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Lamprey Parasitism of Sharks and Teleosts: High Capacity Urea Excretion and Ornithine Urea Cycle Activity in an Extant Vertebrate Relic

Michael P. Wilkie*1, Steven Turnbull2, Jonathan Bird3, Yuxiang S. Wang4, Jaime F. Claude1, and

John H. Youson5

1. Department of Biology, Mount Allison University, Sackville, New Brunswick, E4L 1E4.

2. Department of Biology, University of New Brunswick Saint John, Saint John, New

Brunswick, E2L 4L5.

3. Oceanic Research Group, 59 Old Andover Road, North Reading, Massachussets, 01864.

4. Department of Biology, Queen’s University, Kingston, Ontario, K7L 3N6.

5. Department of Life Sciences, University of Toronto at Scarborough, Toronto, Ontario,

M1C 1A4.

* To whom all correspondence should be addressed.

Present address: Department of Biology, McMaster University, 1280 Main Street West,

Hamilton, Ontario, L8S 4K1.

Phone: 905 525-9140 ext. 27257. FAX: 905 522-6066

Email: wilkie@zoo.utoronto.ca

Running Title: Nitrogen Excretion by Parasitic Lampreys

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ABSTRACT

Although phylogenetically ancient sea lampreys (Petromyzon marinus) have plagued Great

Lakes’ fisheries for many years, little is known about the diet of rarely seen anadromous sea

lampreys during their marine phase. We observed 10 sea lampreys parasitizing basking sharks

(Cetorhinus maximus), the world’s second largest fish, in the Bay of Fundy. Due to the high

concentrations of urea in the blood and tissues of ureosmotic elasmobranchs, we hypothesized

that sea lampreys would require a mechanism(s) to eliminate co-ingested urea while feeding on

basking sharks. Initial urea excretion rates (JUrea) in two lampreys removed from the sharks by

divers were approximately 450 (≈ 9,000 µmol⋅kg-1⋅h-1) and 75 times (≈ 1,500 µmol⋅kg-1⋅h-1)

greater than basal (non-feeding) rates (≈ 20 µmol⋅kg-1⋅h-1). In contrast, JUrea increased by 15-fold

after parasitic lampreys were removed from non-ureosmotic rainbow trout (Oncorhynchus

mykiss). Since activities of the ornithine urea cycle enzymes carbamoyl phosphate synthetase III

(CPSase III) and ornithine carbamoyl transferase (OCT) were relatively low in liver, and below

detection in intestine and muscle, the excreted urea unlikely arose from de novo urea synthesis.

High arginase activities suggested that hydrolysis of dietary arginine made a minor contribution

to JUrea.. Post-feeding ammonia excretion rates (JAmm) were 15-25 fold greater than basal rates in

lampreys removed from both basking sharks and rainbow trout, suggesting that parasitic

lampreys have a high capacity to deaminate amino acids. We conclude that the sea lamprey’s

ability to penetrate the dermal denticle armour of sharks, an ability to rapidly excrete large

volumes of urea, and a high capacity to deaminate amino acids, represent adaptations that have

contributed to the evolutionary success of these phylogenetically ancient vertebrates.

KEY WORDS: arginase, ammonia, basking shark, carbamoyl phosphate synthetase, dermal

denticle, facilitated urea transport, osmoregulation, sea lamprey, trimethylamine oxide, urea,

uricolysis

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INTRODUCTION

Lampreys are extant representatives of the superclass Agnatha, an ancient group of

jawless vertebrates whose life style and body structure has been highly conserved for at least 350

million years (Forey and Janvier, 1993). After a filter-feeding larval phase lasting several years,

lampreys undergo a complex metamorphosis characterized by major changes in external and

internal body structure including a re-organization of the nervous, reproductive, respiratory,

digestive, hepatic and renal systems (reviewed by Youson, 1997). Following metamorphosis, the

sexually immature young of parasitic species begin feeding on the blood and/or flesh of fishes,

while non-parasitic species by-pass feeding and immediately begin sexual maturation. It is

generally accepted that these non-parasitic species arose from anadromous, parasitic ancestors

(Potter, 1980).

