Variable Effects of Goitrogens in Inducing Precocious Metamorphosis in Sea Lampreys (Petromyzon Marinus)

290 R.G. JOURNALMANZON ET OF AL. EXPERIMENTAL ZOOLOGY 289:290–303 (2001)

Variable Effects of Goitrogens in Inducing Precocious Metamorphosis in Sea Lampreys (Petromyzon marinus)

RICHARD G. MANZON, JOHN A. HOLMES, AND JOHN H. YOUSON* Department of Zoology and Division of Life Sciences, University of Toronto at Scarborough, Toronto, Ontario, M1C 1A4 Canada

ABSTRACT The ability of different goitrogens (anti-thyroid agents) to induce precocious metamorphosis in larval sea lampreys (Petromyzon marinus) was assessed in four separate experiments. Two of these goitrogens (propylthiouracil [PTU] and methimazole [MMI]) are inhibitors of thyroid peroxidase-catalyzed iodination, and three (potassium perchlorate [KClO4], potassium thiocyanate [KSCN], and sodium perchlorate [NaClO4]) are anionic competitors of iodide uptake. Be- cause, theoretically, all of these goitrogens prevent thyroid hormone (TH) synthesis, we also measured their influence on serum concentrations of thyroxine and triiodothyronine. All goitrogens except PTU significantly lowered serum TH concentrations and induced metamorphosis in some larvae. The incidence of metamorphosis appeared to be correlated with these lowered TH concentrations in that KClO4, NaClO4, and MMI treatments resulted in the lowest serum TH concentrations and the highest incidence of metamorphosis in sea lampreys. Moreover, fewer larvae metamorphosed in the KSCN and low-KClO4 treatment groups and their serum TH concentrations tended to be greater than the values in the aforementioned groups. MMI treatment at the concentrations used (0.087 and 0.87 mM) was toxic to 55% of the exposed sea lampreys within 6 weeks. The potassium ion administered as KCl did not alter serum TH concentrations or induce metamorphosis. On the basis of the results of these experiments, we have made the following conclusions: (i) In general, most goitrogens other than PTU can induce metamorphosis in larval sea lampreys, and this induction is coincident with a decline in serum TH concentrations. (ii) The method by which a goitrogen prevents TH synthesis is not directly relevant to the induction of metamorphosis. (iii) PTU has variable effects on TH synthesis and metamorphosis among lamprey species. (iv) Unlike in protochordates, potassium ions do not induce metamorphosis in sea lampreys and are not a factor in the stimulation of this event. J. Exp. Zool. 289:290–303, 2001. © 2001 Wiley-Liss, Inc.

The stimulatory role of the thyroid gland on the tiformes (lampreys) represent the one exception to onset and progression of amphibian metamorpho- this trend (Youson, ’97). sis were first described by Gudernatsch (’12). Fol- The nature of the involvement of TH in lam- lowing this initial report, several studies have prey metamorphosis has yet to be resolved; how- shown that a rise in serum thyroid hormone (TH) ever, some data are consistent with the idea that titers (thyroxine [T4] and triiodothyronine [T3]), from their function differs from that observed in other prometamorphosis to their peak at metamorphic cli- vertebrates. Serum TH concentrations in the sea max, is critical to metamorphosis. Furthermore, lamprey (Petromyzon marinus) increase gradually treatment with exogenous TH stimulates precocious metamorphosis, and anti-thyroid agents (goitrogens) R.G. Manzon’s current address: Department of Biology, Univer- can prevent or dramatically delay the onset of spon- sity of Michigan, Ann Arbor, MI 48109-1048. taneous metamorphosis in amphibians. The stimu- J.A. Holmes’ current address: Axelrod Institute of Ichthyology, De- partment of Zoology, University of Guelph, Guelph, Ontario, N1G 2W1, latory role of TH on metamorphic development has Canada. been observed in most amphibians (for review see Grant sponsor: Natural Sciences and Engineering Research Coun- cil of Canada (NSERC); Grant number: A5945. Grant Sponsor: Great Dodd and Dodd, ’76; White and Nicoll, ’81; Galton, Lakes Fisheries Commission. ’83) and bony fishes (flat fishes, eels, and tarpons) *Correspondence to: John H. Youson, Division of Life Sciences, Uni- versity of Toronto at Scarborough, 1265 Military Trail, Toronto, (Just et al., ’81; Inui et al., ’94) studied to date. Ontario, M1C 1A4, Canada. E-mail: [email protected] Within the vertebrate subphylum, the Petromyzon- Received 22 May 2000; Accepted 27 October 2000 © 2001 WILEY-LISS, INC. GOITROGEN-INDUCED METAMORPHOSIS IN LAMPREYS 291 throughout the 3- to 7-year larval period, peak tively, very low TH titers may permit or even trig- prior to the onset of metamorphosis, and decline ger the onset of metamorphosis if the appropriate sharply concomitant with the first external signs physiological and environmental conditions have of metamorphosis (for review see Youson, ’97). been met (Youson, ’97; Manzon and Youson, ’99). Throughout metamorphosis and for the remain- The absence of induced metamorphosis in PTU- der of the sea lamprey life cycle, serum TH con- treated lampreys of two different species, how- centrations are maintained at these reduced ever, provides compelling evidence to challenge concentrations. The universal nature of this de- these notions. Whether the induction of precocious cline in serum TH concentrations at the onset of metamorphosis is due to a decline in serum TH con- metamorphosis in lampreys is supported by simi- centrations or to a phenomenon related to extra- lar observations in other lamprey species. Signifi- thyroidal effects of certain goitrogens (i.e., KClO4) cant declines in serum T4 and T3 concentrations remains to elucidated. Alternatively, PTU may have from larval values were observed in the southern extrathyroidal effects that are either inhibitory to hemisphere lamprey, Geotria australis (Leather- metamorphosis or toxic to lampreys. Perhaps the land et al., ’90), and the American brook lamprey, ability of a particular goitrogen to induce metamor- Lampetra appendix (Holmes et al., ’99), by stages phosis is related to the mechanism through which 1 and 2 of metamorphosis, respectively. it inhibits TH synthesis. Lampreys also differ from other vertebrates in In the current study we attempted to resolve that goitrogens induce rather than inhibit meta- some of the aforementioned uncertainties sur- morphosis. Hoheisel and Sterba (’63) first reported rounding goitrogen-induced metamorphosis in the induction of an incomplete metamorphosis in lampreys. Our approach was to assess the ability Lampetra planeri following exposure to the goitro- of various goitrogens to lower serum TH concen- gen potassium perchlorate (KClO4). The induction trations and induce precocious metamorphosis in of a complete metamorphosis following treatment larval sea lampreys. This study consisted of a se- with KClO4, sodium perchlorate (NaClO4), propy- ries of four experiments in which larval sea lam- lthiouracil (PTU), or thiourea was later reported preys of different sizes were exposed to various in a series of preliminary studies on Lampetra goitrogens. The goitrogen treatments included reissneri (Suzuki, ’86, ’87, ’89). More recent stud- three anionic competitive inhibitors of iodide up- ies conducted at a time of year when spontaneous take (KClO4, NaClO4, and potassium thiocyanate, metamorphosis does not occur resulted in the in- KSCN) and two goitrogens that inhibit the pro- complete metamorphosis of P. marinus (Holmes and cess of thyroid peroxidase-catalyzed iodination Youson, ’93; Manzon and Youson, ’97; Manzon et (PTU and methimazole, MMI) (Gentile et al., ’95). al., ’98) and L. appendix (Holmes et al., ’99). In In addition, one experiment was designed to ex- + these studies, KClO4-treated larvae had serum T4 amine the effects of potassium ions (K ) associ- and T3 concentrations that were 18–80% and 72– ated with KClO4 and KSCN treatment on the 95% lower than control values, respectively. The induction of metamorphosis in lampreys, since K+ observed declines in serum TH concentrations at ions induce metamorphosis in larval protochor- the onset of both KClO4-induced and spontaneous dates (Degnan et al., ’97). metamorphosis are consistent with the idea that these declines may be involved in the initiation of MATERIALS AND METHODS metamorphosis (Youson et al., ’95; Manzon et al., Experimental protocols and animals ’98; Holmes et al., ’99). In contrast, PTU did not induce metamorphosis in G. australis (Leatherland This study consisted of four experiments con- et al., ’90) or L. appendix (Holmes et al., ’99) de- ducted in different years, at a time of year that spite its ability to significantly lower serum TH spontaneous metamorphosis does not occur; meta- concentrations. Serum T4 and T3 concentrations morphosis occurs in sea lampreys between July were reduced by 95% and 75%, respectively, in G. and October (Potter et al., ’78). A description of autralis and 54% and 80%, respectively, in L. ap- the procedures for animal collection and experi- pendix. mental setup, monitoring and maintenance used The correlation between lowered serum TH con- in all four experiments follows. Any variations centrations and the induction of metamorphosis from these procedures are provided in the detailed in goitrogen-treated lampreys indicates that high description of each individual experiment. Larval TH titers may have an anti-metamorphic effect, sea lampreys were collected from various streams preventing the onset of metamorphosis. Alterna- in the Great Lakes drainage basin (Canada and 292 R.G. MANZON ET AL.

