Skin permeability of the amphibious mangrove rivulus Kryptolebias
marmoratus in response to emersion
by
Quentin Heffell
A Thesis
presented to
The University of Guelph
In partial fulfillment of requirements
for the degree of
Masters of Science
in
Integrative Biology
Guelph, Ontario, Canada
© Quentin Heffell, January, 2016
ABSTRACT
Skin permeability of the amphibious mangrove rivulus Kryptolebias marmoratus in
response to emersion
Quentin Heffell
University of Guelph 2016
Advisor:
Dr. Patricia Wright
The skin of the amphibious fishes is known to be a physiologically important
tissue for maintaining homeostasis during emersion. I tested the hypothesis that skin
permeability would be altered to maintain water balance during salinity acclimation
and/or emersion. Osmotic permeability of the skin was assessed using 3H2O fluxes, and
bulk osmotic flow was quantified using a novel dye-dilution technique. No regional
variability between the dorsal and ventral skin region was found, although fish more
often positioned themselves on the ventral surface when emersed. Hypersaline
acclimation resulted in a decrease in 3H2O influx across the skin relative to hyposaline
acclimation. Acute emersion (1 day) induced a decrease in bulk osmotic water loss in
hypersaline-acclimated fish, whereas prolonged emersion (7 days) resulted in an increase
in 3H2O influx. These findings suggest that K. marmoratus alter skin permeability to
maximize water uptake and minimize water loss while emersed in hypersaline conditions.
TABLE OF CONTENTS
Abstract……………………………………………………………………………………ii
Table of Contents…………………………………………………………………………iii
Acknowledgements……………………………………………………………………….iv
List of Figures……………………………………………………………………………..v
Introduction………………………………………………………………………………..1
Methods…………………………………………………………………………………..13
Results……………………………………………………………………………………22
Discussion………………………………………………………………………………..50
General Discussion………………………………………………………………………64
Literature Cited………………………………………………………………………......67
iii
ACKNOWLEDGEMENTS
First and foremost I would like to recognize Dr. Patricia Wright for her guidance,
patience, and seemingly endless drive. Pat, you have been an amazing partner throughout
this process and have instilled skills and qualities of a researcher that I value highly. You
are a continuous motivation and inspiration, with an advisory style to be admired. Thank
you for making my masters experience meaningful, productive, and transformative.
I would also like to recognize the members of the Wright lab for their academic
support. In particular, I would like to recognize Andy Turko and his intellectual
contributions to this work. Andy’s innovation and passion for his work have provided me
a great model for thinking outside of the box. I look forward to seeing what you
accomplish.
Finally, I would like to extend thanks to members of my personal life including
friends and family who have been nothing but supportive throughout my time in Guelph.
Namely, I would like to thank Jen Bernard and Kayla Deasley for their camaraderie on
and off campus. I will also take this opportunity to formally thank my parents and loved
ones on Prince Edward Island, who have been a tremendous foundational body. I’m sure
GG has burned through a few sets of beads on my behalf. Finally, I would like to
formally thank Dan Hamilton, who has been a tremendous motivator and reminder of
what is to come.
To all, I sincerely thank you.
iv
LIST OF FIGURES
Figure 1
Time series of 3H2O movement (dpms) across isolated K. marmoratus
skin, indicating linear transfer of 3H2O over 10 minutes. All data are
means ± SEM (N=4)………………………………………………..…p. 27
Figure 2
Spectral scan of 1% food dye solution showing peaks of optical density
(absorbance) with a suggested optimal wavelength at 190 nm. This
suggested reading value is indicative of the minimum sensitivity of the
machine, rather than an optimal reading value. A more optimal peak in
absorbance at 425 nm was chosen for dye-dilution sample reading,
resulting in minimal error……………………………………....……p. 29
Figure 3
Standard curve of absorbance values from diluted 1% green food dye
solution. Samples were diluted using 12.5 µL of pure water and all
samples were read at a 425 nm wavelength. Absorbance values from bulk
osmotic flow experiments were used to obtain a volume of water moved
via the osmotic gradient using similar standard curves..………….…p. 31
Figure 4
Behavioural observation of intact K. marmoratus held out of water for 1
day. The time (%) spent with various skin regions in contact with the
moist filter paper substrate in 15 cm3 polystyrene wells (A, combined data
B) or 700 cm3 plastic containers (C, combined data D). All data are means
± SEM (N=5: bracketed values). Groups that do not share a common letter
v
are significantly different as determined by One Way ANOVAs, while a *
symbol indicates significant differences as a result of a paired T-test
(P<0.004: 15 cm3; P<0.001: 700 cm3)………………………………..p. 33
Figure 5
3
H2O influx and efflux rates (µL/cm2/min) across the isolated dorsal and
ventral skins of K. marmoratus acclimated to brackish water (15 ‰). All
data are means ± SEM (N=7: bracketed values). Data analyzed using a
Two Way ANOVA (P<0.05) yielding no statistical significance……p. 35
Figure 6
3
H2O influx (A) and efflux (B) rates in the isolated skin of K. marmoratus
acclimated to 0.3 and 45 ‰ water for 7 days and either kept in water or air
exposed for 1 day. All data are means ± SEM (N=6-11: bracketed values).
An * symbol indicates statistical significance as a result of a two-way
ANOVA and Holm-Sidak post hoc test (P<0.05).……….…………p. 37
Figure 7
PEG permeability (P-PEG, cm/s) of the skin to 14C-PEG-4000 influx (A)
and efflux (B) in the isolated skin of K. marmoratus acclimated to 0.3 and
45 ‰ water for 7 days and either kept in water or air exposed for 1 day.
All data are means ± SEM (N=4-11: bracketed values). All data were
analyzed using a two-way ANOVA (P<0.05).…………………….….p. 39
Figure 8
3
H2O flux (A: influx, B: efflux) and PEG permeability (P-PEG, cm/s) of
the skin to 14C-PEG-4000 flux (C: influx, D: efflux) in the isolated skin of
vi
K. marmoratus acclimated to 45 ‰ water for 7 days and either kept in
water or air exposed for 7 days. All data are means ± SEM (N=5-11:
bracketed values). An * symbol indicates statistical significance as a result
of a t-test (P<0.05)…………………………………………….……...p. 41
Figure 9
Bulk osmotic flow of water across the isolated skin of K. marmoratus
(mucosal to serosal) acclimated to 0.3 ‰ water for 7 days followed by
24-hours of water or air exposure. Data are means ± SEM (N=5: bracketed
values) and were analyzed using a t-test.………………...……………p. 43
Figure 10
Bulk osmotic flow of water across the isolated skin of K. marmoratus
(serosal to mucosal) acclimated to 45 ‰ water for 7 days followed by 24hours of water or air exposure. All data are means ± SEM (N=4-5:
bracketed values). An * indicates statistical significance as a result of a ttest (P<0.05)………………………………………………..……….p. 45
Figure 11
Representative images of fluorescein-stained (FITC – green) and
tetramethylrhodamine-stained (TRITC – red) skins of K. marmoratus
acclimated to their respective salinities for 7 days and emersion time was 1
day. A: 0.3 ‰ in water, B: 0.3 ‰ air, C: 45 ‰ in water, D: 45 ‰ air.
FITC staining localizes to Na+/K+-ATPase rich cells while the TRITC
staining is specific for claudin 10e. 20x magnification; scale bar = 100µm.
S: Skin; M: Muscle; Arrowhead: mucous cells……………………….p. 47
vii
INTRODUCTION
Amphibious fishes are a morphologically diverse group of semi-aquatic
organisms living at the boundary between water and land (Sayer and Davenport,
1991). These fishes vary in their tolerance of terrestrial environments, as moving
between aquatic and terrestrial habitats places burdens on physiological systems to
maintain homeostasis (Sayer, 2005). Amphibious fishes have various adaptations to
maintain respiration, iono- and osmoregulation, and acid–base regulation out of
water. My MSc thesis addressed osmoregulatory challenges in a model euryhaline,
amphibious fish, the mangrove rivulus, Kryptolebias marmoratus. This species
tolerates extended emersion and encounters variable salinities in its natural habitat,
especially with changing seasons. My goal was to identify the mechanisms of water
balance that K. marmoratus use when out of water on either hypo- or hypersaline
substrates.
Iono-Osmoregulation
The main osmoregulatory epithelium in fishes is the gill. It functions as the
primary exchange surface for ions and water between the internal blood plasma and
the external environment. The dynamics of ion and water movement differ between
the freshwater and seawater fishes due to differences in ionic and osmotic gradients
(Isaia 1984; Payan, Girard and Mayer-Gostan 1984).
Seawater fishes. Seawater fishes maintain an internal osmotic concentration
approximately one third that of their external environment. Generally, the blood
plasma in marine fishes is ~400 mOsm, while their hypersaline environment is ~1000
1
mOsm (Evans 2008). This generates a strong osmotic gradient, favoring the outward
passive diffusion of water toward the hyperosmotic environment. To maintain
osmotic equilibrium, saltwater fish drink seawater and absorb water across the
intestinal epithelium. The relatively low internal osmolality of saltwater fishes also
generates electrochemical gradients that favor the movement of salts from the
ambient water into the relatively hypoosmotic blood plasma. To oppose this
diffusional gradient, saltwater fish actively excrete excess NaCl through
mitochondrial rich cells, or ionocytes, in the gills. The kidney also aids in salt
excretion, producing low volumes of urine rich in divalent ions (Krogh 1939; Evans
1980; Evans et al. 1999; Evans et al. 2005; Marshall and Grosell 2006; Evans 2008;
Whittamore 2012; Edwards and Marshall 2013).
