RESEARCH ARTICLE

2014 | VOLUME 30 | PAGES 33-41

Physiological response of Bufo viridis (Laurenti, 1768)

populations across an aridity gradient
Joy Hoffman1*
1.

Department of Biology, Technion, Israel Institute of Technology, Haifa 32000, Israel.

Bufo viridis (also known as Pseudepidalea viridis), a semi-terrestrial anuran found in climates ranging from
temperate to semi-desert, has both behavioural and physiological defences against the threat of dehydration; it is also able to tolerate a wide range of salinities. We examined the prediction that those populations
inhabiting drier environments will have a compensatory physiological response to dehydration. To that end
we compared laboratory-acclimated samples of toads collected from three sites in Israel, one in north-west
Italy, and one in Kyrgyzstan differing in the level of annual rainfall in their natural habitats. When kept on
damp soil without access to free water, measurements of changes in body mass, plasma osmolality, and the
concentrations of urea and major electrolytes in the plasma revealed no significant differences in the physiological responses of toads from the different sites. This contrasts with the negative correlation that had
previously been found between the frequency of a behavioural, water-seeking response and annual rainfall
at these same sites. The results are discussed in the light of what is known about the evolutionary history of
the species. It is suggested that, because drought resistance and euryhalinity are both features of general osmotic stress tolerance, these properties may have evolved initially in the species area of origin as a response
to fluctuations in the salinity of inland water bodies; later, when the species migrated into arid regions, it was
an exaptation enabling the toads to tolerate water shortage.

INTRODUCTION
The naked amphibian skin offers little or no protection against evaporation (Bentley, 1971; Noble, 1931; Shoemaker, 1988), so semi-terrestrial species, in which adults very rarely enter water except for breeding, are particularly
vulnerable to dehydration. The rate of water loss is often reduced behaviourally: many amphibians are nocturnal, seeking
cool, moist shelter during the day, but even so, except in humid habitats, water is inevitably lost by evaporation. Usually,
it is replaced during the animals occasional returns to water; how frequently they return and for how long depend on local
climatic conditions.
Despite their vulnerability to dehydration, some amphibian species are successful in hot, dry regions. Most of
these fall into one of three main groups. First, there are species that have no particular specialisations for water conservation, depending on permanent water at oases or living in places where the physical properties of the local soil type allow
the release of soil-water, even when its moisture content is relatively low (Cartledge et al., 2006a). Those species that are
now confined to oases may be relict populations which formerly had more widespread distributions (Shoemaker, 1988).
Amphibian species in the second group (mainly tree-frogs) reduce evaporation by coating themselves with waterproofing wax (Loveridge, 1976; Wygoda, 1984). Those in the third group aestivate in cocoons in deep burrows for the greater
part of the year (Ruibal et al., 1969; Shoemaker et al., 1969; Cartledge et al., 2006b). Species in all three groups are
confined to arid or semi-arid regions, and most have distributions limited to this biome. In contrast, the green toad Bufo
viridis (Laurenti, 1768), (also known as Pseudepidalea viridis) which is also found in arid regions, has an extremely wide
geographic distribution. It is successful in climates ranging from cool and temperate in north-west Europe (Jrgensen,
1984) to hot and arid in the Arabian peninsular (Mendelssohn & Steinitz, 1945). Because it is also euryhaline, it occupies