United States of America) in the spring and sum- Experiment 1: Propylthiouracil and mer months using electrofishing equipment. Sea triiodothyronine lampreys were transported to the University of Larval sea lampreys 65–95 and 105–119 mm in Toronto at Scarborough and housed in large fi- length were collected from Fish Creek, NY (Sep- berglass aquaria supplied with substrate (7–10 cm tember 1993) and Pigeon River, MI (June 1993), of industrial sand) and continuously flowing, aer- respectively. Thirty larvae from each size group ated, dechlorinated tap water. Larvae were main- (10 larvae per aquarium and three replicate tanks tained at seasonal water temperatures and fed a per experimental group) were randomly assigned suspension of baker’s yeast once weekly (1 g of to each of the following experimental groups: con- yeast per animal). trol (untreated), PTU (6-n-propyl-2-thiouracil), T , Prior to the onset of each experiment, larval sea 3 and PTU plus T3 (PTU+T3); see Table 1 for nomi- lampreys were anaesthetized in a 0.05% solution nal ambient aquarium concentrations. In addition, of tricaine methanesulfonate (MS-222, Syndel at the onset of the experiment we sampled 30 lar- Laboratories Ltd., Vancouver, British Columbia, vae 65–95 mm in length (Fish Creek) and 20 lar- Canada). Their lengths and weights were re- vae 105–119 mm in length (10 larvae from each corded, and groups of 10 larvae were randomly stream population) to serve as baseline estimates × × assigned by lottery to 21-l aquaria (40 20 25 of larval size and TH status and as a control be- cm). Each aquarium was supplied with 7–10 cm tween the two populations. We chose a PTU treat- of substrate and 10 l of dechlorinated water. ment concentration of 0.059 mM (10 mg/l) because Aquaria were maintained static (i.e., not on a flow- this concentration was previously shown to lower through system) on a 15 hr light, 9 hr dark light serum TH concentrations by 75–95% in G. aus- cycle and were continuously aerated. These hous- tralis (Leatherland et al., ’90). The experiment be- ing conditions were similar to those previously gan in January 1994 with the addition of the used to study lamprey metamorphosis (Holmes aforementioned treatments to the appropriate and Youson, ’93; Manzon and Youson, ’97; Manzon aquaria (Table 1). Treatments were administered et al., ’98; Holmes et al., ’99). Water temperature to each aquarium as 50 ml of a stock solution pre- varied with the ambient room temperature (14– pared in 0.1 M sodium hydroxide (NaOH). Sodium ° 22 C). Routine monitoring and maintenance in- hydroxide was used to increase the solubility of PTU cluded recording water temperatures twice daily; and T3 when making the stock solutions. Sodium adding water as needed to account for evapora- hydroxide did not have any effects on animal size, tion; feeding larvae once weekly; cleaning aquaria, TH status, or the incidence of metamorphosis in an changing aquaria water and adding fresh treat- experiment conducted on larval sea lampreys from ments to aquaria every 2 weeks; and changing Fish Creek (Manzon and Youson, ’97), so a NaOH the substrate every 4 to 8 weeks. All chemicals control treatment was omitted in this study. All ex- used for experimental treatments were obtained perimental animals were sampled 23 weeks after from Sigma-Aldrich, Canada. The onset of each the experiment began. experiment was designated as the first day that treatments were added to the appropriate aquaria Experiment 2: Propylthiouracil and the experiment was terminated when the last sea lamprey was sampled. Treatments were Larval sea lampreys were collected from Beaver- added to aquaria from stock solutions prepared dam Brook, NY, in May 1995. Lampreys were in dechlorinated water unless otherwise indi- housed at various water temperatures and ani- cated. Sampling of sea lampreys was carried out mal densities throughout the spring and summer as follows: anaesthetizing in 0.05% MS-222; re- months to assess the effects these variables have cording animal lengths and weights; assigning a on the incidence of spontaneous metamorphosis. stage of metamorphosis (1 to 7) based on exter- In November 1995 we pooled the larvae from this nal morphology (Youson and Potter, ’79); and col- population that were greater than 129 mm in lecting serum. Animals that died during the length. Larvae from this pool were randomly as- experiment were promptly removed and replaced signed to glass aquaria (10 larvae per aquarium), with larvae marked by latex dye injection into and three replicate aquaria were randomly as- the caudal sinus. Marked larvae were used to signed to each of the control (untreated), NaOH, maintain animal density within an aquarium but and PTU experimental groups (Table 1). Treat- were excluded from all experimental and data ments were administered following the procedures analyses. used in Experiment 1. The study began in No- GOITROGEN-INDUCED METAMORPHOSIS IN LAMPREYS 293

TABLE 1. Baseline and experimental groups in four separate experiments, the nominal ambient aquarium concentration of the various experimental treatments, and mean sea lamprey (P. marinus) size at the time of sampling1

2 Experiment Size Mean animal size at sampling number group Group N DS Length ± 2 SE Weight ± 2 SE Treatment concentration 1 (65–95 mm) Baseline 30 0 93.1 ± 1.0a 1.12 ± 0.05a – Control 30 0 80.3 ± 2.4b 0.72 ± 0.03b – PTU 30 0 76.2 ± 3.3b 0.55 ± 0.08c 0.059 mM (10 mg/l) b b –3 PTU+T3 30 15 77.8 ± 3.2 0.60 ± 0.06 0.059 mM + 1.5 × 10 mM b b –3 T3 30 0 78.4 ± 2.6 0.65 ± 0.06 1.5 × 10 mM (1 mg/l)