Freshwater fishes. Freshwater fishes have blood that is hyperosmotic to their
ambient environment. Generally, the blood plasma of freshwater fish is ~300 mOsm
while their ambient environment is ~1 mOsm (Evans 2008). In contrast to seawater
fishes, freshwater fishes face a gradient favoring ion loss and must transport Na+ and
Cl- ions inward via active transport mechanisms in gill ionocytes. There is also an
osmotic gradient across the gill that causes the passive uptake of water. Excess water
uptake, as well as ion loss, is compensated for by the kidney, through NaCl
reabsorption and the production of copious amounts of dilute urine. It is obvious how
important a permeable surface is for compensatory diffusion and exchange to
maintain osmotic and ionic balance in both saltwater and freshwater fishes (Krogh
1939; Evans, 1980; Evans et al. 2005; Marshall and Grosell 2006; Evans 2008;
Whittamore 2012).
2
Terrestrial animals. Maintaining water balance is also a challenge for terrestrial
animals. Evaporation of water directly from the surface of the skin accounts for 5080% of total water loss in resting terrestrial animals (reptiles, birds, and mammals),
followed by pulmonary evaporation occurring across respiratory surfaces (Kirschner
1991). Variation in evaporative water loss across the skin is dependent upon the
animals’ environment, as individuals from more arid environments have adaptations
to minimize water loss to cutaneous evaporation. Inhabiting burrows and high
altitudes allow animals to decrease the stress of evaporative water loss, as these
environments generally have higher relative humidity and are cooler and moister
(Kirschner 1991). The skin of many reptiles is also adapted to arid habitats with a
very low permeability to water to prevent evaporative water loss. Even scaleless
aquatic sea snakes in air lose less water across the skin than a typical amphibian
(Bennett and Licht 1975). Altering skin permeability to prevent evaporation and
maintain water balance is thus obviously important to animals inhabiting terrestrial
environments.
When amphibious fishes leave water or emerse, gill function may not be
maintained (Graham, 1997). Thus, many fishes moving to land must employ an
alternative exchange surface to maintain homeostasis of ions and water. Some
amphibious fish use the cutaneous surface for ion and water regulation.
Mitochondrial-rich cells similar to gill ionocytes have been discovered in the skin of
the mudskippers Periophthalmus modestus (Yokota et al. 1997) and Periophthalmus
cantonensis (Yokoya and Tamura, 1992), the ventral surface of the African lungfish
(Strula et al. 2001), and the mangrove rivulus K. marmoratus (LeBlanc et al. 2010).
3
Skin ionocytes increase in size after emersion in K. marmoratus with no change in
sodium efflux rate, suggesting the recruitment of the skin for ionoregulation (LeBlanc
et al. 2010). In the amphibious swamp eel (Synbranchus marmoratus), cutaneous ion
transport represents 75% of ionoregulation with no effect of emersion on sodium
influx or efflux (Stiffler et al. 1986). Similar results were found in estivating lungfish
(Protopterus dolloi), with no effect of long-term emersion on plasma osmolality or
ion flux (Wilkie et al. 2007). Thus, there is evidence that the skin plays a critical role
in maintaining ion gradients in fish out of water.
Water flux
The balance of water across the skin of amphibious fish is a topic that has not
been thoroughly investigated. Many studies of water balance in fishes focus on whole
body water flux experiments (Evans 1967; Potts et al. 1967; Rudy 1967; Evans
1969a,b; Potts and Rudy 1971; Loretz 1979; Wilkie et al. 2007; LeBlanc et al. 2010).
Whole body fluxes incorporate drinking rates and exchange across the gill, kidney,
and skin. Drinking during emersion in amphibious fishes, however, would not be
possible. In addition, a terrestrial environment with a hypersaline substrate may result
in decreased water content of both intra- and extracellular body compartments,
resulting in dehydration via osmotic water loss (reviewed by Takei 2015). Because
fish out of water are unable to drink to rehydrate these compartments, alternative sites
of water exchange must be used.
The specific sites of water exchange and the cellular mechanisms involved in
water flux have not been explored in amphibious fishes in detail. It is possible that the
4
skin is an important site for water balance in amphibious fish. In the African lungfish
(P. dolloi), chronic emersion (6 months) caused water efflux across the skin to
decrease by 60% (Wilkie et al. 2007), potentially as a means to conserve body water.
Although water flux across the dorsal skin was never measured, it was assumed that
water moved primarily across the ventral skin in this fish. This was because water
fluxes were comparable between the immersed and emersed groups, despite the
emersed fish being limited to only ventral contact with the water source. This
potentially high ventral permeability of the lungfish mirrors the highly permeable
pelvic patch common in anuran amphibians (McClanahan and Baldwin 1968; Bentley
and Main, 1972; Bentley and Yorio 1976). It is possible that P. dolloi and other
amphibious fishes use the ventral skin for water exchange during emersion and alter
skin permeability to water during prolonged emersion to prevent excessive water loss.
Lungfish are known to enter a metabolic depression during estivation, which may
account for the observed decrease in cutaneous water flux (Smith 1930; Janssens
1964). Many amphibious teleost fish do not enter an estivation during emersion and
could respond to emersion differently than the lungfishes. It is largely unknown how
teleost skin permeability is altered during emersion.
Water balance in amphibious fishes has also been studied by measuring tissue
water loss. In air-exposed (1 day) barred mudskippers (Periophthalmus sobrinus),
body mass decreased as a result of evaporative water loss (Gordon 1969). This
decrease however was relatively low compared to more terrestrially adapted
amphibians (Gordon 1969). A related species of mudskipper, P. cantonensis uses a
hierarchy of tissue water loss when exposed to desiccation pressures (Gordon et al.
5
1978). These fish lost the greatest proportion of water from the heart, followed by the
blood and white muscle, with the liver and brain actually increasing in water content
(Gordon et al. 1978). This strategy seems to utilize internal redistribution of water to
maintain a hydrated central nervous system. Low rates of water loss were found in the
amphibious Chilean clingfish Sicyases sanguineus (Gordon 1970). The gut has been
suggested as an osmoregulatory surface in this latter species, as they ingest large
volumes of water before extended emersion (Marusic et al. 1981). S. sanguineus
retain intestinal water for up to 6 hours, but by 1 day water was no longer in the
intestine, suggesting it’s use for water balance.
Taken together, previous studies suggest that amphibious animals have
adaptations to maximize water balance and prevent water loss during emersion.
Although much is known about amphibian integumentary function, no work has
examined water flux across amphibious fish skin in isolation. Whole body
experiments do not allow us to examine the relative contribution of the skin to overall
osmoregulation. How do different skin regions differ in terms of skin permeability?
And how does cutaneous water permeability change with acclimation to a terrestrial
habitat, particularly under hypersaline conditions when fish are unable to replace
water loss by drinking?
Measures and mechanisms of water flux. The movement of water is driven by
osmotic gradients. The movement of ions down chemical and/or electrical gradients
generates changes in osmotic potential, thus creating osmotic gradients. Water
movement across biological membranes is often quantified with two measures of
membrane permeability: the diffusional permeability (unidirectional flux measured
6
with tracers) and hydraulic conductivity (bulk water flux in an osmotic experiment)
(Danity and House 1966; Kirschner 1991). Tracers are substances that can be tracked
through an organism or other biological system, such as radiolabelled water (tritiated
water: 3H2O). Diffusional permeability is a measure of water movement as water
molecules obey Fick’s law of diffusion. Water movement, here, is solely due to
diffusion, as diffusional permeability experiments are conducted under isosmotic
conditions. Hydraulic conductivity measures bulk water movement as water moves
down osmotic gradients and obeys fluid dynamics (Isaia 1984). The literature
surrounding the permeability of biological membranes to water is often inconsistent
with regard to terminology and technique. Hydraulic conductivity, for example, is
often referred to as “osmotic permeability” as it will be in the current study. Such
variation requires that terms are explicitly defined to avoid inappropriate
comparisons. In the current study, water flux down an osmotic gradient was
examined, and the osmotic permeability of amphibious fish skin was calculated.
Although tracer experiments were used to measure unidirectional water flux,
diffusional permeability could not be quantified, as an osmotic gradient was always
present.
Water movement has been examined at the cellular level (between the intraand extracellular fluid), as well as across the whole body or specific epithelia
(Kirschner 1991). Water may move down osmotic gradients through epithelia by
transcellular or paracellular pathways (Brandner 2007; Kawedua et al. 2007).
Transcellular. Water molecules can move across a cell membrane by simple
diffusion, that is, directly between the lipid molecules that make up a phospholipid
7
bilayer (Zeuthen 2010). Water may also move freely though transmembrane protein
channels incorporated directly into the cellular membrane (facilitated diffusion).
These channels were discovered and termed aquaporins (Agre et al. 1993).
Aquaporins make up a superfamily of membrane protein channels (Edwards and
Marshall, 2013), and orthologs of these proteins are widespread throughout both the
plant and animal kingdoms with the teleost fish clade having the largest variety
(Cerdà and Finn 2010). Aquaporins exist in the membrane as homotetramers, made
up of four monomeric channels that provide a route for the movement of water down
the osmotic gradient.
There is evidence that aquaporin isoforms have specialized roles within
tissues. Aquaporin-3 expression in the gill has been found to change with salinity
acclimations (increased expression in freshwater; decreased in seawater) in European
sea bass (Dicentrarchus labrax) (Giffard-Mena et al. 2007), the silver sea bream
(Sparus sarba) (Deane and Woo 2006) and the European eel (Anguilla anguilla)
(Cutler and Cramb 2002). These results suggest that aquaporin-3 specifically, and
broadly the transcellular route via aquaporins, is important for osmoregulation in fish
and responds to changes in the environment by altering membrane permeability. It is
unknown how an aerial environment and the risk of water loss would influence the
transcellular route in the skin of amphibious fishes.
Water moving against a transepithelial osmotic gradient (or ‘uphill’ water
movement) has been observed in classic studies of water movement across
amphibious skin (Reid 1892; Koefoed-Johnsen and Ussing, 1953; House 1964).
