Lactose causes heart arrhythmia in the water flea Daphnia pulex

Abstract

The cladoceran Daphnia pulex is well established as a model for ecotoxicology. Here, we show that D. pulex is also useful for investigating the effects of toxins on the heart in situ and the toxic effects in lactose intolerance. The mean heart rate at 10 degrees C was 195.9+/-27.0 beats/min (n=276, range 89.2-249.2, >80% 170-230 beats/min). D. pulex heart responded to caffeine, isoproteronol, adrenaline, propranolol and carbachol in the bathing medium. Lactose (50-200 mM) inhibited the heart rate by 30-100% (K(1/2)=60 mM) and generated severe arrhythmia within 60 min. These effects were fully reversible by 3-4 h. Sucrose (100-200 mM) also inhibited the heart rate, but glucose (100-200 mM) and galactose (100-200 mM) had no effect, suggesting that the inhibition by lactose or sucrose was not simply an osmotic effect. The potent antibiotic ampicillin did not prevent the lactose inhibition, and two diols known to be generated by bacteria under anaerobic conditions were also without effect. The lack of effect of l-ribose (2 mM), a potent inhibitor of beta-galactosidase, supported the hypothesis that lactose and other disaccharides may affect directly ion channels in the heart. The results show that D. pulex is a novel model system for studying effects of agonists and toxins on cell signalling and ion channels in situ.

Full text

Lactose causes heart arrhythmia in the water flea Daphnia pulex
Anthony K. Campbell
a,
*, Kenneth T. Wann
b
, Stephanie B. Matthews
c
a
Department of Medical Biochemistry and Immunology, Wales College of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK
b
Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cathays Park, Cardiff, CF10 3XF, UK
c
Department of Medical Biochemistry and Immunology, Llandough Hospital, Cardiff and Vale NHS Trust, Penarth, Vale of Glamorgan, CF64 2XX, UK
Received 17 April 2004; received in revised form 24 June 2004; accepted 20 July 2004
Abstract
The cladoceran Daphnia pulex is well established as a model for ecotoxicology. Here, we show that D. pulex is also useful for
investigating the effects of toxins on the heart in situ and the toxic effects in lactose intolerance. The mean heart rate at 10 8C was 195.9F27.0
beats/min (n=276, range 89.2–249.2, N80% 170–230 beats/min). D. pulex heart responded to caffeine, isoproteronol, adrenaline, propranolol
and carbachol in the bathing medium. Lactose (50–200 mM) inhibited the heart rate by 30–100% (K
1/2
=60 mM) and generated severe
arrhythmia within 60 min. These effects were fully reversible by 3–4 h. Sucrose (100–200 mM) also inhibited the heart rate, but glucose
(100–200 mM) and galactose (100–200 mM) had no effect, suggesting that the inhibition by lactose or sucrose was not simply an osmotic
effect. The potent antibiotic ampicillin did not prevent the lactose inhibition, and two diols known to be generated by bacteria under
anaerobic conditions were also without effect. The lack of effect of l-ribose (2 mM), a potent inhibitor of h-galactosidase, supported the
hypothesis that lactose and other disaccharides may affect directly ion channels in the heart. The results show that D. pulex is a novel model
system for studying effects of agonists and toxins on cell signalling and ion channels in situ.
D2004 Elsevier Inc. All rights reserved.
Keywords: Daphnia pulex; Lactose intolerance; h-Galactosidase; Heart; Arrhythmia
1. Introduction
The cladocerans Daphnia pulex and Daphnia magna
have been established as useful model systems in ecotox-
icology and for investigating the ecological impact of toxic
substances in freshwater (Persoone and Van de Vel, 1988;
Baird et al., 1989a,b; Diamantino et al., 2000; Guilhermino
et al., 2000; De Coen and Janssen, 2003). Daphnia can be
used to study nutrition and starvation responses (Tessier et
al., 1983; Elendt, 1990a,b; Elendt and Storch, 1990).
Daphnia have been reported to have a myogenic heart
(Bekker and Krijgsman, 1951), though this now needs
confirming using modern electrophysiological and pharma-
cological techniques. The Daphnia heart responds to a
range of agonists and antagonists that affect heart rate and
rhythm in humans (Viehoever and Cohen, 1937; Sollman
and Webb, 1941; Postmes et al., 1973; Villegas-Navarro et
al., 2003). The aim of the experiments described here was to
show whether Daphnia could be used to investigate the
effects of sugars on the heart and to establish Daphnia as a
model for investigating the role of toxins in human disease.