Received 28 August 2012

*Corresponding author

Accepted 23 February 2013

Published Online 31 January 2014

ISSCA and authors 2014

jhoffman@tx.technion.ac.il

JOY HOFFMAN

a unique position among known anuran species; no other exhibits such versatility (Hoffman & Katz, 1997).
In the semi-arid zone of Israel, approaching the southern limit of the distribution of B. viridis, the toads
remain nocturnally active during a six to seven months dry season (Degani et al., 1981). Pre-dawn dew occurs
frequently during mid summer (Zangvil & Druian, 1980), and B. viridis, like some other bufonids, has a waterseeking response in which it moves, preferentially, to moist surfaces (Stille, 1958; Hillyard et al., 1998). The
combination of the toads abilities to sense water vapour gradients (Reshetnikov, 1998) and to absorb water from
moist surfaces enables them to detect and utilize this dew to replace water lost during the previous day. However,
in the total absence of free water, when behavioural compensations can only allow for temporally avoidance of
desiccation, their survival depends on additional strategies. Under these conditions, the toads are able to adapt
physiologically by raising the osmolality of their body fluids, mainly by accumulating urea in their blood (Degani
et al., 1984; Katz & Gabbay, 1986). This creates a favourable osmotic gradient across the skin that, together with
an increased rate of capillary blood flow (Viborg & Hillyard, 2005), permits the absorption of soil-bound water.
In laboratory studies, we have shown that this physiological response is biphasic. Within two days after transfer to
damp soil, urine voidance ceases, and, during the following three to four days, the rate of urea synthesis increases
dramatically. The precise timing of this first phase depends on the dampness of the soil. It is followed by a plateau
period lasting one to two weeks during which the rate of urea synthesis remains unchanged, and then a second
phase in which the rate of urea synthesis increases again (Hoffman et al., 1990).
In an earlier study, I showed that the behavioural response of B. viridis to the presence of a patch of moisture differs between populations: in laboratory conditions, when toads were allowed a free choice between sitting
on wet or on dry filter paper, the frequency of the water-seeking response in animals collected from sites in Israel
(Golan, West Bank and Haifa Bay), north-west Italy (Vigone) and Kyrgyzstan (Kokjar) was inversely related to the
level of annual rainfall in their natural habitats (Hoffman, 2006). It was therefore of interest to see if the magnitude
of the physiological response to the threat of desiccation, in which the osmolality of the body fluids is raised, is
also related to local rainfall. In this study, I have focussed on the first phase of the physiological response to the
absence of free water by following the changes in body mass, plasma osmolality, and the concentrations of urea
and major electrolytes in the plasma.

MATERIALS AND METHODS

In Israel, a large increase in the human population and consequent urbanization had reduced the size and
quality of native amphibians natural habitats. Conservationists recommended a reduction in the capture of fresh
specimens for experimental use and, when practical, a greater re-use of limited numbers of those already in captivity (Ortal et al., 2004; Hoffman et al., in press).
The 27 toads (tab. 1) used in this study had been subjects of earlier experiments in which differences between the frequency of the water-seeking responses of animals collected from geographical regions with different
climates were investigated (Hoffman, 2006). They had therefore been in captivity for 3-5 months. On completion of the behavioural observations, they were maintained in laboratory conditions, with natural daylight hours
and room temperature controlled at 222 C, for at least a further two weeks before the beginning of the present
experiments. They had free access to a source of tap water and a dry area, and were fed mealworms, ad lib, once
a week. It had been found previously that the behavioural response of toads maintained in laboratory conditions
remains stable over time (Hoffman, 2006), and it was assumed that this is also true of the physiological response
being studied.

Table 1. Changes in body mass of laboratory acclimated toads from sites with different annual rainfall when kept without access to free water.
n is the number of animals.

Gross mass as a percentage of initial mass (mean sd)

Annual
rainfall
(mm)

Initial
mass (g)