Many studies focusing on uphill water movement examine the intestinal lumen during
8
digestion (Curran, 1960; Skadhauge 1969, Genz et al. 2011; Whittamore 2012), but
little is known about this property in skin. Uphill water movement is accomplished by
means of cotransporters: proteins that move water alongside solutes. These proteins
use downhill ion gradients to move water molecules against uphill osmotic gradients
(Zeuthen 2010). Water molecules can also become engulfed within a membrane
protein, and during this protein’s conformational change to transport target solutes,
water is secondarily moved (Whittamore 2012). As in the intestine, it is possible that
water cotransport is important to cutaneous water dynamics.
Paracellular. Tight junctions are regions in the paracellular space where
membrane associations are made between neighboring cells. The tight junction
complex proteins are used to regulate paracellular transport (Tsukita and Furuse,
1999; Kolosov et al. 2013). The major protein constituents of the tight junction
complex are the claudins, occludins, tricellulins, junctional adhesion molecules
(JAM), and zonula occludens (ZO) proteins (Chasiotis et al. 2012; Gonzalez-Mariscal
et al. 2003). More specifically, claudin proteins are often tissue specific and, along
with occludin and tricellulin proteins, dictate passage through the paracellular space
(Gunzel and Yu, 2013). The JAM molecules function primarily to traffic larger cells
like lymphocytes (Gonzalez-Mariscal et al. 2003). Finally, the ZO proteins provide a
link between the cell membrane and the cytoskeleton, creating support for the
complex protein assemblage (Bauer et al. 2010).
The claudin family is extensively studied in fish. There have been a total of 56
claudin proteins discovered in a single fish species (Fugu rubripes; Loh et al. 2004).
Some of these isoforms are comparable to mammals, while some are the result of
9
genetic duplication events. In general, high concentrations of tight junction proteins
are found in the gills of hyperosmotic fish (to prevent the excessive paracellular
diffusion of ions) and low numbers of tight junction proteins are found in ‘leaky
junctions’ of hypoosmotic fish (to allow for ease of Na+ paracellular diffusion)
(Evans et al. 2005; Kolosov et al. 2013).
Tight junction proteins have been discovered in the skin of multiple fish
(pufferfish Takifugu rubripes: Loh et al. 2004; green spotted puffer Tetraodon
nigroviridis: Bagherie-Lachidan et al. 2009; zebrafish Danio rerio: Kumai et al.
2011; goldfish Carassius auratus: Chasiotis and Kelly 2012; carp Cyprinus carpio:
Syakuri et al. 2013) though little work has been done to detail their role in water
balance. In the fully aquatic T. nigroviridis, it has been found that salinity has a
significant effect on tight junction tightness through the mRNA expression of
claudin-3, claudin-8 and claudin-27 in the skin (Bagherie-Lachidan et al. 2008;
Bagherie-Lachidan et al. 2009). Under these conditions there was also an
upregulation of claudin proteins, reducing skin and gill permeability. Yet, in the
European eel (A. anguilla), claudin-27 gene expression declined in the gills after
seawater acclimation (Kalujnaia et al. 2007). It appears that there is some degree of
species specificity to claudin regulation in response to salinity changes.
To date, there is no information on how epithelial tight junctions or water flux
across the skin may be altered in amphibious fish out of water. LeBlanc et al. (2010)
discovered that hypersaline seawater (45 ‰) acclimated K. marmoratus exhibited a
decrease in water efflux, while freshwater (1 ‰) acclimated fish demonstrated an
increase in water efflux after 9 days of air exposure. Changes in water efflux could be
10
related to branchial, cutaneous or renal responses and involve either paracellular or
transcellular flux. LeBlanc et al. (2010) suggested the potential role of tight junction
proteins in osmoregulation in emersed fish, although this has not been explored.
Interestingly, body water content significantly increased by ~1% after 11 days in
emersed K. marmoratus (Litwiller et al. 2006), suggesting that fish may actually take
up water from a moist substrate during emersion.
Objectives and Hypothesis
I designed my experiments under the assumption that tritiated water (3H2O) would
travel across the skin identically to unlabeled water. I also assumed that 3H2O would
move through both paracellular and transcellular routes, while 14C-PEG-4000 would
move exclusively through the paracellular route due to its relatively large molecular
size. I questioned whether there would be regional differences in permeability in K.
marmoratus’ skin, because in preliminary observations, K. marmoratus appeared to
“sit” on their ventral surface when air exposed. Thus, the ventral surface should be
adapted for water uptake from the moist substrate. A porous and water permeable
ventral surface has also been described in other amphibious organisms to facilitate
water balance.
I hypothesized that the permeability of the skin would be altered through
paracellular and transcellular mechanisms during emersion to facilitate water balance.
This hypothesis predicts the following outcomes:
i.
In hypersaline-acclimated fish, emersion will result in an increase in water
influx to combat evaporative water loss, as well as a decrease in water
11
efflux to oppose the osmotic gradient and maintain internal body water. In
freshwater-acclimated fish, water influx and efflux will remain
comparable to immersed fish, as evaporative water loss will be opposed by
passive water uptake due to the osmotic gradient.
ii.
Bulk osmotic flow of water across the skin (efflux) will be lower in
hypersaline-acclimated fish exposed to air relative to control fish in water,
as the skin will be tightened to prevent water loss. Inward bulk osmotic
flow (influx) will be greater in hyposaline-acclimated fish exposed to air
relative to control fish in water because water uptake will combat
evaporative loss and any osmotic water loading may be compensated for
by the renal system.
iii.
In both hypersaline- and freshwater-acclimated fish, emersion will induce
a tightening of the skin’s cellular junctions (paracellular) to maintain body
water content. Thus, 14C-PEG-4000 influx and efflux will decrease in
emersed compared to immersed fish.
iv.
Aquaporins are ubiquitous in animal tissues and facilitate transcellular
water flux. Inhibition of aquaporins will reduce water flux regardless of
salinity, but will have a proportionally larger impact where water flux
rates are higher.
12
METHODS
Experimental Animals
Adult mangrove rivulus, Kryptolebias marmoratus (0.18 g ± 0.01) were
acquired from a colony housed under constant conditions (25°C, 15 ‰, pH 8,
12L:12D cycle) in the Hagen Aqualab at the University of Guelph. The DAN strain,
originally native to Belize, was used in these experiments. All experiments were
conducted following the guidelines of the Animal Utilization Protocol 2239 at the
University of Guelph.
Experimental Design
Four series of experiments were conducted. The first series examined
emersion behavior to see if certain regions of the skin are selected for moist substrate
contact. The second series aimed to identify any differences in 3H2O fluxes across the
dorsal and ventral regions of the skin. The third series was designed to examine
whether air exposure and hyper- or hypo- salinity acclimation had effects on the
overall osmoregulatory function. In the third series, 3H2O and 14C-PEG-4000 fluxes
were used to distinguish the relative importance of the transcellular (3H2O) and
paracellular (PEG) transport routes through the skin. The third series also involved
designing a novel method to identify the degree of osmotic water movement that
occurred across the skin, after acclimation to different salinities and emersion
treatments. In the fourth series, experiments were performed to examine the effects of
HgCl2, a known aquaporin channel blocker, on water flux across isolated K.
marmoratus skins (Savage and Stroud 2007).
13
Series 1: Body orientation during emersion. Behavioral analysis of the body
position of fish out of water (1 day) was conducted using serial still photography. A
GoPro camera (Original HD Hero, CA, USA) was mounted above emersed fish in
containers lined with moistened filter paper. Experiments were originally conducted
using a smaller (15 cm3) emersion environment. The experiments were repeated using
a larger (700 cm3) emersion environment. The small arena was used to parallel leaf
litter or insect galleries inside of logs that K. marmoratus inhabit during dry seasons
(Taylor et al. 2008), while the larger arena mimicked the crab burrows that K.
marmoratus frequently travel between on land (Taylor et al. 1995). Photos were
captured every five seconds for two hours and body position in contact with the
substrate was recorded. Photos were coded based on how fish were orientated (dorsal,
ventral, lateral, dorsolateral, or ventrolateral). These values were then converted into
time (%) to determine how long fish maintained skin-substrate contact per skin
region.
Series 2: Regional variation in skin permeability. To determine if regional
differences in permeability of the skin existed, in vitro experiments were carried out
using isolated pairs of dorsal and ventral skins from the same individual in an Ussing
chamber, following a protocol similar to that of Cooper et al. (2013). Briefly, fish
were euthanized and the skin was carefully dissected. The skin was scraped free of
muscle with a blunt instrument while being bathed in serosal saline solution. The
serosal saline consisted of (mmol/L) 125 NaCl, 2 KCl, 1 MgSO4, 5 NaHCO3, 2
14
CaCl2, 1.25 KH2PO4, and 5.55 glucose and the pH was adjusted to 7.5 (Cooper et al.
2013). The skin was then sandwiched between the two halves of the Ussing chamber
(~0.6 mL each). The skin was then left for one hour to stabilize (Cooper et al., 2013)
in saline (serosal side) or an environment bath (mucosal side; water of the appropriate
salinity). The serosal solution was aerated with a humidified 0.5% CO2/O2 balance
gas mix to mimic the relevant gaseous environment of the blood plasma, while the
mucosal solution was aerated with humidified air. During the one-hour stabilization
period, a dye test was performed using food colouring (Club House, London ON,
Canada; water, propylene glycol, tartrazine, citric acid, and sodium benzoate) to
ensure that there was no leakage. At the end of this stabilization period, the chamber
was fully rinsed and replenished with the appropriate solutions. Triplicate samples of
these solutions were collected from the chamber system to analyze and subtract as
background. 3H2O (Perkin Elmer Ltd., Ontario, Canada) was added to either the
serosal or mucosal side (1 µCi/mL), mixed by gently pipetting, and triplicate samples
(25 µL) were taken at t = 0 min from both the mucosal and serosal chambers.
