the effect of propylthiouracil, triiodothyronine and environmental ... serum thyroxine (T^) and triiodothyronine (T3) fell from 31.73 ± 4.09 and 5.06 ± 0.70 nM in ...
Fish Physiology and Biochemistry vol. 8 no. 2 pp 167-177 (1990) Kugler Pubhcations, Amsterdam/Berkeley
Changes in serum thyroxine and triiodothyronine concentrations during metamorphosis of the Southern Hemisphere Lamprey Geotria australis, and the effect of propylthiouracil, triiodothyronine and environmental temperature on serum thyroid hormone concentrations of ammocoetes J.F. Leatherland', R.W. Hilliard^, D.J. Macey^ and l.C. Potter^ ^Department of Zoology, University of Guelph, Guelph, Ontario, Canada NIG 2 Wl; ^School of Biological and Environmental Sciences, Murdoch University, Murdoch, Western Australia 6150 Keywords: lamprey, metamorphosis, thyroid hormones, Geotria australis, environmental temperature, goitrogen Abstract Serum thyroid hormone concentrations were measured during the seven stages of metamorphosis (1-7) of the southern hemisphere lamprey, Geotria australis. The respective mean concentrations ± SEM of serum thyroxine (T^) and triiodothyronine (T3) fell from 31.73 ± 4.09 and 5.06 ± 0.70 nM in large ammocoetes sampled in February, at the time when metamorphosis was initiated, to 4.54 ± 0.36 and 1.03 ± 0.12 nM at stage 5. Although there was a small, but significant, recovery of serum T^ concentrations during stages 6 and 7, no such corresponding statistically significant rise occurred in serum T3 concentrations. Serum thyroid hormone concentrations in ammocoetes sampled during the period when metamorphosis was taking place, exhibited a marked seasonal increase between February and May-June (late autumn/early winter); serum T3 and T^ concentrations peaked in May-June and were, respectively, > 2 fold and > 8 fold higher than those recorded for samples in late February (mid summer). By mid-July the serum T4 and T3 levels had declined from the peak values. Ammocoetes taken from streams at 16°C in June and acclimated to aquaria water at 25°C or 6°C had significantly lower serum T3 and T^ concentrations at the higher temperature, and also a lower serum T4, but not T3 concentration, at the lower temperature. Treatment of separate groups of ammocoetes with either propylthiouracil or T3 for 70 days significantly depressed and raised respectively, the serum thyroid hormone and hepatic T3 concentrations and caused significant changes in the body weight, but did not induce the onset of metamorphosis. Introduction The thyroid hormones are generally considered to play important roles in the developmental processes of vertebrates (Norris 1985). In lower vertebrates, the thyroid hormones have been shown to play an integral part in such complex events as amphibian metamorphosis (Galton 1988), the development of larval teleost fish, and the part-smolt developmental changes associated with the preparation of
young salmonid teleosts for their seaward migration (see Discussion for references). The metamorphosis of the parasitic species of lampreys, such as Geotria australis, from sedentary, filter-feeding ammocoete to the more active, and essentially predatory, adult involves major physiological and morphological changes (Youson 1980; Potter et al. 1982) which are comparable to those exhibited during amphibian metamorphosis and larval development of teleosts. Since the initiation
168 of the transformation of the endostyle to form a true (follicular) thyroid gland in lampreys occurs during the early stages of lamprey metamorphosis (Wright and Youson 1976, 1980a; Youson 1988), it has been tempting to propose a regulatory role for the thyroid in this process (see Youson 1980, 1988). However, until recently, most studies in lampreys have relied on histological criteria to assess changes in thyroid function, and conclusions drawn from such work in lampreys (and other vertebrates) have been suspect. With the advent of sensitive radioimmunoassays (RlAs), which enable the thyroid hormones to be measured in tissues and small volumes of serum or plasma, it has been possible to employ more direct measures of thyroid gland activity. Such studies have been applied systematically to only one species of lamprey, namely Petromyzon marinus, representing the single holarctic family Petromyzontidae (Wright and Youson 1977; Lintlop and Youson 1983a). Those studies, which used animals collected from the field and retained in captivity under controlled photoperiod and temperature regimes, provided evidence of a marked fall in serum thyroxine (T^) and triiodothyronine (T3) concentrations early in metamorphosis, and showed that the hormone levels remained low throughout the later stages of metamorphosis. Other studies on P. marinus, Lampetra {Entosphenus) japonica and L. lamottenii reported decreases in thyroid hormone levels between ammocoetes and fully metamorphosed lampreys (Suzuki 1982; Lintlop and Youson 1983; Weirich et al. 1987). The purpose of the present study was to examine the changes in serum thyroid hormone concentrations during the metamorphosis of the southern hemisphere lamprey, Geotria australis, the sole representative of the Geotriidae (Potter 1980). The study employed a highly sensitive RIA which enabled the measurement of thyroid hormones in small volumes of serum (2-5 ^1). Measurements were made on representatives of each stage soon after their capture, with the animals being maintained during the short period between capture and samphng under temperature and photoperiod regimes which paralleled those encountered in the streams from which they were taken. Particular attention was paid to the serum thyroid hormone
