Amino acids modulate ion transport and fluid secretion by insect Malpighian tubules
1
Dept of Biology, McMaster University, 1280 Main Street West, Hamilton,
Ontario, L8S 4KI, Canada
2
Dept of Zoology, Downing Street, Cambridge, CB2 3EJ, UK
* Author for correspondence (e-mail: odonnell{at}mcmaster.ca)
Accepted 1 October 2002
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Summary |
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Key words: amino acid, compatible osmolyte, epithelia, ion transport, Malpighian tubule, Rhodnius prolixus, Drosophila melanogaster
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Introduction |
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Amino acids may perform a number of functions and so may be particularly
important for tissues such as the Malpighian tubules (MTs) that are bathed in
the haemolymph. The nonprotein amino acid canavanine has been shown to inhibit
fluid secretion in isolated Locusta MTs but to potentiate the
subsequent response of the tubules to stimulation with cAMP or diuretic
hormone (Rafaeli and Applebaum,
1980). In most cells, amino acids act as intracellular compatible
osmolytes (Yancey et al.,
1982
). Glutamine, for example, is a major compatible osmolyte
engaged in the role of cell volume control as a response to cell shrinkage
(Fumarola et al., 2001
).
Taurine is a non-protein amino acid and compatible osmolyte that modulates ion
transport by many tissues (Guizouarn et
al., 2000
; Law,
1994
). Proline is an important compatible osmolyte in both
intracellular and extracellular fluids of mosquito larvae
(Patrick and Bradley, 2000
).
Some amino acids play pivotal roles in metabolism by insect tissues. Proline
and alanine are equally as important as carbohydrates in supplying energy to
the flight muscles of the African fruit beetle Pachnoda sinuate
(Auerswald et al., 1998
).
Proline secreted into the lumen of Schistocerca MTs is passed into
the rectum downstream, where it acts as a respiratory substrate to drive
electrogenic chloride reabsorption across the lumen-facing membrane of the
rectum. Fluid secreted by isolated MTs of the desert locust Schistocerca
gregaria contains as much as 44 mmoll-1 proline
(Chamberlin and Phillips,
1982
). By contrast, the permeability of the walls of the tubules
of Rhodnius to amino acids is low during diuresis, in spite of the
fact that the tubule cells actively accumulate high concentrations of amino
acids (Maddrell and Gardiner,
1980
). Urine concentrations of amino acids during diuresis are
<2% of those in the haemolymph, whereas in non-diuretic tubules, secreting
1000 times more slowly, the concentrations of amino acids are 70-90% of those
in the haemolymph. It has also been shown that the principal amino acids
(glycine, alanine, proline, serine and valine) are not significantly
metabolized by the tubule cells (Maddrell
and Gardiner, 1980
).
Previous studies have not addressed in detail the role of amino acids other
than proline in acting as metabolites or osmolytes during fluid secretion by
isolated MTs. However, fluid secretion by isolated tubules of
Drosophila has been shown to be enhanced when tubules are bathed in a
1:1 mixture of saline and Schneider's Drosophila medium, and amino
acids are major components of the latter
(Dow et al., 1994).
Drosophila tubules bathed in an amino-acid-replete saline (AARS)
containing seven of the most abundant amino acids in Schneider's
Drosophila medium (1.65 mmoll-1 Gly, 7.35
mmoll-1 Pro, 6.1 mmoll-1 Gln, 1.28 mmoll-1
His, 0.57 mmoll-1 Leu, 4.5 mmoll-1 Lys, 1.28
mmoll-1 Val) secrete approximately 40% faster than tubules bathed
in a saline containing the same concentration of glucose but with no amino
acids (Linton and O'Donnell,
1999
).
The latter result raises the question of whether each of the amino acids at the listed concentration contributes equally to the stimulation of fluid secretion or whether one or a few amino acids are responsible for the observed effects. In the present study, therefore, we have examined the modulatory effects of specific amino acids on fluid and ion transport in isolated MTs of two species, the fruit fly Drosophila melanogaster and the blood-feeding hemipteran Rhodnius prolixus. Rhodnius tubules secrete at high rates when stimulated with serotonin, whereas Drosophila tubules secrete at substantial rates even in the absence of stimulation with diuretic factors or their second messengers. We first examined the effects of individual amino acids at a concentration equal to the total amino acid concentration in AARS (i.e. 20 mmoll-1). We show that individual amino acids may have stimulatory or inhibitory effects on sustained rates of fluid secretion and epithelial ion transport by tubules of both species. Moreover, pronounced stimulatory effects are observed after pre-incubation of tubules for 1-2h in saline containing specific amino acids, even when the tubules are subsequently washed free of amino acids and the secretion assay is performed in a simple saline containing only inorganic salts and glucose. For Rhodnius tubules, we have also examined the effects of amino acids at concentrations approximating those in the haemolymph.
We have also looked at the interaction of amino acids and bathing saline
K+ concentration on secretion rates of Rhodnius MTs.
Previous studies have shown that rates of fluid secretion increase as bathing
saline K+ is increased above 2 mmoll-1, reaching a
maximum at approximately 6 mmoll-1 K+
(Maddrell et al., 1993). High
secretion rates are sustained for 10-30 min after the addition of serotonin,
and then decline to a plateau value of approximately 30% of the peak rate in
saline containing 3 mmoll-1 K+
(Maddrell et al., 1993
).
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Materials and methods |
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Rhodnius prolixus Stål were periodically fed on rabbits and were maintained at 25-28°C and 60% relative humidity in the Department of Biology, McMaster University. Animals in the third, fourth and fifth instar were used 3-30 days after the blood meal. Experiments were carried out at room temperature (20-25°C).
