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Variations of calcitonin-like immunoreactivity in the crustacean Orchestia cavimana during a molt cycle
GENERAL
AND
COMPARATIVE
Variations
F. GRAF*,
ENDOCRINOLOGY
73,80-84 (1989)
of Calcitonin-like lmmunoreactivity in the Crustacean Orchestia cavimana during a Molt Cycle
M. FOUCHEREAU-PERON?,A. VAN-W• RMHOUDT,~ AND J. C. MEYRAN”
*U.A. 674 Universite’ de Bourgogne, Laboratoire de Biologie Animale, 6, Boulevard Gabriel, 21100-Dijon, and fLaboratoire de Biologie Marine, Collkge de France, 29110 Concarneau, France Accepted July 11, 1988 The investigation of calcitonin-like immunoreactivity performed in whole extracts of the terrestrial crustacean Orchestia cavimana using radioimmunoassay revealed large amounts of these peptides varying in concentration according to the stages of the molt cycle. Their level is maximum at the time of the exuviation. The results are then discussed regarding the mtrticular calcium metabolism of this species in function of its terrestrial conditions of life.
0 1989 Academic
Press, Inc.
the calcium previously stored is reabsorbed within 24-48 hr (stage B) to mineralize the newly secreted cuticle (Graf, 1969; Graf and Michaut, 1977; Graf and Meyran, 1985; Meyran et al., 1986). Moreover, the possibility of a precise determination of the successive stages of the relatively short molt cycle is typical of this species (Graf, 1969, 1986). The‘immunoreactive investigation of CTlike molecules during the molt cycle of Orchestia was performed in comparison with what has been previously observed in marine crustaceans (Arlot-Bonnemains et al., 1986; Fouchereau-Peron et al., 1987) and was discussed regarding the particular calcium turnover of this terrestrial species during molting.
The calcitonins (CT) are widely distributed in vertebrates and their role in the regulation of calcium metabolism appears established in mammals. More recently, CTlike molecules have been detected in marine invertebrates especially in crustaceans (Arlot-Bonnemains et al., 1986; Fouchereau-Peron et al., 1987). Theirphysiological function is still unknown in urochordates (Fritsch et al., 1980; Girgis et al., 1980) and, in fish, gonadotrophic functions are suspected (Kline et al., 1987). These peptides are presumed to play a role in crustaceans with calcified cuticle in relation to the calcium turnover according to the different stages of the molt cycle (ArlotBonnemains et al., 1986; FouchereauPeron et al., 1987). However, vertebrate CT seems to have no effect on calcium metabolism in crustaceans (Cameron, 1987). In the terrestrial amphipod Orchestia cavimana, the calcium fluxes during molting are of particular interest: during 12 days before exuviation (stage D), there is an important shift of calcium from the old cuticle to the posterior caeca (PC) which are involved in temporary calcium storage (Graf, 1969; Graf and Meyran, 1983; Meyran et al., 1984). Then, after exuviation (stage A),
MATERIALS
Animals. The crustacean 0. cavimana Heller is the only European terrestrial amphipod living in moist biotopes, with a calcium metabolism during molting adapted to its hygrophile aerial conditions of life (Graf, 1969). In order to avoid possible hormonal diferences between age classes or sexes, only male adults, 18-20 mm long, raised in subaquatic terrariums (Graf, 1%9), were used in this study. They were alloted according to their molt stages following the staging method of Graf correlated with the cuticular depo80
0066480/89 Copyri&t AlI rights
$1.50
B 1989 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
CT-LIKE
MOLECULES
sition (Graf, 1986). For an average duration of 46 days, the whole molt cycle was divided into 14 stages. Just after exuviation, the animals were considered to be at stage A (early postmolt), and 12 to 36 hr after exuviation, at stage B (late postmolt). Hormonal measurements. Groups of I.5 whole animals exactly at the same molt stage (average fresh weight: 2 g) were analyzed: samples were sonicated and homogenized in 0.1 N hydrochloric acid. After centrifugation, supernatants were lyophylized and stored at -20” until assayed. The CT-like concentrations of the total extracts were determined with the heterologous radioimmunoassay of Treilhou-Lahille et al. (1984) using ‘251-labeled synthetic salmon CT (SCT) as described in Arlot-Bonnemains et al. (1986). Samples were analyzed with different dilutions until reproducible results were found. SCT antibodies (kindly donated by Dr. M. S. Moukhtar, Paris) were used at a l/20,000 dilution. Standards for SCT (lot 54 F.0335) were obtained from Sigma Chemical Company. Results of radioimmunological assay were expressed as the percentage of initial binding BIB0 where Bo represents the binding of labeled CT in the absence of unlabeled CT. Linearization of the standard curve was obtained by plotting ln (B/Bo)/l - (B/Be) as a function of In of CT concentration (Feldman and Rodbard, 1971).
