Kazumasa Ikuta

Effects of sex steroids on the smoltification of masu salmon, Oncorhynchus masou

Testosterone (T; 25-micrograms/g diet), estradiol-17 beta (E2; 2.5-, 25-micrograms/g diet), and 5 alpha-dihydrotestosterone (DHT; 2.5-, 25-micrograms/g diet) were administered to yearling masu salmon from February 3 through May 8. T and E2 inhibited the natural smoltification in spring and stimulated gonadotropin (GtH) accumulation in the pituitary, but DHT had no effect. T (25-micrograms/g diet) was administered to underyearling masu salmon from September 30 through December 14. At the end of the T treatment, fish body color darkened and pituitary GtH content increased in the T-treated group. On May 8 in the next spring, however, plasma T levels and pituitary GtH contents already decreased and smoltification occurred in both control and T-treated groups. On October 29, underyearling masu salmon reared under the short daylength (8 L:16 D) for 4 months were switched to a long daylength (16 L:8 D) and simultaneously administered with T (25-micrograms/g diet) or 11-ketotestosterone (11-KT; 25-micrograms/g diet). On December 17, the proportion of smolts was 52.8% in the control, whereas T and 11-KT administration inhibited smoltification under the artificially increased daylength. Pituitary GtH content significantly increased both in the T-treated and 11-KT-treated groups, though it was much lower in the latter. These results indicate that the springtime smoltification which is induced by increasing daylength is inhibited by biologically active sex steroids such as E2, T, and 11-KT.

Effects of thyroxine and methyltestosterone on smoltification of masu salmon (Oncorhynchus masou)

Abstract Administration of thyroxine (T 4 ; 250 μg/g diet) from 16 July to 24 December induced silvery skin in underyearling masu salmon, but failed to induce seawater adaptability, darkening of dorsal and caudal fin margins and slimness of the body. There was no difference in the percentage of precocious males in autumn between T 4 -treated and untreated groups. Administration of methyltestosterone (MT; 2.5 μg or 25 μg/g diet) from 10 February to 27 April inhibited natural smoltification both in T 4 -pre-treated and untreated yearling masu salmon. Fish treated with MT resembled parr or dark parr, and seawater tolerance did not develop. In contrast, approximately 90% of the fish in the control group became smolts. Increases in body length and weight were greatest in the control group and MT 2.5 μg group, respectively. Condition factor decreased in control fish but remained unchanged in the groups treated with MT. The MT 25 μg group showed the slowest growth. A dose-dependent decrease in plasma T 4 level was observed in the MT-treated groups. On the other hand, a dose-dependent increase was found in plasma and pituitary GtH content.

The involvement of sex steroid hormones in downstream and upstream migratory behavior of masu salmon.

From May through July when masu salmon, Oncorhynchus masou, commence downstream migration under natural conditions, yearling precocious male masu salmon (resident form) showed higher GSI and plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) in contrast to immature smolts (migratory form). From March through September coinciding with the upstream migration period, 2-year-old male and female adults also showed higher GSI and plasma levels of T, estradiol-17beta (E(2)) 11-KT, 17alpha-hydroxyprogesterone and 17alpha,20beta-dihydroxy-4-pregnene-3-one (DHP). In order to test the effects of steroid hormones on migratory behaviors, silascone tube capsules containing 500 microg of T, E(2), 11-KT, DHP, or a vehicle was implanted into smolts, castrated precocious males, or immature parr, and downstream and upstream behavior were observed in artificial raceways in spring and autumn. Downstream behavior of smolts was inhibited significantly by T, E(2) and 11-KT. Upstream behavior was stimulated by T and 11-KT in castrated precocious males and stimulated by T, E(2) and 11-KT in immature parr. These results indicate that T, E(2) and 11-KT are the factors regulating downstream and upstream migratory behavior. In particular, because of its changing patterns in plasma and significant effects, T, the common precursor hormone of E(2) (female) and 11-KT (male), is considered to play central roles in both types of behavior.

Ontogenic development of salmon GnRH and chicken GnRH-II systems in the brain of masu salmon (Oncorhynchus masou)

Abstract Ontogenic development of salmon gonadotropin-releasing hormone (GnRH) and chicken GnRH-II systems in masu salmon (Oncorhynchus masou) was examined. Salmon GnRH was first detected by radioimmunoassay in the embryo on day 36 after fertilization. Salmon GnRH-immunoreactive fibers were detected initially by immunocytochemistry in the vicinity of the olfactory placode of the embryo (day 36) and were distributed widely in the brain as well as in the pituitary gland of fish just after hatching (day 80). Salmon GnRH-immunoreactive neuronal somata were first detected about 6 months after fertilization in the rostroventral brain area, ranging from the olfactory nerve to the preoptic area. Salmon GnRH neuronal somata were detected earlier by in situ hybridization than by immunocytochemistry. Neuronal somata expressing salmon GnRH mRNA were first detected in the vicinity of the olfactory epithelium on day 40 and then were seen to be migrating from the olfactory epithelium, along the olfactory nerve, on day 60 and in the transitional area between olfactory nerve and olfactory bulb on day 80. In the brain, these neurons were first detected in the ventral olfactory bulb on day 80, and thereafter they were also detected in the caudal brain regions. The chicken GnRH-II system was detected later than the salmon GnRH system; chicken GnRH-II was first detected by radioimmunoassay on day 57, and chicken GnRH-II-immunoreactive fibers were first detected on day 67. Chicken GnRH-II-immunoreactive neuronal somata were not detected during the observation period. These results suggest that salmon GnRH neurons derive from the olfactory placode and then migrate into the brain and that salmon GnRH is synthesized before chicken GnRH-II.