Experimental protocols
Tubules were dissected under saline and were transferred to saline droplets
under paraffin oil for measurement of fluid secretion rates using the Ramsay
technique. Drosophila anterior Malpighian tubules (MTs) were
dissected out under Drosophila saline containing 117.5
mmoll-1 NaCl, 20 mmoll-1 KCl, 2 mmoll-1
CaCl2, 8.5 mmoll-1 MgCl2, 10.2
mmoll-1 NaHCO3, 4.3 mmoll-1
NaH2PO4, 15.0 mmoll-1 Hepes and 20.0
mmoll-1 glucose and adjusted to pH 7.0. Isolated tubules were then
transferred to 10 µl droplets of saline under paraffin oil in a
Sylgard-lined Petri dish. The paired tubules were arranged so that one tubule
was pulled out of the saline droplet and wrapped around a metal pin embedded
in the Sylgard base of the dish approximately 1 mm from the edge of the
droplet, while the other tubule was left in the saline droplet. Secreted
droplets formed on the common ureter, which was positioned just outside the
bathing droplet, and were removed with a glass probe every 20 min and allowed
to settle to the bottom of the Petri dish. The diameter (d) of the
droplet was measured using an ocular micrometer and the droplet volume
calculated as (d3)/6. Secretion rate was calculated by
dividing secreted droplet volume by the time over which it formed
(Dow et al., 1994
).
Rhodnius tubules were dissected under saline containing 129 mmoll-1 NaCl, 8.6 mmoll-1 KCl, 8.5 mmoll-1 MgCl2, 2.0 mmoll-1 CaCl2, 10.2 mmoll-1 NaHCO3, 4.3 mmoll-1 NaH2PO4, 8.6 mmoll-1 Hepes and 20.0 mmoll-1 glucose and adjusted to pH 7.0. For secretion assays, the fluid-secreting upper segment and a short length of the lower MT were isolated and transferred to 100 µl droplets of Rhodnius saline that were held under paraffin oil in depressions cut into the base of a Sylgard-lined Petri dish. The cut end of the lower tubule was pulled out and wrapped around a metal pin that had been pushed into the Sylgard base. The entire upper MT remained within the saline droplet. In most experiments, tubules were stimulated to secrete at high rates by addition of 10 µmol l-1 serotonin [5-hydroxytryptamine (5-HT)]. Secreted fluid droplets that formed on the cut end of the tubule were pulled off at intervals with a glass probe. Secretion rates were calculated as described above.
For all secretion assays, one of three protocols was followed:
Continuous exposure
Malpighian tubules were set up in a Ramsay assay
(Dow et al., 1994) immediately
after isolation and were exposed to a specific amino acid throughout the
course of the experiment. We first examined the effects of individual amino
acids at a concentration equal to the total amino acid concentration in AARS
(i.e. 20 mmol l-1). Threonine and tyrosine were applied at 10 mmol
l-1 and 0.5 mmol l-1, respectively, because of limited
solubility. For Rhodnius, we also examined the effects of continuous
exposure to all 17 of the predominant amino acids at the concentrations
normally present in the haemolymph 18 days after the blood meal
(Barrett, 1974
). In these
experiments, 4th instar tubules were exposed to control saline or to saline
containing, in descending order of concentration, Pro (16.0 mmol
l-1), Val (4.9 mmol l-1), Gly (4.3 mmol l-1),
Tyr (4.0 mmol l-1), Ala (3.2 mmol l-1), His (3.1 mmol
l-1), Leu (2.7 mmol l-1), Gln (2.5 mmol l-1),
Ser (2.3 mmol l-1), Thr (2.0 mmol l-1), Lys (1.9 mmol
l-1), Iso (1.8 mmol l-1), Phe (1.3 mmol l-1),
Asp (1.1 mmol l-1), Arg (0.8 mmol l-1), Glu (0.3 mmol
l-1) and Cys (0.2 mmol l-1). The sum of the
concentrations of these 17 amino acids was 52.4 mmol l-1.
Pre-incubation
Isolated tubules were bathed in saline with a specific amino acid present
at a given concentration for 1 h for Drosophila and 1-2 h for
unstimulated Rhodnius tubules before being transferred to an
amino-acid-free saline and set up in a secretion assay. Rhodnius
tubules were stimulated with 5-HT, and secretion rates were measured for 1-2
h.
Rescue
Isolated Rhodnius tubules were stimulated with serotonin in
amino-acid-free saline, and secretion rates were measured for 1-2 h. After
secretion rates had decreased to a stable low value, approximately 15% of the
maximal stimulated rate, a specific amino acid from a stock solution was added
to the bathing droplets and secretion rate was then measured for an additional
1-2 h.
Measurement of K+ and Na+ concentrations and pH
in secreted droplets
K+ and Na+ concentrations and pH of the secreted
droplets were measured using ion-selective microelectrodes as described
previously (Maddrell and O'Donnell,
1992; Maddrell et al.,
1993
; O'Donnell and Maddrell,
1995
). The pH microelectrodes were based on H+
ionophore I, cocktail B (Fluka Chemical Corp. Ronkonkoma, NY, USA) and were
calibrated in droplets of saline adjusted to pH 6.5 and 7.5, as determined
with a macro pH electrode. K+-selective microelectrodes were based
on potassium ionophore I, cocktail B (Fluka) and were calibrated in solutions
of 15 mmol l-1 KCl: 135 mmol l-1 NaCl and 150 mmol
l-1 KCl. Na+-selective electrodes were based on sodium
ionophore I, cocktail A (Fluka) and were calibrated in 15 mmol l-1
KCl: 135 mmol l-1 NaCl and 150 mmol l-1 NaCl. Electrodes
were acceptable for use when the slope of the response to a 10-fold change in
K+ or Na+ or a 1 unit pH change concentration was >50
mV and the 90% response time of the ion-selective barrel to a solution change
was <30 s. Typical slopes for K+, Na+ and pH
microelectrodes were 54 mV, 52 mV and 57 mV, respectively. The reference
electrode for K+ measurements was filled with 1 mol l-1
Na+ acetate at the tip and lower one-third of the barrel and 1 mol
l-1 KCl for the upper two-thirds of the barrel. The reference
electrode for pH and Na+ measurements was filled with 1 mol
l-1 KCl.
The concentration of ions in secreted droplets was calculated using the
formula:
[Ion]droplet=Cx10(/slope),
where [Ion]droplet is the ion concentration in the secreted
droplet, C is the ion concentration in one of the calibration
solutions (150 mmol l-1 or 15 mmol l-1),
is
the voltage difference between the secreted droplet and the same calibration
solution, and the slope is the change in electrode voltage measured in
response to a 10-fold change in ion activity.