RESULTS
Similar displacement of (‘251)-SCTbound to antibody was observed with serial dilutions of whole extracts from Orchestia (stage A) and SCT (Fig. 1). Analysis of the immunoreactivity of the logit
t to
$1
IN Orchestiu
whole extracts revealed on average relatively large amounts of CT-like material with important variations according to the different stages of the molt cycle (Fig. 2). The lowest concentrations were observed at the beginning of the inter-molt (stage Cd (about 25 pg/mg of fresh weight animal), before a weak increase at the end of this period (CJ. Then, after some decrease at the beginning of the preexuvial period (stages De-Dr”‘), the concentrations rapidly increased during the end of this period (stages D2-Ds) up to a maximum just at the time of the exuviation (stage A) (on average 2675 pg/mg of fresh weight animal). This sharp peak is finally followed by a rapid decrease in postexuvial period (stage B). The staging method is so precise in 0. cavirrzana as to enable us to assumethat the peak occurs exactly at the end of stage D3, i.e., just when the exuviation begins; a few hours later, the CT-like levels are signifrcantly lower. Moreover, the amplitude of the variation is remarkable. pg/mg
f.w.
t
intermolt 1
premolt I
1st inierm. !
6 B/Be)
I
1 I I11111100 log
I (pg
salmon
I I ,lll,
loo0
calcitonin)
FIG. 1. Immunoreactive salmon CT-like molecules in whole extracts of Orchestia during stage A.
FIG. 2. Variations of immunoreative CT-like peptides (&rng of fresh weight animal) in whole extracts of Orchestia during the different phases of the molt cycle.
82
GRAF ET AL.
ing hormone (Keller and Sedlmeier, 1988). In this respect, the maximum of CT-like peptides just at the time of the exuviation in Orchestia may be interpreted as a storage of the hormone in D,-A before its release in B-C as observed in the shrimp (ArlotBonnemains et al., 1986). So, it is still impossible to assign a precise role for these CT-like molecules in the calcium metabolism of Orchestia, as in other crustaceans. Moreover, it may not be sure that CT-like material acts in the same way in all crustaceans, owing to the differences of dynamics of the calcium turnover, according to their conditions of life. In marine decapods, where calcium shifts occur among water, cuticle, and hemolymph (Graf, 1978), the maximum of hemolymphatic CT-like peptides was observed during postmolt, which corresponds to a fall of the circulating calcium concentration (Arlot-Bonnemains et al., 1986). Such a hypocalcemia is typical of numerous aquatic crustaceans (Graf, 1978), and may be the result of massive absorption of water in the growing body. In the terrestrial amphipod Orchestia, calcium turnover takes place among cuticle, hemolymph, and PC which
DISCUSSION
Although the present results may be considered as preliminary, they contribute to give evidence that immunoreactive CT-like molecules are widely distributed among invertebrates, e.g., in crustaceans with calcilied cuticle (Arlot-Bonnemains et al., 1986; Fouchereau-Peron et al., 1987). However, it is obviously not easy to compare our measurements on whole specimens of a terrestrial amphipod with those previously reported on the hemolymph of a marine decapod (Arlot-Bonnemains et al., 1986). Still, both results suggest that the variations of concentration of these peptides are related to differences in calcium metabolism according to the stage of the molt cycle, thus indicating a possible role of these substances in the regulation of calcium shifts. If the levels of CT-like material seem to be very high in the total extracts of Orchestiu, it is important to stress that, in our results, both storage and functional forms of these peptides are measured. For comparison, in the crab, the CHH storage is about 2 pg per sinus gland while, in hemolymph, there are only a few nanograms of circulat-
Preexuvial
CT-like
peptides
hemolymphatic
Postexuvial
period
concentration
calcium
concentration
deposition cuticular
period
/
,'
1
I4 >
calcium (
resorption
storage calcium
within
PC resorption
I
FIG. 3. Comparison between the variations of CT-like peptides concentration and the variations of the different factors of the calcium turnover in Orchestia during the molt cycle.