Preliminary experiments showed that 3H2O flux across the skin was linear up to 30
minutes (Figure 1), so a 10-minute flux period was chosen. Immediately following
this flux, the chamber was rinsed and replenished with unlabeled serosal/mucosal
solutions. 3H2O was then added to the opposite side and samples were taken as
before. The direction of flux was randomized, providing both influx and efflux data
on each skin without an effect of order. Following the bidirectional fluxes, an
additional dye test was performed to ensure the skin had not been compromised
during the experimental period. The serosal/mucosal saline samples were mixed with
15
aqueous counting scintillant (5 mL; 667 ml toluene, 333 ml Triton X-100, 4 g 2,5diphenyloxazole, and 0.2 g 1,4-bis[5-Phenyl-2-oxazolyl]ben- zene; 2,2′-pPhenylene-bis[5-phenyloxazole]; Sigma) and subsequently counted in a scintillation
counter (Beckman Coulter LS6500 Multi-Purpose Scintillation Counter, California,
USA).
Series 3: Influence of salinity and air exposure on skin permeability. To
determine the potential effects of salinity and emersion on water transport through the
skin, fish were divided into four experimental conditions: hypersaline water in water,
hypersaline water in air, hyposaline water in water, and hyposaline water in air. These
salinities are also ecologically relevant, as K. marmoratus populations have been
found at and between these extremes in the wild (0.3 ‰: Wright, unpublished data;
45 ‰: Frick and Wright 2002). Fish were transferred from brackish water (15 ‰) to
0.3 ‰ or 45 ‰ and were acclimated for one week prior to experiments. These
salinities represented osmotic differences of ~380.63 mOsM in the freshwater
treatments and ~1373.27 mOsM in the hypersaline treatment. Air-exposed fish were
emersed for 1 day in an aerial environment of high humidity as previously described
(Ong et al. 2007). The air-exposure time was chosen because of the reported acute
nature of claudin upregulation, (Kwong et al. 2013) and preliminary evidence of
cutaneous claudin protein expression in K. marmoratus (F. Galvez, P. Wright and S.
Kelly, unpublished data). An additional hypersaline emersion group with a seven-day
aerial exposure period was also tested to determine the effects of prolonged air
exposure. Following their respective acclimations, fish from each of the five
16
experimental treatments were euthanized and the skins were mounted in Ussing
chambers, as described above.
Unidirectional water flux was measured through a dorsal section of the skin in
either the mucosal-to-serosal (influx) or serosal-to-mucosal (efflux) direction. 3H2O
fluxes were sampled after 10 minutes as described above. Following the 3H2O flux,
each half of the chamber system was thoroughly rinsed with their respective solutions
and replenished. The paracellular permeability marker 14C-PEG-4000 was added to
the same side as 3H2O had been previously added (1 µCi/mL). As before, background
samples were collected, and the aerated chamber system was left for 1 hour. Final
samples were taken (t = 60 min) and a dye test was used to ensure skin viability was
maintained. An hour-long flux period was chosen for 14C-PEG-4000 due to the larger
molecular mass and size of PEG-4000 compared to 3H2O. Experiments on isolated
tissues have also measured 14C-PEG-4000 over hourly periods previously (Genz and
Grosell 2011; Wood and Grosell 2012).
To determine if the skin tissue held on to trace amounts of radionucleotide
between influx and efflux experiments, digestion of skin and subsequent scintillation
counter analysis was preformed. Methods followed a modified protocol of Wood and
Laurent (2003), where tissues were submerged in 1N HNO3 and left at 60 °C for 1
day with occasional vortexing. Samples were then centrifuged at 500 x g for 5
minutes and 25 µL of supernatant was mixed with scintillation cocktail and counted.
Transepithelial potential (TEP) was also measured during flux experiments.
Electrodes were constructed using 4% agar bridges, 0.5 mol/L KCl, and ferric
chloride treated silver wires (A-M Systems, Washington, USA; Cooper et al. 2013).
17
The electrode bridges were placed on either side of the skin while a modified BNC
cable was submerged in the KCl solution, completing the electrical circuit. The signal
was recorded in mV using a pH meter (Orion 520, Thermo Fisher Scientific Inc.,
Massachusetts, USA).
To assess the distribution of claudin proteins in the skin,
immunohistochemical staining was used. Staining procedures followed closely the
methods of Chasiotis and Kelly 2008 and Bui and Kelly 2014. Briefly, tissues were
dissected and fixed in Bouin’s fixative for 1-4 hours. Tissues were then dehydrated in
a series of ethanol rinses (70-100%), followed by cleaning with xylene. Tissues were
then embedded in paraffin and sectioned (5 µm) on a Leica RM 2125RT manual
rotary microtome (Leica Microsystems Inc., Richmond Hill, ON, Canada). Sections
were then placed on glass slides and deparaffinized with xylene. Tissue slides were
rehydrated with an ascending series of ethanol rinses (100-50%) Heat-induced
epitope retrieval (HIER) was accomplished by repeatedly heating samples while
being bathed in sodium citrate buffer followed by washes in phosphate buffered
saline (PBS). Samples were then outlined with a hydrophobic pen and quenched for
30 minutes in 3% hydrogen peroxide. Slides were rinsed with distilled water and
washed with Kodak Photo-Flo 200 in PBS, TritonX-100 in PBS, and antibody
dilution buffer (ADB) in PBS sequentially. Slides were incubated overnight in
primary antibodies for Na+-K+-ATPase (NKA; mouse anti-NKA; 1:10 dilution) and
claudin-10e (rabbit anti-Cldn; 1:10 dilution). Claudin 10e was chosen because of its
localization in the skin of a teleost fish as well as its potential role in osmoregulatory
function (Bui and Kelly 2014). Following this primary incubation, slides were rinsed
18
in PBS containing 0.05% Triton X-100 and probed for one hour at room temperature
with secondary antibody for NKA and Cldn (fluorescein isothiocyanate (FITC)labeled goat anti-mouse in 1:500 dilution and tetramethyl rhodamine isothiocyanate
(TRITC)-labeled goat anti-rabbit in 1:500 dilution, respectively (Jackson
ImmunoResearch Laboratories, Inc., USA). All antibodies were diluted in PBS
containing 0.05% Triton X-100, 10% goat serum and 0.1% BSA. Slides were then
rinsed in a series of PBS washes containing 0.05% Triton X-100 and Kodak PhotoFlo 200 before being carefully wiped and mounted with Molecular Probes ProLong
Antifade (Invitrogen Canada Inc., Canada).
The relative influence of the osmotic gradient on water movement across K.
marmoratus skin was determined using a novel dye-dilution method. First, green food
dye (Club House, London ON, Canada) solutions (1%) were made using either
mucosal (hypersaline groups) or serosal (hyposaline groups) saline. This skins from
fish previously acclimated to hypo- or hypersaline water (control, emersion: 1 day)
were mounted in the Ussing chamber as previously described. In hyposalineacclimation experiments, the serosal saline was replaced with the 1% food dye serosal
saline solution. In hypersaline acclimation experiments, 45 ‰ seawater was replaced
with the 1% food dyed hypersaline solution. Duplicate samples (100 µL) of equal
volume were collected at t = 0 min from both sides of the Ussing chamber to maintain
hydrostatic equilibrium across the skin. The system was then left for 1 hour to allow
for water to move via the osmotic gradient across the skin. Following this time
period, replicate well-mixed 100 µL samples were taken (t = 60 min). Food dye
19
solutions represented the environment into which water would move osmotically,
thus diluting the colour and reducing the absorbance of the sample.
To determine the optimal wavelength at which to measure the absorbance of
the diluted samples, a spectral scan of the 1% dye solution was conducted on a
SpectraMax® 384 Plus microplate reader (Molecular Devices, Sunnyvale CA, USA).
The scan yielded multiple peaks in absorbance with a suggested maximal absorbance
value of 190 nm (Figure 2). Measuring samples at this wavelength, however, yielded
a large degree of error. This was because 190 nm was just at the minimal sensitivity
of the microplate reader. Therefore, 425 nm was used as the optimal absorbance
wavelength and a standard curve of various dye dilutions consistently yielded a R2 of
>0.98 (Figure 3). The experimental 100 µL samples were loaded into a 96 well plate
and read at 425 nm. Absorbance values obtained from the samples were compared to
a standard curve to determine how much water moved osmotically across the skin
over the one-hour period (Figure 4). These values were precise down to the µL range.
This technique allowed me to measure true bulk water movement across the skin as a
result of ecologically-relevant osmotic gradients.
Series 4: Influence of HgCl2 on 3H2O flux. Skins were mounted in the Ussing
chamber setup as previously described. All HgCl2 treated 3H2O fluxes were
performed in the mucosal to serosal (influx) direction. An initial 3H2O flux was taken
over 10 minutes as above, followed by a 30-minute incubation in HgCl2 enriched
solutions of serosal saline and hypersaline water (0.03 or 3 mol/L). These solutions
were refreshed following the incubation and used for the post-incubation flux media.
20
This protocol allowed for the comparison of pre- and post-incubation 3H2O flux rates
of the same skin to quantify the effect of HgCl2 treatment.
Calculations and Statistical Analysis
Analytical procedures to calculate 3H2O flux rate (J3H2O) followed the methods
and formulae detailed in Pärt et al. (1999). The 3H2O flux formula is represented as
below:
𝐽3𝐻2𝑂 =
∆ 𝑑𝑝𝑚
𝑇 × 𝐴 × 𝑆𝐴
(1)
where ∆ dpm is change in disintegrations per minute, T is time (s), A is area
(cm2), and SA is specific activity. Specific activity was calculated using Equation 2
below:
𝑑𝑝𝑚𝑖 + 𝑑𝑝𝑚𝑓
)
2
𝑆𝐴 =
𝑉𝑜𝑙𝑢𝑚𝑒
(
(2)
where the initial (dpmi) and final (dpmf) disintegrations per minute values per
sample volume (µL) are averaged and divided by sample volume. A PEG
permeability (cm/sec) formula similar to Gilmour et al. 1998 is detailed below:
𝑃 − 𝑃𝐸𝐺 =
∆ 𝑑𝑝𝑚 × 𝑉𝑜𝑙𝑢𝑚𝑒
𝑇 × 𝐴 × 𝑆𝐴 × 3600
(3)
where ∆ dpm is change in disintegrations per minute, Volume is sample
volume, T is time (h), A is area (cm2), SA is specific activity as calculated in Equation
2, and 3600 to convert hours to seconds.