Calculations and statistics
Values are expressed as mean ± S.E.M. for the indicated number
(N) of tubules. Significance of differences between means were
measured by Student's t-test using P<0.05 as the level of
significance. Experimental and control groups were compared using unpaired
t-tests assuming equal variances. The responses of the same group of
tubules before and after an experimental treatment were compared using a
paired t-test. In those figures where secretion rate is expressed as
a percentage of the control rate, the secretion rate for each experimental
tubule was divided by the mean rate for the corresponding set of controls and
the result was multiplied by 100. Where the percentage change in the value of
a measured parameter is referred to, all statistical tests were done on the
scalar values (secretion rate or ion concentration) not on the percentages.
Doseresponse curves relating stimulation or inhibition of fluid
secretion rate to amino acid concentration were fitted using a commercial
graphics and analysis package (Igor, WaveMetrics Inc., Lake Oswego, OR, USA)
and an associated set of procedures written by Dr F. Mendez (Patcher's Power
Tools,
http://www.wavemetrics.com/Users/ppt.html).
The iterative procedure allowed estimation of the baseline response, the
maximum response, the slope and the amino acid concentration that produced a
response halfway between baseline and maximum (EC50).
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Results |
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The extent of stimulation by glutamine was less variable than that produced by methionine, and we therefore examined the effects of glutamine in more detail. A doseresponse curve shows near maximal stimulation by glutamine at 2 mmol l-1 (Fig. 1, inset). Glutamine did not alter the secreted fluid concentrations of Na+ or K+. Fluid secreted by Drosophila MTs bathed in saline containing 20 mmol l-1 glutamine contained 54.5±3.9 mmol l-1 Na+ and 126.6±5.7 mmol l-1 K+ (N=10 tubules). These values did not differ significantly from concentrations of 50.0±4.1 mmol l-1 Na+ and 121.1±4.4 mmol l-1 K+ in fluid secreted by tubules bathed in glutamine-free saline.
Stimulation by tyrosine appeared to be independent of that produced by glutamine. Fluid secretion by tubules bathed in 0.5 mmol l-1 tyrosine increased further from 0.68±0.07 nl min-1 to 0.79±0.07 nl min-1 with the subsequent addition of 20 mmol l-1 glutamine (N=8). Moreover, glutamine significantly stimulated fluid secretion (P<0.05) in both Na+-replete and Na+-free saline, whereas there was no significant stimulation by tyrosine in Na+-free saline. Secretion rates after 40 min with and without glutamine in Na+-free saline were 0.64±0.06 nl min-1 (N=10) and 0.37±0.03 nl min-1 (N=12), respectively. By contrast, secretion rates with and without 0.5 mmol l-1 tyrosine in Na+-free saline were 0.35±0.04 nl min-1 (N=20) and 0.32±0.02 nl min-1 (N=10), respectively.
In contrast to the other amino acids tested, fluid secretion was completely inhibited by 20 mmol l-1 cysteine (Fig. 1) and was reduced to 0.014±0.04 nl min-1 by 5 mmol l-1 cysteine, 69% less than the corresponding control rate of 0.45±0.04 nl min-1 (N=6). Inhibition of fluid secretion by cysteine could be reversed almost completely with the addition of glutamine to the bathing solution, suggesting that the cysteine effect was not due to simple toxicity (Fig. 2).
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Drosophila MTs have previously been shown to secrete at high rates
when stimulated with cAMP and/or the peptide leucokinin
(O'Donnell et al., 1996).
Tubules were further stimulated by addition of glutamine. Addition of 20 mmol
l-1 glutamine to tubules 40 min after stimulation with high
concentrations of cAMP (1 mmol l-1) and leucokinin (10 µmol
l-1) significantly (P<0.05) increased secretion rate
after a further 50 min by 93±15% (N=10) relative to cAMP- and
leucokinin-stimulated controls in glutamine-free saline.
Rescue experiments: Rhodnius
Fig. 3 shows that in saline
containing 8.6 mmol l-1 K+ and 20 mmol l-1
glucose, secretion rates peaked 15-30 min after the addition of serotonin, and
then began a slow decline to a plateau value of approximately 15% of the peak
value between 65 min and 110 min. Secretion rates were restored to within
approximately 5% of the peak value over the course of 45-60 min
(Fig. 3) after addition of 20
mmol l-1 glutamine (Fig.
3). In two similar experiments (N=14 tubules), secretion
rates were maintained within approximately 10% of the peak value for a further
100-120 min after the addition of glutamine. Secretion rates then slowly
declined to control values after 140 min. Similar effects were seen in tubules
isolated from 3rd, 4th or 5th instar Rhodnius. Glutamine restored
secretion rates to within 5% of the peak value of approximately 20 nl
min-1, approximately 40 nl min-1 and approximately 75 nl
min-1 for 3rd, 4th, and 5th instar Mts, respectively
(N10 tubules for all instars). Fluid secretion was also
stimulated by nine other amino acids at 20 mmol l-1, although not
to the same extent as glutamine (Fig.
4). Lysine and arginine inhibited fluid secretion
(Fig. 4).
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The effects of concentrations of glutamine closer to the concentration of
2.5 mmol l-1 found in Rhodnius haemolymph
(Gringorten, 1979) were also
examined. Secretion rates in saline containing 2 mmol l-1 and 5
mmol l-1 glutamine were 118±24% (N=8 tubules) and
242±45% (N=8 tubules), respectively, above those of the
corresponding controls run in Rhodnius saline. A doseresponse
curve indicates an EC50 of approximately 1.3 mmol l-1
(Fig. 4, inset). The
EC50 values for inhibition by arginine and lysine were 1.4 mmol
l-1 and 9.2 mmol l-1, respectively
(Fig. 4, insets).