Although sea lampreys are anadromous, migrating to sea during their parasitic phase

(Scott and Scott, 1988), a landlocked population is also present in the Great Lakes. Indeed,

invading sea lampreys decimated commercial and recreational fisheries in this region in the early

20th century, and continue to threaten Great Lakes fisheries (Klar and Shleen, 2000). The sea

lamprey has several adaptations that make it a highly effective parasite, or a predator when it

ultimately kills its host. Through rhythmic rasping with its tongue, and by creating negative

pressure pulses with its sucker-like oral disc, blood is drawn into the lamprey’s oral cavity

(Rovainen, 1996). Blood flow is promoted by lamphredin secreted by paired buccal glands,

which has anticoagulant and cytolytic properties (Farmer, 1980). In addition, the gills are tidally

ventilated, a respiratory strategy likely selected due to the difficulty associated with drawing

water into the oral cavity while attached to a fish during feeding, or to the substrate (Randall,

1972; Rovainen, 1996).

Despite such adaptations, it is not clear if sea lampreys can parasitize sharks, which

would be ideal hosts due to their large size. Potential obstacles to parasitism could include the

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shark’s sharp dermal denticles (scales), which could act as a barrier to sea lamprey attacks, and

the very high osmolarity of the shark’s blood, which is due in part to high concentrations of urea

(e.g. Smith, 1936; Goldstein et al. 1968; Schmidt-Nielsen et al., 1972; Wood et al., 1995). In

contrast to ureosmotic elasmobranchs, sea lampreys are hyposmotic and use mechanisms of

osmoregulation similar to marine teleosts, in which water is extracted from ingested sea water in

the gut, and the co-ingested Na+ and Cl- actively excreted across the gills (Beamish et al., 1978).

Consumption of shark blood would therefore represent an additional osmotic challenge, but

toxicity could also result from urea’s perturbing effects on macromolecules such as proteins

(Yancey, 2001). A sea lamprey feeding on an elasmobranch therefore requires a mechanism(s)

to prevent internal urea accumulation, and/or an ability to eliminate co-ingested urea from their

blood. Accordingly, two objectives of the present study were to (i) confirm that sea lampreys

were able to penetrate the dermal denticle armour of sharks, and (ii) test the hypothesis that sea

lampreys have mechanisms to eliminate co-ingested urea from their blood while feeding.

The presence of basking sharks (Cetorhinus maximus) in the Bay of Fundy in late

summer and early autumn made it possible to determine if they were attacked and parasitized by

sea lampreys that are known to inhabit this area (Scott and Scott, 1988). Basking sharks, which

may attain lengths exceeding 10 m (Scott and Scott, 1988), migrate into this region in search of

zooplankton, the main food item of these giant filter-feeders (Sims and Quayle, 1998). In the

present study, lamprey parasitism of basking sharks was photographically documented. This was

followed by measurements of post-feeding urea and ammonia excretion rates in lampreys after

their removal from the ureosmotic basking sharks, or from freshwater acclimated rainbow trout,

which served as non-ureosmotic controls. To establish if endogenous urea production via

uricolysis or the ornithine urea cycle (OUC) contributed to post-feeding urea excretion patterns,

key enzymes of enzymes of these pathways were also measured in tissues of parasitic sea

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lampreys. These latter measurements also enabled us to examine the long-standing hypothesis

that the OUC was present in ancestral agnathans (Griffith, 1991; Walsh, 1997).

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MATERIALS AND METHODS

Experimental Animals and Protocol

All experiments were approved by the Mount Allison University, University of New

Brunswick at Saint John, and the University of Toronto Animal Care Committees, and followed

Canadian Council on Animal Care guidelines. Two parasitic sea lampreys (Petromyzon

marinus) were removed from basking sharks (Cetorhinus maximus) and were identified based on

the dentition of their oral disc and body structure (Scott and Scott, 1988). Following removal, the

lampreys were rapidly transported to the University of New Brunswick’s research vessel,

weighed, and transferred to separate, aerated, darkened flux chambers for immediate

determination of urea excretion (JUrea) and ammonia excretion (JAmm) rates (Wilkie and Wood,

1991). Water samples (15 mL) were taken at regular intervals (e.g. 0, 0.5, 1.0, 1.5, 2.0, 3.0 h)

using a 20 mL plastic syringe. In one lamprey, JUrea and JAmm were also monitored between 16

and 20 h post-capture. The lampreys were then transported in a 90 L cooler containing aerated

sea-water to Mount Allison University, where they were held for 2 weeks in a 500 L Living

Stream (Frigid Units Inc), containing sea water at 12ºC, without access to food. Basal JUrea and

JAmm were then determined and the animals were then killed with buffered anaesthetic (3 g

buffered tricaine methanesulfonate⋅L-1; Syndel laboratories), and blood samples (5 mL) taken

from the heart using 5 mL plastic syringes, coated with Na+-heparin (Sigma), for later

determination of ammonia and urea.