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Kokjar

375

51.7 6.38

95.7 2.42

90.4 3.22

87.5 3.52

84.1 3.19

80.9 3.21

81.0 3.06

Haifa Bay

399

34.9 4.84

96.6 0.80

91.8 1.71

86.7 2.61

85.5 1.15

82.1 1.15

81.3 2.46

West Bank

450

33.5 3.20

95.1 2.47

94.8 2.55

87.5 2.68

86.1 2.08

85.1 0.78

83.1 2.05

Vigone

680

19.8 1.72

94.5 2.05

89.2 3.85

84.1 2.69

82.0 1.38

79.3 2.14

76.9 1.67

Golan

800

37.8 4.84

97.5 0.99

96.3 0.86

91.6 0.88

88.7 0.80

85.8 0.76

83.5 1.29

Site

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ALYTES 2014 | 30

The annual rainfall at the sites at which B. viridis were collected is given in table1. The data for sites in
Israel (Haifa Bay, West Bank and Golan) were obtained from meteorological stations each within 25 km of the
specific collection sites (unpublished Meteorological Services data, pers. comm.); for north-west Italy (Vigone)
and Kyrgyzstan (Kokjar) the rainfall data are those given in Castellano and Giacoma (1998) and Castellano et al.
(1999), respectively.
The effects of the absence of free water was followed by measuring the changes in gross mass, plasma
osmolality, and concentrations of plasma urea and major electrolytes of toads kept in mesh-covered plastic boxes
(40x15x35 cm3) filled with garden soil to a depth of 25 cm. Each group of five to six animals from the same locality was placed in an individual box. The soil used had been sifted, washed, oven-dried and repulverized. Its water
content (ca 11%) was determined by drying to constant mass at 95 C, and its water potential (5-5.5 atms) was
estimated from a soil moisture curve produced by ceramic plate extraction (Kramer et al., 1966; see Hoffman &
Katz, 1989, fig. 1). When not in use, the soil was stored in sealed polythene bags.
Before transfer to the experimental condition, the standard (i.e., bladder empty) mass (0.1 g) of each
toad was determined after catheterization through the cloaca, as described in Hoffman and Katz (1999). Samples
of blood (~100 l) were taken from the vena angularis into heparinized capillaries, and the cells were removed by
centrifugation. Following this procedure, to minimise the effect of any stress, the animals were allowed to recover
for two days, during which they received no further food.
The gross mass of each animal was determined just prior to transfer to the soil-filled boxes where they
remained for six days. Room temperature was controlled at 22 C 2 C. The toads gross mass was determined
daily between 07:00 and 08:00 am, but otherwise they were left undisturbed. When on soil, the toads do not void
urine, and HOFFMAN (1989) showed that when B. viridis is kept for two to three weeks without access to free
water on soil having an 11-12% water content, more than 95% of the total loss of mass is due to loss of water.
Therefore, during the six days in the experimental conditions, the difference between mass loss and water loss is
considered to be negligible, and the loss in mass can be attributed to water loss. At the end of the six-day period,
blood was sampled again.
Plasma osmolality, chloride concentration and urea concentration (always in that order) were determined
for the fresh samples taken two days before and at the end of the period in the experimental conditions. When
sufficient plasma was available, the remaining plasma was frozen for the later measurement of sodium and potassium. Osmolality was measured using a Wescor 5500 Vapor Pressure osmometer (Logan, UT); the concentration
of Cl was determined on a Radiometer CMT10 chloride titrator, and that of urea was determined colourimetrically
(Sigma bulletin No 640). Na+ and K+ were determined on a Corning 480 flame photometer (Halstead, UK).
One-way ANOVA and Tukeys HSD test were used to compare the rates of mass loss of different groups.
Measurements made on blood samples taken at the beginning and end of the experimental period were compared
using Students paired t-tests. The degree of relationship between annual rainfall and the magnitude of the physiological response was assessed using Spearmans two-tailed rank correlation coefficients.

RESULTS
After six days in the experimental conditions, all groups lost 16.5-23.1% of their initial mass (tab. 1).
There was a significant difference between groups (F = 7.8302, df = 4.22, P = 0.0004), but Tukeys HSD test
indicated that the only group that differed was that from Vigone. When this group was excluded, there was no significant difference between the other four (q = 2.5917). The greater loss of mass in toads from Vigone is consistent
with their small size (Warburg, 1972). This explanation was supported when a heavier group of seven toads (mean

Table 2. Initial plasma osmolality and urea concentration and values at the end of the experimental period on damp soil without access to free
water. d is the actual differences between the initial and final values, and % is the increase from the initial value.

Site

Osmolality (mean s.d. mosm kg-1)

Initial

Final

Urea concentration (mean sd mmol l-1)