We examined whether the decline in secretion rate was due to insufficient
levels of either 5-HT or glucose in the bathing saline and whether a similar
decline was observed if tubules were stimulated with the second messenger cAMP
rather than 5-HT. Increasing the concentration of glucose from
20mmol
l-1 to 40
mmoll-1 did not
significantly change the plateau secretion rate of
5.4±0.8
nl
min-1 in 4th instar tubules
(N=5). Similarly, increasing 5-HT concentration from
10-5
mol
l-1 to
2x10-5
mol
l-1 in the saline bathing
stimulated tubules whose secretion rates had declined over the course of
120
min did not increase the rate of fluid secretion. Tubules stimulated
with 10-5
mol
l-1 5-HT secreted at
4.2±0.4
nl
min-1 (N=7). The rate of
4.6±0.4
nl
min-1 measured 45
min after
increasing the 5-HT concentration to
2x10-5
mol
l-1 5-HT was not significantly
different (N=10 tubules). Secretion rate also declined to a similar
extent when tubules were stimulated by addition of cAMP instead of 5-HT and
were again restored by addition of glutamine. In response to stimulation with
1
mmol
l-1 cAMP, secretion rate declined from a peak value
of 42.56±2.6
nl
min-1 to a plateau value of
18.52±1.01
nl
min-1 90
min later and was
restored to 34.5±1.6
nl
min-1 60
min after the
addition of glutamine (N=8). Moreover, tubules whose secretion rate
had declined after previous stimulation with
10-5
mol
l-1 5-HT were not rescued by addition
of cAMP. Secretion rate declined to 7.3±0.5
nl
min-1
60
min after the addition of 5-HT and was not significantly different from
the value of 6.7±0.4
nl
min-1 measured after a
further 30
min in the presence of 1
mmol
l-1 cAMP
(N=7).
The effects of glutamine were distinct from those associated with changes
in bathing saline K+ concentration. Secretion rates in
glutamine-free saline declined to approximately 15% of the peak values in
saline containing 3 mmol l-1, 4 mmol l-1 or 8.6 mmol
l-1 K+ with half-times of approximately 11 min, 21 min
and 48 min, respectively (N=6-11 tubules at each K+
concentration). The more rapid decline in secretion rate in saline solutions
containing lower levels of K+ confirms previous findings
(Maddrell et al., 1993). The
addition of 20 mmol l-1 glutamine restored secretion rates to
within 19%, 12% and 5% of the peak rate in 3 mmol l-1, 4 mmol
l-1 and 8.6 mmol l-1 K+, respectively. The
rundown in amino-acid-free saline does not appear to reflect changes in
activity of the basolateral Na+/K+-ATPase. Secretion
rates of 5-HT-stimulated 4th instar tubules declined from 38.2±4.2 nl
min-1 to 6.9±0.8 nl min-1 in control saline and
from 36.7±3.3 nl min-1 to 5.7±0.6 nl min-1
in saline containing 0.1 µmol l-1 ouabain (N=12-13
tubules).
The effects of glutamine were also distinct from the inhibition of tubule
secretion rates in the presence of exogenous cGMP (0.5 mmol
l-1) and the reversal of such inhibition by addition of cAMP (1
mmol l-1; Quinlan et al.,
1997
; Quinlan and O'Donnell,
1998
). Fourth instar tubules inhibited by 0.4 mmol l-1
cGMP did not recover in response to subsequent addition of 20 mmol
l-1 glutamine. Secretion rates were reduced from 36.6±3.9 nl
min-1 before cGMP to 0.74±0.19 nl min-1
(N=5) after addition of cGMP. Rates of 0.47±0.06 nl
min-1 measured 45 min after addition of glutamine indicated that
there was no recovery.
Continuous-exposure experiments: Rhodnius
The results of Fig. 3
indicated that increased secretion rates after addition of glutamine were
maintained for >40 min. This indicated that glutamine minimized the rundown
of tubules seen in the absence of amino acids, and we therefore examined the
effects of continuous exposure to amino acids for longer periods. For 3rd
instar tubules continuously exposed to 20 mmol l-1 glutamine, there
was no significant difference in secretion rates at 20 min (25±2.3 nl
min-1; N=8) compared with 120 min after stimulation with
5-HT (22.6±1.8 nl min-1). Secretion rates of control tubules
declined by 79% from 39.3±3.8 nl min-1 (N=7) at 20
min after addition of 5-HT to 8.3±1.3 nl min-1 at 120 min.
There was no significant rundown of secretion rate when 4th instar tubules
were bathed in saline containing 17 amino acids at the concentrations normally
present in the haemolymph. These tubules secreted at rates of 39.4±4.1
nl min-1 at 20 min after addition of 5-HT and rates of
35.6±3.8 nl min-1 after a further 100 min.
Pre-incubation experiments: Rhodnius and
Drosophila
Unstimulated tubules of Rhodnius prolixus secrete at very low
rates, typically << 1.0 nl min-1 for 5th instar tubules
(Maddrell, 1963).
Surprisingly, unstimulated 4th instar tubules in the presence of 20 mmol
l-1 glutamine secreted fluid at a rate of 0.12±0.02 nl
min-1 (N=8), approximately 70% less than the rate of
0.37±0.03 nl min-1 for tubules incubated in saline without
glutamine (N=7). However, when MTs were pre-incubated in control
saline plus glutamine and subsequently transferred to glutamine-free saline
and stimulated with serotonin, they secreted at rates of up to 40 nl
min-1 (4th instar), which are 3- to 4-fold higher than
secretion rates of tubules pre-incubated for the same period in saline without
glutamine (Fig. 5). For
Rhodnius tubules, all the amino acids, with the exception of
methionine, that significantly increased fluid secretion rates in the rescue
protocol also stimulated tubules in the pre-incubation experiments. Moreover,
several amino acids (Val, Ser, Phe, Thr and Asp) that were not stimulatory in
the rescue experiments significantly increased secretion rate in
pre-incubation experiments (Fig.
6).
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There were also differences with respect to inhibition of fluid secretion. Four amino acids (Tyr, Trp, His and Cys) that had no effect or were mildly stimulatory for Rhodnius MTs in the rescue experiments (Fig. 4) were inhibitory in the pre-incubation experiments, dramatically so for 20 mmol l-1 cysteine (Fig. 6). Inhibition of secretion by Rhodnius tubules by low concentrations of cysteine (0.25 mmol l-1, 0.5 mmol l-1 or 1 mmol l-1) was almost completely reversed by the subsequent addition of 20 mmol l-1 glutamine to the bathing solution. Inhibition by higher concentrations of cysteine was partially reversible (Fig. 6, inset); the EC50 for inhibition was 1.3 mmol l-1. Only lysine was inhibitory in both protocols. Arginine, which partially inhibited secretion in the rescue experiments, had no effect in the pre-incubation protocol.
