J Comp Physiol B (2010) 180:475–493
DOI 10.1007/s00360-010-0447-0
R EV IE W
Freshwater elasmobranchs: a review of their physiology
and biochemistry
James S. Ballantyne · J. W. Robinson
Received: 16 October 2009 / Revised: 13 January 2010 / Accepted: 14 January 2010 / Published online: 9 February 2010
© Springer-Verlag 2010
Abstract Only 5% of elasmobranch species live in fresh-
water (FW) compared to more than 40% of known teleost
species. The factors aVecting the poor penetration of elas-
mobranchs into FW environments are currently unknown,
however, an important consideration may be the high urea
requirement of many proteins in marine elasmobranchs.
Urea is an important osmolyte in marine elasmobranchs
and must be reduced in dilute environments. There are
three identiWable stages in the successful colonization of
FW. The euryhaline marine species freely entering and
leaving FW represent the initial stage of FW colonization.
In this group, there is an apparent inability to eliminate all
urea due to protein integrity issues and this results in energy
and nitrogen losses that may constrain growth and repro-
duction. The second stage is represented by those species
that live entirely in FW but must also retain some urea. This
group also suVers from the same constraints as the Wrst
group. These two groups have kidneys and sensory organs
that more closely resemble strictly marine forms. The third
and Wnal stage is represented by the Potamotrygonid sting-
rays where the need for urea in FW has been eliminated.
Consequently nitrogen and energy losses are reduced and
those sections of the kidney needed for urea conservation
have been eliminated. The driving force for such modiWca-
tions is a reduction in urea levels and the concomitant
saving of energy needed for urea synthesis. Other physio-
logical adaptations associated with survival in FW include
Communicated by I. D. Hume.
J. S. Ballantyne (&) · J. W. Robinson
Department of Integrative Biology,
University of Guelph, Guelph, ON N1G 2W1, Canada
e-mail: jballant@uoguelph.ca
giving birth to live young, the capacity of sperm to be acti-
vated in freshwater and modiWcations of the electrosensory
system to function in a low conductivity environment. The
need for many anatomical, metabolic and physiological
modiWcations for FW existence may constrain the rapidity
and hence the frequency of FW colonization, compared to
the situation in the more advanced osmoregulating teleosts.
Once optimally adapted to FW, recolonization of sea water
by elasmobranchs is problematic due to the loss of urea
synthetic capacity and renal structures for urea retention.
Keywords Elasmobranch · Freshwater · Metabolism ·
Osmoregulation · Salinity · Physiology
Introduction
While the vast majority of elasmobranchs reside in sea-
water (SW) for their entire life, a few species [5% of all
species (Helfman et al. 1997)] routinely enter freshwater (FW)
and only 3–4% of all species spend their entire lives in FW
(Martin 2005). All of these cases occur in tropical or sub-
tropical environments. By contrast, 41% of known teleost
Wsh species live in FW from the poles to the equator (Cohen
1970). The problems faced by an animal transitioning from
a marine to a FW environment include osmoregulatory,
ionoregulatory, acid–base, sensory and energetic con-
straints. FW is more dilute, contains lower levels of most
ions, is a poor conductor of electricity and can be more
acidic.
The basis for the poor penetration of FW habitats by
elasmobranchs is not known and one of the objectives of this
review is to discuss the possible bases for this phenomenon.
One important diVerence between marine elasmobranchs
and marine teleost Wsh lies in their osmoregulatory strategies.
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476
There are several recent reviews on this subject including
Evans et al. (2005) and Hammerschlag (2006). Marine
elasmobranchs use large quantities of organic solutes to
maintain their osmotic status slightly hyperosmotic to SW.
In particular, they retain very high levels of urea (40% of
their solutes) and methylamines or amino acids (up to 20%
of solutes). This heavy investment in nitrogen-containing
compounds for osmoregulation resulted in large diVerences
in the metabolic organization of marine elasmobranchs
compared to their teleost counterparts. In FW, the need for
these solutes decreases substantially but many aspects of their
metabolic organization are retained.
On physiological grounds, we can identify three stages
in the colonization of FW. The Wrst stage involves euryha-
line marine species that can routinely enter and leave FW
[e.g. bull shark (Carcharhinus leucas) and some popula-
tions of Dasyatis sabina]. The second stage includes spe-
cies that can live permanently in FW but are able to tolerate
higher salinities. Examples of this group include some popu-
lations of D. sabina in the St. Johns River system of
Florida (Johnson and Snelson 1996), D. garouaensis from
western Africa and Himantura signifer from southeast
Asia. The third and Wnal stage includes the species found
only in FW that have no capacity to tolerate higher salini-
ties. These are the stenohaline FW stingrays of the Family
Potamotrygonidae that have been isolated in the FWs of
South America for millions of years (Lovejoy et al. 1998)
and lack the ability to tolerate higher salinities. The physi-
ology of some groups found in FW is completely unknown
and so it is not possible to assign them to one of these
groups (e.g. the FW shark Glyphis spp.). The Potamotrygo-
nids should be considered the most successful elasmo-
branch group to adapt to FW with more than 25 related
species. The FW stingrays from southeast Asia (D. laosen-
sis, D. bennetti, H. signifer and H. chaophraya) have only
marine species as their nearest relative (Sezaki et al. 1999)
implying multiple colonizations rather than speciation
within FW. The review by Compagno (1995) provides a
more complete list of species names and known salinity
tolerances of all elasmobranchs found in FW either perma-
nently or transiently.
The earliest studies of the eVects of salinity on an elas-
mobranch are those of Quigley (1928) who exposed
Squalus acanthias to low salinity and found that gill move-
ments and heart function ceased in FW after about 2 h.
Studies of osmoregulation in FW elasmobranchs really
began with those of Homer Smith who examined the south-
east Asian FW sawWsh Pristis microdon (Smith 1931).
Subsequent studies of the FW populations of bull shark
C. leucas in Lake Nicaragua showed that this species was
euryhaline and established some aspects of its FW biology
(Thorson 1962a, b, 1971; Thorson et al. 1966, 1973). Thor-
son also studied FW stingrays (Family: Potamotrygonidae)
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J Comp Physiol B (2010) 180:475–493
from South America and was the Wrst to show that the
members of this group diVered physiologically from other
elasmobranchs in FW (Brooks et al. 1981; Gerst and
Thorson 1977; Thorson et al. 1967, 1978, 1983; Thorson
1970; Thorson and Brooks 1983). More recently, the dis-
covery of a resident population of FW stingrays (D. sabina)
in the St. Johns River system in Florida has led to further
studies of this more accessible species (Janech et al. 2006b;
Johnson and Snelson 1996; Piermarini and Evans 2000).
Most recently studies of FW stingrays from rivers in south-
east Asia have begun to characterize the physiology of this
group (Ip et al. 2003; Raschi et al. 1997; Tam et al. 2003).
This review focuses primarily on those aspects of the
physiology and metabolism of elasmobranchs that are
relevant to their existence in FW.
Osmoregulation
It is widely accepted that all FW elasmobranchs are derived
from marine forms (Thorson and Brooks 1983) and hence
to understand the physiology of elasmobranchs in FW, it is
Wrst necessary to understand the relevant aspects of the
physiology of marine forms. The physiology of marine
elasmobranchs is very diVerent from that of marine teleosts.
Marine elasmobranchs maintain the osmolarity of their
Xuid compartments slightly above that of SW while marine
teleosts are substantially hypotonic to SW. Thus, although
both marine teleosts and elasmobranchs face the inXux of
salt (NaCl), the elasmobranchs also face a water inXux
while marine teleosts lose water. Marine teleosts drink SW
to gain back water and expel Na+ and Cl¡ ions via the gills.
Marine elasmobranchs on the other hand do not drink SW
and they have a specialized organ, the rectal gland, that
helps eliminate excess salt. This organ provides much of
the same osmotic function as the marine teleost gill. When
marine elasmobranchs encounter lower salinities, initially
the greater hypertonicity causes an inXux of water and a
transient increase in Xuid compartment volumes. For exam-
ple, blood volume as a percentage of body weight increases
in Scyliorhinus canicula (Anderson et al. 2007). To reduce
this, the rectal gland and kidney act to excrete Na+ Cl¡ ions
and increase urine Xow to reduce salt and urea levels,
respectively. After an acclimation period, the volumes of
various Xuid compartments of elasmobranchs in FW or SW
are similar with only slightly higher total water content in
FW (Thorson 1962b) and the need for the rectal gland is
eliminated. The turnover of water in marine and FW
adapted species is similar and higher than those of marine
and FW teleosts (Table 1).
The distinguishing feature of the marine elasmobranch
osmoregulatory system is the accumulation of high levels
of urea in all body Xuid compartments. Urea has many
J Comp Physiol B (2010) 180:475–493 477