concentrations found in animals towards the end of metamorphosis. In addition, some ammocoetes of a metamorphosing size were acclimated to different temperatures and others were treated with propylthiouracil (PTU) to determine whether these treatments affected serum thyroid hormone concentrations and/or induced morphological changes. The aim of these studies was to determine whether depressed serum T^ or T3 concentrations might be a controlling factor in the regulation of the onset of metamorphosis in G. australis, as has been claimed for L. reissneri (Suzuki 1987). Materials and methods Collection and maintenance of animals Ammocoetes and the seven stages (1—7) in the metamorphosis of Geotria australis were collected between February and June 1988 by electric fish shocking in tributaries of the Donnelly and Warren Rivers in southwestern Austraha. The animals were taken to Murdoch University in river water and maintained in a temperature- and photoperiodcontrolled room in aquaria supplied with substrate and water from their natal river systems. The room temperature was adjusted so that the water temperature of the aquaria paralleled that recorded in the field and the photoperiod was adjusted to parallel that of the conditions in the field. The animals used for the determination of serum T^ and T3 concentrations were sacrificed within two days of capture. Details of the areas of collection and the classification of stages in metamorphosis are given in Potter et al. (1980). Blood collection The animals were anaesthetized in a 0.01% solution of benzocaine and bled by caudal severance using the method described by Macey and Potter (1981). The blood was centrifuged, the haematocrit recorded, and 2 to 10 yl aliquots of the serum dispensed into microcentrifuge tubes for subsequent measurement of serum thyroid hormone content. The serum
169 was then frozen in the microcentrifuge tubes and stored at -20°C until it was assayed (usually within 5 days). Thyroid hormone radioimmunoassay methods
(RIA)
Serum thyroid hormone levels were measured using the Amersham Amerlex RIA for triiodo-L-thyronine (Tj) and L-thyroxine (T^). In these assays the antibodies, which are bound to magnetized polymer beads, are incubated with the appropriate tracer hormone and unknown sample. Antibody-bound hormone can then be separated from the unbound hormone using either a magnet or centrifugation. In the present study, the incubation was performed at 4°C for 16-18 h in Eppendorf microcentrifuge tubes which were then centrifuged for 5 - 6 min at 14,000 X g and the supernatant removed from the pellet of polymer beads by means of a 50 /xl Drummond microcap tube. The extreme base of the microcentrifuge tube (which contained the pellet) was then removed using a razor blade and placed in a gamma counter to measure radioactivity. This 'microassay' method permitted thyroid hormone concentrations to be measured in serum volumes as low as 2 jA for Tj and 10 /xl for T^. Intra- and interassay coefficients of variation for both assays were < 12%; assay sensitivities were 2.0 and 0.2 nM of serum for T^ and Tj respectively. Wherever possible, duplicate samples of serum from a single animal were assayed for both Tj or T4. In some instances, however, the volume of serum obtained from a single animal did not permit this, and samples from 2 - 4 animals were pooled to obtain the necessary volume. Effect of propylthiouracil (PTU) and T j Forty large larvae of metamorphosing length (> 80 mm), which had been maintained in a laboratory aquarium for 30 days and had not entered metamorphosis by the end of March 1988, were weighed (to the nearest 0.01 g) and their length measured (to the nearest 1 mm). These animals were then randomly assigned in approximately
equal numbers to one of three identical 41 aquaria containing dechlorinated tap water and an equal volume of clean river sand. The larvae, which immediately burrowed into the substrate, were acclimated to the conditions of the aquaria (16°C) for 5 days. The animals were not fed for the duration of the experiment. The animals in each aquarium were then subjected to one of the following treatments. L-T3 (Sigma, St. Louis, MO) was added to one of the aquaria as a solution in 0.1 M NaOH to make a final solution in the aquarium water of 1 mg/1. PTU (Sigma, St. Louis, MO) was supended in 0.1 M NaOH and an appropriate volume of the mixture added to another aquarium to make a final solution of 10 mg/1. A similar volume of 0.1 M NaOH was added to the third (control) aquarium. T3, PTU and/or NaOH was added to each aquarium every fourth day, and the water siphoned off and replaced without disturbing the animals buried in the substrate, every 10-14 days. The exposure to PTU and T3 was continued for 70 days. On day 35 and at the termination of the study, the animals were removed from the substrate, lightly-anaesthetized with 0.01% benzocaine. Under doubleblind conditions, each animal was carefully examined for any external signs of the onset of metamorphosis. At the termination of the study, the length, weight and stage of metamorphosis of each animal was recorded. Blood samples were taken for measurement of hematocrit and serum T^ and T3 concentrations. In addition, the livers of 10 animals from each group were removed, homogenized in 4 volumes of 0.1 M phosphate buffer (pH 7.5) and extracted overnight with 2 volumes of absolute ethanol for determination of the hepatic T3 content. The extraction efficiency of this method, determined using samples spiked either with '^^I-labelled T3 or unlabelled T3 and measured by RIA, was > 85%. The protein content of the homogenate was measured using the Lowry method (with bovine serum albumin as standards), and the T3 content was expressed as nM Tj/mg protein. The eviscerated carcasses of the control animals and PTU-treated animals were weighed, freezedried and reweighed to determine the carcass water content.