We also examined the effects of pre-incubation in saline containing amino acids on secretion rates of Drosophila MTs (Fig. 7). Tubules pre-incubated in saline containing glutamine or methionine secreted at rates of 147% and 162%, respectively, of control tubules pre-incubated in Drosophila saline containing no amino acids (Fig. 7). In contrast to the stimulatory effect of tyrosine, phenylalanine or alanine in the continuous-exposure experiments (Fig. 1), none of these three amino acids had any significant effect in pre-incubation experiments (Fig. 7). Pre-incubation in 20 mmol l-1 cysteine completely inhibited fluid secretion for at least 60 min after transfer of Drosophila MTs to amino acid-free saline (Fig. 7).
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Effects of glucose-free saline on the effects of glutamine
Secretion assays in glucose-free saline indicated that the stimulation of
fluid secretion by glutamine is not simply due to its role as a preferred
metabolite by Drosophila or Rhodnius MTs. For 3rd instar
Rhodnius MTs, fluid secretion in saline with glutamine but no glucose
(3.8±0.43 nl min-1) was only 10-15% that of tubules in
saline with glucose and glutamine (25.5±2.3 nl min-1;
N=8 tubules). Similarly, Drosophila MTs bathed in saline
containing glutamine but no glucose (0.5±0.05 nl min-1)
secrete at two-thirds the rate of tubules in saline containing glucose and
glutamine (0.75±0.03 nl min-1). Moreover, the continuous
presence of 100 µmol l-1 amino-oxyacetic acid (a potent
inhibitor of glutamine metabolism) in the bathing droplet did not block the
glutamine-dependent recovery of fluid secretion by 4th instar
Rhodnius MTs set up in a rescue experiment. Tubules exposed to
amino-oxyacetic acid recovered to a rate of 50.5±4.7 nl
min-1 upon addition of glutamine. This rate was not significantly
different from the rate of 48.5±3.3 nl min-1 for tubules
exposed to glutamine in the absence of amino-oxyacetic acid (N=7
tubules).
Effects of amino acids on secreted fluid pH and Na+ and
K+ concentrations
Glutamine resulted in an increase in Na+ concentration
(approximately 30 mmol l-1) and a corresponding decrease in
K+ concentration relative to controls in the secreted fluid of
Rhodnius MTs for both the rescue
(Fig. 8) and the pre-incubation
(Fig. 9) protocols. It is
important to note that in the rescue protocol there was no significant
alteration in Na+ or K+ concentration in the secreted
fluid during the decline in secretion rate prior to addition of glutamine
(Fig. 8), indicating that the
effects of glutamine are not a simple reversal of the rundown process. Tubules
pre-incubated in the presence of glutamine also produced fluid with a
dramatically higher pH when stimulated with serotonin compared with tubules
pre-incubated in glutamine-free saline
(Fig. 10). Alkalinization was
sustained for more than an hour. Moreover, there was no change in secreted
fluid pH for tubules pre-incubated in arginine. For tubules set up in the
rescue protocol, the rundown in secretion rate is associated with a gradual
acidification of the secreted fluid. When secreting at the peak rate, 15-30
min after stimulation with 5-HT, Rhodnius tubules secrete fluid with
a pH near neutral (7.0±0.04; N=8), whereas 60-80 min after
stimulation the pH of the secreted fluid has dropped to 6.37±0.06.
Taken together the results of Figs
8,9,10
indicate that glutamine does not simply increase the rate of fluid secretion
but it leads to changes in the pH and in the proportions of Na+ and
K+ in the secreted fluid.
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Discussion |
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Physiological versus pharmacological effects
Some of the amino acids used in this study were used at concentrations
close to the physiological ranges found in insect haemolymph, whereas others
were clearly at levels many times greater than those found in the haemolymph.
We initially used a concentration of 20 mmol l-1 for studying
individual amino acids because this level approximates to the total
concentration of the seven amino acids included in an amino-acid-replete
saline (AARS) used in previous studies of Drosophila MTs
(Linton and O'Donnell, 1999).
Tubules bathed in AARS secrete at high and stable rates, equivalent to those
found for MTs bathed in a 1:1 mixture of Drosophila saline and
Schneider's medium (Dow et al.,
1994
).
The EC50 for stimulation of Drosophila MTs by glutamine
is 1.0 mmol l-1 (Fig.
1). Although the concentrations of amino acids in
Drosophila haemolymph are unknown, the latter value is similar or
less than the concentrations of glutamine found in haemolymph of other
dipterans such as Simulium venustum (6.4 mmol l-1;
Gordon and Bailey, 1974),
Calliphora vicina (8.2 mmol l-1;
Evans and Crossley, 1974
) and
Glossina austeni (9-11 mmol l-1;
Tobe, 1978
). Similarly, the
concentration of tyrosine that we used (0.5 mmol l-1) is similar to
the haemolymph levels of 0.6 mmol l-1 in Chironomus
tentans (Firling, 1977
),
0.9-3.7 mmol l-1 in G. austeni
(Tobe, 1978
) and 0.1-0.24 mmol
l-1 in three species of blackflies
(Gordon and Bailey, 1974
). The
levels of glutamine and tyrosine that significantly stimulate fluid secretion
by Drosophila MTs are thus close to expected physiological levels. By
contrast, the level of 20 mmol l-1 methionine is well above the
haemolymph levels of 0.13 mmol l-1 (C. vicina;
Evans and Crossley, 1974
), 0.14
mmol l-1 (C. tentans;
Firling, 1977
) and <0.42
mmol l-1 (G. austeni;
Tobe, 1978
). We did not
examine the effects of lower concentrations of methionine because the effects
were quite variable at 20 mmol l-1. However, although tyrosine and
glutamine appear to stimulate MT fluid secretion independently, it is quite
possible that other amino acids may exert their effects in concert, in which
case the concentrations of 20 mmol l-1 used in this study should be
compared with total concentrations of 16-34 mmol l-1 in haemolymph
of C. tentans (Firling,
1977
), approximately 40 mmol l-1 in S.
venustum (Gordon and Bailey,
1974
) and approximately 50 mmol l-1 in C.
erythrocephala (Evans and Crossley,
1974
).
The EC50 for stimulation of Rhodnius MTs by glutamine
is 1.3 mmol l-1 (Fig.
4), which is below the haemolymph level of 2.5 mmol l-1
(Gringorten, 1979). Haemolymph
levels of proline at various times after the blood meal range from 14 mmol
l-1 to 21 mmol l-1 in 5th instars
(Barrett and Friend, 1975
) and
19 mmol l-1 to 30 mmol l-1 in adults
(Barrett and Friend, 1975
).
