Animals

Monthly surveys were made between 1997 and 1998 at five survey sites near Sapporo, named Bankei 1, Bankei 2 (100 m elev.); Teine (200 m elev.), Jozankei (800 m elev.) and Asari (1000 m elev.), where H. retardatus breeds (Fig. 1). In order to document the spawning season and larval growth and development, embryos and larvae were collected and measured every month from April 1997 to November 1998. Because the survey sites were completely covered with snow during the winter season, surveys were not done from December to next March. Water temperature of the ponds or pools 5–10 cm under the surface, where almost all egg sacs were spawned, was measured. Developmental stages were determined according to the normal table for H. nigrescens (Iwasawa and Yamashita, 1991). Measurements of snout-vent length (SVL) were made from the tip of the snout to the anterior corner of the cloaca using a slide caliper to the nearest 0.05 mm. Data were expressed as means±SD. For statistical analyses, Student's t-tests was utilized after Schaffe's F test for variance. The significance level was at 0.05. Four to 6 larvae each were collected and used for laboratory studies.

Fig. 1

Locations of the breeding sites of Hynobius retardatus. Approximate elevations are 100 m at Bankei 1 and Bankei 2, 200 m at Teine, 800 m at Jozankei and 1000 m at Asari.

Temperature effects on metamorphosis

In order to know effects of temperature on the metamorphosis of H. retardatus, degrees of morphological metamorphosis were analyzed in larvae which had been reared at low temperature (4°C) and then transferred to 18°C. Twenty four newly hatched larvae, which had been hatched from a pair of egg sacs collected at Bankei 2, were cultured at 4°C at first. Six larvae each were then transferred to 18°C at 30 days intervals. SVL and the maximal tail height (TH) including tailfins were measured at every 10 days, and a proportion of SVL to TH (SVL/TH) was calculated. Because the SVL and TH similarly increased before the metamorphosis, the proportion of SVL to TH did not change (SVL/TH was approximately 3.0). During the metamorphosis, however, the proportion of SVL to TH gradually increased because of a gradual decrease in TH, and reached finally the level of 6.0–6.5 at the end of the metamorphosis.

Electrophoresis

Four to 6 animals from each collection were anesthetized in an aqueous solution of MS222 (Sandoz) and then they were bled in ice-chilled 50% PBS with 10 mM EDTA, either by puncturing the heart or cutting the tail. Procedures for preparation of hemolysates were previously described (Arai and Wakahara, 1993; Wakahara et al., 1994). After the amount of protein in each hemolysate was determined using BCA Protein Assay reagent (Pierce Chem Co.), each sample from individual animals was electrophoresed or frozen at −80°C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970), using 15% separating gels. All electrophoresed gels were stained with Coomassie Brilliant Blue and then photographed.

Breeding habitats

Geographical and ecological characteristics of each survey site (Fig. 1) were as follows.

Bankei 1: A transitory pool of water formed by melted ice and water which totally or partially dried up by autumn. At the breeding season (early April), the size of the pool was 7 m × 20 m in diameter with approximately 1.5 m depth. It was located by a side of a car road, and surrounded by bushes and small trees planted on steep slope of a mountain in the other side. The earliest, first spawning was observed on 30th, March in 1994 and the latest one on 9th, April in 1992 during recent 7 years (Table 1). Seasonal water temperatures did not exceed 20°C even in the mid summer (Fig. 2). Whereas this pond completely dried up by September in 1997, it did not dry up throughout the year in 1998, probably because of a lot of rainfalls in autumn of this year.

Table 1

Dates of the earliest spawning or spawned egg sacs found of Hynobius retardatus in different breeding sites during recent 7 years*, **.

Fig. 2

Yearly fluctuations in water temperatures from April 1998 to November1998 in the ponds of Bankei1, Bankei 2, Teine, Jozankei and Asari. Water temperatures were measured at 10:00 to 10:30 am at Bankei 1, Bankei 2 and Teine, and at 11: 00 to 11:30 am at Jozankei and Asari. The water temperature at Jozankei in April was not measured, accidentally. Because Asari in April was covered with snow, the water temperature could not be measured.