A long-term objective was to test our hypothesis that the gut
and systemic symptoms in people with lactose intolerance
were caused either by lactose itself of by bacterial toxins
produced in the gut. This paper provides the first step in this
objective, showing effects of lactose on Daphnia heart that
are similar to those in humans.
Lactose intolerance is caused by an inability to digest
lactose, galactose h1,4 glucose the sugar in milk, because
of an inadequate level of the gut enzyme lactase–lactase
phlorizin-hydrolase, LPH, EC 3.21.23/62 (Flatz, 1987;
Sahi, 1994; Carper, 1995; Campbell and Matthews, 2001;
Tolston, 2000; Swallow, 2003). h-Galactosidase also
1096-4959/$ - see front matter D2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpc.2004.07.004
* Corresponding author. Tel.: +44 29 20742951; fax: +44 29
20745440.
E-mail address: campbellak@cf.ac.uk (A.K. Campbell).
Comparative Biochemistry and Physiology, Part B 139 (2004) 225 – 234
www.elsevier.com/locate/cbpb

cleaves lactose into galactose and glucose and is ubiq-
uitous in pro- and eu-karyotes. Lactose intolerance causes
a wide range of gut and systemic symptoms (Srinivasan
and Minocha, 1998; Matthews and Campbell, 2000, 2004;
Campbell and Matthews, 2001), but a particularly dramatic
symptom is the occurrence of heart palpitations and
arrhythmia. We propose that the symptoms in lactose
intolerance are caused either by lactose itself, inappropri-
ately absorbed, or by toxins generated by bacteria in the
small intestine. These toxins include diacetyl, acetoin, 2,3
butandiol, 1,3 propandiol, d-lactate, hydrogen and pep-
tides. In order to test this hypothesis, a model system was
required that could be used to examine the effects of
lactose and these putative toxins on the heart, gut and
other tissues, and that could be used to image effects on
intracellular signals in live cells within an intact organism.
Thus, a microscope system was established to measure the
effect of lactose and other agents on heart rate and rhythm
when the agents were added to the water in which the
Daphnia were swimming.
Here, we report that lactose, at concentrations found in
products containing milk, caused a dramatic decrease in
Daphnia heart rate and induced severe arrhythmia. Our
results provide further evidence of Daphnia as a unique
model system in biology and medicine.
2. Materials and methods
2.1. Materials
All chemicals were Analar grade and were obtained
either from Fisher Chemicals or Sigma Chemicals (Lon-
don). Lactose is not as easy to dissolve as sugars such as
sucrose, glucose and galactose. It was made up as a molar
stock in water or the appropriate buffer and stored frozen,
when some of it came out of solution. The stock was
warmed gently in a microwave to ensure that all the
lactose had dissolved before aliquoting it into lower
dilutions.
2.2. Daphnia
D. pulex were obtained from our pond in Pembroke-
shire, Wales, UK. They were kept in 10- or 80-l tanks on
the window ledge of a laboratory at approximately 22 8C.
The tanks generated enough microalgae to sustain a
colony of several hundred for at least 6 months. The
walls of the tanks were scraped clean every 2–3 months
and topped up alternatively with pond water or distilled
water to replenish water lost by evaporation. The Daphnia
were observed in a specially constructed cooled chamber,
maintained in most experiments between 10 and 11 8C,
using a binocular dissecting microscope, magnification
20–40. The D. pulex were approximately 1–2 mm in
length, with a heart 100–200 Am long and 50 Am across.
The heart is situated above the brooding chamber, though
the animal normally swims what appears at first glance
upside down, with the feelers that move food into the
mouth facing upwards. The highest magnification (40)
was required in order to see the heart beat adequately for
counting. Each D. pulex was maintained within a 50-Al
droplet throughout the experiment.
2.3. Measurement of heart rate
A wide range of artificial media have been developed to
study Daphnia sp. (Banta, 1921; Keating, 1985; Elendt,
1989, 1990a,b; Kluttgen et al., 1994). Here, D. pulex were
incubated in 50-Al drops of either pond water or a
specially designed simple salt medium A (25 mM MOPS,
25 mM NaCl, 2 mM KCl, 2 mM CaCl
2
, 1 mM MgCl
2
,1
mM NaHCO
3
, pH 7.0) containing the agent being studied.
