Variation of crustacean hyperglycemic hormone (cHH) level in the eyestalk and haemolymph of the shrimp Palaemon elegans following stress
BRAIN Center, Department of Biology, University of Trieste, via Giorgieri 7, I-34127 Trieste, Italy
* Author for correspondence (e-mail: ferrero{at}univ.trieste.it)
Accepted 27 August 2004
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Summary |
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Key words: Crustacea, cHH, glucose, heavy metals, lipopolysaccharide, water quality
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Introduction |
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Our studies (Lorenzon et al.,
2000) on the effect of heavy metals on blood glucose levels in
P. elegans showed that the intermediate sublethal concentrations of
Hg, Cd and lead (Pb) produced significant hyperglycaemic responses, while the
highest concentrations elicited no hyperglycaemia in the 24 h following
treatment. By contrast, animals exposed to Cu and zinc (Zn) showed
hyperglycaemia, even at high concentrations. This difference in response can
probably be explained by the physiological roles of the essential elements Cu
and Zn in crustaceans, and consequent tolerance adaptations, as opposed to the
toxic xenobiotic heavy metals Cd, Hg and Pb. Hyperglycaemic responses to both
groups of heavy metals are not elicited in eyestalk-ablated animals,
therefore, they involve MTXO-SG hormones, probably cHH.
However, in spite of the richness of information about blood glucose
variation following stress, much less is known about the stress-induced
variation of cHH content in the sinus gland and circulating in the haemolymph.
In the crayfish Orconectes limosus undergoing hypoxia, cHH titres of
around 120 pmol l1 are reached within 15 min
(Keller and Orth, 1990). In
Cancer pagurus, emersion induced an increase of the haemolymph cHH
after 4 h (Webster, 1996
).
Chang et al. (1998
) used ELISA
to monitor the blood cHH variation in Homarus americanus following
various environmental stresses. Emersion proved to be a potent stimulator for
elevation of cHH, while temperature and salinity variations were less
effective.
Moreover, in C. maenas it has been shown that the concentration of
cHH in the haemolymph increases dramatically during moulting: from 15
fmol 100 µl1 in the intermoult, up to 150200 fmol
100 µl1 during ecdysis
(Chung et al., 1999). More
recently, variation of cHH titre was reported in the haemolymph of
Nephrops norvegicus infected by the parasitic dinoflagellate
Hematodinium sp. (Stentiford et
al., 2001
). Finally, increased water temperature in Cancer
pagurus and Procambarus clarckii induced an increment in blood
cHH (Wilcockson et al., 2002
;
Zou et al., 2003
). The aim of
this paper was to monitor the variation of cHH in the eyestalks and haemolymph
of P. elegans (Decapoda, Caridea) following various stresses (heavy
metals and LPS), and to relate cHH levels to the variation of amount and time
course of blood glucose.
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Materials and methods |
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Haemolymph sampling and determination of glycaemia
The animals were blotted dry and haemolymph (50 µl) was withdrawn from
the pericardial sinus into a sterile 1 ml syringe fitted with 25 g needles.
Animals (N=10 for each treatment) were bled at 0 h, usually between
910 a.m., to reduce possible interference due to circadian change in
blood-glucose level (Kallen et al.,
1990).
Haemolymph glucose content was quantified by using One touch® II Meter (Lifescan, Miltipas, CA, USA) and commercial kit test strips (precision of strips±3% coefficient of variation in the tested range). Owing to the speed of processing, no anticoagulant was needed. In the results, variations of glycaemia defined as increments are given as the mean of: [(experimental value)/(value displayed by the same animal at 0 h)]1.
Effect of HgCl2, CuCl2 and lipopolysaccharide (LPS) on blood glucose level
Variation of glycaemia was tested on groups of intact P. elegans
(N=10 for each treatment) following exposure to Hg2+ (0.1,
0.5 and 5 mg l1, administered as HgCl2),
Cu2+ (0.1 and 5 mg l1, administered as
CuCl2) in seawater, and the controls maintained in uncontaminated
water. At 0, 0.5, 1, 2, 3 and 24 h after exposure to heavy metals, animals
were bled as described above.