Taken together, then, our results suggest that physiologically relevant levels
of amino acids stimulate MT fluid secretion in both the rescue and
pre-incubation protocols. Our data thus indicate a physiologically important
role for amino acids in the long-term (minutes to hours) regulation of tubule
secretion rate.
Haemolymph concentrations of lysine are in the range of 0.18-1.8 mmol
l-1 in adult females and 0.7-2.4 mmol l-1 in 5th instars
(Barrett, 1974;
Barrett and Friend, 1975
).
Concentrations of 1-2 mmol l-1 lysine were associated with minimal
inhibition (Fig. 4), suggesting
that the effects of lysine are probably pharmacological. The effects of
cysteine also appear to be primarily pharmacological. The EC50 for
inhibition of Rhodnius MTs by cysteine
(Fig. 6) is approximately
6-fold higher than the haemolymph concentration of 0.2 mmol l-1
(Barrett and Friend, 1975
), and
there was no significant inhibitory effect of 0.25 mmol l-1
cysteine. Given that haemolymph concentrations of the inhibitory amino acids
are normally well below the EC50 values for inhibition of MTs
in vitro, it is tempting to speculate that the regulatory processes
that control haemolymph amino acids may be designed, in part, to avoid
inhibition of MT secretion and consequent impairment of haemolymph ion and
osmoregulation.
Effects of amino acids on Rhodnius MTs
Stimulation of fluid secretion in the pre-incubation experiments outlasted
the duration of exposure of Rhodnius MTs to glutamine by >2 h,
indicating that glutamine per se is not transported into the lumen.
The increase in secretion rate does not, therefore, reflect a flow of
osmotically obliged water in response to transepithelial glutamine transport.
This is in contrast to the finding that approximately 10% of the secretion
rate of isolated Schistocerca tubules was osmotically coupled to
transepithelial proline transport
(Chamberlin and Phillips,
1982). It is also unlikely that the increased secretion rate seen
when Rhodnius MTs are stimulated after pre-incubation in glutamine
can be explained as an osmotic consequence of release of glutamine or its
metabolites following their sequestration within the tubule during
pre-incubation. Rhodnius tubules secrete a volume of near iso-osmotic
fluid equivalent to their own cell volume every 15 s when stimulated with
serotonin (Maddrell, 1991
). It
is therefore improbable that sufficient levels of glutamine or its metabolites
could be sequestered to explain the dramatic and prolonged increases in
secretion rate. Amino acids do not act to draw significant volumes of fluid
into the lumen by osmosis, because amino acid concentrations in the fluid
secreted by 5-HT-stimulated tubules are typically <2% of those in the
bathing saline (Maddrell and Gardiner,
1980
). Consistent with this view is the finding from both rescue
and pre-incubation experiments that the sum of the concentrations of
Na+ and K+ in the secreted fluid (approximately 180 mmol
l-1) does not change in response to glutamine (Figs
8,
9). If secreted fluid
osmolality was maintained and the increase in fluid secretion rate of up to
7-fold was an osmotic consequence of glutamine transfer into the lumen then we
would expect to see a corresponding decline in the sum of the concentrations
of Na+ and K+, the major cations in the secreted
fluid.
In the rescue experiments, secretion rates peak approximately 30 min after
stimulation with 5-HT, then gradually decline to a stable plateau value. The
rundown is not due to depletion of metabolic substrates in the bathing saline,
as addition of more glucose does not restore secretion rates. There is no
change in the concentrations of Na+ and K+ in the
secreted fluid during this rundown (Fig.
8). This indicates that the drop in secretion rate is not simply
due to the tubules reverting back to an unstimulated state due to a lack of
5-HT, as unstimulated tubules secrete a high K+ and low
Na+ fluid (Maddrell,
1991). Furthermore, the rundown is associated with a gradual
acidification of the lumen (Fig.
10). At maximal secretion rates, Rhodnius tubules
secreted a near-neutral fluid, whereas 60 min after stimulation, the pH of the
secreted fluid had dropped to approximately 6.4. The addition of glutamine to
the bathing solution during the rundown process appears to prevent or mitigate
further acidification, and is also associated with an increase in secreted
fluid Na+ concentration of approximately 30 mmol l-1 and
a nearly equimolar decrease in K+ concentration
(Fig. 8). In the preincubation
protocol as well, glutamine increased secretion and secreted fluid
Na+ concentration and decreased secreted fluid K+
(Fig. 9). Moreover, tubules
pre-incubated in glutamine secreted fluid 0.7 pH units more alkaline than
control tubules preincubated in glutamine-free Rhodnius saline
(Fig. 10). These results
suggest that glutamine is not simply acting as a metabolite for the tubules or
as a significant contributor to secreted fluid osmolality but is instead
having specific effects on apical ion transporters. If the presence of
glutamine augments apical Na+/H+ exchange, for example
luminal pH and sodium concentrations would increase, as observed. Glutamine
has been shown to stimulate an apical Na+/H+ exchanger
in piglet ileum (Rhoads et al.,
1997
).
The rundown of secretion rate in the absence of glutamine does not appear
to reflect a decline in intracellular levels of cAMP. Previous studies have
shown that inhibition of fluid secretion with exogenous cGMP (0.5 mmol
l-1; Quinlan and O'Donnell,
1998) is associated with an increase in [K+] and a
decrease in [Na+] in the secreted fluid. These effects on secretion
rate and on cation concentrations are reversed by the addition of 1 mmol
l-1 cAMP. By contrast, concentrations of Na+ and
K+ in secreted fluid do not change as secretion rates decline in
amino-acid-free saline (Fig.
8), and addition of cAMP does not restore secretion rate. In
addition, the rundown is distinct from that produced by lowering the level of
K+ in the bathing saline
(Maddrell et al., 1993
).
Irrespective of the rate of rundown of salines with different concentrations
of K+, tubule secretion rate can be restored by the addition of
glutamine.