Disturbance of plasma melatonin profile by high dose melatonin administration inhibits testicular maturation of precocious male masu salmon.

Abstract We have previously shown that the testicular development of underyearling male masu salmon Oncorhynchus masou reared under a long photoperiod was accelerated by oral melatonin treatment (0.5mg melatonin/kg body weight/day), suggesting that melatonin mediates photoperiodic signaling. In this study, we further examined the effects of a disturbance in the plasma melatonin profile on gonadal development in underyearling male masu salmon by administering a higher dose of melatonin. Fish randomly selected in June were divided into two groups. They were reared under a light:dark (LD) cycle of 16:8 (lights on 04:00–20:00 hr) and fed with pellets sprayed with melatonin or vehicle twice a day at 08:30 and at 15:30 hr (7.5mg melatonin/kg body weight/day) until October. Fish were sampled on Day 0, 25, 60, 90 and 120. The plasma melatonin levels were high in the dark phase and low in the light phase in the control group, while they were constantly high with no significant change in the melatonin-treated group. Melatonin treatment had inhibitory effects on the gonadosomatic index and plasma testosterone levels. Pituitary salmon gonadotropin-releasing hormone content and luteinizing hormone content were significantly lower in the melatonin-treated group on Day 60 and 90, respectively. These results indicate that the plasma melatonin profile is important for mediating photoperiodic signals that regulate brain-pituitarygonadal axis in underyearling precocious male masu salmon.

Effects of photoperiod on smolting and hormonal secretion in masu salmon, Oncorhynchus masou

Abstract Underyearling masu salmon were reared under a 8L:16D photoperiod for approximately 3 months, and subsequently transferred to 8L, 10L, 11L, 12L, 13L, 14L or 16L photoperiod in October, in order to determine the suitable photoperiod for induction of smolting (Experiment 1). Smolt was judged by the external appearance of fish, i.e., silvery body color, black margin on the dorsal and caudal fins, and slim body shape. As early as 1 month following this transfer, smolts appeared in the 16L group. After 2 months, the 13L group had the highest frequency of smolts. After 3 months, most smolts were found in the 11L group and presmolts appeared even under constant short daylength (8L:16D). Overall, the largest number of smolts appeared in the group transferred from 8L:16D to 13L:11D. Underyearling fish reared under natural photoperiod were also transferred to 11L, 12L, 13L, 14L, 15L or 16L photoperiod in October (Experiment 2). Only a few smolts appeared during this experiment. In order to determine a suitable short daylength for pretreatment prior to the transfer to long daylength, underyearling masu salmon were reared under 6L, 7L, 8L, 9L, 10L, 11L, 12L and 13L photoperiods for approximately 5 months, and subsequently transferred to a 13L:11D photoperiod in October (Experiment 3). One month after the transfer, a small number of smolts appeared in only the group transferred from 6L. After 2 months, a large number of smolts appeared in the groups transferred from 10L and downward. After 3 months, a small number of smolts newly appeared in the groups transferred from 12L and 11L. A few smolts appeared in the group under constant 13L after 6 months. Finally, effects of increasing daylength on the secretion of growth hormone (GH), thyroxine (T 4 ) and triidothyronine (T 3 ) were investigated (Experiment 4). Seven days after transfer from short (8L:16D) to long (13L:11D) daylength, plasma GH levels were significantly higher than those in the control group under constant 8L. Plasma GH levels tended to increase gradually in both groups throughout the experiment. Plasma T 4 and T 3 levels were not affected by increasing daylength. These results indicate that it is necessary to rear fish under short daylength for a sufficient period of time before daylength can be changed to long-day for purposes of artificial induction of smolting, and the larger the difference in daylength after changing photoperiod to long daylength, the earlier smolts appear. Furthermore, an increase in daylength may stimulate the transient secretion of GH, which may participate in the process of smolting.

Inhibitory effects of testosterone on downstream migratory behavior in masu salmon, Oncorhynchus masou.