A separate series of experiments were conducted at the University of Toronto at

Scarborough, on landlocked, parasitic sea lampreys, provided courtesy of the Hammond Bay

Biological Station, U.S. Fish and Wildlife Service, Millersburg, Michigan. These animals were

held in freshwater at 15ºC for a minimum of 1 week before JUrea and JAmm were determined under

basal (non-feeding) conditions or after feeding on rainbow trout (Oncorhynchus mykiss). One

objective was to determine if similar post-feeding increases in JUrea took place after the ingestion

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of blood from a non-ureosmotic fish. A second goal was to determine if parasitic lampreys could

produce urea via the ornithine urea cycle (OUC) or uricolysis.

The rainbow trout, weighing approximately 300 g, were held in a 500 L Living Stream

receiving fresh-water at a rate of 1-2 L⋅min-1, and were fed three times per week with

commercial trout pellets. Parasitic sea lampreys (N = 8) were then introduced. Three days after

initial attachment of lamprey was noted, the trout, with the lamprey still attached, were removed

from the water, and placed in a 5 L bath containing anaesthetic (tricaine methanesulfonate; 1:10

000 dilution) for 1 minute, which rendered the trout unconscious, but not the sea lamprey. The

sea lamprey were then removed and placed in individual flux chambers for measurement of JUrea

and JAmm over 1 h time intervals for 8 h to 72 h. After 24 h or 72 h, the lampreys were killed

with anaesthetic, and blood, liver, intestine and muscle samples were collected. Tissues were

snap-frozen using liquid nitrogen cooled aluminum tongs. Non-feeding lampreys ( N = 10)

served as controls.

Analytical Techniques and Calculations

Measurements of JAmm and JUrea for both anadromous and landlocked, parasitic lampreys

were based on changes in the concentration of each waste product appearing in the water over 30

minutes to 2 hours, after correcting for the animal’s body weight and the volume of water in the

chamber (Wilkie and Wood, 1991). Water and plasma urea concentrations were determined

using a modification of the diacetyl monoxime method (Crocker, 1967) and commercial reagents

(Sigma). Water ammonia was measured using the salicylate-hypochlorite assay (Verdouw et al.,

1978), while plasma ammonia concentration was determined enzymatically (glutamate

dehydrogenase) using a commercial kit (Sigma).

Determination of OUC enzyme activities were optimized and analyzed as previously

described (Mommsen and Walsh, 1989; Barber and Walsh, 1993; Walsh, 1996; Wilkie et al.,

1999). Briefly, chunks of frozen tissues (liver, muscle, intestine) were transferred to a chilled 1.5

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mL centrifuge tube and ground for 30 seconds in 4 volumes of homogenization buffer (pH 7.5;

20 mmol.L-1 KH2PO4, 10 mmol.L-1 HEPES, 0.5 mmol.L-1 EDTA, 1 mmol.L-1 dithiothreitol; 50

percent glycerol) using a PowerGen model 125 tissue homogenizer. Following

homogenization, the samples were centrifuged for 3 minutes at 13 000 G and 4oC, afterwhich

enzyme activities were determined on the supernatant using a 96 well plate spectrophotometer at

the appropriate wavelengths (Molecular Devices, Spectramax340). Carbamoyl phosphate

synthetase III (CPSase III) determinations were based on the appearance of citrulline in the

reaction buffer following a 60 minute incubation of homogenate in a cocktail containing N-

acetyl glutamate, ornithine, glutamine and ornithine carbamoyl transferase. To ensure potential

CPSase II activity did not interfere with CPSase III determinations, uridine triphosphate (UTP)

was included in the cocktail to inhibit the former enzyme (Anderson, 1995). Ornithine

carbamoyl transferase activity, also based on citrulline generation, was determined in the

presence of saturating concentrations of ornithine and carbamoyl phosphate. Arginase activity

was based on observed increases in urea concentration following incubation of the liver

homogenate with arginine. Glutamine synthetase activity was based on the rate of appearance of

γ-glutamylhydroxamate, in the presence of glutamine, hydroxylamine and ferric chloride (Walsh,

1996). All enzyme activities are expressed as µmol product formed per gram of wet tissue per

minute at 20°C (µmol⋅g-1⋅min-1).