Table 1 Permeability of marine

Species Turnover (% per hour)
and FW elasmobranchs and tele-
osts to ions, water and urea Sodium Chloride Water Urea

Marine elasmobranchs
Ginglymostoma cirratum 0.46 § 0.16b 1.52 § 0.41b 81 § 17b 0.14 § 0.07b
c
Scyliorhinus canicula 156.6 § 13.08
Raja montagu 167 § 11.1c
Raja erinacea 64.2 § 1.91d
Torpedo marmorata 97c
Poroderma africanum 97 § 8.5e
Freshwater elasmobranch
Potamotrygon sp. 0.28 § 0.16a 0.22 § 0.13a 96 § 22a
Freshwater teleosts
Carassius auratus 51.1 § 3.9f
Oncorhynchus mykiss 28g
Anguilla anguilla 13g
a
Carrier and Evans (1973), Platichthys Xesus 20g
b Marine teleosts
Carrier and Evans (1972),
c
Payan and Maetz (1971), Platichthys Xesus 36g
d
Payan et al. (1973), e Haywood
Serranus scriba 23.8 § 1.2f
(1974), f Motais et al. (1969),
g
Evans (1969) Anguilla anguilla 34g

functions in organisms including acting as a waste product
in terrestrial mammals, as a nitrogen transport molecule in
ruminants, as an osmolyte in estivating lungWsh and
amphibians and for acid–base regulation [see Withers
(1998) for review]. In elasmobranchs, its main function is
osmotic. Numerous adaptations are required to help retain
this molecule. When marine elasmobranchs enter dilute
environments, solute levels must be reduced. The main
eVector of this reduction is loss of urea, although concentra-
tions of other organic solutes (methylamines and amino
acids) also decline.
Freshwater elasmobranchs display two diVerent patterns
of urea retention (Table 2). Euryhaline marine forms have
high levels in the 280–450 mM range in SW and lower lev-
els (80–200 mM) in FW. Some species found primarily or
exclusively in FW also have lower urea levels in the same
range. In the exclusively FW Potamotrygonids, urea is
found at negligible levels (<1 mM) (Table 2).
Changes in water movement are obviously important
when elasmobranchs move from SW to FW and vice versa.
The role of aquaporins in this has not been examined but
several forms have been identiWed in C. leucas (Cutler and
Cramb 2009; Meischke et al. 2007). Important issues to
resolve include the type of aquaporins, the tissue distribu-
tion and their regulation. The selective permeability of
these aquaporins to urea and water also needs to be deter-
mined since the relative rates of transport of these two mole-
cules are critical in determining cell volume changes.
Inorganic ion levels in the plasma and intracellular com-
partment also diVer between the two FW groups with the
Potamotrygonids having lower levels of Na+ and Cl¡ in
blood compared to the other group of FW elasmobranchs
(Table 3). Turnover of Na+ and Cl¡ are lower in FW elasmo-
branchs compared to marine species in SW as would be
expected (Table 1). Potamotrygonid stingrays do have
higher plasma Cl¡ levels (155–192 mM) than water breath-
ing Amazonian teleosts (Johansen et al. 1978; Mangum
et al. 1977).
Most inorganic ions and ionic strength remain relatively
constant when sharks move from SW to FW (Urist 1962)
but at least in the initial stages (Wrst 8 days) of the transition
back from FW to SW Na+, Cl¡, K+, Mg+2 and Ca+2 increase
(Pillans et al. 2006). K+, Ca+2, HCO3¡, phosphorus and
protein in plasma are not diVerent between FW, estuarine
and marine bull sharks (Coelho and Erzini 2006).
The following sections outline the roles of the three tis-
sues involved in osmo- and iono- regulation: the rectal
gland, the kidneys and the gills.
Rectal gland
The rectal gland is a specialized structure unique to elasmo-
branchs that aids in salt excretion. This organ produces a
highly concentrated Xuid containing primarily NaCl that is
channeled into the rectum for excretion. While it has an
important role in the early stages of FW adaptation, it is not

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Table 2 Urea concentrations in
Species
plasma of elasmobranchs
Marine elasmobranchs
Dasyatis americana
Raja erinacea
Heterodontus portusjacksoni
Sphyrna tiburo
Euryhaline elasmobranchs in seawater
Carcharhinus leucas
Dasyatis sabina
Euryhaline elasmobranchs in freshwater
Pristis microdon
Dasyatis Sabina
Dasyatis uarnak
Carcharhinus melanops
Hypolopus sephen
Carcharhinus leucas Lake Nicaragua
Carcharhinus leucas
Carcharhinus leucas
Carcharhinus leucas
Himantura signifer
Freshwater elasmobranchs
Dasyatis benneti
Dasyatis garouaensis
Dasyatis garouaensis
Potamotrygonids
Potamotrygon hystrix
Potamotrygon motoro
Potamotrygon magdalenae
needed in ion poor FW. The importance of the rectal gland
diVers among the three groups of elasmobranchs in FW.
For those species that enter and leave FW routinely, the
rectal gland is retained but produces no salt secretion in FW
and may decrease slightly in size. Rectal glands of FW bull
sharks are smaller than those of their marine conspeciWcs
(Oguri 1964) but in bull sharks in Australia, there was no
diVerence in rectal gland size between FW and estuarine
forms (Pillans et al. 2008; Pillans and Franklin 2004). The
rectal glands of the euryhaline D. sabina in FW were 80%
smaller than those of marine D. sabina (Piermarini and
Evans 1998). For those species residing permanently in
FW, it is substantially reduced in size and in Potamotrygo-
nids it is non-functional (Thorson et al. 1978).
Interestingly, euryhaline species such as bull sharks
(Thorson et al. 1978) and Dasyatis guttata (Thorson and
Brooks 1983) have substantially smaller rectal glands even
in SW compared to their stenohaline congeners. The basis
for this has not been examined but may imply a greater role
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J Comp Physiol B (2010) 180:475–493
Plasma urea Reference
concentration (mM)
444 Cain et al. (2004)
359.4 § 17.9 Treberg et al. (2006)
269 § 16 Cooper and Morris (1998b)
289.6 § 30.3 Mandrup-Poulsen (1981)
370 § 9.5 Pillans and Franklin (2004)
394.5 + 5.5 Piermarini and Evans (1998)
130 Holmes and Donaldson (1969)
195.9 § 7.9 Piermarini and Evans (1998)
104 Holmes and Donaldson (1969)
103 Holmes and Donaldson (1969)
81 Holmes and Donaldson (1969)
132
180 Holmes and Donaldson (1969)
169 § 5.4 Thorson et al. (1973)
151 § 5 Pillans et al. (2005)
78 § 2 Chew et al. (2006)
140.0 § 23.6 Ballantyne (unpublished)
69.3 Taniuchi (1991)
212 Thorson and Watson (1975)
0.68 Bittner and Lang (1980)
0.65 § 0.17 Tam et al. (2003)
0.71 § 0.08 Ogawa and Hirano (1982)
for the gills in salt transport than in other marine elasmo-
branchs.
The mechanism of extrusion of NaCl by the rectal gland
is in principle similar to that of the chloride cells of marine
teleosts or the salt glands of marine birds and reptiles. A
basolateral Na+/K+/2Cl¡ cotransporter acts to bring Na+, K+
and Cl¡ into the rectal gland cell driven by the electro-
chemical gradient produced by the basolateral Na+/K+-
ATPase (EC 3.6.3.9). Na+/K+-ATPase transfers Na+ back to
the blood and K+ is also transferred back via a basolateral
K+ channel. Cl¡ is secreted into the lumen via an apical
chloride channel. The electrochemical gradient produced
by the extrusion of Cl¡ through the Cl¡ channel causes Na+
to move into the lumen via a paracellular route. Na+/K+-
ATPase activity in rectal gland decreases in FW D. sabina
(Piermarini and Evans 2000) and bull sharks (Pillans et al.
2008) in FW and dilute SW in leopard sharks Triakis semi-
fasciata (Dowd et al. 2010). In D. sabina, this was accom-
plished by a decrease in protein abundance (Piermarini and
J Comp Physiol B (2010) 180:475–493 479

Table 3 Plasma concentrations of inorganic ions, urea, methylamines and osmolality

Species Na+ Cl¡ K+ Ca2+ Mg2+ Methyl- Osmolality
amines (mM) (mOsm)