170 Effect of acclimation temperature Three groups of approximately 10 ammocoetes were removed from a common pool of larvae which had been held in laboratory aquaria for at least 8 weeks and acclimated to the gradually decreasing water temperature of early winter conditions (from 20°C to 16°C). The first group was retained at the initial water temperature (16°C) and the remaining two groups were respectively either cooled to 6°C, or warmed to 25°C; the temperature changes were made in a step-wise fashion (2-3°/day) over a 6 day period. The animals were acclimated to these conditions for 14 days before they were removed from the substrate, anaesthetized and bled as described above. Hematocrits and serum T3 and T4 concentration were measured using the method described above. Statistics The data on serum T4 and T3 levels were log-transformed prior to statistical analysis to satisfy the criterion of homogeneity of variance. Data on serum T4 and T3 levels and hematocrits of ammocoetes and the seven stages of metamorphosis were subjected separately to one-way analyses of variance. Predetermined means were then compared using the least significant difference and Tukey's W tests (Steel and Torrie 1980). Data on serum T3 and T^ concentration, hepatic T3 content and hematocrit in the PTU- and Tj-treated ammocoetes were subjected separately to one-way analysis of variance. Where F values indicated significance (p < 0.05), the means of the treated groups were compared with those of the controls using the least significant difference test. Because individual larvae were not identified from the beginning to the end of the PTU/T3 treatment experiment, a repeated-measures statistical design could not be used. However, their log-transformed length and weight data were subjected to a cross-classified analysis of covariance designed to correct for differences in body length, and the treatment by time interaction terms were examined to determine significant variations in changes in adjusted weights between treatments over time. Two
analyses were performed using the Manova procedure of the SPSSX package, one assuming parallel slopes, the other incorporating a log length by treatment interaction term to allow for differences between the length and weight relationship among the three groups. Data on serum T3 and T4 concentrations in the ammocoetes acclimated to ambient temperatures of 6, 16 or 25°C were subjected separately to one-way analysis of variance. Where F values indicated significance (p < 0.05), the hormone concentrations of the 6°C and 16°C accHmated groups were compared with those of the 25 °C acclimated animals using the least significant difference test. Results Haematocrit There were no significant differences between the haematocrits of ammocoetes and metamorphosing stage 1 animals sampled in late February 1988. However, the haematocrits of stages 2, 3, 4, 5 and 6 were significantly lower (p < 0.01) than in larvae sampled at the same time (Table 1). There were no significant differences between the haematocrit of stage 7 animals and that of the comparable collection of ammocoetes (Table 1). The haematocrit of stage 7 was significantly higher (p < 0.01) than in stages 1, 2, 3 and 5 (Table 1). In addition, the haematocrits of ammocoetes sampled from late March until early June were significantly greater (p < 0.01) than those of ammocoetes sampled in late February. Changes in serum thyroid hormone during metamorphosis
concentrations
The mean serum T4 concentration in ammocoetes sampled in February 1988, at the time when metamorphosing individuals first appeared in the population, was 31.73 ± 4.09 nM (Fig. 1). These levels fell progressively and reached minimal values of less than 5 nM at stage 5. Thereafter, there was a modest, but significant, increase in the levels in stages 6 (p < 0.01) and 7 (p < 0.05) and in young adults (p < 0.01) (see Note added in proof) over those in
171 Table 1. Haematocrit values of ammocoetes and metamorphosing stages of Geotria australis used in this study Month(s)
Metamorphosing animals
Ammocoetes
late February
28.1 ± 2.4 (13)
March late March-early April mid April mid May early June
34.2 40.0 45.2 39.8 40.7
± ± ± ± ±
2.0 (15) 1.5 (25)** 2.0 (10)** 1.3 (22)** 1.4 (14)**
Stage
Haematocrit
1 2 3 4 5 6 7
25.5 18.2 25.6 30.3 24.3 31.1 35.0
± 1.8 (15) ± 1.1 (15)* ± 2.0 (19)* ±1.1 (38)* ± 0.9 (18)* ± 1,2(31)* ± 3.3 (8)***
Data are shown as mean + SEM (number of animals); measurements of haematocrit were made for each individual animal; * significantly lower (p < 0.01) than in ammocoetes sampled at the same time; ** significantly higher (p < 0.01) than in ammocoetes sampled in late February; *** significantly higher (p < 0.01) than in stages 1, 2, 3 and 5. serum T4 concentration
35-302520151050
6-
serum T3 concentration InMl
543210
32
arnm
1
15
14
23
2
3
4
16 5
12
6
stage of metamorphosis
1-
14
11
24
14
10
27
amm
la
lb
2
3
4
stage of metamorphosis
5
17
12
6
7
Fig. 1. Serum L-thyroxine (T4) concentration (nM) in Geotria australis at different stages of metamorphosis. Stages of metamorphosis are shown on the X-axis; the histograms represent means ± SEM; the number of determinations is shown at the base of each histogram.