Abstract The effects of testosterone (T) on downstream migratory behavior of yearling masu salmon, Oncorhynchus masou, were studied during its downstream migratory period using artificial raceways. In experiment 1, 22 and 19 smolts were implanted with a medical silicone tube capsules containing 500μg of T or vehicle only. These groups were transferred together to the upper pond of artificial raceway which was connected to the lower pond through fish-way. Downstream migratory behavior was then observed for 1 week. In experiment 2, T500μg-, T50μg-, and T5μg-treated smolts, control smolts, and precociously mature males were transferred to the upper pond of the raceway and downstream migratory behavior was observed for 2 months. In experiment 3, 40 smolts were implanted with capsules containing 500μg of T or vehicle only. These groups were transferred separately to the upper ponds of raceways and downstream migratory behavior was observed for 3 weeks. In each experiment, injection of T caused increases in plasma T levels within physiological levels. In experiment 1, frequency of downstream migratory behavior was 89.5% in the control group and 31.8% in the T500μg-treated group. In experiment 2, the frequency was lower in the T500μg- and T50μg-treated groups than in the control group. T5μg implantation failed to inhibit downstream migratory behavior. Precocious males were not observed to go down the raceway. In experiment 3, frequency of downstream migratory behavior of the control group and the T500μg-treated group was 100 and 40%, respectively. In each experiment, plasma levels of T in T500μg-treated smolts which did not show downstream migratory behavior was higher than those of migrants. These results indicate that the downstream migratory behavior of masu salmon smolts is inhibited by physiological levels of T.

Acidification severely suppresses spawning of hime salmon (land-locked sockeye salmon, Oncorhynchus nerka)

A laboratory study was conducted to investigate the effects of an acidic environment on the female sexual behavior of hime salmon (land-locked sockeye salmon, Oncorhynchus nerka). Spawning hime salmon were extremely sensitive to the acidity of ambient water. Nest-digging behavior was severely inhibited (P<0.05) by very slight acidification (pH below 6.4) of the water. Below pH 6.0, females showed almost no diggings. When the ambient water was returned to nearly neutral (pH 6.6), diggings reappeared in four of the six fish tested, whereas in two individuals, none was observed. Avoidance of slightly acidic water in selection of spawning site or cessation of spawning behavior in weakly acidic environments may be the most potent cause of the reduction of salmonid populations in the early stages of acidification.

Ontogenic origin of salmon GnRH neurons in the ventral telencephalon and the preoptic area in masu salmon

During the ontogeny of masu salmon Oncorhynchus masou, neurons producing the salmon type of gonadotropin-releasing hormone (sGnRH) were first detected in the olfactory epithelium of the eyed egg and, subsequently, in the brain, suggesting a migration of these cells. Among sGnRH neurons distributed from the olfactory nerve (ON) through the preoptic area (POA), those in the ventral telencephalon (VT) and the POA are indicated to regulate gonadotropin secretion. Thus, it is of interest to know whether all the sGnRH neurons originate from the olfactory epithelium. In the present study, we examined by in situ hybridization whether sGnRH neurons are present in the VT-POA of fish, whose olfactory epithelia including sGnRH clusters were cauterized just after hatching (44 days after fertilization). Fish were sampled in June (212 days after the operation). Neurons expressing sGnRH mRNA were detected in the VT-POA as well as in the ON, ventral olfactory bulb, and transitional area between the olfactory bulb and telencephalon (which is considered to correspond to the terminal nerve ganglion) in the control group. In contrast, neurons expressing sGnRH mRNA were not detected in the VT-POA in the olfactory epithelium lesioned (OEL) group. Furthermore, pituitary sGnRH content in the OEL group was just above the detectable limit and was significantly lower than that in the corresponding control group in both sexes. These results indicate that sGnRH neurons in the VT-POA are derived from the olfactory epithelium in masu salmon, although the possibility cannot be ruled out that sGnRH neurons in the VT-POA arise from the VT-POA, but were delayed in expressing sGnRH because of the trauma of cauterization.

Effects of Acidification on Salmonid Spawning Behavior

We studied the effects of acidification on female sexual behavior in brown trout (Salmo trutta) and compared the results with those in hime (land-locked sockeye) salmon (Oncorhynchus nerka) (Kitamura and Ikuta, 2000). The results were similar to those of sockeye salmon. Spawning brown trout were extremely sensitive to the acidity of ambient water, and nest-digging behavior was severely inhibited (p<0.05) by very slight acidification (pH below 6.4). However, there were some differences between the two species. Female trout and salmon showed almost no digging below pH 5.0 and 6.0 (Kitamura and Ikuta, 2000), respectively. When the ambient water was returned to nearly neutral (pH6.6) conditions, digging in hime salmon reappeared in 4 of the 6 fish tested (Kitamura and Ikuta, 2000), whereas digging in brown trout reappeared in all 6 fish tested. The above-mentioned differences in behavioral response between the two species appear to reflect the species difference in terms of vulnerability to acidification (Ikuta et al., 1992). Avoidance of slightly acidic water in selection of spawning site or cessation of spawning behavior in weakly acidic environments may be the most potent cause of the reduction of salmonid populations in the early stages of acidification.