Marine elasmobranchs
Dasyatis americana 315k 342k 5.0k 4.12k 1,065k
l l l l
Heterodontus francisci 235 § 6.9 230.0 § 9.8 10.0 § 0.9 5.0 § 0.2
Heterodontus portusjacksoni 359 § 4n 310 § 3n 4.4 § 0.1n 5.1 § 0.1n 1.2 § 0.1n 57 § 12 TMAOn 987 § 13n
m m m m m m
Sphyrna tiburo 258.3 § 3.3 279.1 § 8.1 6.0 § 1.8 4.5 § 0.9 6.6 § 1.8 67.1 § 17.3 TMAO
Euryhaline elasmobranchs in seawater
Carcharhinus leucas 289 § 3h 296 § 6h 3.8 § 0.7h
i i
Dasyatis sabina 310 § 5 300 § 4.5 6.95 § 0.7i 3.1 § 0.2i 1,034 § 75i
Euryhaline elasmobranchs in freshwater
Carcharhinus leucas 245 § 4b 219 § 4b 6.4 § 0.3b 4.5 § 0.15b 1.4 § 0.09b 673.3 § 12.0b
e e e e e e
Carcharhinus leucas 221 § 4.1 220 § 4.9 4.2 § 0.2 3.0 § 0.15 1.3 § 0.1 19.1 § 1.3 TMAO 595 § 11e
a a a a
Dasyatis sabina 211.9 § 2.8 207.8 § 3.4 5.2 § 0.25 4.3 § 0.25 621.4 § 10.8a
Pristis perrotteti 217j 193j
Freshwater elasmobranchs
Potamotrygon spp. 164 § 5.6c
Potamotrygon magdalenae 141 § 2.4d 147 § 4.5d 8.5 § 0.29d 3.0 § 0.09d 1.3 § 0.06d 358 § 6.7d
f f
Potamotrygon sp. 178.2 § 4.8 146.2 § 2.1 319.6 + 8.5f
Potamotrygon motoro 0.39 § 0.16 TMAOg
Freshwater teleosts
Oncorhynchus mykiss 147l 122l 3.7l 2.5l 1.3l 246l
l l l l l
Carassius auratus 133 118 3.3 1.8 1.5 274l
l l l
Ictalurus punctatus 142 110 3.1 259l
a
Piermarini and Evans (1998), b Thorson et al. (1973), c GriYth et al. (1973), d Ogawa and Hirano (1982), e Pillans et al. (2005), f Wood et al.
(2002), g Treberg et al. (2006), h Pillans and Franklin (2004), i de Vlaming and Sage (1973), j Thorson (1967), k Cain et al. (2004), l McDonald and
Milligan (1992), m Mandrup-Poulsen (1981), n Cooper and Morris (1998b)

Evans 2000). It also increases when C. leucas moves from
FW back to SW (Pillans et al. 2005). A relatively rapid
increase in Na+/K+-ATPase activity can occur in the rectal
gland such as those observed in Scyliorhinus canalicula
(Dowd et al. 2008; MacKenzie et al. 2002) in response to
feeding due to the increased intake of salt water. The apical
and basolateral membranes of rectal glands of marine elas-
mobranchs have a much lower permeability to urea than
water. Since this diVerence in permeability apparently does
not involve aquaporins, it may be due to membrane lipid
components (Zeidel et al. 2005). DiVerential permeability
of the membranes of the rectal gland to urea and Na+ and
Cl¡ may play an important role in secreting salt without the
concurrent loss of urea.
Kidney
While the rectal gland primarily plays an important role in
salt excretion, the kidneys have at least two functions.
Since water inXux is a problem for marine elasmobranchs,
the kidneys produce a hypoosmotic urine (Boylan 1967) to
oVset water intake. Kidneys are also important in urea
(Janech et al. 2006b) and TMAO (Cohen et al. 1958) reten-
tion. These dual functions require a complex kidney.
Kidneys of marine and euryhaline species have two
regions: a bundle zone with two loops and a collecting duct
forming a countercurrent system for urea recovery and a
sinus zone with two loops. The collecting duct has been
implicated as the main site of urea reabsorption (Hyodo
et al. 2004a). The eYciency of the urea retaining mecha-
nisms is high. In SW 97% of the urea is reabsorbed. In 50%
SW only 84% is recovered due to the higher rate of urine
Xow (Janech et al. 2006b). In marine species, urea reab-
sorption is isoosmotic (Kempton 1953) and kidney urea
transport may involve both facilitated and non-facilitated
processes (Morgan et al. 2003b). At high concentrations,
uptake is non-saturable while at low concentrations a satu-
rable component has been identiWed with a Km of 0.7 mM
(Morgan et al. 2003b). Urea transporters in the dorsal
(bundle) region diVer from those in the ventral (sinus)

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region (Morgan et al. 2003b). In the dorsal region, uptake is Table 4 Comparisons of ammonia and urea excretion rates in FW and
not saturable and is inhibited by phloretin and HgCl2 con- marine elasmobranchs
sistent with a facilitated transporter such as an aquaporin Species Ammonia Urea eZux rate
(Morgan et al. 2003b). Urea transport in the ventral region eZux rate
mol kg¡1 h¡1
is stimulated by a Na+ gradient suggesting a Na+ dependent
mol kg¡1 h¡1
transporter (Morgan et al. 2003b). Thus, while urea recov- Marine Elasmobranchs
ery in the bundle countercurrent region employs isoosmotic Squalus acanthias 60a 275b
urea uptake this may be augmented by a sodium dependent
Scyliorhinus canicula 250c
high aYnity system in the sinus region. Four types of urea
Raja erinacea 50d 240e
transporters (UT) have been identiWed (Anderson et al.
Raja erinacea 570f
2007). Multiple transcripts of a single gene product UT are
Negaprion brevirostris 190g
produced by splicing and polyadenylation and one of these
Ginglymostoma cirratum 210–380h
(strUT-2c) was expressed at lower levels in dilute (50%)
Poroderma africanum 280i
SW (Janech et al. 2006a). In dilute environments, urea con-
Hemiscyllium plagiosum 670j
centrations in the kidney decrease and at least some of the
Freshwater Elasmobranchs
urea transporters are downregulated (Janech et al. 2006a;
Himantura signifer 208k 104.2k
Morgan et al. 2003a). One of these urea transporters l
Potamotrygon sp. 507 § 80 60l
(strUT-1) has been implicated in the high degree of euryha-
linity in D. sabina (Janech et al. 2003). P. motoro 11.9k
The urea reabsorbing function retains its importance in Marine Teleosts
euryhaline forms entering FW since these species retain Myoxocephalus 343.2 § 67.2m
octodecimspinosus
signiWcant (albeit lower) urea levels (Table 2) and these
Opsanus beta 248.4 § 36m
must be maintained constant. In Potamotrygon sp. where
Freshwater teleost
urea retention is not required, the kidney is simpler and the
Oncorhynchus mykiss 263 § 35.5n
bundle zone and countercurrent system are absent (Lacy
a
and Reale 1995). Due to the low levels of urea, the rates of Wood et al. (2005), b Wood et al. (1995), c Payan and Maetz (1971),
d
excretion of urea in Potamotrygon are low, 12– Cameron (1986), e Goldstein and Forster (1971a), f Payan et al.
(1973), g Goldstein et al. (1968), h Carrier and Evans (1972), i
60
mol kg ¡1 h¡1 (Ip et al. 2003; Wood et al. 2002) Haywood (1974), j Wong and Chan (1977), k (Ip et al. 2003), l Wood
compared to euryhaline species in FW (Table 4). Whole et al. (2002), m Goldstein et al. (1982), n Payan and Matty (1975)
body and renal loss of urea are not aVected by salinity in
Potamotrygon (Gerst and Thorson 1977). Although the
structure of the kidney of the FW species H. signifer has species such as D. sabina, there is a fourfold increase in
not been examined, transfer from FW to 50% SW results glomerular Wltration rate (GFR) in 50% SW (Janech et al.
in reduced rates of urea excretion and retention of signiWcant 2006b). Elasmobranchs in FW have an almost tenfold bet-
amounts of urea (Chew et al. 2006) implying a more ter capacity for urinary dilution (1/10 of plasma osmolarity)
complex kidney with countercurrent mechanisms in place than FW teleosts and mammals (Janech et al. 2006b;
for urea retention. Janech and Piermarini 2002). Thus, at least initial renal
The reduction in urea levels is matched by a reduction in clearance of both NaCl and urea increases in dilute environ-
methylamines partly due to renal mechanisms since the ments (Goldstein et al. 1968; Goldstein and Forster 1971b;
clearance of TMAO increases upon environmental dilution Sulikowski and Maginniss 2001; Wong and Chan 1977).
in R. erinacea (Goldstein and Forster 1971a). Nothing is Once optimally adapted to FW, recolonization of SW by
known of the renal transport mechanisms for recovery of elasmobranchs such as the Potamotrygonids would be
TMAO. problematic due to the loss of renal structures for urea
The collecting duct is also the main site of urine dilution retention.
and is responsible for sodium retention in dilute environ-
ments (Stolte et al. 1977). It has been suggested that the Gills
kidney plays a more important role in ion retention in FW
than in SW since Na+/K+-ATPase activities in that tissue The gills likely play only a small role in salt balance in
are higher in FW in C. leucas (Pillans et al. 2005). In FW, marine elasmobranchs and are more important in acid–base
the kidney of elasmobranchs produces large amounts of regulation (Shuttleworth 1988). In FW, the gills are more
dilute urine (Thorson and Brooks 1983). Urine Xow rates of important as sites of salt uptake. The mitochondria-rich
FW elasmobranchs are much higher than those of FW teleosts (chloride) cells of marine elasmobranchs lack the extensive
(Janech and Piermarini 2002; Smith 1931). In euryhaline infolding of the basolateral membrane found in teleost Wsh