Bankei 2: An artificial pond originally dwelled as a fishing pond which had been abandoned about 30 years ago. The size of this pond was 20m×30 m in diameter with about 2.0 m depth. This pond never dried up throughout the year, even though there was no water supply by a stream or a spring. The earliest, first spawning was observed on 10th, April in 1995 and the latest one was presumed to be 5 or 6 days before 25th, April in 1996, when we found gastrula embryos within egg sacs which had previously been spawned. The seasonal water temperatures were considerably higher than in Bankei 1 throughout the year, even though both ponds are at the same altitude of 100 m, probably because of no water supply in Bankei 2.

Teine: A small (5 m×8 m) and shallow (approximately 0.5 m in depth) pond with an influx of melted snow and a spring water, situated by a path through a forest on the foot of Mt. Teine. Although this breeding site was very small in size and shallow in depth, water never dried up throughout the year, due to constant supply of a spring water. The earliest, first spawning was presumed to be 3 or 4 days before 21st, April in 1998 and the latest one on 13th, May in 1997. The seasonal water temperatures did not exceed 17°C even in the summer.

Jozankei: A relatively small (6 m×9 m) and shallow (about 1.0 m in depth) pool with an influx of water from melted snow and a stream through mountains, situated by a path surrounding an artificial lake for water supply service, Lake Sapporo. Similarly to Teine, the water never dried up throughout the year. The earliest, first spawning was observed on 29th of April, 1998, and the latest one was presumed 3 or 4 dyas before 22nd of May, 1997. Maximal water temperature was approximately 12°C during the summer season (from July to early September).

Asari: A relatively large (8 m×25 m) and deep (about 2.0 m in depth) pond located near the highest point on the Asari Pass, with an influx of water from melted snow and a mountain stream, surrounded by bushes and trees on mountainous land. The earliest, first spawning in this pool was presumed to be 3 or 4 days before 8th of May, 1998 and the latest one was 3rd of June, 1996. Water temperature was similar to Jozankei.

Larval growth in different habitats

Fig. 3 shows patterns of growth in SVL in larval H. retardatus collected from the 5 survey sites described above. In Bankei 1 (Fig. 3A) and Bankei 2 (Fig. 3B), newly hatched larvae were observed in mid May. They gradually grew during the summer season, and metamorphosed by August (Bankei 1) or September (Bankei 2), when no larvae were collected. It was confirmed that all larvae in Bankei 1 and Bankei 2 metamorphosed by autumn during recent 7 years. In these sites, only larvae with similar size (i.e., the same age) were observed throughout the collection times. Earlier metamorphosis was observed in Bankei 1 where the water dried up completely by August (1997) or partially by October (1998), than in Bankei 2 where the water never dried up throughout the year, though the water temperature in Bankei 1 was lower than in Bankei 2 (Fig. 2).

Fig. 3

Patterns of growth in SVL in larval H. retardatus in different habitats. (A) Bankei 1, (B) Bankei 2, (C) Teine, (D) Jozankei and (E) Asari. Whereas all hatched larvae metamorphosed until September or October of the year in Bankei 1 and Bankei 2, one-year (Teine) or two-yearoverwintered larvae (Jozankei and Asari) were observed and metamorphosed in the second or third years. Data express means ±SD. Letters represent sample sizes. Open and closed rectangles indicate approximate time of spawning and of metamorphosis at each breeding site, respectively.

Contrary to these, large larvae were observed even in the breeding season (early May) at Teine, where embryos within egg sacs were concurrently encountered (Fig. 3C). They must be overwintered larvae which had been developed from egg sacs spawned in the year before. Those large larvae could not be observed in the next month, probably because they metamorphosed and moved from aqueous to terrestrial habitats. Newly hatched larvae were observed in June. They grew during the summer season, but could not metamorphose by late autumn when the water temperature drastically fell (Fig. 2). They were full grown larvae (stage 63) of 29.7±2.9 mm in SVL in 1997. They might hibernate during the winter season in the muddy ground under snow, and then metamorphosed in the next year.