%

Initial

Final

Kokjar

363 18.8

554 34.2

191 27.6

53 6.0

68 17.7

188 22.1

120 20.0

176 67.3

Haifa Bay

346 21.5

516 39.0

170 31.5

49 6.8

63 25.1

191 26.5

128 25.8

203 109.0

West Bank

354 21.5

510 36.0

156 29.7

44 5.8

64 16.4

174 20.2

110 18.4

171 63.7

Vigone

326 16.2

538 36.2

212 28.0

65 7.6

33 7.0

125 14.2

92 11.2

280 91.2

Golan

334 18.2

496 33.4

162 26.9

48 5.8

45 8.1

141 18.0

96 14.0

212 65.2
35

JOY HOFFMAN

mass 22.1 5.00 g), collected from the same site in Vigone and subjected to the same experimental conditions,
lost only 19.6% of their mass in six days (unpublished). Providing that general morphology is similar, surface area
is related to gross mass and, therefore, so is the rate of evaporative water loss (EWL). Skull size & limb length
can distort the relationship &, although the samples in all five groups appeared to be roughly the same shape, bone
mass cannot be determined in live animals As loss of mass is almost entirely due to water loss, the data in tab.1
provide a good measure of the rate of water loss during the experimental period.
In all groups, plasma osmolality increased significantly (P < 0.001) from an initial value of ca 350 mosm
kg-1 to ca 500 mosm kg-1 after six days; there were also significant (P < 0.001) increases in all plasma urea concentrations (tab. 2). These were sufficient to account for the greater part of the rise in osmolality. No evidence was
found that annual rainfall in the natural environment had a significant influence on either the increase in osmolality (Rs[5] = 0.20, P > 0.05) or on that of the urea concentration (Rs{5] = 0.60, P > 0.05), both increases expressed
as percent initial value (tab. 2). Among the measured electrolytes, the concentration of potassium did not change
significantly (P > 0.05), but there were significant increases in both sodium and chloride (P < 0.001) (tab. 3). Together, these could account for the remaining rise in plasma osmolality.
To determine how much of the increase in plasma urea and electrolytes can be explained by the concentrating effect of water loss, the expected percentage increases were calculated making the following assumptions:
1.

The loss of water is the same as the loss in mass.

2.

Total body water is 72.7% of standard (bladder empty) mass (Hoffman, 1989).

3.
In laboratory conditions, urine accounts for ca 5% of the gross mass, and on damp soil renal
function continues and urine volume does not change significantly (Hoffman, 1989).
With these assumptions, the water content as a percentage of gross mass is estimated to be 72.7 x 0.95 =
69.1, and the percentage water loss in the experimental conditions is (100/69.1)w, where w is the percentage mass
loss. From this it follows that the expected percentage increase in the concentration of substances in the plasma
due to water loss can be expressed by (100 x 1.45w)/(100 - 1.45w). As shown in table 4, the measured concentration of urea greatly exceeds the value calculated from this expression, while the observed and expected values for
chloride are similar.

Table 3. Changes in the concentration of major plasma electrolytes during the experimental period on damp soil without access to free water.
d is the difference between the initial and final values; n.m. means that the values were not measured; asterisk indicates plasma from only two
animals was measured.

Site

Na+ (mean sd mmol l-1)

K+ (mean sd mmol l-1)

Initial

Final

Initial

Final

Kokjar

125 21.2

168 27.8

43 24.7

4.4 1.24

4.8

Haifa Bay

129 11.8

154 28.3

25 21.7

5.3 1.24

5.6 1.54

West Bank

Cl- (mean sd mmol l-1)

Initial

Final

0.4 1.1

105 13.4

145 18.0

40 13.6

0.3 1.4

107 13.9

140 13.9

33 13.9

n.m.

n.m.

n.m.

n.m.

96 10.2

124 6.0

28 8.4

Vigone

117 7.4

172 12.4

55 10.2

6.4 0.42

6.9 0.42

0.5 0.4

107 7.9

159 24.8

52 18.4

Golan

118 7.1

140 9.8

22 8.6

6.5 1.44

5.4 1.53

-1.1 1.5

94 15.1

124 6.9

30 10.7

DISCUSSION
The results show no statistically significant differences in the measured physiological response to the
absence of free water in toads collected from five climatically different sites. All lost mass was through water loss
(tab. 1) while, at the same time, their plasma osmolality increased, largely due to the accumulation of urea (tab. 2).
The plasma concentration of potassium, which makes only a negligible contribution to extracellular osmolality but
whose stability is vital for many vertebrate physiological functions, remained stable, whereas the concentrations of
sodium and chloride increased (tab. 3). Hoffman and Katz (1997) showed that sodium concentrations appear to be
under limited physiological control, but chloride simply follows the concentrating and diluting effects of changes
in body fluid volume. For this reason, chloride can be taken as the indicator of the concentrating effects of water
loss. Relative to this indicator, the increase in the concentration of urea far exceeded the value expected as a result
of water loss alone (tab. 4). It is known that when Bufo viridis is free to burrow, the toads lose only a third of the
36