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J Comp Physiol B (2010) 180:475–493
(Wright 1973) and activities of Na+/K+-ATPase are about
tenfold lower than in marine teleost Wsh (Jampol and
Epstein 1970). Unlike the situation in teleost Wsh, the Na+/
K+-ATPase activity and abundance in the gills of FW
D. sabina is higher than that of SW adapted Wsh (Piermarini
and Evans 2000). There is no need for high Na+/K+-ATPase
activity in the marine elasmobranch gill since salt excretion
is carried out by the rectal gland.
In FW, uptake of ions such as Na+ and Cl¡ becomes
important and the gill of elasmobranchs functions more like
that of an FW teleost. An Na+/K+-ATPase is required and
an H+-K+-ATPase (EC 3.6.3.10) has been demonstrated in
the gills of D. sabina with the expression increasing in FW
presumably to help in potassium uptake (Choe et al. 2004).
Na+ uptake against a concentration gradient has been dem-
onstrated in Potamotrygon (Pang et al. 1977).
The gills of elasmobranchs play an important role in
acid–base regulation. Ion regulation and acid–base regula-
tion are connected in that the uptake of Cl¡ can be coupled
to the extrusion of HCO3¡ via the Cl¡/HCO3¡ exchanger
and Na+ uptake is coupled to the extrusion of H+ via the
Na+/H+ exchanger. It has been suggested (Evans 1984)
that the role of gills in SW was for acid–base regulation
and acquired an ion transport function only when they
entered FW. Euryhaline elasmobranchs such as D. sabina
compensate for hypercapnia better in SW than in FW
(Choe and Evans 2003). This is likely due to the reliance
on external Na+ for acid excretion at the gills (Choe and
Evans 2003).
Elasmobranch gills have two types of mitochondria-rich
cells (Fig. 1). One type has a basolateral Na+/K+-ATPase
and an apical Na+/H+ exchanger and the other a V-H+-ATP-
ase (EC 3.6.3.14) with an apical HCO3¡/Cl¡ exchanger.
V-H+-ATPase has been localized in the mitochondria-rich
cells in gills of both marine and FW elasmobranchs
(Piermarini and Evans 2001). The number of cells with H+-
ATPase increases in FW (Wood et al. 2003). V-H+-ATPase
has been further localized to the basolateral membrane in
the same cells with an apical Cl¡/HCO3¡ exchanger (Pier-
marini et al. 2002) where it likely functions in HCO3¡
excretion and Cl¡ uptake (Evans et al. 2004; Wood et al.
2003). The Na+/H+ exchanger has been found in apical
membranes of the gills of S. acanthias where it is thought
to have a role in acid–base regulation (Choe et al. 2007).
This same carrier could play a role in Na+ uptake during
FW acclimation. Na+/H+ exchanger is not found in the
same cells as V-H+-ATPase (Choe et al. 2007). The current
model for elasmobranch gill ion movement indicates simi-
lar arrangements of ion exchanges and transporters in both
SW and FW (Fig. 1). In FW, higher expression of HCO3¡/
Cl¡ exchanger (Piermarini et al. 2002), H+/K+ ATPase
(Choe et al. 2004) and Na+/K+-ATPase (Piermarini and
Evans 2000) occur to enhance ion uptake. In some cases
481
Fig. 1 Diagram of known transporters in the two types of mitochon-
dria-rich cells of the gills of marine and FW elasmobranchs. 1 Na +/
K + -ATPase, 2 Na+/H+ exchanger, 3 H+/K+-ATPase, 4 V-H+-ATPase,
5 HCO3¡/Cl¡ exchanger. FW acclimation results in enhanced expres-
sion of 1, 3, 4, 5
increased ion transport capacity may take the form of more
cells (Wood et al. 2003).
Although ion uptake is important in FW, in some situa-
tions acid–base issues can also be important. The pH of FW
is frequently lower than that of SW and sometimes substan-
tially lower such as in the blackwaters of the Amazon
where Potamotrygon spp. are found (Wood et al. 2003).
Some protection from the acid-load is provided by black-
water dissolved organic carbon (Wood et al. 2003). This
helps explain the practice of adding blackwater extract to
aquarium water to improve survival of FW stingrays. The
capacity for acid–base regulation in FW may be a limiting
factor in successful entry into that habitat. It has been sug-
gested that inability to deal with the acid–base conse-
quences of low salinity limits the distribution of species
like the Port Jackson shark (Heterodontus portusjacksoni)
(Cooper 2004).
The large surface area of the gills and rapid Xow of water
over them make them important sites of loss or gain of
water. The permeability of the gills of S. acanthias to water
is similar to that of teleost Wsh (Part et al. 1998) (Table 1).
Water permeability decreases in dilute environments in
some species (Payan and Maetz 1971) but not in others
(Payan et al. 1973). The superior tolerance of dilute envi-
ronments of some species has been attributed to enhanced
control of water permeability and ion regulation (Cooper
and Morris 1998a).
Gills are important sites of urea retention. Although
most (>90%) urea is lost at the gills of marine elasmo-
branchs like S. acanthias (Wood et al. 1995), the gills are
highly impermeable to urea compared to those of teleost
Wsh (Boylan 1967; Fines et al. 2001; Payan et al. 1973;
Walsh and Smith 2001; Wood et al. 1995). This has been
123
482
attributed to both barrier and transporter related adapta-
tions. A high cholesterol content in the basolateral mem-
branes of the gills of S. acanthias is thought to lower
permeability to urea (Fines et al. 2001). Consistent with
this concept is the fact that the gills of S acanthias are
highly impermeable to urea from 1 to 15°C but this
increases sharply at temperatures above 15°C as the phase
properties of the physical barrier abruptly change (Boylan
et al. 1963). Potamotrygon is 20–50 times more “leaky” to
urea than S. acanthias (Goldstein and Forster 1970; Gold-
stein and Forster 1971b) but there have been no studies of
the basis for this. Studies of FW and marine stingrays indi-
cate lower cholesterol content in gill basolateral mem-
branes in FW compared to marine species (Ballantyne,
unpublished). Lower salinity does not alter gill membrane
permeability to urea in some species (Goldstein et al. 1968;
Goldstein and Forster 1971a; Payan et al. 1973). In addition
to structural barriers to urea loss, a sodium dependent urea
transporter with a low Km for urea has been found in the gill
basolateral membrane that would function to scavenge any
urea that enters the branchial cells and transports it back to
the blood (Fines et al. 2001).
Hormonal Regulation of osmo- and ionoregulation
Sensing salinity change in elasmobranchs likely involves
the polyvalent cation receptor protein found in kidney, rec-
tal gland, stomach, intestine, gill, olfactory organs and
brain (Nearing et al. 2002). There have been no studies of
the regulation of this protein in euryhaline or stenohaline
FW elasmobranchs. At least four hormone systems have
been identiWed as being involved in regulating water and
osmolyte balance in elasmobranchs (Gelsleichter 2004).
These are (a) the hypothalamus–pituitary–interrenal (HPI)
axis, (b) vasoactive peptides, (c) cardiac natriuretic pep-
tides (CNP) and (d) the renin–angiotensin system (RAS).
The complexity of the role of HPI axis has not been fully
explored. Hypophysectomy decreases water permeability in
S. acanthias (Payan and Maetz 1971) perhaps mediated by
aquaporin recruitment. The HPI axis in elasmobranchs
relies on a single corticoid, 1
-hydroxycorticosterone
(AHC) that is thought to have both glucocorticoid and min-
eralocorticoid functions (Nunez et al. 2006). AHC synthe-
sis is stimulated by adrenocorticotropic hormone (ACTH)
in D. sabina (Nunez and Trant 1999). Levels of plasma
AHC increase in S. acanthias exposed to dilute SW (50%)
as a mechanism to reduce sodium loss (Armour et al.
1993). In the euryhaline D. sabina angiotensin II (ANG II)
does not induce AHC synthesis but it does in stenohaline
marine species (Nunez et al. 2005). AHC may play a role in
regulating NaCl or urea transport across the gills (Hazon
et al. 2003). The steroidogenic acute regulatory (StAR) pro-
tein is involved in regulating synthesis of AHC and expres-
123
J Comp Physiol B (2010) 180:475–493
sion in non-interrenal tissues (nerve, muscle, gill and heart)
of elasmobranchs may indicate a regulatory role in ion regu-
lation (Nunez et al. 2005). Salinity challenge in P. motoro
had no eVect on expression of StAR perhaps due to the poor
ability to respond to salinity challenge (Nunez et al. 2006).
The hormonal regulation of kidney function relies on
some of the same eVectors [ANG II, arginine vasotocin
(AVT) and CNP] as in other vertebrates. AVT has been
implicated in regulating renal urea retention in elasmo-
branchs (Acher et al. 1999). Urine Xow is decreased by
AVT and ANG II which act by vasoconstricting vessels to
some nephrons reducing the number Wltering blood (Hazon
et al. 2003). Plasma AVT concentrations correlate posi-
tively with salinity in Triakis scyllium consistent with a role
in reducing Xuid loss in high salinities and increasing loss
in low salinities (Hyodo et al. 2004b). Due to glomerular
vasoconstrictor eVects (Pang et al. 1983; Wells et al. 2002)
AVT has been suggested to aVect collecting duct UT in
elasmobranch kidney (Hyodo et al. 2004b). While the roles
of CNP in FW or SW acclimation are not clear (Anderson
et al. 2006), it has been implicated in increasing clearance
of Na+, Cl¡ and urea in the kidneys (Wells et al. 2006).
Acute transfer from FW to SW resulted in an increase in
ANG II and AVT but no change in CNP in C. leucas. After
acclimation, AVT levels matched FW levels but CNP
remained elevated (Anderson et al. 2006). Levels of CNP in
plasma are lower in C. leucas at low salinities (Anderson
et al. 2005). The mechanism of stimulation of salt secretion
in SW involves C-type natriuretic peptide (CNP) produc-
tion with a volume load (Hazon et al. 2003). The function
of CNP in FW is not known. Hypotonic stimulation also
causes increased cytosolic calcium levels and hyperpolarized
cells that may play a role in regulation (Thiele et al. 1998).
In teleost Wsh cortisol plays a role in stimulating chloride
cell salt secretion but elasmobranchs lack this hormone.
The role of cortisol is played by AHC. Receptors for AHC
have been found in rectal gland (Idler and Truscott 1967)
but its role in regulating rectal gland secretion has not been
established.
The RAS plays an important role in regulating drinking
in elasmobranchs (Hazon et al. 1999). There is some evi-
dence that the RAS diVers in euryhaline compared to steno-
haline species. Vasoactive intestinal peptide stimulates salt
secretion in the rectal gland in S. acanthias (Epstein et al.
1983) via a cyclic AMP mediated pathway (Olson 1999)
but the role in response to environmental dilution has not
been established.
Metabolism
The large amount of urea needed for osmotic purposes
aVects amino acid, methylamine, ketone body and lipid
J Comp Physiol B (2010) 180:475–493 483