(105–119 mm) Baseline 20 0 113.6 ± 2.5a 2.20 ± 0.17a – Control 30 1 106.9 ± 2.3b 1.68 ± 0.13b – PTU 30 1 104.5 ± 2.2b 1.52 ± 0.12b 0.059 mM b b –3 PTU+T3 30 1 104.7 ± 2.0 1.60 ± 0.12 0.059 mM + 1.5 × 10 mM ab b –3 T3 30 0 108.2 ± 3.0 1.76 ± 0.16 1.5 × 10 mM

2 (>129 mm) Control 30 0 128.1 ± 2.0a 2.81 ± 0.12a – NaOH 30 0 125.3 ± 2.0a 2.72 ± 0.17a 0.5 mM PTU 30 0 124.8 ± 2.0a 2.57 ± 0.14a 0.059 mM

3(≥115 mm) Baseline 30 0 127.0 ± 4.2a 2.70 ± 0.28a – Control 30 0 122.4 ± 3.8ab 2.69 ± 0.32a – ab ab L-KClO4 30 0 122.0 ± 4.6 2.34 ± 0.26 0.072 mM (0.001%) b b H-KClO4 30 0 117.1 ± 3.5 1.99 ± 0.24 0.72 mM (0.01%) L-KSCN 30 0 123.5 ± 4.8ab 2.48 ± 0.35ab 0.051 mM (0.0005%) H-KSCN 30 0 123.1 ± 5.2ab 2.38 ± 0.32ab 0.51 mM (0.005%) L-MMI 30 18 120.7 ± 5.5ab 2.59 ± 0.44ab 0.087 mM (0.001%) H-MMI 30 17 122.4 ± 6.0ab 2.56 ± 0.46ab 0.87 mM (0.01%)

4 (110–119 mm) Baseline 9 0 111.6 ± 2.9a 1.81 ± 0.16a – Control 9 0 110.4 ± 2.9a 1.73 ± 0.24ab – bc bc KClO4 9 0 104.2 ± 3.7 1.42 ± 0.16 3.6 mM (0.05%) b c NaClO4 9 0 101.3 ± 2.3 1.33 ± 0.16 3.6 mM L-KCl 9 0 108.0 ± 3.3ac 1.67 ± 0.16abc 3.6 mM H-KCl 9 9 – – 18 mM

(>119 mm) Baseline 9 0 122.9 ± 4.1a 2.49 ± 0.34a – Control 9 0 119.9 ± 6.1a 2.46 ± 0.48a – a a KClO4 9 0 121.2 ± 5.9 2.55 ± 0.39 3.6 mM (0.05%) a a NaClO4 9 0 118.6 ± 5.6 2.36 ± 0.39 3.6 mM L-KCl 9 0 117.6 ± 3.6a 2.26 ± 0.21a 3.6 mM H-KCl 9 9 – – 18 mM

1 Abbreviations: N, sample size at onset; DS, number of deaths at sampling; SE, standard error; PTU, propylthiouracil; T3, triiodothyronine; NaOH, sodium hydroxide; L, low; H, high; KClO4, potassium perchlorate; KSCN, potassium thiocyanate; MMI, methimazole; NaClO4, sodium perchlorate, KCl, potassium chloride. 2Animal sizes between groups within a size group are statistically different (P > 0.05) if labeled with different letters.

vember 1995 and was terminated following 18 low-KClO4 (L-KClO4); high-KClO4 (H-KClO4); low- weeks of treatment, at which time all animals KSCN (L-KSCN); high-KSCN (H-KSCN); low- were sampled. MMI (2-mercapto-1-methylimidazole, L-MMI); and high-MMI (H-MMI) (Table 1). In addition, 30 lar- Experiment 3: Potassium perchlorate, val sea lampreys were sampled at the onset of potassium thiocyanate, and methimazole the experiment to serve as baseline data for lam- Larval sea lampreys were collected from the prey size and TH status. The experiment began Platte River, MI (August 1996). Larvae ≥115 mm in January 1997 and was terminated 16 weeks in length were randomly sorted into glass aquaria later. However, due to the high mortality in both (10 larvae per aquarium), and three replicate MMI treatment groups, all surviving MMI-treated aquaria were randomly assigned to each of the animals were sampled 6 weeks after the study following experimental groups: control (untreated); began. Sea lampreys in one randomly chosen tank 294 R.G. MANZON ET AL.

from the control, H-KClO4, and H-KSCN experi- Tukey–Kramer’s post-hoc test using individual mental groups were assessed for any signs of aquaria and/or experimental groups as indepen- metamorphosis at the time that the MMI-treated dent variables and animal length, weight, and se- lampreys were sampled. rum TH concentrations as dependent variables. Prior to these statistical analyses, all data were Experiment 4: Potassium perchlorate, sodium tested for homoscedasticity with Cochran’s Q test; perchlorate, and potassium chloride data that did not meet this assumption were

Larval sea lampreys were collected from Fish transformed (log10) to minimize the heterogeneity Creek, NY (May 1998) and sorted into two size of the variances (Sokal and Rohlf, ’80). All statis- groups, 110–119 mm and >119 mm in length. Lar- tical analyses were performed using Statistix for vae from each size group were randomly sorted into DOS. The data are presented as mean ± 2 SE glass aquaria (9 larvae per aquarium). One aquar- (standard errors), and differences were accepted ium from each size group was randomly assigned as statistically significant if P < 0.05. Any devia- to each of the following experimental groups: con- tions from these statistical analyses are indicated

trol (untreated); KClO4; NaClO4; low potassium chlo- as they are presented in the Results section. ride (L-KCl); and high potassium chloride (H-KCl) (Table 1). The experiment began in March 1999 and RESULTS was terminated following 13 weeks of treatment, at which time all surviving sea lampreys were Experiment 1: Propylthiouracil and sampled. At the start of the experiment we sampled triiodothyronine 9 larvae from each size group to serve as baseline Aquaria water temperatures ranged from 14 to values for animal size and TH status. 18°C with a mean of 16 ± 0.1°C. In the PTU+T3 experimental group (65–95 mm size group) we ob- Serum collection and TH measurement served a 50% mortality rate; however, 67% of these Blood was collected from anaesthetized sea lam- deaths occurred in a single aquarium in which preys by caudal severance using heparinized all larvae died (Table 1). Metamorphosis was not haematocrit tubes and was allowed to clot over- observed in any of the treated larval sea lampreys, night at 4°C. The following morning the blood was but one control animal was at stage 3 of meta- centrifuged at 7,000g for 3–5 min, and sera were morphosis at the time of sampling. collected and stored at –70°C until analyzed. Ani- At the start of the experiment, larval length and mals were sacrificed by decapitation immediately weight in the 65–95 mm size group did not differ after blood collection. between the four experimental groups; the over-