Daphnia thrived in both media for several hours with no
significant change in heart rate. Between six and nine
droplets could be examined in each experiment on a
cooled Petri dish. The Daphnia were free to swim around
in each droplet. It was necessary to cool the Daphnia
since the heart rate was too fast at room temperature to
obtain accurate measurements. The heart rate was counted
manually for three 15-s intervals and the mean converted
to beats per minute. These were consistently within 5% of
each other. The mean was then converted to % of time 0
for each condition. In the case of the lactose dose response
(Fig. 4a,b), the results were plotted as % inhibition (i.e. the
% of time 0 was subtracted from 100%) to produce a dose
response similar to a conventional plot. Each condition
involved three separate Daphnia. The temperature chosen
for the majority of experiments was 10–11 8C. Great care
was taken to maintain the temperature of the droplets
constant. The temperature (meanFS.D.) of 276 Daphnia
measurements was 10.6F0.3 8C (range 9.8–12.0). Daph-
nia kept in the 50-Al droplets showed no significant
change in heart rate over 2 h under these conditions,
where the heart rate was the same in either pond water or
the simple salt medium A. In order to measure the heart
rate the majority of the liquid surrounding each Daphnia
was removed for 1–2 min, thereby preventing the animal
moving about. There was no evidence visually that this
affected the heart rate, the feelers that move food into the
mouth, eye movement, gut motility or defecation which
was observed as the animal flicked the spine at the end of
its intestine. The temperature of the drops was monitored
throughout each experiment using a small thermocouple
and remained within F0.5 8C of the initial temperature.
This was important in view of the sensitivity of the heart
rate to temperature. Although some experiments were
carried out blind, it was difficult to dblindTexperiments
routinely. The dramatic nature of the results, together with
the unexpected timing and magnitude of many of the
effects, could not be explained by bias in the measurement
of heart rate.
A.K. Campbell et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 225–234226

Statistical analysis was carried out as paired t-tests using
SPSS and the results are expressed as probability ( p)oftwo
values being significantly different when pb0.05.
3. Results
3.1. Effect of temperature on heart rate
The temperature in the pond normally varies from
approximately 2–14 8C throughout the year. Thus, normal
room temperature (approx. 22–24 8C) was considerably
higher than that in the pond where the Daphnia live.
However, during the year, the laboratory temperature varies
from about 17 8C to over 30 8C, hopeless for reproducible
heart measurements. Furthermore, the heart rate at room
temperature was too high to obtain routine accurate
measurements. The effect of temperature was therefore
investigated (Fig. 1) in order to determine a suitable
temperature for investigating the effect of lactose and other
agents, and to see how rigorously this had to be controlled in
order to prevent artefacts due to temperature changes when
substances were added. The temperature was first reduced,
leaving the Daphnia to stabilise at each temperature for 5
min. The heart rate decreased down to 5 8C, below which
the heart usually stopped beating. The heart beat appeared
regular at all temperatures with no apparent arrhythmia,
even at the lowest temperature. Raising the temperature
enabled the heart to start beating again. The heart rate
increased at approximately 24 beats/min/8C, equivalent to
approximately 10% of the heart rate at 10 8C, the temper-
ature chosen for the remainder of the experiments. It was
therefore necessary to maintain the temperature at F0.5 8C
so that changes of 5% in heart rate could be detected. No
changes in temperature were observed as a result of removal
or addition of water from and to the droplets. As a result,
there were no changes in heart rate when changes were
made to the droplets in which the Daphnia were swimming.
At approximately 10.5 8C in either pond water or medium
A, the heart rate (meanFS.D.) of 276 separate Daphnia was
195.9F27.0 (range 89.2–249.2) beats/min, and in the
controls with no added substances remained within 5–10%
of the time 0 measurement in most experiments. There was a
considerable variation in normal heart rate between indi-
vidual Daphnia (Fig. 2), approximately 80% being between
170 and 230 beats/min, the peak in the distribution being
200–210 beats/min.