Fig. 2. Serum triiodo-L-thyronine (T3) concentration (nM) in Geotria australis at different stages of metamorphosis. Stages of metamorphosis are shown on the X-axis; the histograms represent means ± SEM; the number of determinations is shown at the base of each histogram.
Stage 5 animals (Fig. 1). Serum T4 concentrations in all the metamorphosing stages were significantly lower than in ammocoetes measured at the time when metamorphosis commenced (p < 0.01). The mean serum T3 concentration in ammocoetes sampled in February 1988, at the time when metamorphosing animals first appeared, was 5.06 ± 0.70 nM (Fig. 2). These concentrations dechned between stage 1 animals sampled in mid-February and those examined approximately 10 days later (Fig. 2). Serum concentrations in the latter group were significantly (p < 0.01) lower than in ammocoetes sampled at the same time (Fig. 1). Thereafter, serum T, levels were significantly lower in
stages 2 to 7 than in the February sample of ammocoetes (p < 0.01, except for stage 3, where p < 0.05). There were no significant differences in serum T3 concentrations in metamorphosing animals between late stage 1 and stage 7. The serum T3 concentration of young adults (4.7 ± 0.9 nM) was significantly (p < 0.01) higher than that of stages 2 to 7 (see Note added in proof). Seasonal changes in serum thyroid hormone concentrations in ammocoetes The serum T^ concentrations in ammocoetes increased significantly between February and May-
172 Table 2. Log body weight (BW) vs. log body length (BL) relationships in propylthiouracil (PTU)- and Tj-treated ammocoetes of Geotria australis Treatment
N
initial (day 0)
final (day 70)
Control PTU
14 14 12
log BW = - 6 . 3 1 + 3.19 log BL (r = 0.96) log BW = - 5 . 6 8 + 2.88 log BL (r = 0.92) log BW = - 3 . 2 1 -1- 1.59 log BL (r = 0.85)
log BW = -5.73 + 2.90 log BL (r = 0.92) log BW = -6.88 + 3.55 log BL (r = 0.95)* log BW = -2.53 + 1.21 log BL (r = 0.79)
T3
' significantly different (p < 0.01) from the control group. serum T4 concentration W
70-
1
60504030-
10
20-
February
March-April
21
10
May-June
July
serum T3 concentration (nMl
40 3020 10 0
14
February
19
March-April
13
May-June
10
July
Fig. 3. Seasonal changes in serum L-thyroxine (T4) and triiodoL-thyronine (T3) concentration in Geotria australis ammocoetes. The histograms represent means ± SEM; the number of determinations is shown at the base of each histogram.
June (Fig. 3). Concentrations in animals sampled in March-April, May-June and July were each significantly higher than in the February sample of ammocoetes (p < 0.05, p < 0.01 and p < 0.05, respectively), and that of the May-June sample was significantly higher than in the March-April (p < 0.01) and July samples (p < 0.05). As with serum T4 concentrations, there was a sig-
nificant increase in serum T3 levels in ammocoetes sampled in the months immediately after February when no further ammocoetes would enter metamorphosis (Fig. 3). Serum T3 concentrations in the ammocoetes sampled in the May-June period were over 8 fold higher than in the animals collected in February (p < 0.01). In ammocoetes sampled in mid-July, the serum T3 concentrations were lower than those of the May-June samples, but still significantly higher (p = 0.05) than those of the animals sampled in February (Fig. 3). Effects of propylthiouracil (PTU) and T^ on ammocoetes Under the conditions used in this study, neither PTU nor T3 produced any external morphological signs indicative of the onset of metamorphosis. The cross-classified analyses showed that, while the length/weight relationship differed among the three groups, the relationship within each group did not change over the course of the experiment. However, the analyses also demonstrated that, compared to the control group, significant variations in body weight (p < 0.01) had occurred to the two groups of treated animals over time, with the PTU and T3 treatments leading to a gain and loss in body weight respectively. Thus, after inserting in the equation given in Table 2 a body length of 90 mm (the mean length at metamorphosis), the corresponding weight in the control group only changed from 0.84 to 0.87 g, whereas that of the PTU-treated group increased from 0.89 to 1.14 g, and that of the T3-treated group decreased from 0.79 to 0.68 g. Serum T4 and T3 concentrations in the PTU- and T3-treated groups were significantly (p < 0.01) lower and higher, respectively, than in the control group (Table 3).