Total serum T4 and T3 concentrations were de- all mean size of these larvae was 84.4 ± 1.4 mm termined in duplicate using the Amersham Amer- and 0.89 ± 0.04 g. However, baseline larvae in this lex TT4 and TT3 radioimmunoassay (RIA) kits, size group had a mean length and weight of 93.1 respectively (Johnson and Johnson, Markham, ± 1 mm and 1.12 ± 0.05 g, respectively, which were Ontario, Canada). The RIA kits were used accord- significantly greater than those recorded for each ing to Leatherland et al. (’90) to accommodate for of the experimental groups. Significant differences low serum volumes. Intra- and inter-assay coeffi- in size between larvae of the four experimental cients of variance for both TH RIAs were less than groups and/or the baseline group were not ob- 9% and 14%, respectively. Assay sensitivities served in the 105–119 mm size group at the on- ranged from 7 to 11 nmol/l for T4 and from 0.15 to set of the experiment; the overall mean size of 0.4 nmol/l for T3. Serum T4 and T3 concentrations these larvae was 113.3 ± 0.7 mm and 2.20 ± 0.05 were determined for Experiments 1, 3, and 4 but g. At the end of the experiment, larvae within a not for Experiment 2 (serum samples were lost size group tended to be smaller in size than at when our freezer failed). In some instances, equal the onset and experimental larvae were signifi- volumes of serum from two or more sea lampreys cantly smaller than baseline larvae, indicating a from the same experimental aquarium were decrease in animal size over the course of the ex- pooled to produce a single sample of sufficient vol- periment (Table 1). ume to assay both TH. At the end of the experiment, 65–95 mm larvae in the control, PTU, PTU+T , and T experimental Statistical analyses 3 3 groups had serum T4 concentrations (40 ± 6.2, 35 The data from all four experiments were ana- ± 6.6, 48 ± 11.2, and 113 ± 19.7 nmol/l, respectively) lyzed with analysis of variance (ANOVA) and that were significantly lower than values measured GOITROGEN-INDUCED METAMORPHOSIS IN LAMPREYS 295

Fig. 1. Mean (± 2 standard errors) serum thyroxine (T4; sampled at the start of the experiment, but control groups A and C) and triiodothyronine (T3; B and D) concentrations were sampled at the same time as treated lampreys. Serum in sea lampreys of two different groups (based on length). T4 and T3 concentrations are significantly different (P < 0.05) Lampreys were either untreated as in the baseline and con- if labeled with different letters. Sample size for each group trol groups or treated with propylthiouracil (PTU), PTU and is indicated in parentheses below the abscissa. T3 (PTU+T3), or T3 for 23 weeks. Baseline groups were in baseline larvae at the start of the experiment trations of baseline animals were significantly (154 ± 14.5 nmol/l) (Fig. 1A). Treatment of 65–95 greater than those of the experimental groups mm larvae with exogenous T3 significantly elevated (Fig. 1B,D). serum T concentrations relative to values in the 4 Experiment 2: Propylthiouracil control, PTU, and PTU+T3 experimental groups (Fig. 1A). Aquaria water temperatures ranged from 15 to

In the 105–119 mm size group, serum T4 con- 21°C with a mean value of 17 ± 0.1°C. At the start centrations in baseline larvae (145 ± 44.4 nmol/l) of the experiment, significant differences in sea were significantly greater than values in the con- lamprey length or weight were not observed be- trol and PTU-treated animals (40 ± 4.9 and 41 ± tween the various experimental groups; the over- 4.4 nmol/l, respectively) but did not differ from all mean animal size was 133 ± 0.9 mm and 3.42 values in the T3- or PTU+T3-treated (192 ± 26 and ± 0.08 g. As was the case for Experiment 1, lar- 120 ± 26.7 nmol/l, respectively) animals (Fig. 1C). vae tended to be smaller at the time of sampling

Exogenous T3 treatment, in the presence or ab- than at the start of the experiment (Table 1). sence of PTU, significantly elevated serum T4 con- Metamorphosis was not observed, and no deaths centrations relative to values in the control and occurred in any experimental groups. PTU experimental groups (Fig. 1C). PTU did not alter serum T concentrations relative to control Experiment 3: Potassium perchlorate, 4 potassium thiocyanate, and methimazole values in either size group (Fig. 1A,C). Serum T3 concentrations of animals in either size group did Aquarium water temperature ranged from 18 not differ significantly between any of the experi- to 22°C with a mean of 20 ± 0.1°C. Mortalities mental groups (i.e., control, PTU, PTU+T3, or T3), were not observed in the control, KClO4, or KSCN but within each size group the serum T3 concen- experimental groups; however, a high incidence 296 R.G. MANZON ET AL. of mortality in the L-MMI and H-MMI treatment groups precipitated the sampling of all surviving sea lampreys from these groups 6 weeks after the experiment started (Table 1). At this time 18/30 and 17/30 larvae had died in the L-MMI and H- MMI experimental groups, respectively. All com- parisons between experimental groups at the time of sampling were made using MMI larvae sampled 6 weeks after the experiment began and control,

KClO4, and KSCN larvae sampled 10 weeks later, unless otherwise indicated. Significant differences in mean larval length or weight were not observed between the seven experimental groups and/or baseline larvae at the start of the experiment; the overall mean size was 130.3 ± 1.6 mm and 3.23 ± 0.1 g. The mean size of all experimental animals at the time of sampling was 121.6 ± 1.8 mm and 2.40 ± 0.12 g (Table 1), indicating that animal size had decreased over the course of the experiment. The three goitrogen treatments used in this experiment induced precocious metamorphosis in sea lampreys; however, the number of metamorphosing lampreys varied depending on the type and concentration of goitrogen used. Treatment of larval sea lampreys with L-MMI or H-MMI for 6 weeks induced metamorphosis in 33% and 46% of surviving sea lampreys, respectively (Fig. 2A; Table 1). When the MMI-treated sea lampreys were sampled, we also examined 10 larvae from the control, H-KClO4, and H-KSCN experimental groups and found that only 2 sea lampreys from Fig. 2. Stage and total number of metamorphosing sea the H-KClO4 treatment group had commenced lampreys in untreated (control) and goitrogen-treated indi- metamorphosis (Fig. 2A). viduals following 6 weeks (A) or 16 weeks (B). Goitrogen treat- After 16 weeks of treatment, metamorphosis ments included: methimazole (MMI), potassium perchlorate (KClO4), and potassium thiocyanate (KSCN); each goitrogen was not observed in the control group, but 10%, was administered at a high (H) and low (L) concentration 66%, 20%, and 43% of lampreys had begun to (see Table 1). Sample size is equal to 30 unless otherwise metamorphose in the L-KClO4, H-KClO4, L-KSCN, indicated in parentheses below the abscissa. and H-KSCN experimental groups, respectively (Fig. 2B). We observed sea lampreys in each of concentrations between larval and metamorphos- the seven stages of metamorphosis (Fig. 2B), and ing lampreys within an experimental group (Table

these goitrogen-treated lampreys were clearly un- 2). Serum T4 concentrations in baseline sea lam- dergoing external morphological changes reminis- preys were significantly greater than concentra- cent of spontaneous metamorphosis. However, in tions in sea lampreys from the seven experimental most instances the timing of these morphological groups (Fig. 3A). Sea lampreys in the H-KSCN, L-

changes was asynchronous compared to what is KClO4, H-KClO4, L-MMI, and H-MMI experimen- observed during spontaneous metamorphosis, and tal groups had serum T4 concentrations that were goitrogen-induced metamorphosis did not proceed significantly lower than control values, but L-KSCN

to completion. treatment did not alter serum T4 concentrations To determine the effects of the various treat- (Fig. 3A). Within the L-KSCN and H-KSCN experi-

ments on serum TH concentrations, we tested for mental groups, the serum T4 concentrations of statistically significant differences between experi- metamorphosing lampreys were significantly lower mental groups and/or baseline animals (Fig. 3). than values for lampreys which did not metamor- In addition, we tested for differences in serum TH phose (Table 2). GOITROGEN-INDUCED METAMORPHOSIS IN LAMPREYS 297

serum T3 concentrations did not differ significantly between the baseline, control, L-KSCN, and L-