Effects of amino acids on Drosophila MTs
Drosophila MTs were stimulated by either glutamine or methionine
in both continuous exposure and pre-incubation protocols. Tyrosine, alanine
and phenylalanine were stimulatory but only in continuous-exposure
experiments. The effects of glutamine and methionine thus outlast the duration
of exposure and may thus act through a different mechanism to stimulate fluid
secretion. Canavanine inhibits secretion by Locusta MTs
(Rafaeli and Applebaum, 1980)
but has no effect on Drosophila MTs. Taurine, a compatible osmolyte
that modulates ion transport by many tissues
(Guizouarn et al., 2000
;
Law, 1994
), is also without
effect on Drosophila MTs.
In particular, there appear to be important differences in the effects of
tyrosine versus glutamine on Drosophila tubules. Firstly,
the effects of glutamine and tyrosine appear to be independent, as the
addition of a saturating concentration of glutamine (20 mmol l-1)
to tubules first stimulated with 0.5 mmol l-1 tyrosine results in a
further increase in fluid secretion rate (approximately 15%). Secondly,
glutamine effects were apparent in both Na+-replete and
Na+-free saline, whereas tyrosine had no stimulatory effect when
tubules were bathed in Na+-free saline. It is also worth noting
that the addition of tyrosine has previously been shown to be required for
characteristic oscillations in transepithelial potential in
Drosophila MTs (Blumenthal,
2001), suggesting that tyrosine may play a pivotal physiological
role in this epithelium.
Species differences
Glutamine stimulates secretion by tubules of both species and in all three
protocols. However, there may be differences in the mechanism of stimulation.
Secreted fluid Na+ increases and secreted fluid K+
decreases in Rhodnius tubules in the presence of glutamine, whereas
there is no change in secreted fluid Na+ or K+ when
glutamine is added to the saline bathing Drosophila tubules.
For other amino acids, there are different effects in the two species. In the pre-incubation experiments, there were more amino acids that were stimulatory (11) for Rhodnius tubules than there were for Drosophila (2). Five amino acids, including cysteine, inhibited Rhodnius tubules, whereas only cysteine inhibited Drosophila tubules. Methionine stimulated tubules of Drosophila but not those of Rhodnius. In contrast to the stimulatory nature of tyrosine for Drosophila tubules, it appears to have a slight inhibitory effect for Rhodnius MTs. Lysine and arginine, which inhibited Rhodnius MTs, had no effect on Drosophila tubules. Differences in inhibition by amino acids such as lysine, arginine and cysteine between the two species and in different protocols may relate to differences in rates of uptake and loss of each amino acid.
How do amino acids modulate Malpighian tubule ion transport?
Given the differences in the effects of different amino acids on fluid
secretion by tubules of Rhodnius and Drosophila, it seems
likely that there is more than one mechanism by which amino acids modulate
transepithelial ion transport. Although full analysis of such mechanisms is
beyond the scope of this paper, it is possible to rule out a number of
possibilities. As noted above, it is unlikely that stimulatory amino acids
such as glutamine act as important osmolytes in driving transepithelial fluid
secretion. In addition, stimulation by glutamine does not appear to reflect an
important role for glutamine in metabolic energy production. For
Drosophila MTs, glutamine has been shown to support fluid secretion
in the absence of glucose at a rate of 66% of that of MTs running in control
saline. This suggests that glutamine may play a limited role as a metabolite
in Drosophila tubules. By contrast, glutamine is not a good metabolic
substrate for Rhodnius MTs; tubules secreting in saline with
glutamine and no glucose secrete at a rate of only 10-15% of those of the
controls. Previous studies indicate that Rhodnius tubules do not
metabolize amino acids at significant rates
(Maddrell and Gardiner, 1980).
Our experiments using inhibitors of glutamine metabolism confirm and extend
this conclusion. Moreover, pre-incubation experiments for tubules of both
species clearly show that the effects of glutamine are still apparent for more
than an hour after the tubules have been transferred to an amino-acid-free
saline.
Free amino acids such as glutamine and proline play important roles as
compatible osmolytes in the regulation of cell volume. Isolation of tubules in
amino-acid-free saline may thus compromise cell volume regulation as amino
acids are gradually lost from the cells. Under these circumstances, the
slowing of transepithelial ion transport may be protective of cell function,
in that further loss of intracellular osmolytes may well also be slowed.
Changes in cell hydration, i.e. in cell volume, can act as important
regulators of cell function, and changes in cell volume through the effects of
hormones and amino acids can thus alter cell function (e.g.
Häussinger, 1996). In
particular, changes in cell volume can alter mitogen-activated protein (MAP)
kinases and related kinases such as stress-activated protein kinase (SAPK;
Häussinger, 1996
). Future
studies will address in detail the cellular mechanisms involved in modulation
of ion transport by insect Malpighian tubules.
The mechanisms by which high concentrations of certain amino acids inhibit
fluid secretion may relate to their physicochemical properties. Lysine and
arginine are not compatible osmolytes, and accumulation of high intracellular
levels in response to elevated bathing saline concentrations may perturb the
functioning of proteins required for rapid ion transport and fluid secretion.
High intracellular levels of cysteine may perturb intracellular redox status.
Cysteine is a thiol-containing amino acid and a rate-limiting precursor of
glutathione but it can also exert effects on cell function independent of
effects on intracellular glutathione levels
(Noda et al., 2002;
Hildebrandt et al., 2002
).
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Auerswald, L., Schneider, P. and Gade, G.
(1998). Utilisation of substrates during tethered flight with and
without lift generation in the African fruit beetle Pachnoda sinuata
(Cetoniinae). J. Exp. Biol.
201, 2333
-2342.
Barrett, F. M. (1974). Changes in the concentration of free amino acids in the haemolymph of Rhodnius prolixus during the fifth instar. Comp. Biochem. Physiol. B 48, 241 -250.[Medline]
Barrett, F. M. and Friend, W. G. (1975). Differences in the concentration of free amino acids in the haemolymph of adult male and female Rhodnius prolixus. Comp. Biochem. Physiol. B 52, 427 -431.[Medline]
Blumenthal, E. M. (2001). Characterization of
transepithelial potential oscillations in the Drosophila Malpighian
tubule. J. Exp. Biol.
204, 3075
-3084.
Chamberlin, M. E. and Phillips, J. E. (1982). Regulation of hemolymph amino acid levels and active secretion of proline by Malpighian tubules of locusts. Can. J. Zool. 60, 2745 -2752.
Dow, J. A., Maddrell, S. H., Gortz, A., Skaer, N. J., Brogan, S.
and Kaiser, K. (1994). The Malpighian tubules of
Drosophila melanogaster: fluid secretion and its control.