The JUrea and JAmm are presented separately for each lamprey removed from the basking

sharks. All other data are expressed as the mean ± 1 standard error of the mean (SEM).

Unpaired and paired data were analyzed using analysis of variance (ANOVA). When significant

variability was noted, statistical significance was determined using the Tukey-Kramer post-test at

the P < 0.05 level.

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RESULTS

Five basking sharks were observed south of the Wolve’s Islands, Bay of Fundy (Water T

=12°C), with a total of 10 lampreys attached to 4 of the sharks. The lampreys were generally

positioned behind the dorsal fin in groups of 1 to 4 (Fig. 1), and numerous lamprey scars and

fresh wounds in the vicinity of the dorsal fin indicated active feeding (Fig. 1). Divers

subsequently removed 2 lampreys from the basking sharks (Fig. 1 inset) and returned them to the

research vessel, where on-board measurements of urea excretion (JUrea) and ammonia excretion

(JAmm) rates were immediately initiated.

In the first lamprey, post-removal JUrea approached 9,000 µmol⋅kg-1⋅h-1 (Fig. 2A) over the

first 30 minutes, which was approximately 450 times greater than basal rates measured in the

laboratory after a two week fasting period, under identical saltwater conditions (full strength sea

water, 12°C). JUrea rapidly decreased over the next 2 h, and stabilized at 1,000-1,500 µmol⋅kg-1⋅h-

1 (Fig. 2A). Compared to basal rates, post-removal JAmm was 20-25 times greater during the first

hour. However, JAmm was 25 to 50 % lower than the corresponding measurements of JUrea during

the same period (Compare Fig. 2A to Fig. 2B). In the second lamprey, JUrea fluctuated between

500 and 1,000 µmol⋅kg-1⋅h-1 over 3 h, but JAmm was 2-3 fold greater (Fig. 2A, 2B). Due to rough

seas, it was not possible to monitor post-removal JUrea and JAmm further. Plasma ammonia and

urea concentrations in the two lampreys were similar after 2 weeks, approximately 1.2 mmol⋅L-1

(Table 1) and 130 µmol⋅L-1 (data not shown), respectively.

When JUrea and JAmm were measured in landlocked, laboratory held parasitic lampreys,

under control conditions, in which they were denied access to food for 7-10 days, basal JUrea was

approximately 15 µmol⋅kg-1⋅h-1 (Fig. 3A), while JAmm fluctuated around 100 µmol⋅kg-1⋅h-1 (Fig.

3B). After parasitizing rainbow trout, JUrea was stimulated 15-fold (Fig. 3A), and remained

elevated by 5- to 10-fold over 8 h before returning to pre-feeding levels. Similarly, post-feeding

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JAmm increased by approximately 25-fold, before returning towards basal levels after 8 h (Fig.

3B). Basal (non-feeding) plasma urea and ammonia concentrations averaged 0.7 ± 0.1 mmol⋅L-1

(N = 15; Table 1), and 685 ± 18 µmol⋅L-1 (N = 15; data not shown), respectively.

Key enzymes of the OUC and uricolytic pathways were monitored in the liver, intestine

and muscle of landlocked parasitic sea lampreys. Analysis of four key OUC enzymes revealed

that each was present in the liver (Table 2). However, CPSase III, and ornithine carbamoyl

transferase were below levels of detection in the intestine and muscle. Arginase, which catalyzes

the hydrolysis of arginine to urea in the final step of the OUC, exhibited 10-fold greater activity

in the liver compared to the intestine, but was below levels of detection in the muscle (Table 2).

Glutamine synthetase activity was detected in all three tissues, but was greatest in the intestine.

The two terminal enzymes in the uricolytic pathway, allantoinase and allantoicase, were also

detected in the liver of parasitic lampreys (Table 2).

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DISCUSSION

The very high JUrea of anadromous sea lampreys provides convincing evidence that these

parasites were feeding sometime prior to their removal from the basking sharks. Earlier studies

and anecdotal observations suggested possible lamprey parasitism of basking sharks (Bigelow

and Shroeder, 1948), and more recently it has been reported that sea lampreys also attack

sandbar (Carcharhinus plumbeus) and dusky sharks (Carcharhinus obscurus; Jensen and

Schwartz, 1994), as well as tiger sharks (Galeocerdo cuvier; Jensen et al., 1998). It is not clear if

lampreys were feeding in these other instances, due to the difficulty and danger associated with

removing lampreys from these more active, predacious shark species. However, open wounds

were reported on the bodies of the sandbar and dusky sharks (Jensen and Schwartz, 1994).