KClO4 lampreys (Fig. 3B). Significant differences in serum T3 concentrations between larval and metamorphosing animals within an experimental group were observed in the L-KSCN, H-KSCN, and L-MMI experimental groups (Table 2). Sur-

prisingly, serum T3 concentrations in metamorphosing L-MMI sea lampreys were significantly greater than the values in larval L-MMI sea lam-

preys; in both cases serum T3 concentrations were much lower than control values (Table 2). Experiment 4: Potassium perchlorate, sodium perchlorate, and potassium chloride Aquaria water temperatures ranged from 17 to 22°C with a mean value of 19.6 ± 0.2°C. With the exception of the H-KCl experimental groups, in which all animals died within 4 days of the start of the experiment, no deaths occurred in either size group (Table 1). There were no differences in larval size between experimental and/or baseline groups within either size group at the onset of the experiment. The mean animal size at this time was 127.7 ± 2.1 mm and 2.77 ± 0.18 g in the >119 mm size group and 113.1 ± 0.1 mm and 1.89 ± 0.06 g in the 110–119 mm size group. Larvae of both size groups were smaller in size at the time they were sampled than at the start of the experiment (Table 1). Metamorphosis was not observed in control or L-KCl-treated lampreys of either size group. How- ever, in the 110–119 mm size group, 33% of

NaClO4- and KClO4-treated sea lampreys were in either stage 1 or 2 of metamorphosis. The incidence of metamorphosis in lampreys >119 mm in

length following KClO4 or NaClO4 treatment was 89%, and these animals were in stages 1, 2, 3, and 4 of metamorphosis (Fig. 4). Fig. 3. Mean (± 2 standard errors) serum thyroxine (T4) and triiodothyronine (T3) concentrations in sea lampreys. In general, serum TH concentrations in baseline Lampreys were either untreated, as in the baseline and con- animals were significantly greater than those of ex- trol groups, or treated with potassium perchlorate (KClO4) perimental lampreys; serum T3 in L-KCl sea lam- or potassium thiocyanate (KSCN) for 16 weeks or with me- preys (>119 mm size group) was the only exception thimazole (MMI) for 6 weeks. Goitrogen treatment concentrations were either high (H) or low (L) as indicated on the to this trend (Fig. 5). Serum T4 and T3 concentra- abscissa (see Table 1). Baseline groups were sampled at the tions did not differ significantly between control and start of the experiment but control groups were sampled at L-KCl sea lampreys within a size group (Fig. 5). the termination of the experiment (16 weeks). Sample size is The goitrogens KClO4 and NaClO4 significantly low- equal to 30 unless otherwise indicated in parentheses below ered serum TH concentrations in lampreys from the abscissa. Concentrations labeled with different letters are significantly different (P < 0.05). both size groups relative to values in the baseline, control, and L-KCl groups (Fig. 5). DISCUSSION Serum T3 concentrations in the H-KSCN, H- KClO4, L-MMI, and H-MMI experimental groups Over the past four decades several laboratories were significantly lower than concentrations in the have studied the effects of goitrogens on lamprey control and baseline groups (Fig. 3B). However, physiology and metamorphosis but the results have 298 R.G. MANZON ET AL.

TABLE 2. Comparison of mean serum thyroxine and triiodothyronine concentrations in larval (A) and metamorphosing (M) sea lampreys (P. marinus) following various goitrogen treatments (Experiment 3)1,2

Thyroxine (nmol/l) Triiodothyronine (nmol/l) Group A M AM A M AM Baseline 169 – 169 30.21 – 30.21 (30, 28.9) (30, 28.9) (30, 3.6) (30, 3.6) Control 80.7 – 80.7 29.25 – 29.25 (29, 12.9) (29, 12.9) (30, 4.7) (30, 4.7) L-KClO4 35.2 41.8 35.6 24.32 13.36 23.56 (27, 4.14) (2, 12.0) (29, 3.95) (27, 3.68) (2, 7.15) (29, 3.6) H-KClO4 22.9 26.5 25.2 4.75 4.70 4.72 (10, 4.76) (19, 3.11) (29, 2.6) (10, 1.01) (18, 1.14) (28, 0.8) L-KSCN 87.7 48.0* 79.7 27.66 2.15* 22.19 (24, 16.18) (6, 34.4) (30, 15.9) (22, 6.49) (6, 0.84) (28, 6.5) H-KSCN 44.6 32.8* 39.1 31.13 1.26* 16.75 (15, 6.35) (13, 4.50) (28, 4.53) (14, 10.69) (13, 0.39) (27, 8.08) L-MMI 24.8 29.1 26.6 2.18 3.95* 2.92 (7, 10.48) (5, 2.07) (12, 6.1) (7, 1.03) (5, 0.88) (12, 0.8) H-MMI 24.7 30.1 27.41 3.79 3.58 3.68 (6, 2.69) (6, 3.85) (12, 2.77) (6, 0.31) (6, 0.40) (12, 0.23)

1 Abbreviations: AM, mean of combined larval and metamorphosing data; L, low; H, high; KClO4, potassium perchlorate; KSCN, potassium thiocyanate; MMI, methimazole. 2Values in parentheses are sample size and two standard errors of the mean, respectively. *Significantly different from larval values within a group.

been variable. The goitrogen KClO4 depresses se- prey serum TH concentrations and metamorpho- rum TH concentrations and can initiate the pro- sis are not consistent and tend to challenge the

cess of metamorphosis in several species of lampreys interpretation of the results from KClO4 induc- (Hoheisel and Sterba, ’63; Suzuki, ’86; Holmes and tion experiments. The induction of metamorpho- Youson, ’93; Manzon and Youson, ’97, ’99; Manzon sis following PTU treatment was first reported in

et al., ’98; Holmes et al., ’99). Serum T4 and T3 con- L. reissneri by Suzuki (’87). Subsequently, both centrations in larval lampreys following KClO4 Leatherland et al. (’90) and Holmes et al. (’99) treatment were 18–88% and 72–95% lower than reported a significant decline in the serum TH con- control values, respectively (Youson et al., ’95; centrations of G. australis and L. appendix, re- Manzon and Youson, ’97; Manzon et al., ’98; Holmes spectively, following treatment with PTU. Despite et al., ’99). The magnitudes of these TH declines in serum TH concentrations that were 54–95% lower