ALYTES 2014 | 30

water that they lose when on a solid surface (Hoffman & Katz, 1989). Nevertheless, the physical barrier of soil
particles is not as efficient as a cocoon, which can reduce the rate of evaporative water loss to one similar to that
found in waterproof frogs (Withers et al., 1982). However, B. viridis has other adaptations that reduce water loss
in potentially dehydrating conditions: they are able to raise the osmolality of their body fluids through the synthesis
and accumulation of urea (Balinsky, 1981; Katz, 1989). All other known anuran species with the ability to do this
inhabit areas in which there are long seasons without rainfall, but B. viridis has a wide distribution, encountering
aridity only in its southernmost reaches. Most populations of B. viridis are found in mild climates and coexist with
other anuran species, and although few of these species have been investigated, none of those that have been studied responds to dehydrating conditions with a large increase in plasma urea (Katz & Hoffman, 1990).

Table 4. Observed and expected percentage increases in concentrations of chloride and urea due to water loss. The expected value was calculated on the assumption that the increase was due solely to water loss (for formula, see Results section).

Site

Expected

Measured
Chloride

Urea

Kokjar

42.2

38.1

176

Haifa Bay

35.5

30.8

203

West Bank

32.5

29.2

172

Vigone

53.1

48.6

279

Golan

31.4

31.9

213

In an earlier study of the groups of toads used in the present investigation, the frequency of a waterseeking response to the absence of free water was shown to be related to the annual rainfall in their natural habitat
(Hoffman, 2006). While local levels of humidity commonly fluctuate and are the outcome of the interaction of a
number of factors, annual rainfall is probably the best indicator of the availability of fresh water over periods of
several toad-generations, and reliable rainfall data are published for the regions of the collection sites. The absence
of evidence in the present study for site-related differences in the toads ability to raise their plasma urea is inconsistent with the evidence presented by Young et al. (2005), which suggests a link between ecological habits and
water loss rates. The simplest explanation of an adaptive response that is common to all populations of a species,
despite differences in their habitats, is that it evolved early in the lineages history, before the species radiated from
its area of origin. If so, the selection pressures that led to the appearance in B. viridis of the ability to synthesise
and accumulate urea may be found in early events in this species evolution.
According to Savage (1973), the immediate predecessors of the Bufo viridis complex arrived in Northern Asia from North America during the Oligocene, and fossil evidence suggests that B. viridis migrated from Asia
to Western Europe during the Miocene (Bhme, 2003; Borkin, 1986; Rage & Roek, 2003). During this period,
Eurasia remained warm temperate, and the major threat to amphibian life probably came from the results of earlier
tectonic movements that had created the Paratethys Basin, today represented by the Black, Caspian and Aral seas.
A land route was established between Asia and Europe and an open ocean changed into an enclosed series of lakes
with reduced salinity. The boundaries of the lakes were unstable, with regional openings and close-offs continuing
into the middle Miocene (Rgl, 1998). There were also short-term changes in sea levels associated with the size
of the polar ice sheets that affected the concentrations of sodium chloride and other minerals in the water bodies:
fossil evidence of fish faunas of the Paratethys shows alternating strata of marine, brackish, and freshwater species
throughout the late Oligocene and early Miocene (Reichenbacher, 1996), and lakes within the same region had
different salinities (Reichenbacher et al., 2004). I suggest, therefore, that during and soon after the emergence of
B. viridis, the frequently changing (on a geological time scale) salinity of water sources made euryhalinity (salinity
tolerance) an advantage to the adult, terrestrial, mobile phase of these former freshwater-dependent toads. Through
strong selection for this characteristic, it became a feature of the species.
Few present-day populations of B. viridis, with the notable exception of those inhabiting the region of the
brackish Baltic lakes in Sweden (LIFE-BaltCoast, 2009), rely totally on saline water sources. Nonetheless, some
of those that depend on freshwater in their natural habitats can be acclimated to high salinity in experimental conditions (Katz, 1973). Of the many amphibian species that have been studied, only B. viridis and two others, Rana
cancrivora and Xenopus laevis, have been found to be truly euryhaline in that they can survive for considerable
periods in salt solutions of a concentration equivalent to that of half-seawater (Balinsky, 1981). In comparison,
the inappropriately named B. marinus can survive for only about six days, even when experimentally subjected to
37