Parasitism of elasmobranchs could be one reason why anadromous sea lampreys are

approximately twice as large as their landlocked counterparts, whose diet is restricted to much

smaller teleosts (Farmer, 1980). Indeed, anadromous sea lampreys weigh up to 1 kg and attain

lengths of more than 70 cm (Beamish and Potter, 1975).

The observed JUrea indicate that parasitic sea lampreys can exhibit facultative ureotelism,

in which urea becomes the dominate nitrogenous waste product excreted, following the ingestion

of urea-rich blood. The rapid decrease in JUrea following the first lamprey’s removal from the

basking shark likely reflected the cessation of feeding. During actual feeding, JUrea would need to

be sustained at the high levels initially measured following removal from the shark. The initially

lower JUrea of the second lamprey may because feeding ceased sometime prior to its removal

from the basking shark.

These JUrea’s are amongst the highest ever recorded in fishes; only the Lake Magadi

tilapia (Alcolapia grahami; Randall et al., 1989) and gulf toadfish (Opsanus beta; Mommsen and

Walsh, 1989) have had JUrea in the range reported here. However, the Lake Magadi tilapia, gulf

toadfish, and other ureogenic fishes such as the Indian catfishes (Heteropneustes fossilis; Saha

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and Ratha, 1989), have a fully functional ornithine urea cycle (OUC), which produces urea at

high rates to detoxify ammonia under conditions (e.g. alkaline water, air exposure, crowding)

that oppose ammonia excretion. Moreover, the urea generated by the Magadi tilapia and the gulf

toadfish is excreted across the gills via facilitated urea transport (Walsh et al., 2000, 2001), while

elasmobranchs appear to use similar transport mechanisms to retain urea for osmoregulation (e.g.

Schmidt-Nielsen et al., 1972; Wood et al., 1995; Smith and Wright, 1999; Fines et al., 2001).

Although urea has a low molecular mass (60 Da) and high water solubility, its very low lipid

partition co-efficient precludes urea movement across lipid bilayers, necessitating the use of

facilitated and active urea transporters (reviewed by Walsh and Smith, 2001). Indeed, facilitated

and active urea transporters have been well characterized in mammalian renal tubules, where

they concentrate urea in the inner renal medulla to maximize water reabsorption (Knepper and

Chou, 1995; You et al., 1993; Sands, 1999). The high JUrea following feeding on basking sharks,

as well as the 15-fold increase following feeding on rainbow trout, therefore suggests that similar

urea transport mechanisms are probably present in the gills, kidneys, and/or the gut of sea

lampreys. However, further investigation is required to confirm this hypothesis and to identify

the route(s) of urea excretion in these animals.

It is unlikely that post-feeding JUrea by parasitic lampreys arose from de novo urea

synthesis. For instance, the plasma urea concentrations were relatively low in both seawater and

freshwater adapted parasitic sea lampreys that were not feeding (Table 1). Although OUC

enzymes were present in the livers of the landlocked sea lampreys, the activities of key enzymes

such as CPSase III and OCT were relatively low, and below detection in intestine and muscle

(Table 2). Arginase activities were relatively high (Table 2) compared to other fishes, which

suggests that hydrolysis of dietary arginine contributed to the high post-feeding JUrea observed in

lampreys following their removal from both basking sharks and trout. Given the low blood

arginine concentrations in both sharks and salmonid fishes, it seems unlikely that arginine

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hydrolysis accounted for more than a small fraction of the observed JUrea. For instance, plasma

arginine concentrations are approximately 0.2 mmol·L-1 in elasmobranchs (Gutiérrez et al.,

1987), while urea concentrations range from approximately 600-900 mmol·L-1 (Table 1).

Nevertheless, it would be useful to more precisely quantify the role that dietary arginine plays in

post-feeding urea excretion by parasitic lampreys.