KClO4-treated lampreys are comparable to those ob- than control values, PTU treatment did not in- served in the early stages of metamorphosis. Sea duce precocious metamorphosis in either species lampreys at stage 3 of spontaneous metamorphosis of lamprey. This absence of metamorphosis indi-

have serum T4 and T3 concentrations that are 40– cates that a decline in serum TH concentrations 74% and 90% lower than premetamorphic larvae, may not be sufficient to induce metamorphosis. respectively (Wright and Youson, ’77; Lintlop and Alternatively, PTU may have other effects on the Youson, ’83; Youson et al., ’94). The findings of physiology of some lamprey species that preclude these metamorphosis studies are consistent with the induction of metamorphosis. In this study we the hypothesis that a decline in serum TH con- attempted to clarify (i) whether several goitrogens centrations may be involved in the initiation of can depress serum TH concentrations and induce lamprey metamorphosis. Alternatively, high serum sea lamprey metamorphosis, (ii) whether this in- concentrations of TH can inhibit or retard both duction of metamorphosis is specific to KClO4, and the spontaneous (Youson et al., ’97) and induced (iii) whether PTU is unique among goitrogens in (Manzon and Youson, ’97; Manzon et al., ’98) meta- its variability at inducing metamorphosis in lam- morphosis of lampreys. Whether TH concentra- preys. The variable effects of PTU on lamprey tions must decline below a threshold level or the metamorphosis may involve species differences; decline from premetamorphic concentrations must a possibility which cannot be fully assessed by this be of a particular magnitude remains to be eluci- study. dated. In the current study, a total of 90 larval sea Studies investigating the effects of PTU on lam- lampreys of three different sizes groups (65–95, GOITROGEN-INDUCED METAMORPHOSIS IN LAMPREYS 299 did not affect sea lamprey serum TH concentrations in Experiment 1, but this result may be related to the physiological condition of the sea lampreys in this study. Unfortunately, we were unable to determine serum TH concentrations in PTU-treated larvae greater than 130 mm in length (Experiment 2). Future studies should in- clude dose–response curves to determine the mini- mum PTU treatment concentration that can depress serum TH titers in larval sea lampreys to titers similar to those measured in metamorphic sea lampreys. A change in the physiological condition of larval sea lampreys over the course of the experiment may have contributed to the absence of a decline in serum TH concentrations in PTU- treated larvae. In Experiments 1 and 2 we observed a decrease in mean larval length and weight over the course of the experiment. In general, larvae within a treatment group were smaller in size at the time of sampling than they were when the study began. Although the exact cause of this decrease in size is unknown, it may be related to an inadequate food supply in captivity. TH status also changed over time; serum TH concentrations in baseline larval sea lampreys were significantly greater than the values for control larvae at the time of sampling in both size groups in Experiment 1 (Fig. 1). Similar trends towards a decrease in the mean animal size and serum TH concentrations over the duration of an experiment were also observed in Experiments 3 and 4 (Figs. Fig. 4. Stage and total number of metamorphosing sea 3 and 5). However, goitrogen treatment in each of lampreys in untreated (control) and treated individuals from these experiments depressed serum TH concentra- two different size groups (based on length). Treatments included two goitrogen experimental groups (potassium perchlo- tions and induced metamorphosis. These results rate [KClO4] and sodium perchlorate [NaClO4]), and a low from Experiments 3 and 4 minimize the likelihood potassium chloride (L-KCl) experimental group. Sample size that a decrease in size or a change in TH status for each group is nine. from baseline values was responsible for the absence of metamorphosis or a decline in serum TH 105–119, and >129 mm in length) were exposed concentrations in Experiments 1 and 2. to the goitrogen PTU for 18–23 weeks. Consis- The treatment of larval sea lampreys 65–95 and tent with the findings of Leatherland et al. (’90) 105–119 mm in length with exogenous T3 signifi- and Holmes et al. (’99), but contrary to those of cantly elevated serum T4 concentrations but had Suzuki (’87, ’89), we did not observe any signs of no significant effect on serum T3 concentrations. metamorphosis in PTU-treated sea lampreys. Sur- These findings are similar to those observed by prisingly, PTU treatment did not significantly al- Manzon and Youson (’97) with sea lampreys 110– ter serum TH concentrations relative to our 119 mm in length. In this earlier study, exogenous control group (Fig. 1). Although it is tempting to T3 significantly elevated serum T4 concentrations suggest that the absence of metamorphosis in our in six out of six sampling periods ranging from experiment was due to the fact that PTU treat- 4–24 weeks of treatment. However, serum T3 con- ment did not lower serum TH concentrations, the centrations increased in only two of these six sam- studies of Leatherland et al. (’90) and Holmes et pling periods. In a second study using larval sea al. (’99) provide evidence which could refute this lampreys ≥120 mm in length, Manzon et al. (’98) as a possibility. We cannot determine why PTU found that exogenous T3 consistently elevated both 300 R.G. MANZON ET AL.

Fig. 5. Mean (± 2 standard errors) serum thyroxine (T4) and sodium perchlorate [NaClO4]) and a low potassium chlo- and triiodothyronine (T3) concentrations in untreated (base- ride (L-KCl) experimental group. Sample size for each group line and control) and treated sea lampreys from two differ- is nine. Concentrations are significantly different (P < 0.05) ent size groups (based on length). Treatments included two if labeled with different letters. goitrogen experimental groups (potassium perchlorate [KClO4]

serum T4 and T3 concentrations. These data im- MMI used in the current study (0.087 and 0.87 ply that large larvae (≥120 mm in length) can mM) were lower than the concentration (1 mM) regulate TH synthesis and metabolism differently that Brown (’97) routinely uses to inhibit amphib- than the smaller larvae used in the present study. ian metamorphosis. However, he observed an un- Eales et al. (’97, 2000) have shown that the activ- predictable mortality rate in zebrafish at MMI ity of the deiodinase pathways change during and concentrations greater than or equal to 0.5 mM, after metamorphosis. Similarly, changes in TH and 0.3 mM was the highest MMI concentration deiodinase activity and/or metabolism may occur that was not toxic. Although we do not know the within the larval period; serum TH concentrations cause of the lamprey mortality in the MMI treat- increase throughout the larval period indicating ment, it appears to be consistent with the unpre- the potential for a change in TH regulation. The dictable effects of MMI toxicity observed in ability of exogenous T3 to elevate serum T4 with- zebrafish. out increasing serum T3 is an interesting phenom- As was the case in all four experiments dis- enon from a regulatory standpoint. The exact cussed in this paper, Experiment 3 was conducted regulatory mechanism responsible for elevating at a time of year when sea lampreys do not meta-

serum T4 concentrations is unknown but could in- morphose spontaneously. Thus it was not surpris- volve changes in the regulation of TH synthesis, ing that larval sea lampreys in the untreated secretion, deiodination, and/or degradation. control groups did not undergo metamorphosis; yet

All sea lampreys in the control, KClO4, and metamorphosis was observed in all goitrogen KSCN experimental groups survived for the du- treatment groups in Experiment 3. Following a ration of Experiment 3. However, a large propor- 6-week exposure, MMI was more successful at in- tion (>55%) of MMI-treated larvae died early in ducing precocious metamorphosis in larval sea the experiment; this led to the sampling of all sur- lampreys than either KClO4 or KSCN. At 6 weeks viving MMI-treated sea lampreys 6 weeks after we observed that 10/23 MMI-treated sea lampreys the experiment began. The two concentrations of had commenced metamorphosis, as compared to GOITROGEN-INDUCED METAMORPHOSIS IN LAMPREYS 301