For LPS, groups of 10 animals were injected with 0.1 and 2 mg
g1 live weight of LPS from Escherichia coli
0111:B4; variation of glycaemia was determined as described above at 0, 0.5,
1, 2, 3, 5 and 24 h. Control groups injected with saline were tested at the
same time; sterile saline for marine crustaceans was prepared with
pyrogen-free distilled water and analytical grade chemicals, according to
Smith and Ratcliffe (1978) and
autoclaved for 25 min. All reagents were supplied by Sigma-Aldrich (St Louis,
Missouri, USA).
Eyestalk homogenate and haemolymph treatment for ELISA measurement of cHH variation
Groups of 10 P. elegans were exposed to Hg2+ (0.1, 0.5
and 5 mg l1), Cu2+ (0.1 and 5 mg
l1) or injected with LPS (0.1 and 2 mg g1
live weight); untreated animals and saline-injected animals were used as
controls. Eyestalks were removed at time 0 and then at 0.5, 1, 2 and 3 h of
exposure (and at 5 h only for LPS) from the 10 animals of each different
experimental group. Animals were anaesthetized for 1 min on ice before
ablation. The eyestalk was quickly frozen and the pigmented eyecup dissected.
Eyestalk homogenate was prepared from 20 eyestalks homogenized in 2 ml cold
phosphate-buffered saline (PBS Sigma) pH 8.0, and then centrifuged for 1 h at
930 g and 4°C, and the pellet discarded. Homogenates were
quickly deep frozen at 20°C and stored until required for
study.
Haemolymph was withdrawn from different groups of 10 P. elegans for each treatment and time, as described above at time 0 and then at 0.5, 1, 2 and 3 h (and at 5 h only for LPS), immediately centrifuged for 1 min at 10,300 g and 4°C, and the supernatants then stored at 20°C.
Direct enzyme-linked immunosorbent assay (ELISA) of cHH
The samples of eyestalk homogenate were always tested at the concentration
of 1 sinus gland equivalent (SGe) in 100 µl and at dilutions of 0.5, 0.1,
0.05, 0.01 and 0.001 SGe in PBS (pH 8.0). Haemolymph was tested undiluted and
at dilutions of 0.5, 0.1, 0.05 in PBS. The standards were known amount (from 1
to 0.001 µg in 100 µl of PBS) of 6 xHis-NencHHwt
(Mr=11 KDa) recombinant protein
(Mettulio, 2002).
100 µl of the samples (eyestalk homogenate, or haemolymph, from the
different treatments and standards) were loaded onto a 96-microwell plate
(Costar, Bethesda, MD, USA) and incubated in duplicate overnight at 4°C.
The content of the wells was discarded and the wells were washed four times
with 250 µl of PBS-T (PBS+0.1% Tween20, pH 7.4), then filled with 100 µl
of 3% bovine serum albumine (BSA, Sigma) solution in PBS pH 7.4 plus 5% foetal
calf serum (FCS, Sigma) and left for 2 h at room temperature (RT). The content
was discarded and the plates washed four times as described above. 100 µl
of the biotinylated anti-NencHH (anti N. norvegicus cHH;
Giulianini et al., 2002)
antibody (1 µg µl1) diluted 1:1000 was then added to
each well and the plate was incubated for 3 h at 36°C.
After removal of the biotinylated antibody, plates were washed extensively with PBS-T, followed by the addition of 100 µl of streptavidin-peroxidase solution (Sigma) diluted 1:5000 and incubated for 1 h at RT. The plates were once again washed four times with PBS-T and developed with 2,2'-azinobis-3-ethylbenz-thiazoline-6-sulphonic acid solution (Sigma; liquid substrate ready for use) in darkness for 1 h at RT (100 µl per well). The absorbance was measured in a multiwell plate reader (Anthos 2020 version 1.1; Krefeld, Germany) at 405 nm.
Fig. 1 presents an example of the standard curve obtained from dilutions of a known amount of a 6 xHis-NencHHwt. The graph shows the mean and standard deviations (after subtraction of the background absorption) of the quadruplicate determinations of recombinant cHH plotted against the optical density (OD) with the calculated linear regression (Fig. 1).Sample values were then inserted into the equation and the amount of unknown cHH thereby determined.
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Statistical analysis
All statistics were performed by using a SPSS 9® (SPSS Inc., Chicago,
IL, USA) for Windows package, and data are given as arithmetic means ±
S.D. Effects of experimental treatments on blood glucose levels
were analysed. Analysis of variance (ANOVA) and Student's t-test were
used to test the null hypotheses that all treatment means were equal, and then
all the data were tested by the LSD and Dunnett post hoc test. The
levels of significance were then calculated by Student's t-test for
paired or independent data. A probability value of <0.05 of the statistical
tests between the control and experimental values (mean ±
S.D.) was considered significant. To test the statistical
significance of ELISA values compared between experiments, Student's
t-test was used at P<0.05. Time scale of graphs is not
proportional, to provide a better visual inspection of data and size of the
illustration.