JOY HOFFMAN

gradually increasing salinity (Liggins & Grigg, 1985). All three naturally euryhaline species share the ability to
accumulate and tolerate high plasma urea, thus limiting water loss; consequently, they are capable of withstanding
long-term osmotic stress.
Salinity resistance and drought resistance are both related to general osmotic stress tolerance, and I suggest euryhalinity was an evolutionary exaptation for drought resistance. There are differences between the responses to the two types of stress, however. In experimentally induced adaptation to salinity, plasma osmolality
increases only sufficiently to prevent the loss of water to the salt solution in which the animal bathes (Katz, 1973).
Most of the increase is due to the accumulation of urea, but sodium and chloride also contribute (Katz et al., 1984).
In contrast, in the absence of free water (simulated drought), plasma osmolality is raised considerably above that
needed to establish a gradient favourable to water absorption, and this apparent anomaly is not unique to B. viridis (Tracy, 1976). In the present experiments, the soil water potential of ca 5.5 atms ( 260 mosm kg-1) evoked
a plasma osmolality of ca. 500 mosm kg-1 within a week. The increase was almost entirely due to urea; there was
no source of additional electrolytes. The high osmolality when on soil has been attributed to the low skin/water
contact compared to the maximal contact when the animal is immersed in liquid (Tracy & Rubink, 1978).
The greatest difference between the responses to aridity and to salinity is that during drought the ability to
synthesise and accumulate urea is enhanced. However, this is only a matter of the degree of urea synthesis: no new
mechanisms are involved. Urea production can be energetically expensive, but the process is not continuous, and
is accelerated only when demanded by conditions of osmotic stress. Hoffman and Katz (1998) showed that most
of the urea comes from the catabolism of muscle protein, and because of the low metabolic rate of amphibians, the
by-product, glucose, may be in excess of immediate requirements. When this occurs, it is stored for future use as
glycogen in the liver, thus reducing the overall energy cost.
The present day distribution of B. viridis includes zones of forests, steppes and semi-deserts, together with
anthropogenic habitats. It is the common toad throughout much of Eurasia, and it is also found along the North
African Mediterranean coastal strip and on the large Mediterranean islands, where it may have been introduced
during the Bronze Age as a religious totem (Lloyd, 2008). Not only are these regions themselves extremely different in climatic conditions, but to reach them toads probably had to migrate through areas in which resistance
to either or both salinity and drought would have conferred an advantage. It seems likely that once established,
salinity tolerance preadapted the toads to the arid conditions they encountered on their migrations.
In arid environments, there would have been further selection for modified physiological and behavioural
mechanisms that enable toads to avoid or minimise the threat of dehydration. Water-seeking behaviour was probably one of these. In an earlier study (Hoffman, 2006), it was found that how frequently the toads show waterseeking behaviour is inversely correlated with the annual rainfall in their natural habitats. Whether this is because
populations differ genetically in their sensitivity to water loss, or because selection has led to a plastic response
in which conditions experienced the pre-adult stages influence adult behaviour, is yet to be determined. GomezMestre and Tejedo (2005) found with B. calamita that premetamorphic and adult responses to osmotic stress are
not coupled, suggesting that they evolved independently, but even if this is a general pattern among anurans, the
first vital search for a suitable habitat in the early postmetamorphic stages might well have a long-lasting effect.
Whatever the reason for the behavioural difference among B. viridis populations is, it shows that the water-seeking
response has a degree of flexibility that is not seen in the physiological response to water loss. Together, the two
types of adaptations behavioural, with its property of comparatively rapid reversibility, and physiological, which
enables survival during extended periods of reduced water availability must have been important factors in the
success of B. viridis as a colonizer of harsh environments. They may account for its extremely wide distribution.

ACKNOWLEDGEMENTS
I thank C. Giacoma for a gift of toads collected in Italy, the Meteorological Services (Bet Degan) for
data on rainfall at the collection sites within Israel, the Israel Nature Reserves and National Parks Authority for
permission to import live toads, Zeev Arad for his constructive comments on the manuscript, Marion J. Lamb for
constant encouragement and a careful review of the manuscript, and Yaacov Goldstein of Carmel Hospital for the
gift of extra time.

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ALYTES 2014 | 30

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