2/10 H-KClO4-treated and 0/10 H-KSCN-treated L-KSCN treatment groups. Larvae in these two sea lampreys (Fig. 2A). These data differ from experimental groups had mean serum T3 con- those of the PTU experiments and indicate that centrations which did not differ significantly MMI, a thyroid peroxidase inhibitor, can induce from control values (Fig. 3B) and in the L- metamorphosis in larval sea lampreys. MMI may KSCN experimental group serum T4 concentra- also have the potential to induce metamorphosis tions were also equivalent to values in the in larval sea lampreys more rapidly than the other control group (Fig. 3A). The results of Experi- goitrogens tested to date. However, a cautious in- ment 3 confirm that goitrogens other than terpretation of these data is necessary since only KClO4 can induce precocious metamorphosis in one third of the H-KClO4- and H-KSCN-treated larval sea lampreys. Furthermore, the propor- sea lampreys were examined at this 6-week point, tion of goitrogen-treated lampreys which were metamorphosing sea lampreys in the MMI experi- induced to metamorphose appeared to be cor- mental groups were in the early stages of meta- related to the degree of the decline in serum morphosis, and MMI treatment was toxic to many TH concentrations; this finding differs from that individuals. Perhaps the rapid induction of meta- reported by Youson et al. (’95) for KClO4-treated morphosis in MMI-treated larvae contributed to larval sea lampreys of different sizes. this observed toxicity. Further studies establish- Potassium is a component of two of the goitro- ing a dose–response curve for MMI are required gens (KClO4 and KSCN) that induce precocious to completely assess the capability of MMI to in- metamorphosis in lampreys. This ion has been duce lamprey metamorphosis. shown to induce metamorphosis in several inver- The observed differences in the incidence of in- tebrate species (from five different phyla) includ- duced metamorphosis in the various goitrogen ing some protochordates (i.e., ascidians) (Yool et treatment groups (Fig. 2) can be correlated, to al., ’86; Pearce and Scheibling, ’94; Degnan et al., some extent, to the efficacy of each goitrogen at ’97). Modern protochordates and vertebrates are lowering serum TH concentrations. Following only both members of the phylum Chordata and are be- 6 weeks of treatment, L-MMI and H-MMI induced lieved to have a complex evolutionary relationship proportionally more larval sea lampreys to enter (Whittaker, ’97). One feature consistent with a com- metamorphosis than either the H-KClO4 or H- mon ancestry in protochordates and vertebrates is KSCN treatments. At the termination of Experi- the presence of an endostyle in both larval lam- ment 3, the H-KClO4 and H-KSCN treatment preys and protochordates. The endostyle of larval groups were the most successful at inducing sea lampreys is a TH-producing, subpharyngeal gland lamprey metamorphosis. These treatments (H- that develops into typical, follicular, thyroid tissue

KClO4, H-KSCN, MMI) that resulted in a high during metamorphosis (Wright and Youson, ’80). incidence of induced metamorphosis were also the In contrast, the endostyle of protochordates is pri- experimental groups in which we measured the marily involved in producing mucus to aid in fil- lowest serum TH concentrations (Fig. 3). ter feeding. However, like the larval lamprey Following 6 weeks of treatment, MMI larvae endostyle, the protochordate endostyle can actively had serum T4 concentrations which were as low concentrate iodide and synthesize iodoproteins or lower than larvae exposed to any other goitro- and TH (Eales, ’97). The finding that K+ can in- gen treatment for 16 weeks (Fig. 3A). Further- duce metamorphosis in protochordates necessi- more, 6 weeks of MMI treatment produced serum tated eliminating the possibility that it may have

T3 concentrations that were significantly lower a role in the induction of lamprey metamorphosis than those in all other experimental groups ex- (via KClO4 and KSCN). Experiment 4 was de- cept the H-KClO4 experimental group (Fig. 3B). signed to ensure that goitrogen-induced metamor- H-KClO4 treatment induced precocious metamorphosis in sea lampreys was due to the goitrogenic – – phosis in more sea lampreys than any other ex- anion (i.e., ClO4 or SCN ) and not to the effects + perimental group; serum TH concentrations in of K . A NaClO4 experimental group was also in- this and the MMI experimental groups were the cluded to verify that the observed effects were not lowest measured in Experiment 3 (Fig. 3). Along related to an interaction between the effects of these same lines of reasoning, we measured higher K+ and the goitrogenic anion. serum TH concentrations in those experimental Treatment with 18 mM KCl resulted in the groups in which a lower incidence of metamor- death of all sea lampreys within 4 days. The high phosis was observed. The lowest incidence of meta- mortality rate within this treatment group may morphosis was observed in the L-KClO4 and be related to an increase in the osmolarity of the 302 R.G. MANZON ET AL. aquarium water. In general, larval lampreys can- ACKNOWLEDGMENTS not osmoregulate even in dilute seawater (i.e., This research was supported in part by a Natu- 10‰ NaCl; Mathers and Beamish, ’74), and po- ral Sciences and Engineering Research Council of tassium ions may exacerbate this osmoregulatory Canada (NSERC) postgraduate scholarship and difficulty. Death was not observed in any other an Ontario Graduate Scholarship (to R.G.M.). The experimental group over the course of Experiment authors thank Nancy Manjovski, Lori-Ann Man- 4. In the L-KCl experimental group larvae were zon, Preshi Shanmugathasan, and Brian Peck for exposed to 3.6 mM KCl, the same concentration + their technical assistance. of K that is present in a 0.05% KClO4 treatment. At this concentration, KCl had no effects on ei- LITERATURE CITED ther the incidence of metamorphosis or serum TH Brown DD. 1997. The role of thyroid hormone in zebrafish concentrations (Figs. 4 and 5). However, serum and axolotl development. Proc Natl Acad Sci USA 94:13011– 13016. TH concentrations in the KClO4 and NaClO4 experimental groups were significantly lower than Degnan BM, Souter D, Degnan SM, Long SC. 1997. Induction of metamorphosis with potassium ions requires development all other experimental groups (Fig. 5). Associated of competence and an anterior signaling center in the ascid- with these declines in serum TH concentrations ian Herdmania momus. Dev Genes Evol 206:307–376. was an induction of metamorphosis. In both the Dodd MHI, Dodd JM. 1976. The biology of metamorphosis.