21
Data were analyzed using either a two-way repeated measures analysis of
variance (ANOVA), one-way ANOVAs, or when appropriate Student’s T-tests in
SigmaPlot 11 (Systat Software, San Jose, CA).
RESULTS
Emersion Behaviour
Fish exposed to an aerial environment for one day spent significantly more
time exposing their ventral/ventrolateral skin surfaces to the moist substrate than
dorsal, dorsolateral or lateral regions. This was true for both the 15 cm3 emersion
environment (P<0.004; Figure 4A) as well as the 700 cm3 emersion environment
(P=0.001; Figure 4C). In the larger environment, the ventral skin was exposed 42%
of the time, while dorsal and dorsolateral regions were never exposed to the moist
surface. Fish most often maintained substrate contact of the ventral region while
simultaneously exposing their lateral side to a wall of the container (deemed
“ventrolateral”). This ventrolateral body orientation occurred ~57% of the time. In the
more confined environment (15 cm3), the ventral and ventrolateral surfaces were most
often exposed to the moist substrate, followed by the dorsal, lateral, and finally the
dorsolateral. When dorsal and ventral categories are combined (lateral data omitted),
greater ventral substrate contact was apparent in both emersion environments
(P<0.004;0.001; Figure 4B,D respectively).
22
Regional Variability in Skin Water Flux
3
H2O flux across the skin of K. marmoratus showed no regional variability
(Figure 5). Dorsal and ventral skins from the same individuals were not significantly
different (P=0.680). 3H2O influx and efflux rates across the same skins were also not
significantly different (P=0.729).
Effects of Salinity and Acute Air Exposure on Skin Permeability
Water flux. Salinity had an effect on water influx rates in control fish in water.
Freshwater-acclimated fish had a higher rate of 3H2O influx compared to the
hypersaline-acclimated fish (P=0.026). There was, however, no effect of one-day of
air exposure in either salinity (P=0.204; Figure 6A). Water efflux rates were not
significantly affected by salinity (P=0.207), or one-day of emersion (P=0.281; Figure
6B).
PEG permeability. Inward PEG permeability (mucosal to serosal 14C-PEG4000 flux) was independent of both salinity (P=0.081) and acute air exposure
(P=0.187; Figure 7A). As with inward PEG permeability, outward PEG permeability
(serosal to mucosal 14C-PEG-4000 flux) was independent of both salinity (P=0.220)
and 24-hour emersion (P=0.210; Figure 7B)
Effects of Chronic Air Exposure on Skin Permeability
Water flux. The emersion period was extended to more closely match the
protocol of LeBlanc et al. (2010) who examined whole body water flux in K.
marmoratus. Water influx was significantly higher in fish acclimated to hypersaline
23
water (7 days) and then exposed to air for 7 days (P=0.031; Figure 8A) compared to
control fish in water. There was no significant difference in 3H2O influx between 45
‰ fish in water and fish that had been air exposed for only one day (P=0.906). There
were no significant effects of either acute or prolonged emersion on 3H2O efflux rates
(Figure 8B).
PEG permeability. Both inward and outward PEG permeability were found to
be independent of air exposure of seven days (Figure 8C, D). There was no
significant difference in 14C-PEG-4000 influx (P=0.115) or 14C-PEG-4000 efflux
(P=0.620) between fish in water or fish air exposed for 7 days.
The Effect of Salinity and Acute Emersion on Bulk Osmotic Flow
Osmotic water flux (mucosal to serosal) did not differ between freshwateracclimated fish in water and those air-exposed for one day (P=0.335; Figure 9). In
freshwater fish, the osmotic influx was between 11 and 16 µL over a one-hour period.
In hypersaline-acclimated fish, osmotic water flux (serosal to mucosal) significantly
decreased in fish exposed to air for 1 day (P=0.042; Figure 10). In the control group
in water, ~21 µL of water was lost across the skin, but this flux decreased to ~9 µL
after one day out of water.
IHC expression
Qualitatively, both hypersaline and freshwater-acclimated fish showed general
staining of Cldn-10e and NKA in the epidermis and underlying muscle tissue. Cldn10e staining appeared more prominent in the muscle tissue than in the skin, appearing
24
more diffuse in the skin. NKA-positive cells were irregular in shape and can be seen
in the skin near mucous cells. Although not quantifiable, NKA-positive cell
abundance and cell size appeared greater in hypersaline-acclimated fish (Figure 11).
Transepithelial Potential (TEP)
Hypersaline-acclimated fish exhibited a more negative TEP than freshwateracclimated fish.
HgCl2 inhibition of aquaporins
3
H2O influx rates across isolated K. marmoratus skins were not significantly
affected by 30-minute incubation in either 0.03 or 3 mmol/L HgCl2 (P=0.509 n=5,
0.417 n=3 respectively; data not shown). Prior to incubation in 0.03 mmol/L HgCl2,
influx was 0.42 µL/cm2/min (± 0.09), while post-incubation influx was 0.49
µL/cm2/min (± 0.13). Prior to incubation in 3 mmol/L HgCl2, influx was 0.21
µL/cm2/min (± 0.04), while post-incubation influx was 0.33 µL/cm2/min (± 0.12).
25
Figure 1.
Time series of 3H2O movement (dpms) across isolated K. marmoratus
skin, indicating linear transfer of 3H2O over 10 minutes. All data are means ± SEM
(N=4).
26
1400
1200
R² = 0.9275
dpm
1000
800
600
400
200
0
0
5
10
15
20
Time (min)
25
30
35
27
Figure 2.
Spectral scan of 1% food dye solution showing peaks of optical
density (absorbance) with a suggested optimal wavelength at 190 nm. This suggested
reading value is indicative of the minimum sensitivity of the machine, rather than an
optimal reading value. A more optimal peak in absorbance at 425 nm was chosen for
dye-dilution sample reading, resulting in minimal error.
28
29
Figure 3.
Standard curve of absorbance values from diluted 1% green food dye
solution. Samples were diluted using 12.5 µL of pure water and all samples were read at
a 425 nm wavelength. Absorbance values from bulk osmotic flow experiments were used
to obtain a volume of water moved via the osmotic gradient using similar standard
curves.
30
1.4
y = -0.0125x + 1.3103
R² = 0.9993
1.2
Absorbance
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
µL Water
50
60
70
80
31
Figure 4.
Behavioural observation of intact K. marmoratus held out of water for 1
day. The time (%) spent with various skin regions in contact with the moist filter paper
substrate in 15 cm3 polystyrene wells (A, combined data B) or 700 cm3 plastic containers
(C, combined data D). All data are means ± SEM (N=5: bracketed values). Groups that
do not share a common letter are significantly different as determined by One Way
ANOVAs, while a * symbol indicates significant differences as a result of a paired T-test
(P<0.004: 15 cm3; P<0.001: 700 cm3).
32
60
A.
a
B.
100
90
50
80
ab
70
bc
30
20
Time (%)
Time (%)
40
bc
bc
10
60
50
40
*
30
20
0
10
0
80
70
40
60
30
20
0
Dorsal
D.
80
Time (%)
Time (%)
a
50
10
Ventral
90
70
60
(5)
100
C.
a
(5)
50
40
30
b
20
*
10
0
(5)
(5)
Ventral
Dorsal
33
Figure 5.
3
H2O influx and efflux rates (µL/cm2/min) across the isolated dorsal and
ventral skins of K. marmoratus acclimated to brackish water (15 ‰). All data are means
± SEM (N=7: bracketed values). Data analyzed using a Two Way ANOVA (P<0.05)
yielding no statistical significance.
34
0.5
0.3
Dorsal
Ventral
0.2
3H O
2
Flux (µL/cm2/min)
0.4
0.1
0
(7)
(7)
Influx
(7)
(7)
Efflux
35
Figure 6.
3
H2O influx (A) and efflux (B) rates in the isolated skin of K. marmoratus
acclimated to 0.3 and 45 ‰ water for 7 days and either kept in water or air exposed for 1
day. All data are means ± SEM (N=6-11: bracketed values). An * symbol indicates
statistical significance as a result of a two-way ANOVA and Holm-Sidak post hoc test
(P<0.05).
36
3H O
2
Influx (µL/cm2/min)
0.4
A.
0.3
*
Water
0.2
Air
0.1
(10)
(11)
(10)
(10)
0
0.3 ‰
3H O
2
Efflux (µL/cm2/min)
0.7
45 ‰
B.
0.6
0.5
0.4
Water
0.3
Air
0.2
0.1
(6)
(10)
(7)
(9)
0
0.3 ‰
45 ‰
37
Figure 7.
PEG permeability (P-PEG, cm/s) of the skin to 14C-PEG-4000 influx (A)
and efflux (B) in the isolated skin of K. marmoratus acclimated to 0.3 and 45 ‰ water
for 7 days and either kept in water or air exposed for 1 day. All data are means ± SEM
(N=4-11: bracketed values). All data were analyzed using a two-way ANOVA (P<0.05).
38
P-PEGinflux (cm/s x 10-6)
12
A.
10
8
Water
6
Air
4
2
(9)
(11)
(9)
(10)
0
0.3 ‰
P-PEGefflux (cm/s x 10-6)
30
45 ‰
B.
25
20
Water
15
Air
10
(5)
5
(7)
(6)
(4)
0
0.3 ‰
45 ‰
39
Figure 8.