CT-LIKE
MOLECULES
temporarily store a part of the cuticular calcium before each exuviation (Graf, 1969, 1978; Meyran, 1987). During the molt cycle, the variations of CT-like molecules are compared to the variations of calcium (hemolymphatic, cuticular, and within the PC) in Fig. 3; on the one hand, during the preexuvial period, the increase of these peptides corresponds to an increase of the hemolymphatic calcium, on the other hand, during the postexuvial period, there is a decrease of CT-like material parallel to a fall of the calcemia (Sellem et al., 1988). Such observations may suggest, in first approximation, to hypothesize a hypercalcemic role of CT in Orchestiu, as opposed to that observed in the shrimp (Arlot-Bonnemains et al., 1986). Nevertheless, the preexuvial hypercalcemia may also be related to the concomitant fall of the cuticular calcium which is progressively dissolved in order to be stored within the PC. In the same way, the postexuvial hypocalcemia may be due only to the calcification of the newly secreted cuticle using the hemolymphatic calcium. This calcium provides from the dissolution of the calcareous concretions previously stored within the PC (Graf, 1969, 1978;Meyran, 1987). So, our results are difficult to interpret since they cannot allow to suggesta possible role of CT-like molecules at the epidermic or PC epithelial level. In conclusion, owing to the particularities and the dynamism of its calcium turnover during the molt cycle, the crustacean 0. cavimunu may be an interesting model for further studies on the involvement of CT-like molecules in the calcium balance. REFERENCES Arlot-Bomemains, Y., Van-Wormhoudt, A., Favrel, P., Fouchereau-Peron, M., Milhaud, G., and Moukhtar, M. S. (1986). Calcitonin-likepeptide in the shrimp, Puluemon serrafus (Crustacea, decapoda) during the intermolt cycle. Experientia 42, 419-420. Cameron, J. N. (1987). Triggering of calcification after moult in the blue Crab. Amer. Zool. 21, 69A.
IN
83
Orchestia
Feldman, M., and Rodbard, D. (1971). Statistical analysis of radioligand assay data. In “Principles of Competitive Binding Assays” (W. D. Ode11and W. H. Daughaday, Eds.), p. 158.Lippincott, Philadelphia. Fouchereau-Peron, M., Arlot-Bonnemains, Y . I Milhaud, G., and Moukhtar, M. S. (1987). Immunoreactive salmon calcitonin-like molecule in crustaceans: High concentrations in Nephrops rzorvegicus.
Gen.
Comp.
Endocrinol.
53, 24f-252.
Fritsch, H. A. R., Van Noorden, J. P., and Pearse, A. G. E. (1980). Calcitonin-like immunochemical staining in the alimentary tract of Ciona intestinalis L. Cell Tissue Res. 205, 43M44. Girgis, S. I., Galan-Galan, F., Amett, R., Rogers, M., Bone, G., Ravazolla, M., and Mac Intyre, I. (1980). Immunoreactive human calcitonin-like molecule in the nervous system of the protochordates and a cyclostome, myxine. J. Endocrinol. 87, 375-382.
Graf, F. (1969). Le stockage de calcium avant la mue chez les Crustaces Amphipodes Orchesria (Talitride) et Niphargus (Gammaride hypoge). These Doct. Sci. Nat. Dijon N” 10.5.Impr. Bertmer, Arch. Orig., Centre Document. CNRS N” 2690, pp. 1-216. Graf, F. (1978). Les sources de calcium pour les CrustacCs venant de muer. Arch. Zool. Exp. Gen. 119, 111-129. Graf, F. (1986). Fine determination of the molt cycle stages in Orchestia. J. Crustacean Biol. 6, 666678.
Graf, F., and Meyran, J. C. (1983). Premolt calcium secretion in midgut posterior caeca of the Crustacean Orchestiar Ultrastructure of the epithelium. J. Morphol.
177, 1-23.
Graf, F., and Meyran, J. C. (1985). Post-exuvial calcium reabsorption in midgut posterior caeca of the crustacean Orchestiu cavimana: Ultrastructural changes of the epithelium. Cell Tissue Res. 242, 83-95.