Since ureosmotic elasmobranchs retain urea in their body fluids at concentrations that are

2-3 orders of magnitude greater than in lampreys and teleosts (Table 1), a more likely

explanation for the high post-feeding JUrea is that the sea lampreys were simply clearing urea that

was co-ingested as they parasitized their hosts. For instance, lampreys parasitizing salmonid

fishes consume blood at rates ranging from 3-30 % of their body weight per day (Farmer et al.,

1975). Thus, if a 100 g sea lamprey were to ingest the blood of a typical elasmobranch

(assuming a blood urea concentration of 636 mmol⋅L-1; Wood et al., 1995) at similar rates, a urea

load of 19-190 mmol urea-N·kg-1 body weight per day would result! Although typical blood

urea concentrations are much lower in salmonids, at most 5 mmol⋅L-1 (Wilkie and Wood, 1991),

a lamprey feeding on a trout would experience a urea load ranging from approximately 0.16-1.60

mmol urea-N·kg-1 body weight per day. Such a urea load from a trout would not likely have any

detrimental effects on a parasitic lamprey. However, the 100-fold greater urea load that may

arise from the parasitism of an elasmobranch such as a basking shark could be toxic due to urea’s

perturbing effects on protein structure. Indeed, urea is known to perturb enzyme function,

microtubule assembly, and the structural integrity of membranes (reviewed by Yancey, 2001).

It might be argued that that the high concentrations of counteracting solutes such as

trimethylamine oxide (TMAO) in elasmobranch blood and tissues would confer some degree of

protection to a lamprey parasitizing a shark. Indeed, TMAO is thought to protect elasmobranchs

against urea by stabilizing protein structure (Yancey, 2001), and would likely have similar

benefits if it were co-ingested with urea by a parasitic sea lamprey. However, it should also be

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pointed out that elasmobranchs have additional adaptations that may provide further protection

against urea’s destabilizing effects such as urea tolerant proteins that include actomyosin

ATPases and hemoglobin (Yancey, 2001). The higher percentage of saturated fatty acids in the

mitochondrial membranes of elasmobranchs are also thought to increase their resistance to urea’s

perturbing effects (Glemet and Ballantyne, 1996). Further studies are required to quantify the

urea and TMAO load in the blood and tissues of lampreys parasitizing elasmobranchs.

Additional experiments should also evaluate the possibility that co-ingested TMAO ameliorates

urea’s perturbing effects, as well as probing for lamprey-specific adaptations that might allow

them to withstand large, transient elevations of internal urea.

Although urea synthesis does not substantially contribute to greater post-feeding JUrea,

these findings may shed some light on the origin of the OUC in vertebrates. Most fishes are

ammoniotelic, and readily excrete most of their nitrogenous wastes as highly soluble ammonia

across the gills (Wilkie, 2002). Because it is less toxic than ammonia, urea synthesis is

considered one of the key adaptations that enabled vertebrates to invade terrestrial habitats

(Mommsen and Walsh, 1989; Griffith, 1991). A central hypothesis is that the OUC was present

in ancestral agnathans, but that its expression was subsequently silenced in fishes because it was

energetically less expensive to produce and excrete ammonia (Brown and Brown, 1985; Griffith,

1991; Wright et al.,1995; Walsh, 1997). The presence of key OUC enzymes in many teleosts,

along with elasmobranchs, coelacanths, lungfishes, amphibians and mammals supports the

hypothesis that the OUC is a monophyletic trait in the vertebrate lineage (Mommsen and Walsh,

1989, 1991). However, this hypothesis has not yet been substantiated by measurements of OUC

enzymes in extant agnathans such as the hagfishes or lampreys.

Wilkie et al. (1999) demonstrated that larval sea lampreys excreted physiological relevant

amounts of urea, but were unable to detect the key OUC enzyme, CPSase III, in the liver of these

animals. We have no explanation for the lack of CPSase III activity in larval lamprey livers, but

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it could be related to their much lower metabolic rate, slow rate of digestion (Potter and Rogers,

1972; Sutton and Bowen, 1994; Wilkie et al., 2001) and vastly different diet, which mainly

comprises detritus (Sutton and Bowen, 1994). Although Read (1968) detected significant

CPSase activity in the liver of adult Pacific lamprey (Entosphenus tridentatus), he was unable to

identify other key OUC enzymes such as OCT. Further, it was not clear if he detected CPSase

III, because he did not inhibit CPSase II using uridine triphosphate (Anderson, 1995), as was

done in the present study. The low activity of CPSase III and OCT in the liver of parasitic sea

lampreys suggests that the components of this key urea production pathway were probably

present in an ancestral jawless vertebrate. However, these low activities also suggest that the

OUC produces little, if any, physiological relevant amounts of urea in parasitic sea lampreys.