metabolism. Thus, changes in the importance of urea as an
osmolyte in FW have important energetic and metabolic
consequences. For example, species such as R. erinacea
and Dasyatis americana undergo large decreases in urea,
free amino acids and TMAO in muscle when exposed to
even moderate dilution such as 50% SW (Forster and Gold-
stein 1976). More profound changes are observed in species
inhabiting FW.
Moon 1986; Lewiston et al. 1979). The optimal osmolarity
is similar to that observed for mitochondria from a marine
teleost (Ballantyne and Moon 1986). This implies that
some aspects of mitochondrial respiration are sensitive to
solutes and/or osmolarity. The role this may play in adapta-
tion to lower salinity has not been established.
Carbohydrate metabolism

Respiratory and oxidative metabolism
Attempts have been made to determine the costs of the elas-
mobranch osmoregulatory strategy versus that of teleost
Wsh in SW (Kirschner 1993) but calculations have had to
rely on an incomplete understanding of the eYciencies and
mechanisms of ion transport and urea synthesis. Comparing
the costs of marine versus FW existence have not been
made for elasmobranchs but empirical evidence indicates in
species such as bat rays Myliobatis californica higher respi-
ration rates occur at lower salinities compared to those in
full SW (Meloni et al. 2002). This diVers from the situation
in euryhaline teleosts such as rainbow trout where the low-
est respiration rates occur in SW (Rao 1971). More studies
are needed to establish the applicability of this observation
to other species of elasmobranchs.
Some FW habitats such as parts of the Amazon have
very low oxygen levels. In these situations, regulation of
hemoglobin oxygen aYnity is critical. The oxygen aYnity
of Potamotrygon hemoglobin is higher than that of many
marine elasmobranchs to optimize oxygen binding in the
hypoxic environment (Martin et al. 1979). Organic phos-
phates such as GTP are low in RBCs of Potamotrygonid
stingrays and this would have the additional eVect of allow-
ing higher oxygen aYnities (Johansen et al. 1978). Interest-
ingly, the hemoglobin of D. sabina is insensitive to organic
phosphates (Mumm et al. 1978). There are diVerences in
the eVects of salinity on blood oxygen aYnity that may
limit survival in lower salinities. For example, a reduction
in whole blood oxygen aYnity is observed in the relatively
stenohaline Heterodontus portusjacksoni but not in the
euryhaline C. leucas (Cooper and Morris 2004).
High Cl¡ levels in Potamotrygonid blood have been sug-
gested to increase oxygen aYnity of hemoglobin (Johansen
There have been few studies of carbohydrate metabolism in
relation to adaptation to dilute environments. Levels of
lactate dehydrogenase (EC 1.1.1.27) are low in heart of
Potamotrygon spp. similar to that of a comparable marine
elasmobranch species, L. erinacea (Driedzic and De
Almeida-Val 1996) and the isoforms are similar to marine
species (De Almeida-Val and Val 1993). Gluconeogenesis
seems to occur primarily in liver and kidney of Potomotrygon
magdalenae similar to that of marine elasmobranchs
(Singer and Ballantyne 1989). The only published value for
blood glucose in a Potamotrygonid stingray (GriYth et al.
1973) is similar to that of marine elasmobranchs (Table 5).
Amino acid metabolism
In elasmobranchs, free amino acids have several functions.
They are needed for protein synthesis, as neurotransmitters,
as oxidative substrates, as osmotically active particles for
cell volume regulation and as nitrogen donors for urea
synthesis. While in the extracellular Xuid amino acids
contribute only 1% of the osmolarity they provide 19% in
the intracellular Xuid of muscle of L. erinacea with the non-
protein amino acids taurine, sarcosine and -alanine being
Table 5 Plasma concentrations of glutamine and glucose in marine
and FW elasmobranchs
Species Glutamine (mM) Glucose (mM)
Marine Elasmobranchs
Hemiscyllium ocellatum 0.201a
Nebrius ferrugineus 0.061a
Taeniura lymma 0.129a
Squalus acanthias 0.08b 1.26 + 0.30c
Raja erinacea 0.01 d

et al. 1978). Potamotrygon hemoglobin has a Bohr eVect Raja erinacea 0.08e
(Martin et al. 1979) similar to that of marine elasmobranchs Scyliorhinus canicula 0.12f 1.42 + 0.54g
(Wells and Weber 1983) implying the importance of regu- Dasyatis americana 1.69h
lating hemoglobin oxygen aYnity in diVerent physiological Freshwater elasmobranchs
states. Himantura signifer 0.01 + 0.002i
Mitochondrial oxidative metabolism has not been exam- Potamotrygon sp. 1.22 + 0.17j
ined in any FW elasmobranch. Intact mitochondria from a
Ballantyne (unpublished), b King and Goldstein (1983b), c Richards
marine elasmobranchs respire at higher rates at lower et al. (2003), d Boyd et al. (1977), e Ballatori and Boyer (1988),
osmolarities when osmolarity is varied with physiological f
McDonald and Milligan (1992), g Zammit and Newsholme (1979),
h
ratios of urea and TMAO or sucrose/KCl (Ballantyne and Cain et al. (2004), i Ip et al. (2003), j GriYth et al. (1973)