Graf, F., and Michaut, P. (1977). Les sph&ules cd ciques de l’epithtlium caecal d’Orchesfiu (Custace, Amphipode), forme de transport de caicium dans le sens apico-basal. C.R. Acad. Sci. Fr. 248D, 49-52. Keller, R., and Sedlmeier, D. (1988). A metabolic hormone in crustaceans: The hyperglycemic neuropeptide. In “Endocrinology of selected invertebrates” (W. H. Domner and H. Lanser, Eds.), pp. 315-326. A. R. Liss, New York.. Kline, L. W., Kaneko, T., Cooper, C. W., and Pang, P. K. T. (1987). The presence of a salmon caldtonin-like substance in fish pituitary gland. Amer. Zool. 27, 97A.
84
GEAF ET AL.
Meyran, J. C. (1987). Dynamique calcique transepithehale dans les caecums posterieurs d’Orchestiu cavimana (Heller) (Crustacee, Amphipode) au tours de la mue. These Doct. SC. Dijon, pp. l-52. Meyran, J. C., Graf, F., and Nicaise, G. (1984). Calcium pathway through a mineralizing epithelium in the crustacean Orchestia in pre-molt: Ultrastructural cytochemistry and X-ray microanalysis. Tissue Cell 16, 269-286. Meyran, J. C., Graf, F., and Nicaise, G. (1986). Pulse discharge of calcium through a demineralizing epithelium in the crustacean Orchestiu: Ultrastruc-
tural cytochemistry and X-ray microanalysis. Tissue Cell 18, 267-283. Sellem, E., Graf, F., and Meyran, J. C. (1988). Some effects of salmon calcitonin on calcium metabolism in the crustacean Orchestia during the molt cycle. J. Exp. 2001. (in press) Treilhou-Lahille, F., Jullienne, A., Aziz, M., Beaumont, A., and Moukhtar, M. S. (1984). Ultrastructural localization of immunoreactive calcitonin in the two cell types of the ultimobranchial gland of the common toad (Bufi bufo L.). Gen. Comp. Endocrinol. 53, 241-252.
AND
COMPARATIVE
Variations
F. GRAF*,
ENDOCRINOLOGY
73,80-84 (1989)
of Calcitonin-like lmmunoreactivity in the Crustacean Orchestia cavimana during a Molt Cycle
M. FOUCHEREAU-PERON?,A. VAN-W• RMHOUDT,~ AND J. C. MEYRAN”
*U.A. 674 Universite’ de Bourgogne, Laboratoire de Biologie Animale, 6, Boulevard Gabriel, 21100-Dijon, and fLaboratoire de Biologie Marine, Collkge de France, 29110 Concarneau, France Accepted July 11, 1988 The investigation of calcitonin-like immunoreactivity performed in whole extracts of the terrestrial crustacean Orchestia cavimana using radioimmunoassay revealed large amounts of these peptides varying in concentration according to the stages of the molt cycle. Their level is maximum at the time of the exuviation. The results are then discussed regarding the mtrticular calcium metabolism of this species in function of its terrestrial conditions of life.
0 1989 Academic
Press, Inc.
the calcium previously stored is reabsorbed within 24-48 hr (stage B) to mineralize the newly secreted cuticle (Graf, 1969; Graf and Michaut, 1977; Graf and Meyran, 1985; Meyran et al., 1986). Moreover, the possibility of a precise determination of the successive stages of the relatively short molt cycle is typical of this species (Graf, 1969, 1986). The‘immunoreactive investigation of CTlike molecules during the molt cycle of Orchestia was performed in comparison with what has been previously observed in marine crustaceans (Arlot-Bonnemains et al., 1986; Fouchereau-Peron et al., 1987) and was discussed regarding the particular calcium turnover of this terrestrial species during molting.