More likely, the small amounts of urea excreted under starved conditions represent endogenous

production arising from uricolysis, as was suggested for larval lampreys (Wilkie et al., 1999).

Indeed, the activities of allantoicase and allantoinase in the present study are comparable to

measurements made in the livers of larval lampreys.

Several-fold increases in JAmm are common in fishes following feeding, due to increased

proteolysis and amino acid catabolism (Wood, 2001). However, the 25-fold greater JAmm

observed in parasitic sea lampreys after feeding on both sharks and rainbow trout is unusually

high compared to other fishes, suggesting that parasitic lampreys have a high capacity to

deaminate excess amino acids arising from their protein rich blood diet and high feeding rates.

Further support for this hypothesis is provided by measurements of hepatic glutamate

dehydrogenase activity and expression, which is several-fold higher in the parasitic stage of the

sea lamprey’s life cycle (Wilkie, Wang, Claude, and Youson, unpublished observations). Such a

strategy would be beneficial during the more active, free swimming parasitic stage of the

lamprey life cycle, by providing the raw-materials (α-keto acids) required for ATP production

via oxidative pathways or glycogen synthesis (Mommsen et al., 1980). Muscle glycogen stores

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could be critical for providing energy during episodes of burst exercise during subsequent

upstream migration and spawning by the sea lamprey (Boutilier et al., 1993). Indeed, the muscle

glycogen concentrations of migrating sea lampreys are comparable to stores measured in the

white muscle of salmonids (Milligan, 1996), which undergo similar extensive migrations.

Our observations reveal that anadromous sea lampreys parasitize basking sharks, and that

they rapidly unload co-ingested urea, which likely prevents deleterious increases in internal urea

and additional osmotic stress. Although the life style and body structure of lampreys has been

highly conserved for at least the last 350 million years (Forey and Janvier, 1993), we have little

evidence of their behaviour or physiology to explain their success. The ability of sea lampreys to

penetrate the dermal denticle armor of an elasmobranch, an ability to rapidly excrete potentially

toxic urea, and a high capacity to deaminate amino acids, may represent additional adaptations

that have contributed to the lamprey’s evolutionary success. The presence of key OUC enzymes

such as CPSase III and OCT, also supports the hypothesis that the components of this pathway of

urea production were likely present in an ancestral jawless fish(es).

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ACKNOWLEDGEMENTS

We are grateful to Bob Bozene, Captain of the University of New Brunswick research vessel the

“Mary O”, and to Wayne Anderson who provided invaluable technical assistance. Ed Woods and

Tim Geers are acknowledged for their assistance locating and photographing the basking sharks

and lampreys observed in this study. We thank Dr. P.J. Walsh for his helpful comments. This

research was funded by Natural Sciences and Engineering Research Council (NSERC) of

Canada research grants to MPW and JHY; ST was supported by the University of New

Brunswick at Saint John; YSW held an NSERC postdoctoral fellowship.

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Table 1: Typical Urea-N Concentrations and Osmolarities in the Plasma of Sea Lampreys,

Sharks, and Teleosts.

Medium Urea-N Osmolarity

(mmol N⋅L-1) (mOsm⋅kg-1)

Parasitic Sea Lamprey Sea Water 1.2 ± 0.1 (N=2)a 263b

(Petromyzon marinus)Fresh Water 0.7 ± 0.1 (N = 15)a 300b

Small-spotted Catshark Sea Water 882c 1118 c (Scyliorhinus canicula)

Spiny Dogfish Shark Sea Water 724d 982d

(Squalus acanthias)636e ------

Lemon Shark Sea Water 842f ------ (Negaprion brevirostris)

Carp Fresh Water 1.7c 274c

(Cyprinius carpio)

Rainbow Trout Fresh Water 5.2g ------ (Oncorhynchus mykiss)

(a) Present Study; (b) Beamish et al. (1978); (c) Evans (1993); (d) Schmidt-Neilsen et al.

(1972); (e) Wood et al. (1995); (f) Goldstein et al.(1968); (g) Wilkie and Wood (1991).

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Table 2: Activities of Key Ornithine Urea Cycle and Uricolytic Enzymes in the Liver, Muscle and Intestine of Parasitic Sea Lampreys.