123
484
the most important (King and Goldstein 1983a). Levels of
amino acids are high in brain (170 mM) of marine elasmo-
branchs (Forster et al. 1978).
Upon environmental dilution, some of these amino acids
are released into the plasma to reduce cell volume (Goldstein
and Brill 1991; Haynes and Goldstein 1993; King and
Goldstein 1983a). Most other amino acids are unaVected.
Loss of solutes such as taurine from cells during hypotonic
stress occurs via anion exchangers which are moved to the
plasma membrane from sequestered sites in cholesterol rich
raft regions (Musch et al. 2004). The eZux also involves
binding of ankyrin to band 3 protein linking it to the cyto-
skeleton (Musch and Goldstein 1996). Some of these sol-
utes may be oxidized to reduce their levels and produce
ATP in the process. Isolated liver cells and mitochondria
from L. erinacea oxidize sarcosine at higher rates at lower
osmolarities, consistent with a mechanism to remove osmo-
lytes from the cells for hypoosmotic cell volume regulation
(Ballantyne et al. 1986). Hypoosmotic eVects on amino
acid oxidation include eVects on mitochondrial transport of
speciWc amino acids (Moyes et al. 1986).
Catabolism of amino acids may be an important energy
source in elasmobranchs. The use of the nitrogen atoms for
urea synthesis allows the carbon skeletons to enter the
Krebs cycle for oxidation. A study of T. semifasciata
(Dowd et al. 2010) found evidence for increased catabolism
of amino acids in gill and rectal gland in response to expo-
sure to 50% SW. High levels of phosphate-dependent gluta-
minase (PDG) (EC 3.5.1.2) have been found in many
marine elasmobranch tissues including heart and red mus-
cle (Chamberlin and Ballantyne 1992). This contrasts with
the situation in mammals where muscle tissue is a net
exporter of glutamine. It is somewhat paradoxical, how-
ever, that a high capacity for glutamine oxidation exists
when large amounts of glutamine are needed for urea syn-
thesis. It is not known if FW elasmobranchs have a similar
high capacity for glutamine catabolism in muscle since
there are no measurements of PDG in FW species.
Glutamine synthetase (GS) (EC 6.3.1.2) is a key enzyme
in marine elasmobranchs for its role in scavenging ammo-
nia for urea synthesis. Levels of GS are high in many
marine elasmobranch tissues (Ballantyne 1997) presumably
to optimize the use of ammonia at the site of production.
Levels of GS have been measured in the liver of H. signifer
and are lower than those of a comparable marine ray
T. lymma (Ip et al. 2003; Tam et al. 2003). GS activity is
lower in FW Potamotrygon spp. than in marine species and
is similar to that of FW teleosts (Webb and Brown 1980). A
study of the FW elasmobranch P. motoro (Ip et al. 2009)
found an upregulation of GS when exposed to 15‰ water
and accumulation of amino acids in liver and muscle.
Glutamate dehydrogenase (GDH) (EC 1.4.1.2) is a key
enzyme for deamination of amino acids being funneled
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J Comp Physiol B (2010) 180:475–493
through glutamate. The activities of liver GDH are
inversely correlated with environmental salinity and inter-
nal urea levels implying that GDH has a reduced role in
marine species where glutamine is funneled into the urea
cycle (Speers-Roesch et al. 2006).
Urea synthesis
Marine elasmobranchs must synthesize urea at rates suY-
cient to replace the losses at the gills and in the urine. In
elasmobranchs the cost of synthesizing urea is Wve ATPs
per urea. It has been estimated that 14% of the oxygen con-
sumption of a marine elasmobranch Scyliorhinus canicula
is dedicated to urea synthesis replacing urea lost at the gills
(Kirschner 1993). In FW, this energy expenditure would be
reduced depending on the amount of urea retained. In Pota-
motrygonids that synthesize negligible urea, there would be
a 14% energy advantage to living in FW. In Potamotrygon,
activities of the enzymes of urea synthesis are ½–1/20
(Goldstein and Forster 1971b) and rates of urea synthesis
are 100-fold lower compared to S. acanthias (Perlman and
Goldstein 1988). Potamotrygonid stingrays are thus unable
to produce osmotically signiWcant amounts of urea when
exposed to higher salinities (Thorson 1970) and thus recol-
onization in SW is problematic in this group.
The urea cycle of elasmobranchs is diVerent from that
of mammals in several ways. The compartmentation of the
enzymes diVers with arginase (EC 3.5.3.1) being mito-
chondrial in elasmobranchs and cytosolic in mammals
(Ballantyne 1997). The consequence of this is that arginine
must be transported into the mitochondrion at high rates
and urea is generated inside the mitochondrion rather than
outside. We have established that urea eZux from liver
mitochondria of L. erinacea is dependent on a proton-sen-
sitive process that may be required to rapidly equilibrate
urea across the inner mitochondrial membrane (Rodela
et al. 2008). The characteristics of the arginine carrier
have not been examined in SW or FW elasmobranchs. It is
not known if arginase is mitochondrial in FW elasmo-
branchs.
Another important diVerence between mammalian car-
bamoyl phosphate synthetase (CPS) and the elasmobranch
counterpart is that in elasmobranchs glutamine is the nitro-
gen donor (CPS III) (EC 6.3.5.5) but not ammonia as in
mammals (CPS I) (EC 6.3.4.16). This single diVerence is
the basis for many of the diVerences in nitrogen metabolism
between elasmobranchs and other vertebrates. The need for
large amounts of glutamine requires nitrogen to be removed
from other amino acids and funneled through glutamate to
glutamine. The need for this nitrogen may have conse-
quences on maximal growth rates of marine elasmobranchs
and so the growth rates of FW species should be faster.
Additionally, the high requirement for nitrogen-containing
J Comp Physiol B (2010) 180:475–493
compounds may explain why all marine elasmobranchs are
carnivorous.
The mechanism whereby reductions in urea concentra-
tions occur may diVer among species. Some species rely
solely on urea loss to reduce tissue levels. For example, the
rates of urea synthesis did not change in lemon sharks Neg-
aprion brevirostris exposed to ½ SW although urea clear-
ance increased threefold (Goldstein et al. 1968). Several
studies have indicated that reductions in urea concentra-
tions upon entry into dilute environments are at least in part
due to reductions in urea synthesis (Goldstein and Forster
1971a; Hazon and Henderson 1984; Tam et al. 2003). A
metabolic alkalosis (higher blood pH and accumulation of
HCO3¡) occurs in D. sabina exposed to FW and this has
been suggested to be due to a reduction in the use of
HCO3¡ for urea synthesis (Choe and Evans 2003). This
does not apply to all species since the pH in the plasma of
bull sharks from FW, estuarine or SW are similar (Coelho
and Erzini 2006).
A variety of other factors can inXuence urea synthesis in
those FW species that still retain urea. The dietary intake of
nitrogen aVects the capacity to synthesize urea in H. sig-
nifer in FW but not in ½ SW (Chew et al. 2006). Exposure
to environmental ammonia resulted in increased levels of
GS and urea cycle enzymes in H. signiWer (Ip et al. 2003).
This strategy would not only help detoxify ammonia but
also augment the urea levels maintained in these species in
FW.
Urea recycling may occur in SW and FW species that
retain urea. Urea eZux into the intestinal lumen has been
quantiWed in bamboo sharks at about 250
moles kg¡1h¡1
(Anderson et al. 2007). This is comparable to the losses at
the gills of S. acanthias (Wood et al. 1995). Indeed, con-
centrations of urea in gut can be higher than that of blood
(Anderson et al. 2007). Low levels in the colon (Anderson
et al. 2009) indicate intestinal urea is either taken back up
or utilized by intestinal Xora for amino acid synthesis.
Although urea cycling has not been demonstrated in elas-
mobranchs it occurs in ruminants (Sarraseca et al. 1998),
marsupials (Kennedy and Hume 1978) and hibernating
mammals (Barboza et al. 1997).
Methylamine and counteracting solute metabolism
The concept of the counteracting eVect of methylamines on
the disruptive eVects of urea are well established (Yancey
and Somero 1979). An “ideal” ratio of 2:1 urea:methylam-
ines has been suggested (Yancey 1994; Yancey and
Somero 1979). An analysis of SW, euryhaline and stenoha-
line FW species found a ratio of urea to total methylamines
of 2.5 (Treberg et al. 2006) slightly higher than proposed
by Yancey (1994). Some of the discrepancies may be due
to the role of other amino acids as compatible and counter-
485
acting solutes (Treberg et al. 2006). The lower urea levels
in FW elasmobranchs reduce or eliminate the need for
methylamine counteracting solutes. Indeed FW elasmo-
branchs accumulate taurine instead of methylamines
(Treberg et al. 2006). -amino acids such as betaine
(trimethylglycine) are more important in FW species with
taurine being the predominant compound (Treberg et al.
2006).
The two enzymes involved in betaine synthesis, choline
dehydrogenase (EC 1.1.99.1) and betaine aldehyde dehy-
drogenase (EC 1.2.1.8) have been detected in all elasmo-
branchs with higher levels in FW species (Treberg et al.
2006). The capacity for TMAO synthesis is rare in SW spe-
cies and absent in FW species (Treberg et al. 2006) presum-
ably since it is readily obtained in the diet of SW species