The calcitonins (CT) are widely distributed in vertebrates and their role in the regulation of calcium metabolism appears established in mammals. More recently, CTlike molecules have been detected in marine invertebrates especially in crustaceans (Arlot-Bonnemains et al., 1986; Fouchereau-Peron et al., 1987). Theirphysiological function is still unknown in urochordates (Fritsch et al., 1980; Girgis et al., 1980) and, in fish, gonadotrophic functions are suspected (Kline et al., 1987). These peptides are presumed to play a role in crustaceans with calcified cuticle in relation to the calcium turnover according to the different stages of the molt cycle (ArlotBonnemains et al., 1986; FouchereauPeron et al., 1987). However, vertebrate CT seems to have no effect on calcium metabolism in crustaceans (Cameron, 1987). In the terrestrial amphipod Orchestia cavimana, the calcium fluxes during molting are of particular interest: during 12 days before exuviation (stage D), there is an important shift of calcium from the old cuticle to the posterior caeca (PC) which are involved in temporary calcium storage (Graf, 1969; Graf and Meyran, 1983; Meyran et al., 1984). Then, after exuviation (stage A),
MATERIALS
Animals. The crustacean 0. cavimana Heller is the only European terrestrial amphipod living in moist biotopes, with a calcium metabolism during molting adapted to its hygrophile aerial conditions of life (Graf, 1969). In order to avoid possible hormonal diferences between age classes or sexes, only male adults, 18-20 mm long, raised in subaquatic terrariums (Graf, 1%9), were used in this study. They were alloted according to their molt stages following the staging method of Graf correlated with the cuticular depo80
0066480/89 Copyri&t AlI rights
$1.50
B 1989 by Academic Press, Inc. of reproduction in any form reserved.
AND METHODS
CT-LIKE
MOLECULES
sition (Graf, 1986). For an average duration of 46 days, the whole molt cycle was divided into 14 stages. Just after exuviation, the animals were considered to be at stage A (early postmolt), and 12 to 36 hr after exuviation, at stage B (late postmolt). Hormonal measurements. Groups of I.5 whole animals exactly at the same molt stage (average fresh weight: 2 g) were analyzed: samples were sonicated and homogenized in 0.1 N hydrochloric acid. After centrifugation, supernatants were lyophylized and stored at -20” until assayed. The CT-like concentrations of the total extracts were determined with the heterologous radioimmunoassay of Treilhou-Lahille et al. (1984) using ‘251-labeled synthetic salmon CT (SCT) as described in Arlot-Bonnemains et al. (1986). Samples were analyzed with different dilutions until reproducible results were found. SCT antibodies (kindly donated by Dr. M. S. Moukhtar, Paris) were used at a l/20,000 dilution. Standards for SCT (lot 54 F.0335) were obtained from Sigma Chemical Company. Results of radioimmunological assay were expressed as the percentage of initial binding BIB0 where Bo represents the binding of labeled CT in the absence of unlabeled CT. Linearization of the standard curve was obtained by plotting ln (B/Bo)/l - (B/Be) as a function of In of CT concentration (Feldman and Rodbard, 1971).
RESULTS
Similar displacement of (‘251)-SCTbound to antibody was observed with serial dilutions of whole extracts from Orchestia (stage A) and SCT (Fig. 1). Analysis of the immunoreactivity of the logit
t to
$1
IN Orchestiu
whole extracts revealed on average relatively large amounts of CT-like material with important variations according to the different stages of the molt cycle (Fig. 2). The lowest concentrations were observed at the beginning of the inter-molt (stage Cd (about 25 pg/mg of fresh weight animal), before a weak increase at the end of this period (CJ. Then, after some decrease at the beginning of the preexuvial period (stages De-Dr”‘), the concentrations rapidly increased during the end of this period (stages D2-Ds) up to a maximum just at the time of the exuviation (stage A) (on average 2675 pg/mg of fresh weight animal). This sharp peak is finally followed by a rapid decrease in postexuvial period (stage B). The staging method is so precise in 0. cavirrzana as to enable us to assumethat the peak occurs exactly at the end of stage D3, i.e., just when the exuviation begins; a few hours later, the CT-like levels are signifrcantly lower. Moreover, the amplitude of the variation is remarkable. pg/mg
f.w.
t
intermolt 1
premolt I
1st inierm. !
6 B/Be)
I
1 I I11111100 log
I (pg
salmon
I I ,lll,
loo0
calcitonin)
FIG. 1. Immunoreactive salmon CT-like molecules in whole extracts of Orchestia during stage A.
FIG. 2. Variations of immunoreative CT-like peptides (&rng of fresh weight animal) in whole extracts of Orchestia during the different phases of the molt cycle.