Liver Muscle Intestine Ornithine Urea Cycle Enyzmes Carbamoyl Phosphate Synthetase III 0.0065 ± 0.001 (7) BLD (6) BLD (6)

Ornithine Carbamoyl Transferase 0.13 ± 0.03 (7) BLD (6) BLD (6) Arginase 9.38 ± 2.57 (7) BLD (6) 0.83 ± 0.38 (6) Glutamine Synthetase 3.06 ± 0.59 (7) 0.33 ± 0.01 (7) 8.43 ± 1.77 (6)

Uricolytic Enzymes Allantoicase 0.03 ± 0.00 (4) ------ ------ Allantoinase 0.50 ± 0.02 (7) ------ ------

All enzyme activities are expressed as µmol product formed per gram of wet tissue per minute at

20°C (µmol⋅g-1⋅min-1). Number of animals sampled in parentheses. BLD; below level of

detection.

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FIGURE LEGENDS

Figure 1: Parasitic sea lampreys attached to the dorsal body surface of basking sharks off of the

Wolves Islands, Bay of Fundy, Canada. Inset: Removal of a sea lamprey from the dorsal

body surface of a basking shark.

Figure 2: Individual (A) urea and (B) ammonia excretion rates of two anadromous, parasitic sea

lampreys following their removal from basking sharks, and their basal rates after a two week

fast. Solid circles represent the first lamprey captured (Mass = 80 g), and open triangles the

second (Mass 292 g).

Figure 3: Post-feeding (A) urea and (B) ammonia excretion rates of landlocked, parasitic sea

lampreys (N=8; Mass = 25.2 ± 7.7 g) after removal from rainbow trout. Basal rates were

determined on lampreys (N = 10; Mass = 22.3 ± 2.6 g) that had been fasted for at least 1

week. Asterisks (*) denote statistically significant differences between post-feeding and basal

(non-feeding) excretion rates (P<0.05).

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FIGURE 1 - Wilkie, Turnbull, Bird, Wang, Claude and Youson.

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FIGURE 2 - Wilkie, Turnbull, Bird, Wang, Claude and Youson.

020

1,000

2,000

6,0008,000

10,000

∆

0 1 2 3 16 18 20Basal

∆

∆

Post-Feeding Urea Excretion

Lamprey 1Lamprey 2

A

0200

1,000

2,000

3,000

4,000

5,000

0 1 2 3 16 18 20Time (h)

∆∆

∆

∆

∆

Post-Feeding Ammonia ExcretionB

Basal

µmol

Nkg

-1h-

1µm

olN

kg-1

h-1

∆ ∆

020

1,000

2,000

6,0008,000

10,000

∆

0 1 2 3 16 18 20Basal

∆

∆

Post-Feeding Urea Excretion

Lamprey 1Lamprey 2

A

0200

1,000

2,000

3,000

4,000

5,000

0 1 2 3 16 18 20Time (h)

∆∆

∆

∆

∆

Post-Feeding Ammonia ExcretionB

Basal

µmol

Nkg

-1h-

1µm

olN

kg-1

h-1

∆ ∆

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FIGURE 3 - Wilkie, Turnbull, Bird, Wang, Claude and Youson.

-2 0 2 4 6 8 10 120

50

100

150

200

250

24 48 72

*

*** * **

*

* *

2 4 8 106 120 24 48 72Basal

A

-2 0 2 4 6 8 10 120

500

1,000

1,500

2,000

2,500

3,000

3,500

24 48 72Basal 2 4 8 106 120 24 48 72

*

* * * * *

*

*

**

**

B

Time (h)

µmol

Nkg

-1h-

1µm

olN

kg-1

h-1

Post-Feeding Urea Excretion

Post-Feeding Ammonia Excretion

-2 0 2 4 6 8 10 120

50

100

150

200

250

24 48 72

*

*** * **

*

* *

2 4 8 106 120 24 48 72Basal

A

-2 0 2 4 6 8 10 120

500

1,000

1,500

2,000

2,500

3,000

3,500

24 48 72Basal 2 4 8 106 120 24 48 72

*

* * * * *

*

*

**

**

B

Time (h)

µmol

Nkg

-1h-

1µm

olN

kg-1

h-1

Post-Feeding Urea Excretion

Post-Feeding Ammonia Excretion

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