and is not needed in FW species. TMA oxidase (EC
1.5.8.2) has not been detected in any FW species (Treberg
et al. 2006). TMAO synthesis has only been reported in the
Carcharhiniformes and the Orectolobiformes (Treberg et al.
2006) but several other orders have not been examined.
Food deprived euryhaline species such as C. leucas
increase urea and TMAO together when moving from FW
to SW over 10 days (Pillans et al. 2006) indicating they
have the capacity for TMAO synthesis.
Ammonia excretion
Marine elasmobranchs are relatively impermeable to
ammonia compared to marine or FW teleost Wsh (Table 4).
This may be due to the need to conserve ammonia for urea
synthesis (Wood 2001) and correspondingly, FW elasmo-
branchs like Potamotrygon spp. have much higher rates of
ammonia excretion than marine elasmobranchs (Perlman
and Goldstein 1988) (Table 4). The rate of ammonia excre-
tion increases with exposure to environmental ammonia (Ip
et al. 2003). Excess ammonia is readily excreted in both
FW and marine species (Ip et al. 2005). Endogenous
ammonia can be converted into urea for osmotic purposes
at higher salinities by T. lymma and H. signifer (Ip et al.
2005).
Lipid metabolism
The liver is the main site of lipid storage in marine elasmo-
branchs with very high levels accumulating in non-benthic
species to increase buoyancy. There is no evidence that FW
forms are diVerent although the diVerent density of SW and
FW may require adjustments in liver lipid content. Future
studies of euryhaline forms should examine liver density in
SW and FW to establish if buoyancy regulation occurs
using liver lipids. In most vertebrates, transport of lipid
from reserves to other tissues involves plasma lipid proteins
such as chylomicrons, very low density lipoproteins
123
486
(VLDL), low density lipoproteins (LDL), high density lipo-
proteins (HDL) and nonesteriWed fatty acids (NEFAs)
bound to plasma albumin.
Marine elasmobranchs have VLDL and LDL levels sim-
ilar to those of teleost Wsh but lower levels of HDL than
most vertebrates (Ballantyne 1997). There are no studies of
the plasma lipoproteins of FW elasmobranchs. Marine elas-
mobranchs are unusual among the vertebrates in their lack
of a fatty acid binding protein (albumin) in the plasma
resulting in very low levels of plasma NEFAs (Ballantyne
1997; Speers-Roesch et al. 2008). It has been speculated
that high levels of urea disrupt the hydrophobic binding of
fatty acids to proteins such as albumin but this has not been
demonstrated. Based on electrophoretic mobility of plasma
proteins, it has been suggested that albumin is present
(152 mg% or 19.2% of plasma protein) in Potamotrygon
(GriYth et al. 1973) but deWnitive fatty acid binding has not
been conWrmed. A single study comparing marine and FW
species found very low concentrations of plasma nonesteri-
Wed fatty acids in both groups (Speers-Roesch et al. 2008)
indicating that the absence of urea is not suYcient to restore
NEFA transport. The low levels of NEFAs correlate with
the absence of signiWcant lipid oxidation in many tissues
since plasma NEFAs are the important transport lipid for
rapid oxidation. Both marine and FW elasmobranchs lack
the capacity for lipid oxidation in the heart and red muscles
(Singer and Ballantyne 1989; Speers-Roesch et al. 2006).
Interestingly, mRNA for carnitine palmitoyl transferase 1
(CPT) (EC 2.3.1.21), a key enzyme of lipid oxidation, has
been found in dogWsh heart even though the enzyme is not
expressed (Speers-Roesch 2009). The kidney and rectal
glands of marine elasmobranchs have relatively high levels
of CPT (Ballantyne 1997; Speers-Roesch et al. 2006). The
delivery of lipids to this tissue may be via plasma lipopro-
teins (Speers-Roesch et al. 2008). The kidneys of marine
elasmobranchs have a higher capacity for lipid oxidation
than those of FW species (Speers-Roesch et al. 2006). It
should be mentioned that one species of Potamotrygon,
P. hystrix has detectable levels of CPT in heart (Driedzic
and De Almeida-Val 1996). Further study of this species is
warranted.
Typically the nature of the essential fatty acids (n3 and
n6) in the diet inXuences the lipids of Wsh. In teleost Wsh,
marine species have higher n3/n6 ratios than FW species
due to the higher proportion of n3 fatty acids in the marine
environment. This does not seem to be the case with elas-
mobranchs. The tropical FW species had low (<1) n3/n6
ratios comparable to those of tropical marine species and
much lower than those of temperate marine species
(Speers-Roesch et al. 2008). The main diVerence between
FW and marine elasmobranch species is the low percentage
of docosahexaenoic acid in FW species (Speers-Roesch
et al. 2008). It has been suggested that n3 fatty acids play a
123
J Comp Physiol B (2010) 180:475–493
role in adaptation to higher salinity in H. signifer (Speers-
Roesch et al. 2008).
Ketone body metabolism
The lack of signiWcant capacity for oxidation of fatty
acids in muscle and heart has resulted in a more extensive
use of ketone bodies for routine metabolic needs than is
found in other vertebrates (Ballantyne 1997). Ketone bod-
ies can be derived from lipid oxidation in the liver and are
readily transported in the plasma without carrier proteins.
High levels of the enzymes of ketone body metabolism
and circulating levels of ketone bodies have been found in
many extrahepatic tissues (Ballantyne 1997). FW elasmo-
branchs also rely extensively on ketone bodies in some
tissues based on measurements of enzyme activities
(Speers-Roesch et al. 2006) but circulating levels of
ketone bodies have not been determined in any FW elas-
mobranch.
Sensory biology
The conductivity of SW and FW is substantially diVerent.
Elasmobranchs rely extensively on electroreception, mak-
ing adaptation of this sensory modality a requirement for
successful FW invasion. It has been shown (McGowan and
Kajiura 2009) that electroreception in D. sabina is substan-
tially lower in FW. Given that feeding is a primary use of
electroreception in elasmobranchs, this has implications for
the survival of elasmobranchs in FW. The sensory organs
involved in electroreception are called the “ampullae of
Lorenzini”. The ampullae of Lorenzini from FW bull
sharks are morphologically diVerent from those of marine
elasmobranchs and FW stingrays (Whitehead 2002). The
Potamotrygonid stingrays have much smaller ampullae of
Lorenzini than marine species and these have been termed
“microampullae” (Szabo et al. 1972). The canals leading to
the sensory epithelium do not have to be as long in FW
(Raschi et al. 1997) since short canals allow impedance to
better match the high resistance of FW compared to SW
(Szamier and Bennett 1980). The FW H. signifer has
ampullae which are intermediate in size (miniampullae)
between those of marine species and the potamotrygonids
(Raschi et al. 1997). Miniampullae have also been observed
in Dasyatis garouaensis an African species found only in
FW (Raschi and Mackanos 1989). The three diVerent types
of ampullae found in elasmobranchs in FW correspond to
the three stages involved in the colonization in FW identi-
Wed using other aspects of their physiology. The skin of FW
stingrays has a higher resistance than that of marine species
(Szabo et al. 1972) but the structural basis for this has not
been established.
J Comp Physiol B (2010) 180:475–493
Reproduction
As a group elasmobranchs display several reproductive
modes. All employ internal fertilization but there are ovipa-
rous forms as well as viviparous forms. The viviparous
forms can be aplacental or placental. Most elasmobranchs
(55%) are viviparous by contrast with the small proportion
of teleosts (2–3%) (Wourms and Demski 1993). All elas-
mobranchs spending signiWcant time in FW bear live
young. The basis for this is not known. This may be due to
the permeability of the egg case. Egg cases laid in SW are
highly permeable to water and urea (Foulley and Mellinger
1980). Embryos of marine elasmobranchs laying eggs are
unable to retain signiWcant urea in the early stages (Evans
1981). In FW, an inXux of water may prove energetically
too expensive for a developing embryo. Relying on mater-
nal osmotic support in utero may be important osmotically
and energetically. Except for the skates, all of the Rajifor-
mes (stingrays, guitarWshes and sawWsh) are viviparous.
This may explain why the egg-laying skates have never col-
onized FW. Viviparity is relatively rare in teleosts and
many species lay eggs in FW. Why elasmobranchs are
unable to use this strategy may be due to the fact that the
early stages of the colonization in FW involves retaining
signiWcant urea. Developing FW elasmobranch embryos in
external eggs exposed to FW may Wnd this too diYcult.
Ecological and behavioral considerations
Little is known of the ecology or behavior of freshwater
elasmobranchs. In particular the behavior of elasmobranchs
while entering freshwater is of interest since the duration of
exposure and acclimation period may be critical to under-
stand the factors limiting the capacity of some species to
deal with dilute environments. A few studies have used sta-
ble isotope or atom ratios to infer life history information.
Using otolith Sr/Ca ratios (Otake et al. 2005) found evi-
dence that H. signifer from southeast Asia spends little time
in estuarine situations and females of that species tend to
avoid higher salinities more than males perhaps due to