3
H2O flux (A: influx, B: efflux) and PEG permeability (P-PEG, cm/s) of
the skin to 14C-PEG-4000 flux (C: influx, D: efflux) in the isolated skin of K. marmoratus
acclimated to 45 ‰ water for 7 days and either kept in water or air exposed for 7 days.
All data are means ± SEM (N=5-11: bracketed values). An * symbol indicates statistical
significance as a result of a t-test (P<0.05).
40
0.4
0.4
A.
0.35
0.25
0.2
0.15
Efflux (µL/cm2/min)
*
0.3
0.3
0.25
0.2
0.15
3H O
2
3H O
2
Influx (µL/cm2/min)
0.35
0.1
0.1
0.05
0.05
(10)
(11)
0 days
7 days
6
C.
(5)
0 days
7 days
D.
5
P-PEGefflux (cm/s x 10-6)
5
P-PEGinflux (cm/s x 10-6)
(7)
0
0
6
B.
4
3
2
1
4
3
2
1
(9)
(11)
0
(5)
(5)
0 days
7 days
0
0 days
7 days
41
Figure 9.
Bulk osmotic flow of water across the isolated skin of K. marmoratus
(mucosal to serosal) acclimated to 0.3 ‰ water for 7 days followed by 24-hours of water
or air exposure. Data are means ± SEM (N=5: bracketed values) and were analyzed using
a t-test.
42
30
0.3 ‰
Osmotic Water Influx (µL)
25
20
15
10
5
0
(5)
(5)
Water
Air
43
Figure 10.
Bulk osmotic flow of water across the isolated skin of K. marmoratus
(serosal to mucosal) acclimated to 45 ‰ water for 7 days followed by 24-hours of water
or air exposure. All data are means ± SEM (N=4-5: bracketed values). An * indicates
statistical significance as a result of a t-test (P<0.05).
44
Water
Air
(4)
(5)
0
Osmotic Water Efflux (µL)
-5
-10
*
-15
-20
-25
-30
45 ‰
45
Figure 11.
Representative images of fluorescein-stained (FITC – green) and
tetramethylrhodamine-stained (TRITC – red) skins of K. marmoratus acclimated to their
respective salinities for 7 days and emersion time was 1 day. A: 0.3 ‰ in water, B: 0.3 ‰
air, C: 45 ‰ in water, D: 45 ‰ air. FITC staining localizes to Na+/K+-ATPase rich cells
while the TRITC staining is specific for claudin 10e. 20x magnification; scale bar =
100µm. S: Skin; M: Muscle; Arrowhead: mucous cells.
46
A
B
S
M
S
C
S
M
D
S
M
M
47
Table 1.
Transepithelial potential (TEP) data (mV) from the isolated skin of K.
marmoratus acclimated to 0.3 and 45 ‰ water for 7 days and either kept in water or air
exposed for 1 day. All data are means ± SEM (N=6-12).
48
Salinity (‰)
0.3
45
Emersion (days)
TEP (mV)
0
7
0
7
-5.26 ± 0.65
-5.79 ± 0.5
1.68 ± 0.06
3.31 ± 0.28
49
DISCUSSION
Cutaneous water movement has been identified in other amphibious organisms,
yet this study is the first to quantify water flux dynamics in isolated skin preparations of
fish out of water. Unlike previous studies in amphibians and lungfish, I found no regional
differences in 3H2O flux. I have shown that acclimation to a hypersaline environment
induced changes in skin permeability, decreasing water influx across the skin. I also
developed a novel technique for assessing the bulk osmotic flow through the skin in vitro.
This technique is highly sensitive and I was able to quantify µL differences in water
movement over an hour due to an osmotic gradient. It appears that exposure to air alters
the properties of the skin. Acute emersion (1 day) in air induced a decrease in bulk
osmotic water loss in hypersaline-acclimated fish, whereas more prolonged emersion (7
days in air) increased 3H2O influx. These data suggest that to maintain water homeostasis
on a hypersaline substrate on land, K. marmoratus increases water influx and decreases
water efflux across the skin. Such changes would help maintain water balance during the
dry season when water sources evaporate completely or become increasingly hypersaline.
K. marmoratus are known to enter rotting mangrove logs in the dry season and may
spend weeks inside aerial chambers hollowed out by insects (Taylor et al. 2008). Along
with metabolic water, cutaneous water transport may be a critical source of water, as they
are unable to drink to replace fluid loss.
Regional Water Permeability and Emersion Behaviour
In this study, I expected that K. marmoratus would exhibit regional differences in
skin permeability, having a water permeable ventral surface to allow for water uptake
50
during emersion. I showed that paired 3H2O fluxes across isolated dorsal and ventral
skins from the same individual did not differ. The lack of regional differences does not
support previous studies in amphibious fishes and amphibians (Wilkie et al. 2007,
McClanahan and Baldwin 1968; Bentley and Main, 1972; Bentley and Yorio 1976; Yorio
and Bentley 1977). The ventral pelvic patch in anuran amphibians facilitates water uptake
across the skin from terrestrial substrates during periods away from water and is 10-20
times more permeable to water than the dorsal skin (Yorio and Bentley 1977; Bentley and
Main 1972). In the only other study of cutaneous water balance in a fish out of water,
Wilkie et al. (2007) also found an appreciable flux of 3H2O across the ventral surface in
African lungfish P. dolloi. Here, it was assumed that 3H2O was moved between internal
and external environments solely through ventral surface contact. Equivalent fluxes
across both dorsal and ventral body regions in K. marmoratus suggest that an increased
ventral permeability is not a shared trait among all amphibious fishes. The lungfishes are
considered basal relatives to modern day tetrapods. It is possible that this enhanced
ventral permeability for water exchange on land is a trait that appeared after the
divergence of the teleost fishes.
My experiments of regional variability were carried out on fish acclimated to
control conditions i.e., a brackish (15 ‰) aquatic environment. It is possible that
increased water permeability across the ventral surface is a response to emersion. Further
experimentation would be needed to assess whether ventral permeability increases only
after fish have been chronically air exposed. In the current study, the size of the available
fish limited experimentation on both dorsal and ventral skins from the same individual.
Concerted effort is required to grow larger fish in larger containers in future work.
51
In preliminary observations, I found that K. marmoratus often exposed the ventral
surface to a moist substrate during emersion. In follow up experiments, behavioural
analysis of body position out of water further supported this observation. K. marmoratus
were most often found “sitting” on the ventral and ventrolateral skin. This finding was
true for both smaller confined chambers (15 cm3) and larger more open chambers (700
cm3). The lack of regional variability in skin permeability implies that greater
ventral/ventrolateral skin-substrate contact is a result of one or a combination of other
physiological and/or behavioural factors.
The finding that K. marmoratus spent more time on their ventral surface can be
explained in different ways. Selection of an upright body orientation may be due to a
righting reflex, which animals use to maintain proper body orientation. More simply,
sitting on the ventral surface may prevent obstruction of the dorsally set eyes in this
species. Ventral/ventrolateral-substrate contact may also be support for thigmotaxis in K.
marmoratus as a predator avoidance technique. Thigmotaxis, or wall-following behavior,
has been noted in multiple fish species (Champagne et al. 2010; Sharma et al. 2009).
When fish are introduced to a novel or stressful environment, they show a preference for
the periphery of the arena. This diminishes their “domain of danger” (Hamilton 1970) or
surrounding area from which a predator could attack. This explanation for greater
ventral/ventrolateral substrate contact is supported by observational data of K.
marmoratus readily seeking shelter upon emersion (Taylor et al. 1990). Minimizing
surface area for evaporative water loss may also be an explanation for the greater
ventral/ventrolateral exposure to the substrate. K. marmoratus communally inhabit moist
rotting logs that have been galleried by insects (Taylor 2008), providing maximal contact,
52
probably along dorsal and ventral surfaces, with a moist substrate. This may be a strategy
to limit exposed surface area and minimize evaporative water loss during emersion.
Unidirectional Water Flux
Methodological issues. Two problems that researchers encounter when measuring
permeability via unidirectional fluxes in in vitro systems have been poor mixing
(unstirred boundary layers) and solute/solvent back-flux (Isaia 1984, Loretz 1979).
Unstirred boundary layers here are regions of laminar flow adjacent to the skin where
molecules move only by diffusion (Dainty and House 1966a,b). If present, unstirred
boundary layers would gradually increase the diffusional distance of 3H2O across the
skin. The formation of unstirred layers significantly decreased diffusional water flow in
isolated frog skins (Dainty and House 1966a,b). Solute/solvent back-flux occurs when
interacting solutions are in equilibrium, and the movement of molecules is bidirectional.
Both may arise in a closed system, and could artificially decrease measured flux across a
membrane. In the current study, both of these potential problems were minimized.
Constant aeration of the Ussing chamber system provided adequate mixing and prevented
the formation of significant boundary layers. Back-fluxes were minimized by using a
short, 10-minute flux period, where water flux rates were in the linear part of the curve
(Figure 1).
Rates of influx and efflux were performed on different individuals to minimize
the potential contamination from leftover radioactivity within the Ussing chamber
system. When skin tissue was dissolved and counted after the initial 10-minute flux, it
had significantly higher radioactivity (dpms) than control skins that had not been used in
53
flux experiments (data not shown). It is possible that residual radioactivity within the skin
could artificially increase rates of flux in subsequent experiments. This was avoided by
using separate individuals for influx and efflux measurements.
Effect of Salinity. Regardless of salinity acclimation, overall rates of water flux
across the isolated skin of K. marmoratus were comparable to amphibians. A study of
four amphibian species reported that rates of inward water transport across ventral skins
were between 0.3 and 1.3 µL/cm2/min, whereas across dorsal skins the values were
between 0.0016 and 0.1 µL/cm2/min. (Yorio and Bentley 1977). My values were in the
same range as the ventral skin of amphibians, but at the lower end (0.15-0.50
µL/cm2/min). Slightly lower permeability of K. marmoratus skin is possibly due to the
presence of scales in the dermis, retained in the isolated skin preparations in this study.