173 Table 3. Effect of propylthiouracil (PTU) and T3 on serum thyroid hormone concentrations, hepatic T3 content and haematocrit in ammocoetes of Geotria australis Control Serum T4 concentration (nM) Serum T3 concentration (nM) Hepatic T3 content (pM/mg protein) Haematocrit
PTU-treated
T3-treated
82.82 ± 4.65 (9)
6.78 ± 0.36 (9)*
101.97 ± 12.29 (7)*
37.56 ± 4.71 (8)
9.67 ± 1.32 (9)*
85.36 ± 10.53 (8)*
1.65 ± 0.08 (8)
0.90 ± 0.08 (8)*
14.90 ± 0.82 (8)*
37.8 ± 1.2 (10)
19.7 ± 1.3 (10)*
33.5 ± 2.5 (11)
Data are shown as mean ± SEM (number of determinations); * significantly different (p < 0.01) from the control group. Table 4. Effect of acclimation temperature on serum thyroid hormone levels in ammocoete Geotria
australis
Ambient water temperature Serum T4 concentration (nM) Serum T3 concentration (nM) Haematocrit
6°C
16°C
25°C
24.50 ± 3.20 (5)*
44.54 ± 4.53 (6)
31.83 ± 6.52 (6)*
33.04 ± 4.69 (4)
36.12 ± 6.11 (5)
18.49 ± 1.93 (6)**
33.7 ± 1.6 (6)
32.6 ± 1.4 (6)
32.6 ± 1.5 (6)
The experiment was carried out in June when the ambient temperature was 16°C. The data are shown as mean ± SEM (n); *, ** significantly different (p < 0.05 and p < 0.01 respectively) from the 16°C - acclimated group.
The hepatic T3 contents of the PTU- and T3treated ammocoetes were significantly (p < 0.01) lower and higher, respectively, than in the control animals (Table 3). The haematocrit in the PTU-treated group was significantly lower than in the control ammocoetes (p < 0.01), but there were no differences between the control and Tj-treated groups (Table 3). Effect of acclimation temperature on serum thyroid hormone levels and hematocrits of ammocoetes The serum T^ concentrations in the animals acchmated to 16°C were significantly (p < 0.05) higher than in the ammocoetes acchmated to 6 or 25 °C (Table 4). In addition, serum T3 concentrations
in animals acclimated to 25 °C were significantly lower than in animals retained at 16°C (p < 0.01) (Table 3). The hematocrits of ammocoetes acclimated to 6, 16 and 25°C were not significantly different from one another (Table 4). Discussion The study reported here shows that both serum T^ and T3 concentrations decline markedly during the metamorphosis of Geotria australis. The fall in the serum concentration of both hormones commences in the very early stages of metamorphosis and the levels reach a minimum at stage 5. A similar progressive decline in serum thyroid hormone levels during metamorphosis was reported for Petromy-
174 zon marinus by Youson and co-workers (Wright and Youson 1977; Lintlop and Youson 1983a). Moreover, Suzuki (1982), Lintlop and Youson (1983a) and Weirich et al. (1987) reported that in Lampetra japonica, L. lamottenii and P. marinus, respectively, the thyroid hormone levels in metamorphosed lampreys were markedly lower compared with the ammocoetes. However, in contrast to the situation in P. marinus (Youson 1988), the serum T4 levels of G. australis showed some recovery in the final stages of metamorphosis. The rapid and marked decline in serum thyroid hormone levels very early in the metamorphosis of two species of lampreys from separate families raises the possibility that high concentrations of these hormones prevent the onset of metamorphosis in all lampreys, and that the morphological and physiological processes associated with metamorphosis in lampreys are permitted to take place as a result of thyroid hormone withdrawal. There was some evidence that this is the case in L. reissneri, since treatment of the 'larger larvae' of this species with various goitrogens (potassium perchlorate, sodium perchlorate, propylthiouracil (PTU) or thiourea) for 200 days induced complete metamorphosis. When smaller larvae were used, however, metamorphosis was only partly induced (Suzuki 1987). In the present study, treatment of G. australis with PTU for 70 days did not induce metamorphosis despite the fact that serum T4 and T3 levels were significantly reduced in the PTU-treated group (Table 3). The reasons for the discrepancy between the two studies are not clear. Although there may be species differences, this seems an unhkely explanation given the essentially similar pattern of morphological change undergone by all lampreys (Youson 1980; Potter et al. 1982). Another possibility is that the period of PTU treatment of G. australis (70 days) may have been insufficient. However, there were clear physiological responses to the treatment exhibited even after 35 days, and by the end of the treatment period the animals were severely hypothyroid and had an increased blood volume compared with the controls (see below). Despite these major physiological responses, double-bhnd studies failed to find any evidence to suggest that metamorphosis had commenced. Yet a further possibility is