Two types of overwintered larvae, large and medium, were observed even in the breeding season (late May to early June) at Jozankei and Asari, indicating the presence of 2-year-overwintered larvae (Fig. 3D, E). Newly hatched, small larvae were observed at mid June (Jozankei) and in July (Asari). They grew during the first summer season, but could not metamorphose by late autumn (stage 54–62, SVL=19.7±3.0 mm at Jozankei in 1997; stage 55–62, SVL=20.3±3.4 mm at Asari in 1997),. They might hibernate during the winter season, and thus became wintering larvae. Although the 1-year-overwintered larvae (i. e., the medium sized larvae observed in the breeding season) grew during the second summer season, they could not metamorphose by late autumn (stage 63, SVL=28.6 ±1.9 mm at Jozankei in 1998; stage 63, SVL= 34.7±0.7 mm at Asari in 1998), and thus might hibernate again until next spring. The 2-year-overwintered larvae (i. e., the larger larvae observed in the breeding season) metamorphosed during the summer season in the third year. In these cases, we could collect three age-groups of H. retardatus; eggs or early embryos within egg sacs and two types of overwintered larvae, large (2-year-old) and medium (1-year-old), concurrently.

Body sizes just before the completion of the metamorphosis were significantly different among the three age-groups; 2-year-overwintered observed at Jozankei and Asari, 1-year-overwintered larvae observed at Teine, and larvae under the age of one observed at Bankei 1 and Bankei 2 (Table 2). In Asari and Jozankei, average SVL was 32.6±1.1 mm, and 32.8±1.8 mm, respectively. Contrary to these, average SVL was 28.1±1.8 mm in Bankei 1, and 29.5±2.4 mm in Bankei 2. Although statistical differences between SVLs of the larvae under the age of one and 1-year-overwintered larvae were not significant (p >0.05), those between the larvae under the age of one and 2-year-overwintered larvae, and between 1-year-overwintered and 2-year-overwintered larvae were significant (p <0.01), respectively.

Table 2

Comparisons of snout-vent length (SVL in mm) in larvae just before the completion of the metamorphosis in Hynobius retardatus of different ages at 5 different survey sites.

Effect of temperature on metamorphosis

The effect of temperature on the metamorphosis of H. retardatus was examined under laboratory condition by transferring larvae from 4°C to 18°C. Fig. 4 shows changes in the proportion of SVL to tail height (SVL/TH) in larvae which have been reared initially at 4°C and then transferred to 18°C. As long as the larvae were reared at 4°C, they did not show any sign of the morphological metamorphosis (Arai and Wakahara, 1993; Moriya, 1979; 1983a), and thus the SVL/TH remained unchanged at the fixed level (approximately 3.0). When larvae were reared at 18°C just after their hatching, they showed initial sign of the metamorphosis after 40 days, and completed their metamorphosis at 70 days of rearing. When the larvae were transferred from 4°C to 18°C at 30 days after hatching, they showed initial sign of the metamorphosis at 70 days of rearing (i.e., 40 days after the transfer to 18°C). Contrary to these, when the larvae were transferred from 4°C to 18°C at 60 and 90 days after hatching, respectively, initiation of the metamorphosis became a little earlier: they showed initial sign of the metamorphosis at 30 (transferred to 18°C at 60 days after hatching), and 20 days (transferred to 18°C at 90 days after hatching), after the transfer.

Fig. 4

Effects of temperature on metamorphosis. Newly hatched larvae were placed at 4°C at first, and then 6 larvae each were transferred to 18°C at the intervals of 30 days. Snout-vent length (SVL) and tail height (TH) were measured every 10 days until they completed the metamorphosis. Data expressed means±SD.

Hb transition in overwintered larvae

Fig. 5 shows chronological changes in the transition of globin subunits from larval to adult types in the populations of Bankei 2 and Jozankei. Typical larval globins were separated into 2 bands (L1 and L2) on SDS-PAGE, whereas adult globins were shown to be composed of 3 fractions (A1 to A3)(Arai and Wakahara, 1993). In the Bankei 2 population, larval globin subunits were predominantly observed in larvae in May and June. Adult globin subunits gradually appeared during the summer season (Fig. 5A). Even at the completion of the metamorphosis (Stage 68; Fig. 5A, lane S1), however, larval globins were faintly observed. Contrary to this, in the Jozankei population, the larvae showed typical larval pattern of globin subunits until the autumn season of the first year (stage 58; Fig. 5B, lane O1). In June and October of the second year, adult globin subunits were detected. In May of the third year (stage 63; Fig. 5B, lane M3), adult globins were predominantly detected, showing the Hb transition completely finished even though the larvae did not complete their metamorphosis.