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Results |
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In animals injected with saline, the level of cHH in the eyestalk reached a minimum of 5.3±1.7 pmol SGe1 (N=3) 1 h after injection, a value that was not significantly different from the resting value of the untreated animals (P=0.855); in the haemolymph, a maximum of circulating cHH of 1.23±0.603 pmol ml1 was detected 30 min after injection of saline, which was not significantly different (P=0.930) from the resting value of untreated animals. Sterile saline was also used as control for animals injected with LPS.
Time course of cHH levels in the eyestalk and haemolymph, and of blood glucose, following exposure to Cu2+, Hg2+ and LPS
The effects of exposure to Cu2+ on cHH level in the eyestalk of
P. elegans are shown in Fig.
2, where 0.1 mg l1 is a sublethal concentration
and 5 mg l1 is a lethal concentration (as determined by
Lorenzon et al., 2000).
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The highest Cu2+ concentration induced a massive release of cHH from the sinus gland. After 30 min the cHH content is 0.71±0.21 pmol SGe1, significantly lower (P=0.006) than in the untreated controls (used as time 0) and the hormone level reached a minimum of 0.45±0.27 pmol SGe1 (P=0.004 vs control) after 2 h. Thereafter, the cHH started to recover with a content of 2.70±3.94 pmol SGe1 detected 3 h after exposure and not significantly different (P=0.126) from the control.
The Cu2+ concentration of 0.1 mg l1 (Fig. 2) caused a gradual release of cHH. The value of cHH decreased to 3.37±2.99 pmol SGe1 1 h after exposure, which is not significantly different from the control (P=0.207). At 2 h, the significant minimum content of cHH of 2.29±0.81 pmol SGe1 (P=0.04) was reached. Thereafter, from 3 h onwards the cHH titre returned to the pre-treatment level of 5.04±4.90 pmol SGe1 (P=0.774 vs untreated control).
Exposure to Cu2+ 5 mg l1 caused a significant elevation of haemolymph cHH (Fig. 3) that rose up to 8.67±2.99 pmol ml1 after 2 h, a value that was significantly different (P=0.001) from that of the untreated animals (1.13±0.28 pmol ml1). The circulating cHH remained significantly (P<0.05) high throughout the experimental period. In animals exposed to the lowest Cu2+ concentration, the blood cHH remained on resting levels and only after 2 h increased slightly, but significantly (P=0.013 vs control), up to 1.68±0.34 pmol ml1. Exposure of P. elegans to Cu2+ induced a dose-related release of cHH from eyestalk to haemolymph.
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Fig. 4 shows the consequent dose-related variation of glycaemia. At the highest concentration, in the first 3 h after exposure, a significant increase in blood glucose became evident, with a peak of increment of 2.60±0.49 (33.80±4.89 mg dl1) at 2 h that was significantly different from the initial value (9.50±1.65 mg dl1; P=0.001) and also from the control at the same time (13.30±2.75 mg dl1; P=0.001). Animals exposed to 0.1 mg l1 Cu2+ showed a similar time course of hyperglycaemia but with a lower increment in blood glucose. A maximum increment of 0.77±0.25 (15.00±1.49 mg dl1) was revealed at 2 h, which is significantly different from the control (13.30±2.75 mg dl1; P=0.046) and from the value at the higher concentration (P=0.001) at the same time.
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Fig. 5 shows the time course
of cHH eyestalk content after exposure of P. elegans to
Hg2+ at the sublethal concentrations (as defined by
Lorenzon et al., 2000) of 0.1
and 0.5 mg l1, and at a lethal concentration of 5 mg
l1, compared with the untreated control. At the lethal
concentration, a rapid and massive release of cHH from the sinus gland became
evident; in fact after 30 min from exposure, the hormone content decreased to
1.22±0.39 pmol SGe1, significantly (P=0.012)
lower than the control (5.60±2.6 pmol SGe1). Values
remained significantly (P<0.05) below the resting level for all
the experimental period with a minimum of 0.72±0.25 pmol
SGe1 detected at 2 h.