J. Exp. Biol. 197, 421
-428.
Evans, P. D. and Crossley, A. C. (1974). Free amino acids in the haemoytes and plasma of the larva of Calliphora vicina. J. Exp. Biol. 61, 463 -472.[Medline]
Firling, C. E. (1977). Amino acid and protein changes in the haemolymph of developing fourth instar Chironomus tentans. J. Insect Physiol. 23, 17 -22.[Medline]
Fumarola, C., Zerbini, A. and Guidotti, G. G. (2001). Glutamine deprivation-mediated cell shrinkage induces ligand-independent CD95 receptor signaling and apoptosis. Cell Death Differ. 8, 1004 -1013.[CrossRef][Medline]
Gordon, R. and Bailey, C. H. (1974). Free amino acid composition of the hemolymph of the larval blackfly Simulium venustum. Experientia 30, 902 -903.[Medline]
Gringorten, J. L. (1979). Aspects of the flight physiology of Rhodnius prolixus: wing-beat pattern, water balance, and amino-acid changes during exhaustive flight. PhD Thesis, University of Toronto.
Guizouarn, H., Motais, R., Garcia-Romeu, F. and Borgese, F.
(2000). Cell volume regulation: the role of taurine loss in
maintaining membrane potential and cell pH. J.
Physiol. 523, 147
-154.
Haley, C. A. and O'Donnell, M. J. (1997).
K+ reabsorption by the lower Malpighian tubules of Rhodnius
prolixus: inhibition by Ba2+ and blockers of
H+/K+-ATPases. J. Exp. Biol.
200, 139
-147.
Håussinger, D. (1996). The role of cellular hydration in the regulation of cell function. Biochem. J. 313, 697 -710.[Medline]
Hildebrandt, W., Kinscherf, R., Hauer, K., Holm, E. and Droge, W. (2002). Plasma cystine concentration and redox state in aging and physical exercise. Mech. Ageing Dev. 123, 1269 -1281.[CrossRef][Medline]
Law, R. O. (1994). Taurine efflux and cell volume regulation in cerebral cortical slices during chronic hypernatraemia. Neurosci. Lett. 185, 56 -59.[CrossRef]
Linton, S. M. and O'Donnell, M. J. (1999).
Contributions of K+:Cl- cotransport and
Na+/K+-ATPase to basolateral ion transport in Malpighian
tubules of Drosophila melanogaster. J. Exp. Biol.
202, 1561
-1570.
Maddrell, S. H. P. (1963). Excretion in the blood-sucking bug, Rhodnius prolixus Stål. J. Exp. Biol. 40, 247 -256.
Maddrell, S. H. P. (1991). The fastest fluid-secreting cell known: the upper Malpighian tubules cell of Rhodnius. BioEssays 13, 357 -362.
Maddrell, S. H. P. and Gardiner, B. O. C. (1980). The retention of amino acids in the haemolymph during diuresis in Rhodnius. J. Exp. Biol. 87, 315 -329.
Maddrell, S. H. P. and O'Donnell, M. J. (1992).
Insect Malpighian tubules: V-ATPase action in ion and fluid transport.
J. Exp. Biol. 172, 417
-429.
Maddrell, S. H. P., O'Donnell, M. J. and Caffrey, R.
(1993). The regulation of haemolymph potassium activity during
initiation and maintenance of diuresis in fed Rhodnius prolixus. J.
Exp. Biol. 177, 273
-285.
Noda, T., Iwakiri, R., Fujimoto, K., Rhoads, C. A. and Aw, T. Y. (2002). Exogenous cysteine and cystine promote cell proliferation in CaCo-2 cells. Cell Prolif. 35, 117 -129.[CrossRef][Medline]
O'Donnell, M. J., Dow, J. A., Huesmann, G. R., Tublitz, N. J.
and Maddrell, S. H. (1996). Separate control of anion and
cation transport in Malpighian tubules of Drosophila melanogaster.
J. Exp. Biol. 199, 1163
-1175.
O'Donnell, M. J. and Maddrell, S. H. P. (1995).
Fluid reabsorption and ion transport by the lower Malpighian tubules of adult
female Drosophila. J. Exp. Biol.
198, 1647
-1653.
Patrick, M. L. and Bradley, T. J. (2000). The
physiology of salinity tolerance in larvae of two species of Culex
mosquitoes: the role of compatible solutes. J. Exp.
Biol. 203, 821
-830.
Quinlan, M. C. and O'Donnell, M. J. (1998). Anti-diuresis in the blood-feeding insect Rhodnius prolixus Stål: antagonistic actions of cAMP and cGMP and the role of organic acid transport. J. Insect Physiol. 44, 561 -568.[CrossRef][Medline]
Quinlan, M. C., Tublitz, N. J. and O'Donnell, M. J.
(1997). Anti-diuresis in the blood-feeding insect Rhodnius
prolixus Stål: the peptide CAP2b and cyclic GMP inhibit Malpighian
tubule fluid secretion. J. Exp. Biol.
200, 2363
-2367.
Rafaeli, A. and Applebaum, S. W. (1980). Canavanine potentiates the response of insect Malpighian tubules to diuretic hormone and cyclic AMP. Nature 283, 872 -873.
Rhoads, J. M., Argenzio, R. A., Chen, W., Rippe, R. A.,
Westwick, J. K., Cox, A. D., Berschneider, H. M. and Brenner, D. A.
(1997). L-glutamine stimulates intestinal cell proliferation and
activates mitogen-activated protein kinases. Am. J.
Physiol. 272, G943
-G953.
Tobe, S. S. (1978). Changes in free amino acids and peptides in the haemolymph of Glossina austeni during the reproductive cycle. Experientia 34, 1462 -1463.[Medline]
Wigglesworth, V. B. (1972). The Principles of Insect Physiology, 5th edn. London: Chapman and Hall.
Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D. and Somero, G. N. (1982). Living with water stress: evolution of osmolyte systems. Science 217, 1214 -1222[Medline]
Zanotto, F., Gouveia, S., Simpson, S. and Calder, D.
(1997). Nutritional homeostasis in locusts: is there a mechanism
for increased energy expenditure during carbohydrate overfeeding?
J. Exp. Biol. 200, 2437
-2448.