Fig. 5

Chronological changes in hemoglobin (Hb) transition from larval to adult types in larvae from different habitats. SDS-PAGE were stained with Coomassie Brilliant Blue. A, Hbs of larvae from Bankei 2 at various months of the year (M1, May, stage 48; J1, June, 58; J1, July, 63; A1, August, stage 65; S1, September, stage 68). B, Hbs of larvae from Jozankei at various months of the first (J1, June, stage 49; O1, October, stage 58), second (J2, June, stage 62; O2, October, stage 63) and third (M3, May, stage 63) years. TL, typical larval Hb; TA, typical adult Hb.

Timing of metamorphosis

In the natural habitats of H. retardatus, duration until the completion of the metamorphosis varies according to their different environments, probably depending on water temperatures, differential stability of water level and other factors such as levels of food supply. In ponds where the water temperatures during summer season are relatively high (Bankei 2) or water of the pond dries up partially or totally by autumn (Bankei 1), larvae of H. retardatus metamorphose within a year (Fig. 3A, B). Contrary to these, larvae in a pond where the water is supplied continuously throughout the year (Teine) cannot metamorphosed within a year (Fig. 3C), even though the water temperatures are not different from Bankei 1 (Fig. 2). These facts suggest that the stability of water level is one of the important factors affecting the timing of metamorphosis in H. retardatus, as known in many amphibians in the temporary pool where the larvae or tadpoles are reported to metamorphose before the water has dried up (Newman, 1988; Sprules, 1974b; Travis, 1983). In spadefoot frog (Scaphiopus couchii) living in desert ponds, there was no evidence for genetic variation in plasticity of development; all sibship exhibited faster development and decreased larval period in ponds of short duration (Newman, 1988). In this respect, Denver (1998) recently reported that tadpoles of S. hammondii transferred to a reduced water condition exhibited significant metamorphic changes by 48 hrs after the transfer and preceding elevations of whole-body T₄ and T₃ contents. Endocrinological surveys are absolutely necessary to clarify the physiological mechanisms that underlie the variable larval life histories observed in H. retardatus living at different habitats.

Because different larval life histories can influence post-metamorphic life history traits and adult fitness (Kalezic et al., 1996; Smith, 1987; Sprules, 1974a), studies on the causes and consequences of such variations are crucial. In this respect, Misawa and Matsui (1997) have recently reported marked differences in growth and development of larvae of Kyoto and Tokyo populations of the stream breeding salamander H. kimurae. Differences seen in larval life histories between the two populations may represent adaptations for differential stability of water level and water temperature, and resultant differences in the size of metamorphs possibly induce differential adult body size, and hence fecundity (Misawa and Matsui, 1997). Larger body size just before the completion of metamorphosis in 2-year-overwintered larvae than in larvae under the age one (Table 2) in H. retardatus suggests an adaptive significance of the overwintered larvae in H. retardatus, because larger body size would be profitable in survivorship after their metamorphosis, and thus in fecundity. In order to elucidate the biological significance of the different larval life histories reported here, detailed examination on gonadal development of the two-year-overwintering and one-year-overwintering larvae and the larvae under the age one at their metamorphosis is now in progress.

In the facultatively neotenic salamander, Ambystoma talpoideum, the primary advantages of earlier maturation are increased survival to first reproduction and shortened generation times (Ryan and Semlitsch, 1998). At present, however, there are no data available on possible differences in the maturation times in the different populations of H. retardatus. The maturation time, fecundity, egg size, clutch size, and inclusive fitness in different populations in this animal, are the problems of the next generation to be solved.