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The intermediate Hg2+ concentration (0.5 mg l1; Fig. 5) was even more effective at inducing release of cHH: after 30 min the hormone content in the eyestalk was 0.19±0.07 pmol SGe1 (P=0.003 vs control) and the eyestalk remained massively and significantly (P<0.05) depleted of the hormone content throughout the experimental time considered. Finally, the lowest concentration of 0.1 mg l1 induced a limited release of cHH that was significantly different (P=0.033) from the control at 1 h after exposure with a cHH level of 1.97±0.62 pmol SGe1.
Exposure to Hg2+ (Fig. 6), at the concentration of 5 mg l1, induced a rapid increase of cHH concentration in the haemolymph with a peak of 5.90±0.68 pmol ml1 after 1 h that was significantly different (P=0.001) from the unexposed control (1.13±0.28 pmol ml1). Thereafter, the circulating cHH remained significantly (P<0.05) higher than in the control. The intermediate concentration was most effective in elevating blood cHH: the hormone concentration rapidly increased and remained significantly (P<0.05) higher than in control group, with a maximal concentration of circulating hormone (6.92±1.27 pmol ml1) at 2 h (Fig. 6). At the lowest Hg2+ concentration (0.1 mg l1), a significant increase (P<0.05) of circulating cHH was recorded 1 and 2 h after exposure with a maximum of 2.14±0.74 pmol ml1. Variation of eyestalk and blood cHH in the case of exposure to Hg2+ is not dose related; in fact, the intermediate concentration proved to be the most effective in altering physiological resting values, with a higher significant (P<0.05) release of cHH from the eyestalk.
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This is paralleled by the variation of blood glucose. Fig. 7 shows time course of glycaemia after exposure of P. elegans to different concentrations of Hg. In animals exposed to 0.5 mg l1 Hg, marked hyperglycaemia became evident after 1 h, which peaked at 3 h with an increment of 1.35±0.42 (22.20±5.45 mg dl1), and a concentration that was significantly different from the initial value of 9.40±1.35 mg dl1 (P=0.001) and from the control at the same time 0.28±0.23 (13.10±2.75 mg dl1, P=0.001). By contrast, at the highest concentration (Fig. 7) no significant variation of blood glucose was revealed for all the experimental times: the maximum increment of 0.28±0.22 (13.88±2.38 mg dl1) recorded at 2 h was not significantly different from the initial value (P>0.05) and from the control (P>0.05). Animals exposed to 0.1 mg l1 (Fig. 7) showed slight hyperglycaemia at 2 h with an increment of 0.58±0.20 (16.30±1.83 mg dl1), significantly different from the time 0 value (10.40±1.51 mg dl1; P=0.001) and from the control, which, at the same time, revealed an increment of 0.29±0.24 (13.30±2.75 mg dl1; P=0.009).
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P. elegans injected with 2 mg g1 living weight of LPS from E. coli 0111:B4 showed a massive release of cHH from the eyestalk (Fig. 8). After 30 min, the hormone level was 0.26±0.03 pmol SG1, which is significantly different from the initial value of 5.60±2.6 pmol SG1 (P=0.003) and from the saline control at the same time point (5.47±1.86 pmol SGe1; P=0.008). After 3 h, the cHH level rose, slightly, to 0.75±0.14 pmol SGe1 and, eventually, 5 h after injection the hormone content was 1.42±0.39 pmol SGe1, which was still significantly lower (P=0.007 and P=0.016, respectively) than the saline control. The lower dose of 0.1 mg g1 (Fig. 8) induced the cHH release in the first hour after injection, with a minimum eyestalk content, revealed at 30 min, of 2.39±0.93 pmol SGe1, which is also significantly different from the saline control (P<0.05). Thereafter, we observed a gradual recovery of the hormone level. From 2 h onwards the cHH eyestalk content (4.11±2.59 pmol SGe1) was not significantly different from control (5.41±0.3 pmol SGe1; P=0.435) and after 5 h the value returned back to a normal resting value of 5.21±1.23 pmol SGe1 (P=0.804 vs control).