To my knowledge, no other studies have examined rates of water flux across isolated fish
skin, thus little comparison can be made.
In the current study, salinity significantly affected rates of 3H2O influx. When K.
marmoratus were acclimated to hypersaline conditions, water influx across the skin was
significantly reduced relative to fish acclimated to hyposaline conditions. Intact
Mozambique tilapia, Oreochromis mossambica, acclimated to double strength seawater
had 3H2O turnover rates one third that of freshwater-acclimated fish, suggesting a greater
permeability for water uptake in freshwater-acclimated fish (Potts et al. 1967). When a
number of freshwater, marine, and euryhaline aquatic fish species were compared, influx
of 3H2O was higher in all freshwater compared to saltwater species (Evans 1969b).
Motais et al. (1969) also found that both A. anguilla and the European flounder
Platichthys flesus acclimated to freshwater had greater osmotic and diffusional inflow of
54
water than saltwater-acclimated individuals (Motais et al. 1969). Thus, acclimation to
hyposaline conditions appears to induce an increase in water permeability in fishes, while
hypersaline acclimation results in a decrease in water permeability. My work identifies
the specific contribution of the skin in reducing osmotic water permeability in
hypersaline conditions, a strategy that may be important in preventing osmotic water loss
across the skin.
3
H2O efflux rates in isolated skin were not affected by salinity acclimation (1 or 7
days) in my study. In intact K. marmoratus, 3H2O efflux was significantly lower (two- to
threefold) in fish acclimated to 45 ‰ relative to 0.3 ‰ for 1 month (LeBlanc et al. 2010).
It is highly possible that in these experiments, whole body water efflux included water
elimination by the kidneys, as freshwater fish are known to excrete relatively large
volumes of urine. In other studies in fish where whole body 3H2O flux was determined,
3
H2O fluxes were identical across environmental salinity (Xiphister atropurpureus Evans
1967; Pholis gunnellus Evans 1969a). As well, in isolated anuran amphibian skin,
external salinity had no effect on water efflux across the skin (Koefoed-Johnsen and
Ussing 1953). As with other fish and amphibian species, outward water flux across the
isolated K. marmoratus skin remains relatively constant regardless of salinity.
Adjustments to osmotic permeability have been attributed to both the transcellular
and paracellular routes in other animals. A 97% reduction in aquaporin 3 transcript
abundance after saltwater acclimation in the European eel A. anguilla (Cutler and Cramb
2002) has been suggested as a cause of the 11-fold reduction in osmotic permeability
after saltwater acclimation in this species (Motais and Isaia, 1972). However, I found no
effect of HgCl2 on 3H2O water flux, suggesting that water may move across the skin
55
through alternative transcellular pathways or via the paracellular route. Salinity
acclimation has also been found to influence the expression of claudins 3, 4, 6, 10d, 10e,
27a, and 30 variably. Changes in expression of claudins have been hypothesized to be
important in osmoregulation in fishes (Tipsmark et al. 2008a; Tipsmark et al. 2008b, Bui
and Kelly 2014). The IHC images of claudin in K. marmoratus skin did not reveal any
obvious differences in expression due to salinity or emersion acclimations. More work is
needed to quantify any possible molecular changes in K. marmoratus skin using Western
blot methodology.
Effect of emersion. My hypothesis predicted that hypersaline acclimation with
emersion would result in an increase in water influx, while hyposaline-acclimated fish
would have 3H2O influx rates similar to immersed fish. The data provide support for both
of these predictions. 3H2O influx rates of both hypo- and hypersaline-acclimated fish
were not influenced by 1 day of emersion, while prolonged emersion (7 day) resulted in a
significant increase in cutaneous 3H2O influx in fish exposed to a hypersaline substrate.
This finding suggests that K. marmoratus has some adaptive mechanism to allow for
water uptake in an environment with compounded stresses of osmotic and evaporative
water loss. These results suggest that water is moving actively, occurring “uphill”
against the osmotic gradient. Active water transport through amphibian skin was first
suggested by Reid (1892) after observing “the passage of fluid across the living skin by
virtue of its own unaided activity”. Other studies of water movement through amphibian
skin using a similar experimental setup as the current study have found similar results
(Keofoed-Johnsen and Ussing, 1958; House 1964). Uphill water movement has been
reported in the intestines of marine teleosts and is thought to be a means to absorb pure
56
water from imbibed seawater (Curran, 1960; Skadhauge 1969, Genz et al. 2011;
Whittamore 2012). It has been proposed that ion uniporters and active ion transporters,
such as Na+/K+-ATPase are the vectors of uphill water movement (Zeuthen and Stein,
1994; Zeuthen 2010). This idea is supported by the interdependency observed between
NaCl and water movement across bullfrog skin (Huf et al. 1951; as cited in KoefoedJohnsen 1953). LeBlanc et al. (2010) observed significantly larger Na+/K+-ATPase richcells in the epidermis of K. marmoratus after hypersaline acclimation and 7 days of
emersion, in agreement with IHC images in this study (see below). Thus, K. marmoratus
may use active ion transporters in the skin to move water against the gradient to maintain
water balance.
My hypothesis predicted that hypersaline acclimation would result in a decrease
in water efflux, while hyposaline-acclimated fish would have 3H2O efflux rates
comparable to immersed fish. Unlike 3H2O influx, short or long-term emersion failed to
induce significant differences in water efflux in either hypo- or hypersaline-acclimated
fish. In whole body water efflux experiments in K. marmoratus, 9 days out of water on a
moist hypersaline surface resulted in a marked decrease in 3H2O efflux (LeBlanc et al.
2010). In the freshwater African lungfish P. dolloi and P. annectens, prolonged air
exposure from 6-8 months also resulted in a lower whole body water efflux (Wilkie et al.
2007; Patel et al. 2009). Whole body flux experiments, however, do not provide specific
information regarding cutaneous exchange. Although 3H2O efflux was unaltered by
emersion in K. marmoratus experiments, bulk osmotic loss of water under similar
conditions was decreased. The reason for the discrepancy between 3H2O efflux and bulk
loss of water (efflux) is unknown (see below).
57
Bulk osmotic flow
I designed a novel technique to measure the bulk flow of water across the skin of
K. marmoratus. In freshwater acclimation experiments, the serosal saline was replaced
with a 1% green food dye serosal saline solution. In hypersaline acclimation conditions,
45 ‰ seawater was also replaced with a 1% green food dye hypersaline solution. Wellmixed samples from both sides of the Ussing chamber were taken at t = 0 and t = 60. The
dyed solutions were hyperosmotic to the un-dyed solutions, thus gaining water
osmotically and becoming dilute over the hour. Absorbance values obtained from the
samples were compared to a standard curve to determine how much water moved
osmotically across the skin in the one-hour period. These values were precise in the µL
range.
Previously, water flow across isolated tissues has been measured gravimetrically
(House and Green 1965; Loeschke, Bentzel, and Csaky, 1970; Collie and Bern 1982;
Cornell et al. 1994; Scott et al. 2008; Wood and Grosell 2012). One disadvantage of this
method is the potential mass gain by the cellular absorption of water across the entire
tissue. Here, the tissue may weigh more without any transmembrane movement of water.
In a similar manner, incomplete drying of the gut sac or trapped water in either sealed
end of the preparation may artificially skew perceived water movement. In my new
technique, these two sources of error are eliminated. Gravimetric analysis also requires
tedious attention to leaks and frequent handling of the preparation. My technique for
measuring osmotic water flow requires minimal handling, and the presence of any leaks
becomes known immediately with appearance of dye in the un-coloured chamber.
58
Bulk water flow across isolated skin preparations from freshwater- and
hypersaline-acclimated fish responded differently in fish previously exposed to air. One
day of air exposure in 45 ‰ but not 0.3 ‰ acclimated fish resulted in a significant
reduction in the bulk water flow across the skin compared to controls. Overall water flow
was reduced by ~97 µL/cm2/hr to the environment or a ~43 % decrease. Thus,
hypersaline acclimation in concert with short-term air exposure is likely an osmotically
stressful environment for these amphibious fish. Studies of amphibians during
metamorphosis found that integument permeability decreases as they adopt a more
terrestrial life. Reductions in 3H2O turnover rates (internal to external media) during
metamorphosis have been discovered in the freshwater African clawed frog Xenopus
laevis (Schultheiss, Hanke and Maetz 1972) and green frog Rana clamitans (Mackey and
Schmidt-Nielsen 1969 as cited in Schultheiss, Hanke and Maetz 1972). These findings in
amphibians, along with a decreased bulk water efflux in K. marmoratus imply that
entering a dehydrating environment results in a decrease in skin permeability, potentially
to conserve water.
It is surprising that a reduction in bulk osmotic water efflux after 1 day of
emersion was not mirrored in the 3H2O efflux data, where there was no significant effect
of acute emersion. Bulk osmotic water flux experiments were preformed immediately
after mounting isolated skins in the Ussing chamber, while 3H2O fluxes were given 1hour to stabilize prior to sampling. It is possible that this stabilization period causes airexposed skins to revert to their prior aquatic permeability, resulting in the lack of
difference in osmotic permeability experiments. Preliminary experiments, however,
showed no significant difference between 3H2O fluxes beginning at t = 0 or t = 60
59
minutes. The bulk osmotic water efflux represents net flux across the skin over 1 hour,
whereas 3H2O efflux is only in one direction over 10 minutes. Also, 3H2O flux
experiments only allow for the measurement of the tritium-labeled water molecules that
move across the skin, while the bulk osmotic water flux experiments allow for the
measurement of all transported water molecules. Further experiments are required to
appropriately compare the data from these two techniques.
One day of air exposure in hyposaline-acclimated fish did not result in a
significant difference from the control group in water. Therefore, freshwater-acclimated
fish deal with a gain of water across the skin from the external environment of ~88-119
µL/cm2/hr. To maintain water balance, the kidney likely voids this water load. The lack
of change in freshwater bulk osmotic flow agrees with 3H2O and 14C-PEG-4000 flux data
in hyposaline fish, as there was no observed effect of short-term emersion.