Since Daphnia reproduce both asexually and sexually
they often are found carrying 1–10 eggs or 1–5 young in
their brooding chamber. The eggs were highly pigmented. In
the experiment reported here, 56.8% of the Daphnia had
Fig. 1. Effect of temperature on D. pulex heart. D. pulex were incubated in 50-Al droplets of pond water as described in Section 2. The starting temperature was
24 8C. The temperature in the cooling bath was then lowered stepwise down to 2 8C and then raised again up to 24 8C. The heart rate was measured at each
particular temperature by counting the heart for three 15-s intervals, after allowing the D. pulex to equilibrate for 5 min. The range for the three determinations
was within 2–3 beats. The heart stopped beating below 5 8C, but started beating again as the temperature was raised. Results represent heart beats per minute
and are the meanFS.E.M. of three D. pulex.
Fig. 2. Distribution of heart rate in D. pulex: effect of eggs (0–10) and
young (0–5). The resting heart rate was measured as described in Section 2
at 10–11 8C (mean 10.6F0.3 8C in 276 separate determinations).
No. of
eggs/young
Beats/min
(mean)
Beats/min
(% total)
Beats/min
(S.D.)
0/0 192.4 98.2 2.1
0/1–5 195.8 99.9 4.3
1–10/0 203.3 103.8 3.0
Total 195.9 100 3.4
A.K. Campbell et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 225–234 227

neither eggs nor young, 23.9% had eggs with no young and
19.3% had young with no eggs (Table 1). There were no
significant differences in normal heart rate between these
three groups.
3.2. Effect of pharmacological substances on Daphnia heart
It has been reported that Daphnia responds to substances
that affect the rate of heart beats when added to the
surrounding medium (Viehoever and Cohen, 1937; Sollman
and Webb, 1941; Postmes et al., 1973; Villegas-Navarro et
al., 2003). In order to establish the validity of our Daphnia
system studied at approximately 10 8C, the effects on heart
rate and rhythm of caffeine (1–10 mM), the h-adrenergic
antagonist propranolol (100 AM), adrenaline (10–1000 AM),
isoproteronol (100 AM) and carbachol (100 AM) were
studied (Fig. 3). A total of 1–2 mM caffeine caused a small,
but reproducible rise in heart rate of some 10% within 5–15
min. This was similar to the time observed for uptake of
fluorescent substances such as fluorescein by Daphnia gut
(data not shown). The heart rate remained at this elevated
level for a further 15 min and then declined to approx-
imately 50–60% of the normal heart, significantly different
from the control ( p=0.03). Within 30–60 min, caffeine
induced an arrhythmia indicated by a highly irregular size
and rate of heart beat. A wash out showed that the effect of
caffeine was 50% reversible by 1 h, the heart rate returning
to normal by 4–5 h. The h-adrenergic antagonist proprano-
lol (100 AM) caused a 30–40% decrease in heart rate
detectable within 5–15 min (Fig. 3), p=0.03 at 60 min, but
unlike caffeine did not generate any obvious arrhythmia in
the size or rate of the heart beat. Both adrenaline (100 AM)
and the pure h-adrenergic receptor agonist isoproteronol
(10–100 AM) only caused a small increase in the heart rate
of 10–20% within 5–15 min. The cholinergic receptor
agonist carbachol (100 AM) caused a 10–30% rise in heart
rate detectable within 5–30 min. These data confirmed that
our Daphnia could respond to pharmacological agents that
affect ion channels and cell signalling when added to the
water in which they were swimming.
3.3. Effect of lactose and other sugars on Daphnia heart
In order to investigate whether lactose could affect the
Daphnia heart, the effect of 2–200 mM lactose was
investigated (Fig. 4a), the concentration in cow’s milk
being approximately 140 mM. Lactose (50–200 mM)
caused a significant decrease in heart rate within 30–60
min (Fig. 4a), which appeared to be saturable. The
concentration for half maximum inhibition was approx-
imately 60 mM lactose, with a plateau at approx. 200 mM
(Fig. 4b). Little or no effect of lactose (2–20 mM) was
detected within 60 min. At concentrations of 100–200 mM
lactose, the heart became severely arrhythmic within 30–60
min. This was a clear arrhythmia, where the heart produced
Table 1
Distribution of D. pulex with eggs and young
(a) Young with no eggs
Eggs 0 0 0 0 0 0
Young 0 1.0 2.0 3.0 4.0 5.0 Total no.