82
GRAF ET AL.
ing hormone (Keller and Sedlmeier, 1988). In this respect, the maximum of CT-like peptides just at the time of the exuviation in Orchestia may be interpreted as a storage of the hormone in D,-A before its release in B-C as observed in the shrimp (ArlotBonnemains et al., 1986). So, it is still impossible to assign a precise role for these CT-like molecules in the calcium metabolism of Orchestia, as in other crustaceans. Moreover, it may not be sure that CT-like material acts in the same way in all crustaceans, owing to the differences of dynamics of the calcium turnover, according to their conditions of life. In marine decapods, where calcium shifts occur among water, cuticle, and hemolymph (Graf, 1978), the maximum of hemolymphatic CT-like peptides was observed during postmolt, which corresponds to a fall of the circulating calcium concentration (Arlot-Bonnemains et al., 1986). Such a hypocalcemia is typical of numerous aquatic crustaceans (Graf, 1978), and may be the result of massive absorption of water in the growing body. In the terrestrial amphipod Orchestia, calcium turnover takes place among cuticle, hemolymph, and PC which
DISCUSSION
Although the present results may be considered as preliminary, they contribute to give evidence that immunoreactive CT-like molecules are widely distributed among invertebrates, e.g., in crustaceans with calcilied cuticle (Arlot-Bonnemains et al., 1986; Fouchereau-Peron et al., 1987). However, it is obviously not easy to compare our measurements on whole specimens of a terrestrial amphipod with those previously reported on the hemolymph of a marine decapod (Arlot-Bonnemains et al., 1986). Still, both results suggest that the variations of concentration of these peptides are related to differences in calcium metabolism according to the stage of the molt cycle, thus indicating a possible role of these substances in the regulation of calcium shifts. If the levels of CT-like material seem to be very high in the total extracts of Orchestiu, it is important to stress that, in our results, both storage and functional forms of these peptides are measured. For comparison, in the crab, the CHH storage is about 2 pg per sinus gland while, in hemolymph, there are only a few nanograms of circulat-
Preexuvial
CT-like
peptides
hemolymphatic
Postexuvial
period
concentration
calcium
concentration
deposition cuticular
period
/
,'
1
I4 >
calcium (
resorption
storage calcium
within
PC resorption
I
FIG. 3. Comparison between the variations of CT-like peptides concentration and the variations of the different factors of the calcium turnover in Orchestia during the molt cycle.
CT-LIKE
MOLECULES
temporarily store a part of the cuticular calcium before each exuviation (Graf, 1969, 1978; Meyran, 1987). During the molt cycle, the variations of CT-like molecules are compared to the variations of calcium (hemolymphatic, cuticular, and within the PC) in Fig. 3; on the one hand, during the preexuvial period, the increase of these peptides corresponds to an increase of the hemolymphatic calcium, on the other hand, during the postexuvial period, there is a decrease of CT-like material parallel to a fall of the calcemia (Sellem et al., 1988). Such observations may suggest, in first approximation, to hypothesize a hypercalcemic role of CT in Orchestiu, as opposed to that observed in the shrimp (Arlot-Bonnemains et al., 1986). Nevertheless, the preexuvial hypercalcemia may also be related to the concomitant fall of the cuticular calcium which is progressively dissolved in order to be stored within the PC. In the same way, the postexuvial hypocalcemia may be due only to the calcification of the newly secreted cuticle using the hemolymphatic calcium. This calcium provides from the dissolution of the calcareous concretions previously stored within the PC (Graf, 1969, 1978;Meyran, 1987). So, our results are difficult to interpret since they cannot allow to suggesta possible role of CT-like molecules at the epidermic or PC epithelial level. In conclusion, owing to the particularities and the dynamism of its calcium turnover during the molt cycle, the crustacean 0. cavimunu may be an interesting model for further studies on the involvement of CT-like molecules in the calcium balance. REFERENCES Arlot-Bomemains, Y., Van-Wormhoudt, A., Favrel, P., Fouchereau-Peron, M., Milhaud, G., and Moukhtar, M. S. (1986). Calcitonin-likepeptide in the shrimp, Puluemon serrafus (Crustacea, decapoda) during the intermolt cycle. Experientia 42, 419-420. Cameron, J. N. (1987). Triggering of calcification after moult in the blue Crab. Amer. Zool. 21, 69A.