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Following the massive cHH release from the eyestalk in animals that had been treated with 2 mg g1 of LPS (Fig. 9), high levels of circulating hormone were detected in the haemolymph. After 30 min, the cHH level rose to 7.12±1.81 pmol ml1, which was significantly different from the initial value in untreated (P=0.001) animals (1.13±0.28 pmol ml1) and from the saline-injected control analysed at the same time point (1.23±0.6 pmol ml1; P=0.006). Circulating hormone concentration remained high until 2 h; afterwards the level gradually decreased to 4.45±1.65 pmol ml1 (P=0.001 vs saline control) at 3 h and to 2.02±0.16 pmol ml1 after 5 h, which is still significantly higher than control (P=0.001). In animals injected with 0.1 mg g1 of LPS (Fig. 9), an increase in circulating cHH was detected from 30 min with a significant (P=0.001 vs control) peak of 4.47±1.26 pmol ml1 at 1 h, then the level decreased and after 3 h there was no significant difference from the control (P>0.05; Fig. 9).
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Blood glucose variation after injection of LPS (Fig. 10) follows the time course of haemolymph cHH. At the highest dose, from 30 min after injection, an increment of glycaemia of 1.55±1.09 (26.90±12.40 mg dl1) is revealed a concentration that was significantly different from initial value (10.70±2.71 mg dl1; P=0.001) and from the control at the same time (11.40±2.37 mg dl1, P=0.001). At 2 h, injected animals showed a peak of increment of 4.79±2.06 (58.90±16.93 mg dl1), and in the next 24 h blood glucose level gradually recovered to resting values. The lower dose (0.1 mg g1) induced a slight hyperglycaemia at 1 h with a maximal increment of 1.22±0.44 (19.10±4.01 mg dl1) significantly different from time 0 value (8.70±1.42 mg dl1 P=0.001) and from control (P=0.001). Thereafter, no significant variation (P>0.05) of blood glucose was recorded.
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Discussion |
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The resting value of cHH in P. elegans eyestalk was found to be
5.6±2.6 pmol SGe1 (62±29 ng
SGe1), which is in the range of those already detected in
other crustacean species. Huberman et al.
(1995) identified in
Procambarus bouvieri two isoforms of the cHH (cHH-I e cHH-II) in
approximate concentrations of 60 and 20 ng SG1,
respectively. Chang et al.
(1990
) found in Homarus
americanus a cHH content in the sinus gland of 3 pmol
SG1. More recently, Marco et al.
(2000
) quantified the content
of cHH-I in the spiny lobster Jasus lalandii as 20 pmol
SG1 and cHH-II as 3 pmol SG1. In C.
maenas and C. pagurus, a larger cHH content of 180 pmol
SG1 and 125 pmol SG1, respectively, was
detected (Keller et al., 1985
;
Webster, 1996
). All former
data were obtained by calibration on HPLC purified SG cHH and antibody raised
against it. We are presently unable to relate immunoreactivity of our
recombinant cHH and antibody with the natural purified neuropeptide. Because
of cross immunoreactivity, western blotting, and similar distribution
(Giulianini et al., 2002
) and
quantitative profiles, we are confident that immuno-localization and
quantification is specific for the native cHH as well.
In P. elegans, exposure to Cu induced a dose-related rapid and massive release of cHH from the eyestalk into haemolymph at the higher, lethal concentration, whereas a gradual and reduced discharge was revealed at the lower concentration. The relationship between exposure to toxicant and release of cHH is confirmed by variation of blood glucose with a dose-related hyperglycaemia that peaked 2 h after exposure to Cu.
Animals exposed to previously defined
(Lorenzon et al., 2000)
sublethal concentrations of Hg showed the same quantitative and time-course
relationships between toxicant and release of cHH from the eyestalk, increment
of hormone level in the haemolymph and subsequent hyperglycaemia (as already
described for Cu contamination). Interestingly, however, the highest, lethal
concentration of 5 mg l1
(Lorenzon et al., 2000
)
induced the release of cHH from the eyestalk to the haemolymph but was not
followed by a significant variation of blood glucose. This situation could be
related to the high toxicity of Hg, which may interfere with the finer
mechanisms that regulate the hyperglycaemic response. It is not caused by a
synaptic blockage of the superimposed neuronal release network
(Lorenzon et al., 1999
) or to
a limited release of circulating cHH because high levels of cHH are discharged
from the SG into the blood compartment. Likewise, it is not due to inhibition
of peripheral receptors on glucogenolytic target organs; indeed, native SG
homogenate injected into eyestalk-less shrimps exposed to lethal
Hg2+ for 3 h can still cause hyperglycaemia
(Lorenzon et al., 2000
). High
concentrations of Hg, instead, may change the functionality of the prepro-cHH
processed during secretory steps, due to Hg ability to bind cysteines
six of which represent a highly conserved feature of the peptide structure
(Lacombe et al., 1999
)
thereby altering the active conformation of the peptide, as seen in another
system (Rodgers et al., 2001
),
but not its immunoreactivity. Moreover, Hg is known to impair osmoregulatory
capability in the crab Eriocheir sinensis
(Péqueux et al., 1996
);
and in P. clarkii Hg induces an inhibition of the
acetylcholinesterase activity (Devi and
Fingerman, 1995
). Therefore, the altered response in P.