with eggs
Overall
total
No. 150.0 20.0 20.0 6.0 4.0 1.0 201.0 264.0
% no with eggs 74.6 10.0 10.0 3.0 2.0 0.5 100
% total 56.8 7.6 7.6 2.3 1.5 0.4 76.1
(b) Eggs with no young
Young 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Total no. of young
Eggs 0 0 0 0 0 0 0 0 0 0
No. 12.0 19.0 13.0 9.0 3.0 0 4.0 1.0 0 2.0 63.0
% of total with no young 19.0 30.2 20.6 14.3 4.8 0 6.3 1.6 0 3.2 100
% overall total 4.5 7.2 4.9 3.4 1.1 0 1.5 0.4 0 0.8 23.9
D. pulex were maintained in tanks of pond water as described in Section 2. The number of eggs or young in the brood chamber was counted before the start of
each experiment. In a few experiments, some young were released within the 50-Al droplet. And in two cases, the D. pulex shed their carapace. Results
represent the data from 264 individual D. pulex. Care was taken to ensure that D. pulex was used in all experiments as the pond also contained D. magna that
were easy to identify.
Fig. 3. Effect of caffeine and propranolol on D. pulex heart rate. Individual
D. pulex were incubated at approx. 10.5 8C in 50-Al droplets and the heart
rate was measured as described in Section 2. After determining the resting
heart rate, 2 mM caffeine or 100 Al propranolol were added and the heart
rate measured at 15, 30 and 60 min and compared with controls with no
additions. Each heart rate was the mean of three 15-s determinations.
Results were plotted as % heart rate at time 0 and represent the
meanFS.E.M. of three separate D. pulex under each condition.
A.K. Campbell et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 225–234228

1–2 very large contractions followed by 1–4 tiny contrac-
tions. In some cases, the heart stopped beating by 60 min.
These results were statistically highly significant. At 60 min,
p=0.026 for both 50 and 200 mM lactose relative to the
controls with no added lactose. The effect of lactose was
reversible (Fig. 5), the heart rate and rhythmic beating
returning to normal by 3 h after removal of the lactose.
When lactose was removed by a wash out, the heart rate
became the same as the controls, being not significantly
different within 2–3 h ( p=0.38).
Sucrose (100–200 mM) also appeared to cause a
decrease in heart rate and heart arrhythmia, the effect of
200 mM being greater than 100 mM (Fig. 6a). However,
glucose (100–200 mM) and galactose (100–200 mM), the
sugars into which lactose is cleaved either by the human
gut enzyme lactase-phlorizin hydrolase (EC 3.2.1.23/62) or
h-galactosidase, had no significant effect ( p=0.2 for
glucose and p=0.95 for galactose at 60 min) on heart rate
or rhythm over 90 min (Fig. 6b).
3.4. Did the toxic effects of lactose require metabolism?
A key question in our hypothesis is whether the effects
require metabolism of lactose by intestinal bacteria or by
endogenous h-galactosidase. Disaccharides such as lactose
and sucrose cannot be metabolised directly by pathways
Fig. 4. Effect of lactose on D. pulex heart rate. Individual D. pulex were incubated at approx. 10.5 8C in 50-Al droplets and the heart rate was measured as
described in Section 2. After determining the resting heart rate lactose (2, 20, 50, 100 and 200 mM) was added and the heart rate measured at 15, 30, 60 and 60
min, and compared with controls with no additions. Each heart rate was the mean of three 15-s determinations. Results were plotted as % heart rate at time0
and represent the meanFS.E.M. of three separate D. pulex under each condition. (a) Time course of lactose dose response. (b) Lactose dose response plotted as
% inhibition against lactose concentration. K
1/2
=60 mM.