that the ammocoetes may have to be in a specific, but as yet undefined, physiological state before they will respond to lowered serum thyroid hormone levels. Youson (1988) reviews what is known of the factors related to the onset of metamorphosis in lampreys, and concludes that the event is triggered by an interaction between internal (e.g. condition factor, pineal and pituitary hormones) and external factors such as photoperiod and temperature. The argument for the role of internal factors being critical is supported in part by Suzuki's (1987) report that as a result of his treatment regime the small ammocoetes did not progress as far into metamorphosis as the larger animals. Presumably, the goitrogens will have had a similar effect on thyroid hormone concentrations in the blood of both small and large ammocoetes, but only the large animals were in a condition which permitted them to respond fully to the treatment. An argument against the hypothesis that the high serum thyroid hormone concentrations act as a brake inhibiting metamorphosis, is the observation that the serum concentrations of both T4 and T3 were still high in stage 1 G. australis, which by definition, had begun to metamorphose. This suggests that the declining thyroid hormone levels are concomitant with, and not necessarily responsible for the onset of metamorphosis in this species. It is possible, for example, that the changing serum thyroid hormone concentrations reflect the changing nutritional state of the animals. In adult G. australis, serum T3 concentrations fall progressively whilst the animal is resident in fresh water, prior to spawning. Serum T4 concentrations do not change to any degree (Leatherland et al. 1990b). In several oncorhynchid teleosts, both serum T4 and T3 fall progressively during their spawning migration (see Leatherland et al. 1989a, for references). In both of the above situations, the animals had ceased to feed and were dependent upon stored energy reserves. During metamorphosis, lampreys stop feeding and also rely upon food reserves that have been acquired during the ammocoete period. It is therefore possible that the declining serum thyroid hormone levels during metamorphosis are related to the role of these hormones in energy partitioning strategies associated with a prolonged fast, rather
175 than to the control of developmental processes per se. Temperature appears to have a significant effect on serum thyroid hormone levels in the ammocoetes of both G. australis (this study) and P. marinus (Wright and Youson 1980b; Lintlop and Youson 1983a). In the sea lamprey, serum T3 concentrations in animals maintained at 7-10°C were approximately 50% higher than in animals at 1 9 21 °C. In ammocoetes of G. australis, the serum T^ and T3 concentrations increased, respectively, by more than 2.5 and nearly 9.0 fold between February and May-June (Fig. 3), a period during which the ambient temperature fell from approximately 25 °C to 15°C. The view that this dechne was related to temperature is supported by the fact that the high concentrations of T3 and T^ found at 15-16°C in May-June could be significantly reduced by re-acchmating ammocoetes to ambient water temperature of 25°C (Table 4). Of interest was the observation that an extremely low ambient temperature, below that normally experienced by the animal in the wild, also significantly depressed serum T4 but not serum T3 concentrations. This apparent inverse correlation between ambient temperature and serum thyroid hormone levels might be used as one of the internal controls used to prevent the onset of metamorphosis once the optimal time has past. However, the fall in serum T^ and T3 levels in ammocoetes between May-June and July (Fig. 3), a period during which there was little or no change in environmental water temperature, suggests that a sustained low ambient temperature does not stimulate a sustained elevation in serum thyroid hormone levels. Whatever the role of the thyroid hormones (if any) in the metamorphosis of lampreys, it does not appear to be of a stimulatory nature such as that found in amphibians (Norris 1985) and the developmental stages of some teleost fishes (Lam 1980; Lam and Sharma 1985; Lam et al. 1985; Inui and Miwa 1985; Miwa and Inui 1987a,b). In several species of teleosts, thyroid hormones appear to be sequestered from the maternal circulation during the development of the eggs, and very high levels are found in the egg yolk (Kobuke et al. 1987; Tagawa and Hirano 1987; Brown et al. 1989;
Greenblatt et al. 1989; Leatherland et al. 1989b). These high thyroid hormone levels may be important to the development of the larval fish prior to the animal's ability to manufacture thyroid hormone from its own thyroid gland. Brown and coworkers (1989) showed that the survival of larval white suckers increased markedly when the sexually mature adult females were injected with T3 2 days prior to removal of the eggs for fertilization. Similarly, very low levels of T4 and T3 in the eggs of Lake Erie stocks of coho salmon may be associated with low survival rates of the larvae (Leatherland et al. 1989b). The yolk thyroid hormone levels fall during larval development and reach their lowest levels prior to first exogenous feed. It is therefore tempting to suggest that the high serum thyroid hormone concentrations in the ammocoetes, and the declining concentrations during metamorphosis, reflect the presence of analogous processes to those occurring during the development of some teleosts. On the other hand, there is good evidence to show that the ammocoete is doing more than simply storing the maternal supply of thyroid hormones. The fact that serum thyroid hormone levels in ammocoetes can change markedly with season and temperature provides strong evidence for a change in kinetics between hormone secretion into the blood and clearance from the blood. The increase in serum thyroid hormone levels associated with season and temperature may be explained by either or both of the above