Transepithelial Potential
TEP values collected in the current study fall in line with previous measurements
in isolated K. marmoratus skins. Cooper et al. (2013) recorded TEP values in isolated K.
marmoratus skins of -4.19 mV and 1.45 mV for 1 and 15 ‰ respectively. The values
presented here follow this trend, with fish acclimated to 0.3 ‰ water having a more
negative TEP value of -5.26 mV and hypersaline-acclimated fish having a TEP of 1.68
mV. These values are reflective of the difference in membrane potential as a result of
ionic gradients across the skin. Here, a greater ionic gradient would generate a greater
TEP and vice versa. These measurements were used as a secondary test of membrane
60
viability, as a torn skin would allow for mixing of the serosal saline and saltwater to yield
a TEP of 0 mV.
Paracellular permeability
14
C-PEG-4000 flux. The polyether compound polyethylene glycol 4000 was used
to assess paracellular permeability changes. Studies examining the movement of PEG
largely use cultured tissues (Wood, Gilmour, and Pärt 1998; Van Itaille et al. 2008;
Chasiotis, Wood, and Kelly 2010). In my study, PEG permeability (P-PEG) calculated
from 14C-PEG-4000 efflux across K. marmoratus skin was between 8.14 x10-7 and 5.85
x10-5 cm/s, while PEG permeability from 14C-PEG-4000 influx was between 7.92 x10-7
and 2.40 x10-5 cm/s. PEG permeability (inward and outward) of the mummichog F.
heteroclitus’ intestine was in the lower range of values I measured in K. marmoratus skin
(1-3x10-7 cm/s; Wood and Grosell 2012). PEG permeability of the isolated skin of K.
marmoratus was not significantly affected by salinity or air exposure. These data suggest
that the paracellular route remains relatively unaltered with exposure to strong osmotic
gradients and the stress of evaporative water loss. It has been found previously that
hypersaline acclimation (35 ‰) decreased tight junction “leakiness” (leak conductance)
in the opercular epithelium of O. mossambicus (Kultz and Onken 1993). As well,
freshwater acclimation has been found to promote an increased PEG flux, or loosening of
the paracellular space in cell cultures of rainbow trout Oncorhynchus mykiss gills
(Gilmour et al. 1998; Wood et al. 1998). In some of my experiments sample size was
relatively low (n=4). Greater sample size may reveal a clearer relationship in K.
marmoratus skin. It has been proposed, however, that there is no comparable relationship
61
between PEG flux and water movement, supported by a lack of alteration to PEG
permeability across various osmotic pressure experiments (Wood and Grosell 2012). The
authors suggested that there is a separation between the paracellular water pathway and
the paracellular PEG pathway.
Immunohistochemistry. Although only qualitatively examined, the skin of
hypersaline-acclimated fish showed notably more Na+/K+-ATPase-positive cells in the
epidermis than in freshwater-acclimated fish. This finding is consistent with the work of
LeBlanc et al. (2010) on K. marmoratus skin, and other studies that show Na+/K+ATPase cells increasing in size after hypersaline acclimation (Uchida et al. 2000;
McCormick et al. 2003; Scott et al. 2008). As discussed above, skin Na+/K+-ATPasepositive cells may also provide a route for active water movement inward against the
osmotic gradient through water cotransport. Claudin 10e staining in the skin was quite
diffuse, only allowing for the conclusion that Cldn10e is present in the skin of K.
marmoratus. Tight junction proteins, including Cldn10e, may be important in mediating
the decreased water influx and decreased bulk flow of water observed in hypersaline
acclimated fish. Recently, hypersaline acclimation has been found to increase Cldn10e
mRNA expression in the skin of T. nigroviridis (Bui and Kelly 2014). As well,
unpublished data has identified claudins 6, 10d, and 10e in the gills and skin of K.
marmoratus through Western blot analysis (Galvez, Bui, Wright, and Kelly unpublished).
In addition, a ‘honeycomb-like’ staining pattern was observed using whole mount
confocal microscopy. This pattern localizes these claudin proteins to the junctions
between neighbouring cells, and hints at their potential role in the tightening/loosening of
62
the paracellular space. Further studies are required to understand the role of claudin
proteins in skin permeability in K. marmoratus.
Summary and Conclusions
My data provide evidence that salinity and emersion alter the permeability of K.
marmoratus’ skin to water. In hypersaline-acclimated fish, bulk osmotic efflux was found
to significantly decrease after only 1 day in air, while 3H2O influx was increased after 7
days in air. These findings together suggest that K. marmoratus limit water loss to an
osmotic gradient and increases water uptake, potentially through water cotransport across
the skin. As well, hypersaline acclimation resulted in a lower 3H2O influx across the skin,
further supporting a reduction in skin permeability in an osmotically dehydrating
environment. It is unclear whether skin permeability is altered through changes in the
transcellular or paracellular route, as 14C-PEG-4000 fluxes were unaffected by both
salinity and emersion acclimations and HgCl2 had no effect on water flux. Overall my
findings suggest that K. marmoratus alter skin permeability to maximize water uptake
and minimize water loss while emersed in hypersaline conditions.
63
GENERAL DISCUSSION
Future directions for this work should address the role of water cotransport
through active ion transporters and uniports on overall water balance. It is currently
unknown what degree of total water movement can be attributed to secondary active
transport. This form of transport is responsible for ~30% of total water movement in a
mammalian intestinal lumen (Zeuthen et al. 2001), and may be an important mechanism
of osmoregulation in amphibious fish. Illustrating an interdependence between ion and
water fluxes, as well as linking changes in water flux to changes in uniport/ion transport
protein abundance in the skin may link water transport and ion regulatory proteins.
Hormonal control of water balance is another area of research that remains
unexplored in K. marmoratus. Neurohypophysial hormones such as vasotocin as well as
the steroid hormones aldosterone and cortisol have been recognized to affect membrane
permeabilities and subsequent movement of water (Goldenberg and Warburg 1982;
Alvarado and Johnson 1966; Bentley and Yorio 1976; Yorio and Bentley 1978; Pärt et al.
1999; Hasegawa 2003; Chasiotis, Wood, and Kelly 2010). Assessing hormone
concentrations and hormone receptor distribution and their influences on skin
permeability could shed light onto how water is managed as a whole in amphibious
organisms out of water. Circulating concentrations of hormones in the blood should first
be assessed following salinity and emersion acclimations. Intraperitoneal injections of
hormones and subsequent osmotic and/or diffusional water permeability measures (via
the isolated skin technique) would help us to understand the influence of hormones on
skin permeability specifically. If injection was not possible in the relatively small K.
marmoratus, hormone-enriched baths may be adequate for hormone delivery.
64
A quantification of drinking rate and urine production in K. marmoratus would be
valuable in examining whole body water balance and how these organisms acclimate to
hyper-/hyposaline acclimation. This may indicate how severe the osmotic challenge is for
hypersaline fish out of water and away from a drinking source. Drinking rate could be
determined by quantifying the ingestion of an inert marker such as yttrium oxide as used
in digestibility studies (Storebakken et al. 1999; Hung et al. 1999). Following salinity
and/or emersion acclimation, fish would be left to drink in a yttrium oxide labeled bath.
Higher drinking rate would be indicated by greater imbibed yttrium/ytterbium in the gut,
quantified using ICP spectrophotometry. Using a radiolabelled impermeant would allow
for scintillation counting of the gut contents for quantification. To quantify urine
production, catheterization of large fish may be necessary.
More intensive investigation of regional variability could also be conducted. In
the current study, dorsal and ventral variability was only examined in fish acclimated to
15 ‰ in an aquatic environment. It is still unknown if acclimation to 0.3 or 45 ‰ water
or emersion induces changes in regional permeability in K. marmoratus. To test this, the
flux of 3H2O across paired dorsal and ventral skins from K. marmoratus should be
conducted following prolonged emersion in hypo- and hypersaline environments.
Further study of regional variability or general skin permeability could focus on
epithelial lipid content. In hylid tree frogs (Agalyhnis dacnicolor, Hyla arenicolor, H.
squirrella, and H. femoralis), lipid content was significantly higher in the dorsal skin,
which was less permeable to water as well as unresponsive to permeability stimulating
hormones (Yorio and Bentley 1977). Changes in skin permeability with salinity/emersion
acclimation may be explained by changes in skin cholesterol content. Cholesterol has
65
been found to affect osmotic water flux in isolated gills due to its stiff structure within
cell membranes (Redwood and Hayden 1969). The steroid ring of the cholesterol
molecule increases the rigidness of a phospholipid bilayer by decreasing the space
between bilayer constituents. The resulting decrease in membrane fluidity limits the
spaces through which water can travel (Finkelstein 1976). Cholesterol depleting agents
have been found to increase gill water weight gain (Redwood and Hayden 1969). In the
same study, a specific cholesterol-complexing aqueous pore-forming antibiotic resulted
in a dramatic permeabilitzation of gill arches to osmotic water flux. It is possible that the
mechanism for altering water flux across K. marmoratus skin is the integration of
cholesterol, or other hydrophobic lipid molecules, into epithelial cell membranes. In an
amphibious fish species (Periophthalmodon schlosseri), low permeability of the skin to
NH3 flux was attributed to increased tissue cholesterol and phosphatidylcholine content
(Ip et al. 2004). These membrane constituents stabilize membranes, decreasing
membrane fluidity and thus permeability. Exposure to increased environmental NH3
induced an increase in tissue cholesterol and saturated fatty acid content, presumably to
decrease the permeability of the skin to NH3 influx. These results highlight the
importance of tissue lipid content on amphibious skin permeability specifically. It is
likely that skin permeability to water flux is also affected by tissue lipid content in K.
marmoratus. This proposed work would generate a holistic profile of water balance in
this euryhaline amphibious fish.
66
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