A.K. Campbell et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 225–234 229

such as glycolysis. They have first to cleaved into their
constituent monosaccharides galactose and glucose or
glucose and fructose respectively. These monosaccharides,
but not fructose, are then be absorbed through the sugar
transporter SGLUT1 and then metabolised by cells. There
are only two enzymes known that cleave lactose–lactase-
phlorizin hydrolase (lactase) and h-galactosidase. Lactase is
only found in mammalian gut. But h-galactosidase, which
has no sequence similarity to lactase, is a ubiquitous enzyme
in pro- and eu-karyotic organisms, where in the latter it is
found in high activity in lysosomes. However, to metabolise
lactose the cell and the organelle must have a permease to
allow access of h-galactosidase to lactose. In order to test
whether bacterial metabolism was required for the toxic
effects of lactose on D. pulex heart three experimental
conditions were investigated: first, the effect of an antibiotic
known to kill bacteria efficiently; secondly, the effect of two
known metabolites of anaerobic sugar metabolism in
bacteria; thirdly, the effect of L-ribose a known inhibitor
of h-galactosidase in bacteria (Huber and Brockbank,
1987). Incubation of D. pulex with the antibiotic ampicillin
(100–300 Ag/ml) for 1–4 days had no effect of the resting
heart rate, and no significant effect ( p=0.45 after 70 min) on
the decreased heart rate and arrhythmia induced by lactose
(Fig. 7a). Furthermore, the diols 1,3 propandiol and 2,3
butandiol (20 mM), anaerobic metabolites of sugar metab-
olism in bacteria (Campbell and Matthews, 2001), had no
significant effect ( p=0.98 for butan 2,3 diol and p=0.42 for
propan 1,3 diol at 60 min) on D. pulex heart rate or rhythm
(Fig. 7b). Nor were there any reproducible effects of 2 mM
ribose ( p=0.61 for d-ribose and p=0.11 for l-ribose at 90
min) on the lactose inhibition of D. pulex heart rate (Fig. 8),
a concentration 10 times the K
i
for h-galactosidase (Huber
and Brockbank, 1987).
4. Discussion
Our results show clearly that lactose can affect dramat-
ically the rate and rhythm of the heart beat in the cladoceran
D. pulex. The arrhythmia observed by 60 min at high
lactose concentrations was seen as 1–2 very large contrac-
tions followed by several small ones (see www.uwcm.ac.uk/
med_biochem/akc and then Daphnia). It is interesting that
lowering the temperature to 5–10 8C, or the addition of
propranolol, reduced the heart rate to a similar low level to
that of lactose without causing an arrhythmia. Although
lactose would not normally be expected to be natural dietary
ingredient for D. pulex, our results support the data from
other model systems that many biochemical and ionic
mechanisms are universal across the animal kingdom.
However, further work is now required to confirm that this
is similar to the heart palpitations and arrhythmia we have
observed in humans with lactose intolerance (Matthews and
Campbell, 2000, 2004; Campbell and Matthews, 2001). The
inhibitory concentration range of 50–200 mM lactose was
within that for lactose found in cow’s milk, and foods or
drinks containing added lactose.
There are three possible mechanisms by which lactose
could affect the heart rate and rhythm: (a) a direct effect of
lactose on ion channels and/or gap junctions in the heart; (b)
a direct effect of lactose metabolites generated by gut
microflora on ion channels and/or gap junctions in the heart;
(c) an indirect mechanism involving the generation of
neuropeptides or other transmitters that can modulate ion
channels and/or gap junctions in the heart. But the timing of
the inhibition by lactose (Fig. 4), together with the fact that
the reduction in heart rate and the arrhythmia caused by
lactose on D. pulex heart took 3–4 h to be reversed, was
consistent with the effect of lactose being mediated directly
Fig. 5. Reversibility of the effect of lactose on D. pulex heart rate. Individual D. pulex were incubated at approx. 10.5 8C in 50-Al droplets and the heart rate was
measured as described in Section 2. After determining the resting heart rate, lactose (200 mM) was added and the heart rate measured at 15, 30 and 60 min, and
compared with controls with no additions. The lactose was then washed out with 350 Al pond water, and the heart rate measured for a further 3 h, the medium
being changed at each time interval. Each heart rate was the mean of three 15-s determinations. Results were plotted as % heart rate at time 0 and represent the
meanFS.E.M. of three separate D. pulex under each condition.
A.K. Campbell et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 225–234230

by a metabolite of lactose or by lactose itself on K
+
,Na
+
and/or Ca
2+
channels in the myocyte. The fact that sucrose
was also capable of inducing a reduction in D. pulex heart
rate and arrhythmia suggested that an osmotic effect of
lactose, for example through stretch receptors, could not be
ruled out. However, such an osmotic mechanism is not
supported by the fact that neither glucose nor galactose at
100–200 mM had an effect on D. pulex heart, nor by the
timing of the lactose inhibition and its reversal. Although by
eye there was no obvious visible evidence of swelling either
of the D. pulex as a whole, or the heart itself at times when
large effects of lactose in the heart were observed, video
microscopy will be needed to confirm this.