processes, but regardless of which is involved there is clear evidence for hormone secretion, and that the endostyle is acting as an edocrine tissue long before the true follicular nature of the thyroid tissue becomes evident. Suzuki (1982) made similar conclusions in his study of two species of Entosphenus. The fall in serum thyroid hormone levels associated with metamorphosis in P. marinus is not correlated with the increase in hepatic nuclear binding of T3 reported by Lintlop and Youson (1983b), or with the apparent absence of change reported by Weirich et al. (1987). Since the first step in thyroid hormone activity is thought to operate at the level of gene expression (Oppenheimer 1983), the absence of a correlation of the hepatic binding of T3 with the metamorphic event might be evidence of a lack of
176 involvement of the hormone in the process. Indeed, the lowering of serum thyroid hormone levels during metamorphosis could well be the result of an increased clearance of the hormones from the circulation. One event which might be pertinent is a change in the peripheral monodeiodination of T4 to produce T3, the biologically active form of thyroid hormone. We have preliminary evidence to show that monodeiodinase activity which is apparently absent from the liver of ammocoete G. australis, is present in the liver of stage 1 and stage 6 individuals (unpubhshed data). PTU ehcited a significant increase in body weight without concomitant increase in body length. It appeared that this increase in weight is the result of an increased blood volume relative to that of the control group, since a) the PTU-treated animals gave approximately 2 - 3 times higher volumes of blood than the controls during routine blood collection (data not shown), b) the haematocrit of the PTU-treated animals was extremely low compared with the controls, and c) the the carcass water content of control and PTU-treated ammocoetes measured after blood collection (74.3 ± 2.2% and 75.7 ± 1.3% for control and PTU-treated groups respectively (n = 10)) was essentially similar. This dilution of the blood compartment may well have been a contributory factor in the lowered serum thyroid hormone levels. However, if a reduction in thyroid hormone levels was the necessary stimulus for transformation to commence, then whatever the cause of the lowered levels should have prompted the response. Unlike the situation in P. marinus, in which the low serum thyroid hormone concentrations evident by mid-metamorphosis are still seen in stage 6 and 7 animals (Youson 1988), in G. australis the serum T4 concentrations in stages 6 and 7 were significantly (p < 0.05) higher than in stage 5. Serum T3 concentrations were similar throughout the mid-to late stages of metamorphosis in both P. marinus and G. australis (Youson 1988, and the present study). However, the levels were significantly elevated in the young adults (see Note added in proof). The biological significance of the modest, but significant, increase in serum T^ concentrations in G. australis late in metamorphosis is unclear, al-
though several possibilities exist. 1) They may be caused by alterations in serum binding protein concentration, although if this were the case, one might expect to find parallel, significant alterations in serum T3 concentrations also. However, these were not found. 2) There may be a reduction in the T4 clearance rate in stages 6 and 7 without concomitant changes in secretion rates. This would tend to elevate serum T4 levels. 3) There may be an increased T4 secretion rate. In this context, it is thus worth noting that the endostyle has completed its transformation into the functional thyroid gland by stage 6 in metamorphosis (Wright and Youson 1976, 1980). More work is required to determine whether the increased serum T4 concentrations in stages 6 and 7 and young adults are indicative of increases perse, or whether they represent a reduction of the thyroid secretory 'brake', which was one of the possible causes of the rapid decline in serum T4 concentrations in stages 1 and 2. In summary, this study shows that a marked fall in serum thyroid hormone concentrations occurs during the early stages of metamorphosis of G. australis, with the serum T4 concentrations recovering shghtly in stages 6 and 7. Serum T4 and T3 concentrations in ammocoetes were influenced markedly by environmental temperature. Depression of serum thyroid hormone levels brought about by administration of PTU failed to bring about any of the morphological changes associated with metamorphosis. Acknowledgements The work was supported by a Collaborative Research Fellowship and a grant-in-aid of research from the Natural Sciences and Engineering Research Council of Canada to J.F.L., and a grant from the Australian Research Grants Scheme to I.CP. We wish to thank Ann Leatherland for her excellent technical assistance. References cited Brown, C.L., Doroshov, S.I., Cochran, M. and Bern, H.A. 1989. Enhanced survival in striped bass fingerlings after