Effect of temperature on metamorphosis

The most marked environmental differences between Teine where 1-year-overwintered larvae were observed (Fig. 3C), and Asari and Jozankei where 2-year-overwintered larvae were encountered (Fig. 3D, E) could be temperatures, but not stability of water level. Thus, it is assumed that a limiting factor to cause 2-year-overwintered larvae is lower water temperatures during the summer seasons at Asari and Jozankei. This assumption is consistent with previous observations that the duration until the completion of the metamorphosis in laboratory conditions basically depended on temperature (Arai and Wakahara, 1993; Moriya, 1979; 1983a). Substantial dependency of the timing of metamorphosis on temperatures was confirmed by our laboratory experiments (Fig. 4). There are several possible explanations for the delay or blockage of metamorphosis by low temperature: the synthesis or release of TSH and/or thyroid hormones may be inhibited, or the binding of the hormone to the receptor or the subsequent reactions in the target organs may be suppressed. Moriya (1983a, b) has shown that 1) the thyroid gland was completely inactive at low temperature such as 4°C, 2) tissues could not respond to externally administered thyroid hormones, either by immersion or injection, and 3) prolactin (PRL), a growth promoting hormone in larval amphibians, affect even at 4°C.

Although larvae of H. retardatus could not reach the metamorphic stages as long as they were placed at 4°C (Arai and Wakahara, 1993), they grew very slowly and their development proceeded to a certain degree. This is consistent with the results of our transfer experiments from 4°C to18°C, showing that the duration until the initiation of the metamorphosis became shorter when the larvae were placed longer at 4°C (Fig. 4).

Sprules (1974b) reported that low temperature and reduced food increased the incidence of neoteny in A. gracile. In his report, well-developed testes were observed in untransformed larvae that were cultivated for a long time at low temperature. The observation suggests that the endocrine system of reproduction would be more functional than the endocrine system of metamorphosis at low temperature. In H. retardatus, however, the larvae reared at low temperatures for prolonged duration did not show any sign of gonadal maturation (4°C for 3 years, Wakahara, unpublished observation; 6°C for 6 month, Yamashita et al., 1991). Thus, it seems premature to assume that an experimental induction of neoteny in this species is possible by rearing the larvae in lower temperature.

Morphological metamorphosis vs biochemical metamorphosis

In 2-year-overwintered larvae observed in Asari and Jozankei, Hb transition from larval to adult types has been almost completed (Fig. 5B), even though they do not compete their metamorphosis. This is consistent with the observations that the Hb transition was completed even in metamorphosis-arrested larvae which have been either treated with goitrogens (Arai and Wakahara, 1993; Wakahara and Yamaguchi, 1996) or thyroidectomized (Satoh and Wakahara, 1997, 1999). Furthermore, because the Hb transition was completed in 4-year-old larvae reared at 4°C (unpublished observation) at which temperature the larvae can not metamorphose at all (Moriya, 1983a), either appearance of the adult globins or disappearance of the larval ones is independent of the morphological metamorphosis. The similar independence of the Hb transition has been reported in the axolotl in which globin subunits changed completely to adult types without morphological metamorphosis (Ducibella, 1974).

The separation of the morphological metamorphosis and biochemical “metamorphosis” such as changes in nitrogen excretion system from ammonotelism (larval type) to ureotelism (adult type) has been reported in H. retardatus (Wakahara et al., 1994). It is thus possible that certain biochemical aspects of “metamorphosis” such as the Hb transition and others occur autonomously without thyroid activity and are chronologically regulated by different mechanisms from those which induce morphological metamorphosis. Furthermore, we have recently reported that the epidermis behaves differently from dermis during the metamorphosis in H. retardatus, even though both tissues constitute the same skin (Wakahara and Yamaguchi, 1996; Ohmura and Wakahara, 1998). These heterochronic phenotypic expressions or development will be fundamental causes of the reported neoteny in this species (Wakahara, 1996). Because the larvae can produce morphologically mature spermatozoa when their metamorphosis was arrested by either thyroidectomy or immersion in goitrogenic solution (Kanki and Wakahara, 1999; Wakahara, 1994; Yamaguchi and Wakahara, 1996), it seems true that every population of H. retardatus other than the extinct population in Lake Kuttara has latent faculties of a neotenic reproduction.