Lactose can be hydrolysed by lactase or any h-galacto-
sidase. However, the lactose has no access to these enzymes,
e.g. in intracellular compartments such as lysosomes, unless
the cell and organelle has a lactose permease analogous to
the permease, which is part of the lac operon in Escherichia
coli. We have proposed that the effects of lactose to cause
systemic symptoms in humans with lactose intolerance are
caused either by lactose itself or by metabolites generated by
bacteria in the large intestine, which is essentially anaerobic
(Matthews and Campbell, 2000, 2004; Campbell and
Matthews, 2001). However, the effects of lactose on D.
pulex heart were unlikely to be caused by bacterial
metabolites since the potent antibiotic ampicillin, expected
to kill gut bacteria and thus prevent metabolite synthesis
from lactose, had no effect (Fig. 7a). Furthermore, two diols
known to be anaerobic metabolites of sugar metabolism in
bacteria were without effect (Fig. 7b). This is perhaps not
surprising since microscopic examination of the D. pulex
showed that their guts were full of bright red fluorescent
microalgae unlikely to metabolise lactose in a manner similar
to the microflora in the human large intestine. Further work
Fig. 6. The effect of lactose, sucrose, glucose and galactose on D. pulex heart rate. Individual D. pulex were incubated at approx. 10.5 8C in 50-Al droplets and
the heart rate was measured as described in Section 2. After determining the resting heart rate, (a) sucrose (100 mM) or glucose (100 mM) and (b) lactose (100
mM) or galactose (100 mM) were added and the heart rate measured at 15, 30 and 60 min, and compared with controls with no additions. Each heart rate was
the mean of three 15-s determinations. Results were plotted as % heart rate at time 0 and represent the meanFS.E.M. of three separate D. pulex under each
condition.
A.K. Campbell et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 225–234 231

is now required to find agents that will knock out the gut
flora in D. pulex and thus prevent the lactose effect on the
heart. While the results are consistent with using D. pulex as
a model for the toxic effects of lactose in humans, it is now
necessary to confirm the claim made over 50 years ago that
D. pulex heart, like the human heart, is myogenic (Bekker
and Krijgsman, 1951). We have so far found no evidence for
innervation into the heart. A key question now is what is the
pacemaker, and how does it become arrhythmic, for example
when the animal is exposed to lactose? This will require
identification of the role of K
+
,Na
+
and Ca
2+
channels in the
action potential and heart beat.
All pro- and eu-karyotic cells use d-ribose in their
nucleotides and nucleic acid. However, it has been reported
(Huber and Brockbank, 1987) that the enantiomer l-ribose
is structurally very similar to the shape of galactose within
lactose predicted when it binds to h-galactosidase. l-ribose
is thus a potent inhibitor of h-galactosidase in bacteria and
lysosomes with a K
i
of approximately 0.2 mM and can be
transported into lysosomes by the sugar transporter. Neither
l-ribose nor d-ribose (2 mM) had reproducible effects on
the potency of lactose to reduce D. pulex heart rate and
induce arrhythmia (Fig. 8). This supported the hypothesis
that cleavage of lactose by h-galactosidase was not
required and that lactose itself was likely to be the toxic
agent. The K
i
for d-ribose inhibiting h-galactosidase in
bacteria is some 100 times that of l-ribose, though the K
i
for d-ribose on eukaryotic h-galactosidase may be lower.
Thus, a role for h-galactosidase in the toxic effects of
lactose on D. pulex heart cannot be completely ruled out.
Although it is clear that pharmacological substances such
as caffeine, h-adrenergic agonists and antagonists, and
acetyl choline added to the external fluid affect the heart
rate of D. pulex, some of the effects are not identical to the
effects of these substances on human heart. But the
increased inhibition by the h-adrenergic antagonist propra-
Fig. 7. The effect of ampicillin and diols on D. pulex heart rate. (a) Effect of ampicillin. Individual D. pulex were incubated in pond water at room temperature
(approx. 24 8C) for up to 3 days with or without the antibiotic ampicillin (100 Ag/ml). The D. pulex were then transferred to the incubation chamber at approx.