reproductive considerations. H.signifer has only been found
in freshwater and is reproductively active at all times of the
year (Chatchavalvanich et al. 2005). It is thought that
H. signifer gives birth in freshwater (Otake et al. 2005) and
it is likely that the entire life cycle of this species, including
mating, occurs in freshwater. Other species such as Pristis
perotteti can also successfully mate and give birth in fresh-
water (Jensen 1976). This is not the case with all species.
Bull sharks give birth in freshwater and juveniles can sur-
vive in freshwater but it is uncertain whether mating occurs
in freshwater (Jensen 1976). The eVects of freshwater on
the sensory signals needed for mating may be problematic
487
(Jensen 1976). Since transfer of sperm involves using ambi-
ent water to help pump sperm into the oviducts of the
female (Wourms 1977), sperm survival in freshwater may
be problematic since activation of sperm motility requires
the correct osmolarity and ion levels (Hamlett 1999). Thus,
another limitation to the extent of colonization in freshwa-
ter may involve sperm tolerance of freshwater. Further
research in this area could validate this hypothesis.
Some species found in or near freshwater may actually
be using the estuarine area for reproduction or as nurseries
(e.g. C. leucas, Pristis microdon and Pristis clavata in
northern Australia’s Fitzroy River) (Thorburn 2006). Estu-
aries are particularly productive areas and are important to
some elasmobranch species for this reason. Juveniles of
several elasmobranch species using estuaries have been
found to target high energy foods to enhance growth to
avoid predation when returning to a marine environment
(Thorburn 2006). Leopard sharks (Triakis semifasciata)
exhibit increased activity in the initial phases of introduc-
tion to 50% SW in the lab leading to the suggestion (Dowd
et al. 2010) that behavioral means are employed to amelio-
rate the stresses of environmental dilution. In estuaries,
rapid movement in and out of freshwater may be the best
strategy to take advantage of the food resources but avoid
the detrimental eVects of lower salinity. Estuaries may also
provide favorable sites for osmotic purposes (Heupel and
Simpfendorfer 2008).
The tropical South American freshwater stingrays repro-
duce seasonally based on the hydrologic cycle with copula-
tion during the dry season and birth occurring during the
wet season (Charvet-Almeida et al. 2005). In subtropical
areas, temperature may aVect the time of breeding. Fresh-
water Dasyatis sabina give birth in the warmer months
(Johnson and Snelson 1996; Snelson et al. 1988). This may
give the newborn pups an advantage in dealing with the
osmotic stress of freshwater. In spite of the fact that there
are populations of D. sabina that spend their entire lives in
freshwater, poor reproductive success correlates with low
conductivity in the St. Johns and associated rivers in Flor-
ida (Johnson and Snelson 1996). Some populations of this
species undergo seasonal migrations to avoid suboptimal
conditions (Snelson et al. 1988).
13C and 15N have been used to establish the trophic
relationships of freshwater elasmobranchs in the Fitzroy
River in Northern Australia and the values obtained for N
indicate that both Pristis and C. leucas occupy the highest
trophic levels even though Pristis feeds on more inverte-
brates (Thorburn 2006). The studies by Thorburn sampled
white muscle for the stable isotope analysis. A study of
P. motoro (MacNeil et al. 2006) found that liver tissue 15N
responded most rapidly to dietary change with blood, mus-
cle and cartilage responding at progressively slower rates.
Inferring trophic status using 15N is time sensitive and
123
488
sampling multiple tissues should be used to increase the
time span over which the feeding history can be docu-
mented.
Evolutionary implications
Why do freshwater forms only occur in warm water?
Besides few rare excursions into temperate FW during the
warmer months (Thomerson et al. 1977) elasmobranchs are
found only in tropical or subtropical waters (Compagno
1995). The coldest temperature at which FW elasmo-
branchs are known to live is 14°C for D. sabina in Lake
Jessup in Florida although their thermal preference is 10°
higher (Wallman and Bennett 2006). Bat rays leave brack-
ish water for higher salinities when the temperature falls
below 10°C (Meloni et al. 2002). One possible explanation
is that at low temperatures there is an imbalance in the
ability to supply the energy needed to maintain the high rates
of solute movement in the face of the relatively high rates
of diVusion at low temperatures. This is because there is a
greater reduction in active transport rates compared to the
reduction in diVusion. This diVusion limitation is observed
in some teleosts (Schwarzbaum et al. 1991; Schwarzbaum
et al. 1992; Staurnes et al. 1994) resulting in higher ion lev-
els in plasma being tolerated at low temperatures. Part of
the adaptation to changing temperature and salinity in tele-
osts involves restructuring membrane lipids. This may be
problematic in elasmobranchs where some aspects of mem-
brane structure are designed for urea retention. The inabil-
ity to modify membrane lipids to allow higher transport
activities at lower temperatures may be constrained by the
need to conserve urea permeability.
Are urea requiring proteins an adaptive roadblock?
Urea is a chaotropic agent that disrupts hydrophobic inter-
actions and so has structural eVects on proteins. Although it
is widely believed that a 2:1 ratio of urea:methylamines
allows optimal protein structure and function, the need for
methylamines is not absolute (Ballantyne 1997; Singh et al.
2009). Indeed there is a whole range of protein responses to
urea and methylamines (Ballantyne 1997). Hemoglobin
from S. acanthias lacks the antagonistic eVects of urea and
TMAO and is only sensitive to urea (Scholnick and
Mangum 1991; Weber 1983). The functional properties of
some euryhaline and stenohaline marine elasmobranch
hemoglobins are unaVected by urea (Bonaventura et al. 1974;
Cooper and Morris 2004; Mumm et al. 1978; Scholnick
and Mangum 1991). Interestingly, the hemoglobin of
Potamotrygon is more stable to urea denaturation than that
of other elasmobranchs (Martin et al. 1979).
123
J Comp Physiol B (2010) 180:475–493
Marine elasmobranch M4 lactate dehydrogenase (LDH)
requires urea to maintain an optimal Km for pyruvate
(Yancey and Somero 1978). The only study of the eVects of
urea and methylamines on Potamotrygonid enzymes indicates
that in the absence of urea the Km for pyruvate for Potamo-
trygon sp. M4 LDH is similar to that of non-elasmobranch
species and diVerent from those of marine elasmobranchs
(Yancey and Somero 1978). Even within protein isoforms,
the sensitivity to urea and other osmolytes diVers. Calcium
binding to parvalbumin I is unaVected by urea or most
methylamines (except betaine) in D. sabina while parvalbu-
min II is sensitive to all osmolytes and no counteraction is
observed (HeVron and Moerland 2008).
Considering the large number of proteins involved if
even a small fraction required urea for optimal function,
reduction of tissue urea levels during incursions into FW
may disrupt metabolism and other protein functions. The
retention of osmotically signiWcant amounts of urea in
many FW elasmobranchs such as H. signiWer may thus be
necessary to retain protein function. Only through pro-
longed evolutionary periods could urea sensitivity of all
proteins be eliminated. This has only occurred in the Pota-
motrygonids that invaded FW millions of years ago (Love-
joy et al. 1998).
Summary: why are there so few species
of elasmobranchs in FW?
FW environments place osmotic, sensory and reproductive
constraints on Wshes. The osmotic strategy employed by
marine elasmobranchs (osmoconformation by urea reten-
tion) is less conducive to a FW existence compared to the
osmoregulatory strategy employed by the more advanced
teleost Wshes. Colonization in FW by elasmobranchs
involves three adaptive phases. The Wrst stage involves
euryhaline marine forms that enter and leave FW and retain
some urea to maintain protein function in FW. Maintaining
high urea levels is disadvantageous since inevitable losses of
urea are incurred and replacement of this urea is energetically
expensive. The second stage includes species that reside
almost entirely in FW but these still have the need to
retain suYcient urea to maintain protein function. The third
and Wnal stage involves the complete elimination of urea as
an osmolyte. We suggest that complete elimination of urea
is the main evolutionary roadblock to successful adaptation
to FW. Other factors must play a role but may be less
important than eliminating urea-adapted proteins. These
include adaptation to the lower conductivity of FW with
modiWed electroreceptors with shorter channels. A com-
plete life cycle in FW would require the capacity of sperm
to survive and be activated in FW. The Potamotrygonids
are the only group to completely adapt to FW and their
J Comp Physiol B (2010) 180:475–493
success is evidenced by their extensive speciation (more
than 25 species). The need to make so many anatomical,
physiological and biochemical modiWcations for an FW
existence constrains the rapidity and hence the frequency of
FW colonization.
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