IN
83
Orchestia
Feldman, M., and Rodbard, D. (1971). Statistical analysis of radioligand assay data. In “Principles of Competitive Binding Assays” (W. D. Ode11and W. H. Daughaday, Eds.), p. 158.Lippincott, Philadelphia. Fouchereau-Peron, M., Arlot-Bonnemains, Y . I Milhaud, G., and Moukhtar, M. S. (1987). Immunoreactive salmon calcitonin-like molecule in crustaceans: High concentrations in Nephrops rzorvegicus.
Gen.
Comp.
Endocrinol.
53, 24f-252.
Fritsch, H. A. R., Van Noorden, J. P., and Pearse, A. G. E. (1980). Calcitonin-like immunochemical staining in the alimentary tract of Ciona intestinalis L. Cell Tissue Res. 205, 43M44. Girgis, S. I., Galan-Galan, F., Amett, R., Rogers, M., Bone, G., Ravazolla, M., and Mac Intyre, I. (1980). Immunoreactive human calcitonin-like molecule in the nervous system of the protochordates and a cyclostome, myxine. J. Endocrinol. 87, 375-382.
Graf, F. (1969). Le stockage de calcium avant la mue chez les Crustaces Amphipodes Orchesria (Talitride) et Niphargus (Gammaride hypoge). These Doct. Sci. Nat. Dijon N” 10.5.Impr. Bertmer, Arch. Orig., Centre Document. CNRS N” 2690, pp. 1-216. Graf, F. (1978). Les sources de calcium pour les CrustacCs venant de muer. Arch. Zool. Exp. Gen. 119, 111-129. Graf, F. (1986). Fine determination of the molt cycle stages in Orchestia. J. Crustacean Biol. 6, 666678.
Graf, F., and Meyran, J. C. (1983). Premolt calcium secretion in midgut posterior caeca of the Crustacean Orchestiar Ultrastructure of the epithelium. J. Morphol.
177, 1-23.
Graf, F., and Meyran, J. C. (1985). Post-exuvial calcium reabsorption in midgut posterior caeca of the crustacean Orchestiu cavimana: Ultrastructural changes of the epithelium. Cell Tissue Res. 242, 83-95.
Graf, F., and Michaut, P. (1977). Les sph&ules cd ciques de l’epithtlium caecal d’Orchesfiu (Custace, Amphipode), forme de transport de caicium dans le sens apico-basal. C.R. Acad. Sci. Fr. 248D, 49-52. Keller, R., and Sedlmeier, D. (1988). A metabolic hormone in crustaceans: The hyperglycemic neuropeptide. In “Endocrinology of selected invertebrates” (W. H. Domner and H. Lanser, Eds.), pp. 315-326. A. R. Liss, New York.. Kline, L. W., Kaneko, T., Cooper, C. W., and Pang, P. K. T. (1987). The presence of a salmon caldtonin-like substance in fish pituitary gland. Amer. Zool. 27, 97A.
84
GEAF ET AL.
Meyran, J. C. (1987). Dynamique calcique transepithehale dans les caecums posterieurs d’Orchestiu cavimana (Heller) (Crustacee, Amphipode) au tours de la mue. These Doct. SC. Dijon, pp. l-52. Meyran, J. C., Graf, F., and Nicaise, G. (1984). Calcium pathway through a mineralizing epithelium in the crustacean Orchestia in pre-molt: Ultrastructural cytochemistry and X-ray microanalysis. Tissue Cell 16, 269-286. Meyran, J. C., Graf, F., and Nicaise, G. (1986). Pulse discharge of calcium through a demineralizing epithelium in the crustacean Orchestiu: Ultrastruc-
tural cytochemistry and X-ray microanalysis. Tissue Cell 18, 267-283. Sellem, E., Graf, F., and Meyran, J. C. (1988). Some effects of salmon calcitonin on calcium metabolism in the crustacean Orchestia during the molt cycle. J. Exp. 2001. (in press) Treilhou-Lahille, F., Jullienne, A., Aziz, M., Beaumont, A., and Moukhtar, M. S. (1984). Ultrastructural localization of immunoreactive calcitonin in the two cell types of the ultimobranchial gland of the common toad (Bufi bufo L.). Gen. Comp. Endocrinol. 53, 241-252.