10.5 8C in 50-Al droplets and the heart rate was measured as described in Section 2. After determining the resting heart rate, lactose (200 mM) was added and
the heart rate measured at 15, 30 and 70 min, and compared with controls with no additions. Each heart rate was the mean of three 15-s determinations. Results
were plotted as % heart rate at time 0 and represent the meanFS.E.M. of three separate D. pulex under each condition. (b) Effect of propan 1,3 diol and butan
2,3 diol. Individual D. pulex were incubated at approx. 10.5 8C in 50-Al droplets and the heart rate measured as described in Section 2. After determining the
resting heart rate, propan 1,3-diol (20 mM) or butan 2,3-diol (20 mM) were added and the heart rate measured at 15, 30, 60 and 90 min, and compared with
controls with no additions. Each heart rate was the mean of three 15-s determinations. Results were plotted as % heart rate at time 0 and were plotted as % heart
rate at time 0 and represent the meanFS.E.M. of three separate D. pulex under each condition.
A.K. Campbell et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 225–234232

nolol with lactose supports the hypothesis that the effect of
lactose involved signalling and ion channels.
D. pulex is about 1–2 mm long and its heart appears to
be a single chamber of approximately 50 by 150 Am
controlled by a single layer of myocytes with large
mitochondria (Stein et al., 1966; von Ruland, Newman
and Campbell, unpublished). Unlike other arthropod
hearts, it has been reported that, like the mammalian
heart, the action potential is generated myogenically
(Bekker and Krijgsman, 1951). This claim now needs re-
examination using modern electrophysiological and phar-
macological techniques. Although there is a fine membrane
visible in the light microscope around the waist of the
heart of D. pulex, the heart appeared to be made up of one
compartment. The ultrastructure of D. pulex has not been
extensively studied, but the organism contains several
organs and cell types such as the multi-lens eye, muscle,
gut and secretory and phago-/endocytic cells (Stein et al.,
1966; Rieder, 1987; Elendt, 1989), and has agonists that
regulate intracellular free Ca
2+
and cyclic nucleotides. This
suggests that Daphnia could also be a model system for
studying cell signalling and intra- and inter-cellular
communication in situ. At present, the precise number of
cells making up one Daphnia is not known, but the size
compared with the nematode worm suggest it will be
b1000. Daphnia is well established as a model for
ecotoxicity studies (Persoone and Van de Vel, 1988; Baird
et al., 1989a,b; Diamantino et al., 2000; Guilhermino et al.,
2000; De Coen and Janssen, 2003). Our results support
previous reports that the heart of Daphnia responds to
pharmacological substances that affect the rate and rhythm
of the mammalian heart (Viehoever and Cohen, 1937;
Sollman and Webb, 1941; Postmes et al., 1973; Villegas-
Navarro et al., 2003). This has lead to Daphnia being used
widely as an educational tool. They are relatively easy to
grow in a simple medium and usually reproduce asexually.
When stressed they produce males. Daphnia is thus an in
situ model for the regulation of the heart beat, heart
development, gut motility and digestion, endo- and phago-
cytosis, gene regulation of haem proteins, Ca
2+
signalling
and ion channel regulation. Initial experiments have shown
that the eggs have a resting potential of about 30 mV
(Wann and Campbell, unpublished). The sequence of the
mitochondrial genome of D. pulex has been published
(Crease, 1999) and an international consortium aims to
have the full genome from its 12 chromosomes available
by 2005 (Colbourne, 2004). Daphnia are transparent and
easy to immobilise and are also smaller than many of the
classic model systems in biomedicine such as Drosophila.
Daphnia also has a haemoglobin that is induced by
oxygen stress (Baumer et al., 2002) in a manner similar
to the haemoglobin found in mammalian brain. This and
our results support the argument for Daphnia as a model
system (www.sciencemag.org/feature/plus/sfg/resources/
res_model.shlml) for investigating the mechanisms and
pathology of ion channels and cell signalling in a live
organism, and as an important potential test system for
drug discovery thereby reducing the use of mice and other
mammals. The wide variation in heart rate in the D. pulex
population (Fig. 1) is similar to the large variation in many
physiological and biochemical parameters in humans and
other organisms (Williams, 1998). Consistent and reprodu-
cible conclusions can be drawn by using each individual as
its own control. It is understanding the mechanisms and
evolutionary significance of these hitherto often ignored
variations in the molecular biodiversity of natural pop-
ulations that provides a challenge for contemporary
biology and medicine (Campbell, 2003).
Acknowledgements
We thank Professor David Luscombe, former Head of the
Welsh School of Pharmacy, for his interest and support.
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