177 maternal triiodothyronine treatment. Fish Physiol. Biochem. 7: 295-299. Galton, V.A. 1988. The role of thyroid hormone in amphibian development. Am. Zool. 28: 309-318. Greenblatt, M., Brown, C.L., Lee, M., Dander, S. and Bern, H.A. 1989. Changes in thyroid hormone levels in eggs and larvae and in iodide uptake by eggs of coho and chinook salmon, Oncorhynchus kisutch and O. tschawytscha. Fish Physiol. Biochem. 6: 261-278. Inui, Y. and Miwa, S. 1985. Thyroid hormone induces metamorphosis of flounder larvae. Gen. Comp. Endocrinol. 60: 450-454. Kobuke, L., Specker, J.L. and Bern, H.A. 1987. Thyroxine content in eggs and larvae of coho salmon, Oncorhynchus kisutch. J. Exp. Zool. 242: 89-94. Lam, T.J. 1980. Thyroxine enhances larval development and survival in Sarotherodon mossambicus Riippell. Aquaculture 21: 287-291. Lam, T.J., Juario, J.V. and Banno, J. 1985. Effect of thyroxine on growth and development in post-yolksac larvae of milkfish, Chanos chanos. Aquaculture 46: 179-184. Lam, T.J. and Sharma, R. 1985. Effects of sahnity and thyroxine on larval survival, growth and development in the carp, Cyprinus carpio. Aquaculture 44: 201-212. Leatherland, J.F., Down, N.E., Dye, H.M. and Donaldson, E.M. 1989. Changes in plasma thyroid hormone levels in pink salmon (Oncorhynchus gorbuscha) during their spawning migration in the Eraser River (Canada). J. Fish Biol. 35: 199-205. Leatherland, J.F., Lin, L., Down, N.E. and Donaldson, E.M. 1989b. Thyroid hormone content of eggs and early developmental stages of three stocks of goitred coho salmon (Oncorhynchus kisutch) from the Great Lakes of North America, and a comparison with a stock from British Columbia. Can. J. Aquat. Fish. Sci. 46: 2146-2152. Leatherland, J.F., Macey, D.J., Hilhard, R.W., Leatherland, A. and Potter, LC. 1990b. Seasonal and estradiol-17|3-stimulated changes in thyroid function of adult Geotria australis, a southern hemisphere lamprey. Fish Physiol. Biochem. (In press.) Lintlop, S.P. and Youson, J.H. 1983a. Concentration of triiodothyronine in the sera of the sea lamprey, Petromyzon marinus, and the brook lamprey Lampetra lamottenii, at various phases of the hfe cycle. Gen. Comp. Endocrinol. 49: 187-194. Lintlop, S.P. and Youson, J.H. 1983b. Binding of triiodothyronine to hepatocyte nuclei from sea lampreys, Petromyzon marinus L., at various stages of the Hfe cycle. Gen. Comp. Endocrinol. 49: 428-436. Macey, D.J. and Potter, l.C. 1981. Measurements of various blood cell parameters during the life cycle of the southern hemisphere lamprey, Geotria australis Gray. Comp. Biochem. Physiol. 69A: 815-823. Miwa, S. and Inui, Y. 1987. Effects of various doses of thyroxine and triiodothyronine on metamorphosis of flounder (Paralichthys olivaceus). Gen. Comp. Endocrinol. 67: 356-363. Norris, D.O. 1985. Vertebrate Endocrinology. Lea and Febinger, Philadelphia. Oppenheimer, J.H. 1983. The nuclear receptor-triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism and biological action. In Molecular Basis of
Thyroid Hormone Action, pp. 1-34. Edited by J.H. Oppenheimer and H.H. Samuels. Academic Press, New York. Potter, I.e. 1980. The Petromyzoniformes with particular reference to paired species. Can. J. Fish. Aquat. Sci. 37: 15951615. Potter, I.e., HilHard, R.W. and Bird, D.J. 1980. Metamorphosis in the southern hemisphere lamprey, Geotria australis. J. Zool., London 190: 405-430. Potter, I.e., HiUiard, R.W. and Bird, D.J. 1982. Stages in metamorphosis. In The Biology of Lampreys. Vol. 4B, pp. 137-164. Edited by M.W. Hardisty and LC. Potter. Academic Press, London. Steel, R.D.G. and Torrie, J.H. 1980. Principles and Procedures of Statistics. McGraw-Hill Co., New York. Suzuki, S. 1982. Thyroid function in cyclostomes. In Phylogenic Aspects of Thyroid Hormone Actions. Gumma Symp. Endocrinol. Vol. 19, pp. 13-28. Edited by Institute of Endocrinology, Gunma University. Center for Academic Publications, Japan, Tokyo. Suzuki, S. 1987. Induction of metamorphosis and thyroid function in the larval lamprey. Proc. First Cong. Asia Oceania Soc. Comp. Endocrinol, pp. 220-221. Tagawa, M. and Hirano, T. 1987. Presence of thyroxine in eggs and changes in its content during early development of chum salmon, Oncorhynchus keta. Gen. Comp. Endocrinol. 68: 129-135. Weirich, R.T., Schwartz, H.L. and Oppenheimer, J.H. 1987. An analysis of the interrelationship of nuclear and plasma triiodothyronine in the sea lamprey, lake trout, and rat: Evolutionary considerations. Endocrinology 120: 664-677. Wright, G.M. and Youson, J.H. 1976. Transformation of the endostyle of the anadromous sea lamprey, Petromyzon marinus L., during metamorphosis. I. Light microscopy and autoradiography with i^sj Comp. Endocrinol. 30: 243-257. Wright, G.M. and Youson, J.H. 1977. Serum thyroxine concentrations in larval and metamorphosing anadromous sea lamprey, Petromyzon marinus. L. J. Exp. Zool. 202: 27-32. Wright, G.M. and Youson, J.H. 1980a. Transformation of the endostyle of the anadromous sea lamprey, Petromyzon marinus L., during metamorphosis. II. Electron microscopy. J. Morphol. 166: 231-257. Wright, G.M. and Youson, J.H. 1980b. Variation in serum levels of thyroxine in anadromous larval lampreys, Petromyzon marinus L. Gen. Comp. Endocrinol. 41: 321-324. Youson, J.H. 1980. Morphology and physiology of lamprey metamorphosis. Can. J. Fish. Aquat. Sci. 37: 1687-1710. Youson, J.H. 1988. First metamorphosis In Fish Physiology, Vol. XIB, pp. 135-214. Edited by W.S. Hoar and D.J. Randall. Academic Press, New York.
Note added in proof Serum T3 and T3 concentrations in serum of young adults sampled during their downstream migration in July 1989 were 8.6 ± 0.7 nM (n = 6) and 4.7 ± 0.9 nM (n=6), respectively. Both values were significantly (p < 0.01) higher than those measured in stage 7.
