Putative involvement of crustacean hyperglycemic hormone isoforms in the neuroendocrine mediation of osmoregulation in the crayfish Astacus leptodactylus
Laetitia Serrano1,
Gaëlle Blanvillain1,
Daniel Soyez2,
Guy Charmantier1,
Evelyse Grousset1,
Fabien Aujoulat1 and
Céline Spanings-Pierrot1,*
1 Laboratoire Génome, Populations, Interactions, Adaptation, UMR
5000, Equipe Adaptation Ecophysiologique et Ontogenèse,
Université Montpellier II, Place E. Bataillon, CP 092, 34095
Montpellier Cédex 05, France
2 Groupe Biogenèse des Peptides Isomères, UMR CNRS Physiologie
et Physiopathologie, Université Paris VI, CC 256, 7 Quai Saint-Bernard,
75252 Paris Cédex 05, France
View larger version (28K):
[in a new window]
|
Fig. 4. (A) High-performance liquid chromatography (HPLC) fractionation of an
acetic acid extract from 50 SGequiv of Astacus leptodactylus. A
reverse-phase column (Nucleosil C-18; 250 mm length x 4.6 mm i.d.; 5
µm particle size) was used. Eluant A: 0.1% trifluoroacetic acid (TFA) in
water; eluant B: 0.1% TFA in 100% acetonitrile. The elution gradient of eluant
B is indicated by the dotted line. Flow rate was 750 µl min-1
and fraction duration was 1 min. UV detection was set at 220 nm. Arrows P1, P2
and P3 show the major immunoreactive peaks. Fractions 45-48 were pooled in
zones Z1 (fractions 45+46), Z2 (fraction 47) and Z3 (fraction 48) for
bioassay. (B—D) Results of the enzyme-linked immunosorbent assay (ELISA)
tests performed on 5 µl of each HPLC fraction with different antisera: (B)
anti-Astacus crustacean hyperglycemic hormone (CHH); (C) anti-D
antiserum; (D) anti-L antiserum. The columns represent the absorbance
determined at 405 nm after 120 min.
|
|
View larger version (17K):
[in a new window]
|
Fig. 1. Time course of variations in hemolymph osmolality in control
(N=7-8) and eyestalkless (N=6-11) Astacus
leptodactylus. Among the measurements taken from the beginning of the
experiment, only values of control crayfish that had molted before 35 days are
considered. All eyestalkless crayfish molted within 22-23 days of the start of
the experiment and only six crayfish were still alive after 43 days. Each
column represents the mean ± S.E.M. Different letters indicate
statistical differences: a—b, P<0.01; a—c,
P<0.001; b—c, P<0.05.
|
|
View larger version (10K):
[in a new window]
|
Fig. 2. Variations of mass in control (N=8) and eyestalkless
(N=6-11) Astacus leptodactylus before and after molting of
all animals at 14 days and 43 days, respectively. Only values of control
crayfish that had molted before 35 days are considered. Each column represents
the mean ± S.E.M. percentage increase compared with the initial mass
(preceding the operation). Statistical comparisons with controls:
*P<0.001.
|
|
View larger version (17K):
[in a new window]
|
Fig. 3. Hemolymph Na+ (A) and Cl- (B) concentrations in
control (N=8) and eyestalkless (N=6-11) Astacus
leptodactylus before and after molting of all animals at 14 days and 43
days, respectively. Only values of control crayfish that had molted before 35
days are considered. Each column represents the mean ± S.E.M.
Statistical comparisons with controls: *P<0.005.
|
|
View larger version (15K):
[in a new window]
|
Fig. 5. Changes in glycemia of eyestalkless Astacus leptodactylus 3 h, 8 h
and 24 h following injection of high-performance liquid chromatography (HPLC)
fractions (10 SGequiv) corresponding to zones Z1 (N=8), Z2
(N=8) and Z3 (N=8) (see
Fig. 4A). Each column
represents the mean ± S.E.M. of the increase in hemolymph glucose
concentration compared with the control. Statistical comparison with controls
injected with a Van Harreveld saline (N=6): *P<0.05;
**P<0.01; ***P<0.001.
|
|
View larger version (17K):
[in a new window]
|
Fig. 6. Changes in osmolality of eyestalkless Astacus leptodactylus 8 h
and 24 h following injection of high-performance liquid chromatography (HPLC)
fractions (10 SGequiv) corresponding to zones Z1 (N=8), Z2
(N=8) and Z3 (N=8) (see
Fig. 4A). Each column
represents the mean ± S.E.M. of the increase in hemolymph osmolality
compared with the control. Statistical comparison with controls injected with
a Van Harreveld saline (N=6): *P<0.05.
|
|
View larger version (14K):
[in a new window]
|
Fig. 7. Changes in hemolymph Na+ (A) and Cl- (B)
concentrations of eyestalkless Astacus leptodactylus 8 h and 24 h
following injection of high-performance liquid chromatography (HPLC) fractions
(10 SGequiv) corresponding to zones Z1 (N=8), Z2 (N=8) and
Z3 (N=8) (see Fig.
4A). Each column represents the mean ± S.E.M. of the
increase in ion concentration compared with the control. Statistical
comparison with controls injected with a Van Harreveld saline (N=6):
*P<0.05; **P<0.01; ***P<0.005.
|
|
© The Company of Biologists Ltd 2003