KClO4 and NaClO4 experimental groups, we ob- In: Lofts B, editor. Physiology of the amphibia, Vol III. New served that 33% and 89% of all sea lampreys had York: Academic Press. p 467–599. Eales JG. 1997. Iodine metabolism and thyroid-related func- initiated metamorphosis in the 110–119 mm and tions in organisms lacking thyroid follicles: are thyroid hor- >119 mm size groups, respectively (Fig. 4). This mones also vitamins? Proc Soc Exp Biol Med 214:302–317. experiment has shown that NaClO4 has the same Eales JG, Holmes JA, McLeese JM, Youson JH. 1997. Thy- effects as KClO4 on both serum TH concentrations roid hormone deiodination in various tissues of larval and and metamorphosis, whereas KCl had neither of upstream-migrant sea lampreys, Petromyzon marinus. Gen Comp Endocrinol 106:202–210. these effects. These results clearly indicate that – + Eales JG, McLeese JM, Holmes JA, Youson JH. 2000. Changes ClO4 , and not K , is the ion responsible for de- in intestinal and hepatic thyroid hormone deiodination dur- pressing serum TH concentrations and inducing ing spontaneous metamorphosis of the sea lamprey, Petro- metamorphosis in lampreys. myzon marinus. J Exp Zool 286:305–312. In summary, the present results show that the Galton VA. 1983. Thyroid hormone action in amphibian metamorphosis. In: Oppenheimer JH, Samuels HH, editors. Mo- induction of precocious metamorphosis in larval lecular basis of thyroid hormone action. Toronto: Academic sea lampreys is associated with a decline in se- Press. p 445–483. rum TH concentrations following treatment with Gentile F, Di Lauro R, Salvatore G. 1995. Biosynthesis a goitrogen. The incidence of goitrogen-induced and secretion of thyroid hormones. In: DeGroot LJ, Bes- metamorphosis in sea lampreys is related to the ser M, Burger HG, Jameson JL, Loriaux DL, Marshall JC, Odell WD, Potts JT, Rubenstein AH, editors. Endo- ability of the goitrogen to lower serum TH con- crinology, 3rd edition, Vol 1. Toronto: W.B. Saunders centrations, further supporting the claim by Company. p 517–542. Manzon et al. (’98) that KClO4-induced metamor- Gudernatsch JF. 1912. Feeding experiments on tadpoles. I. phosis requires a decline in serum TH concentra- The influence of specific organs given as food on growth tions. Despite the absence of metamorphosis and differentiation. A contribution to the knowledge of organs with internal secretion. Arch Entwicklungsmech Org following PTU treatment, we feel that the induc- 35:457–483. tion of metamorphosis is a response to goitrogens Hoheisel G, Sterba G. 1963. Über die Wirkung von Kalium- in general and is not dependent on the mecha- perchlorat (KClO4) auf Ammocoeten von Lampetra planeri. nism by which a goitrogen reduces the capacity Bloch Z Mikrosk Anat Forsch 70:490–516. to synthesize TH. This idea is supported by the Holmes JA, Youson JH. 1993. Induction of metamorphosis in landlocked sea lampreys, Petromyzon marinus. J Exp Zool observation that MMI, a thyroid peroxidase-in- 267:598–604. hibiting goitrogen, can induce lamprey metamor- Holmes JA, Chu H, Khanam SA, Manzon RG, Youson JH. phosis. PTU may be unique among goitrogens 1999. Spontaneous and induced metamorphosis in the because it does not appear to induce lamprey American brook lamprey, Lampetra appendix. Can J Zool metamorphosis; however, further studies using 77:959–971. PTU concentrations that depress serum TH con- Inui Y, Miwa S, Yamano K, Hirano T. 1994. Hormonal control of flounder metamorphosis. In: Davey KG, Peter RE, Tobe centrations in sea lampreys are required to con- + SS, editors. Perspectives in comparative endocrinology. Ot- firm this finding. Lastly, we have shown that K tawa: National Research Council of Canada. p 408–411. does not play a role in KClO4- or KSCN-induced Just JJ, Kraus-Just J, Check DA. 1981. Survey of chordate metamorphosis. metamorphosis. In: Gilbert LI, Frieden E, editors. Meta- GOITROGEN-INDUCED METAMORPHOSIS IN LAMPREYS 303

morphosis: a problem in developmental biology, 2nd edition. tion in the larval lamprey. Proc First Congr Asia Oceania New York: Plenum Press. p 265–326. Soc Comp Endocrinol 1:220–221. Leatherland JF, Hilliard RW, Macey DJ, Potter IC. 1990. Suzuki S. 1989. Why goitrogens are chemical triggers of Changes in serum thyroxine and triiodothyronine concen- metamorphosis in the lamprey; relationship between thy- trations during metamorphosis of the Southern Hemisphere roid function and metamorphosis. Gen Comp Endocrinol lamprey Geotria australis, and the effect of propylthiou- 74:277. racil, triiodothyronine and environmental temperature on White BA, Nicoll CS. 1981. Hormonal control of amphibian serum thyroid hormone concentrations of ammocoetes. Fish metamorphosis. In: Gilbert LI, Frieden E, editors. Meta- Physiol Biochem 8:167–177. morphosis: a problem in developmental biology, 2nd edition. Lintlop SP, Youson JH. 1983. Concentrations of triiodothyro- New York: Plenum Press. p 265–326. nine in the sera of the sea lamprey, Petromyzon marinus, Whittaker JR. 1997. Chordate evolution and autonomous and the brook lamprey, Lampetra lamottenii, at various specification of cell fate: the ascidian embryo model. Am phases of the life cycle. Gen Comp Endocrinol 49:187–194. Zool 37:237–249. Manzon RG, Youson JH. 1997. The effects of exogenous Wright GM, Youson JH. 1977. Serum thyroxine concentra- thyroxine (T4) or triiodothyronine (T3), in the presence tions in larval and metamorphosing anadromous sea lam- and absence of potassium perchlorate, on the incidence prey, Petromyzon marinus L. J Exp Zool 202:27–32. of metamorphosis and on serum T4 and T3 concentra- Wright GM, Youson JH. 1980. Transformation of the endo- tions in larval sea lampreys (Petromyzon marinus L.). style of the anadromous sea lamprey, Petromyzon marinus Gen Comp Endocrinol 106:211–220. L., during metamorphosis. II. Electron microscopy. J Manzon RG, Youson JH. 1999. Temperature and KClO4-in- Morphol 166:231–257. duced metamorphosis in the sea lamprey (Petromyzon Yool AJ, Grau SM, Hadfield MG, Jensen RA, Markell DA, marinus). Comp Biochem Physiol 124C:253–257. Morse DE. 1986. Excess potassium induces larval metamor- Manzon RG, Eales JG, Youson JH. 1998. Blocking of KClO4- phosis in four marine invertebrate species. Biol Bull 170: induced metamorphosis in premetamorphic sea lampreys 255–266. by exogenous thyroid hormones (TH); effects of KClO4 and Youson JH. 1997. Is lamprey metamorphosis regulated by thy- TH on serum TH concentrations and intestinal thyroxine roid hormones? Am Zool 37:441–460. outer-ring deiodination. Gen Comp Endocrinol 112:54–62. Youson JH, Potter IC. 1979. A description of the stages in Mathers JS, Beamish FWH. 1974. Changes in serum osmotic the metamorphosis of the anadromous sea lamprey, Petro- and ionic concentration in landlocked Petromyzon marinus. myzon marinus L. Can J Zool 57:1808–1817. Comp Biochem Physiol 49A:677–688. Youson JH, Plisetskaya EM, Leatherland JF. 1994. Concen- Pearce CM, Scheibling RE. 1994. Induction of metamorpho- trations of insulin and thyroid hormones in the serum of sis of larval echinoids (Strongylocentrotus droebachiensis landlocked sea lampreys (Petromyzon marinus) of three lar- and Echinarachnius parma) by potassium chloride (KCl). val year classes, in larvae exposed to two temperature re- Invertebr Reprod Dev 26:213–220. gimes, and in individuals during and after metamorphosis. Potter IC, Wright GM, Youson JH. 1978. Metamorphosis in Gen Comp Endocrinol 94:294–304. the anadromous sea lamprey, Petromyzon marinus L. Can Youson JH, Holmes JA, Leatherland JF. 1995. Serum con- J Zool 56:561–570. centrations of thyroid hormones in KClO4-treated larval sea Sokal RR, Rohlf FJ. 1981. Biometry. New York: W.H. Free- lampreys (Petromyzon marinus L.). Comp Biochem Physiol man. p 859. 111C:265–270. Suzuki S. 1986. Induction of metamorphosis and thyroid func- Youson JH, Manzon RG, Peck BJ, Holmes JA. 1997. Effects tion in the larval lamprey. In: Mederios-Neto G, Gaitan E, of exogenous thyroxine (T4) and triiodothyronine (T3) on editors. Frontiers in thyroidology, Vol I. New York: Plenum spontaneous metamorphosis and serum T4 and T3 levels in Press. p 667–670. immediately premetamorphic sea lampreys, Petromyzon Suzuki S. 1987. Induction of metamorphosis and thyroid func- marinus. J Exp Zool 279:145–155.