries Experimental Station and Gunma Prefectural Fisheries Ex seems to indicate the variety of the exposed core area in the perimental Station for their providing samples and data of water tempera surface. tures. This study was supported in part by Grants-in Aid from the Minis Figure 4 shows the relationship between ambient water try of Education, Science, Sports and Culture of Japan (No. 09660194) and from the Ministry of Agriculture, Forestry and Fisheries of Japan. temperature and otolith Sr:Ca ratios of amphidromous

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