elegans exposed to high concentrations of Hg2+ may be related
to physiological modifications induced by Hg2+ at a different
systemic level.
Contamination with different doses of a bacterial thermostable endotoxin,
such as LPS from E. coli, confirms the dose-related and convergent
chain of events that bring about hyperglycaemia. It suggests that blood
glucose elevation is a general purpose response to stressors and is likely to
perform a protective role. Variation in the level of circulating cHH has been
reported following exposure to various stresses. In Cancer pagurus,
variation of haemolymph cHH is reported after emersion; hormone titre rapidly
increases from undetectable levels to 30 pmol l1 after 4 h
with a simultaneous increase of blood glucose
(Webster, 1996).
Chang et al. (1998) used
ELISA to measure the levels of circulating cHH in Homarus americanus
following three different kinds of stress: emersion, temperature elevation and
salinity change. Emersion was the most potent stimulator for the elevation of
haemolymph cHH increasing the baseline values of 4 fmol ml1
to 168.1 fmol ml1 after 4 h; thermal and salinity stress
caused only a slight increase in circulating hormone. In H.
americanus, the maximum increment of glycaemia is reached at 2 h after
emersion while the maximum cHH level in the haemolymph is revealed at 4 h. By
contrast, in our experiment, the peak of circulating cHH precedes, or at least
coincides with, the maximum level of blood glucose. Recently, Zou et al.
(2003
) demonstrated that
variation of water temperature from 24 to 34°C increases cHH levels from
32.4±4.9 fmol ml1 up to 123.3±21.1 fmol
ml1 after 2 h in P. clarkii.
Variation of blood cHH has been reported during the moult of C.
maenas (Chung et al.,
1999) and, in particular, a dramatic increase of the cHH level
became evident during ecdysis (from 15 fmol 100
µl1 of intermoult to 200 fmol 100
µl1). In this case, the release of cHH is not dependent
on the eyestalk, as an increase in circulating cHH is revealed also in
eyestalk-less animals and release from the gut-associated neuroendocrine
tissue. However, additional eyestalk release could be related with the cells
containing cHH, as identified by Chang et al.
(1999
) in the second roots of
the thoracic ganglia and in the suboesophageal ganglion.
Infection of Nephrops norvegicus by the parasitic dinoflagellate
Hematodinium sp. induces an elevation of the cHH titre in the
haemolymph (Stentiford et al.,
2001). Concentration of blood hormone increases with the severity
of the infection from 32.2 fmol ml1 in uninfected animals to
77.2 and 107.65 fmol ml1. In this case, the concentration of
glucose in the haemolymph is significantly reduced in infected animals,
probably due to the use of glucose as a substrate for the growth of the
parasite in the haemolymph (Stentiford et
al., 2001
).
The basal level of cHH in the haemolymph of P. elegans was 1.13±0.28 pmol ml1 (12±0.3 ng ml1), which is higher than in the species discussed above. This could depend on the species tested or, in the presence of a similar cHH eyestalk content and release, on a smaller volume of the body-fluid compartment and possibly slower turnover of the circulating hormone. Therefore, further information is needed about the time course and fine mechanisms that regulate the release of cHH, its binding to receptors, and about the half-life and the catabolism of this hormone in the haemolymph in species belonging to different taxa and/or of different inner-fluid-compartment volumes.
In conclusion, the results presented in the present study are the first data that: (1) relate the release of cHH from the eyestalk, the circulating hormone level and the consequent glycaemic response to stress; (2) provide evidence of the interference by Hg2+ on the regulation of this mechanism; (3) confirm the dose-related pathway that leads to variation of blood glucose as a quantitative biomarker of environmental quality, even at sublethal toxicant concentration.
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Acknowledgments |
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