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Effect of temperature on haemolymphatic glucose and albumen gland polysaccharides during dormancy in the snail Helix aspersa maxima
Camp. Biochem. Physiof. Vol. 106A, No. 4, pp. 701-705,
0300-9629/93$6.00+ 0.00 0 1993Pergamon Press Ltd
1993
Printed in Great Britain
EFFECT OF TEMPERATURE ON HAEMOLYMPHAT~C GLUCOSE AND ALBUMEN GLAND POLYSACCHARIDES DURING DORMANCY IN THE SNAIL HELLY ASPERSA MAXIA4A JACQURLINEBRIDE, &MY ~NNE~Y-CLAUDET Laboratoire
and LUCIRN C&MOT
de Zoologie et Embryologic, U.F.R. Sciences et Techniques, place Markhal 25030 Besancon Cedex, France
Leclerc,
(Received 22 January 1993; accepted 26 February 1993) Abstract-l. In artificial hibernation at 6”C, a rapid decrease in haemol~ph glucose concentration occurred up to the third month and thereafter decreased slowly. The wet weight of the female albumen gland increased and its glycogen content increased after the third month whereas galactogen levels remained constant. 2. At 18-20°C in dryness (artificial estivation), haemolymph glucose remained constant. The albumen gland sustained a drastic decrease in wet weight. Its glycogen and galactogen were lost after the third month. 3. The results suggest that a low temperature during a &months artificial dormancy of Helix asperse rnuxima would promote ~pr~uction.
laying. Thus, it is necessary to have at one’s disposal a field-collected stock population of adult snails stored in dormancy, to have available egg-laying snails according to the needs. In order to determine whether low or high temperature during the dormancy of the stock population of Helix aspersa maxima maximizes reproductive success, we examined comparatively the haemolymph glucose level and the female albumen gland.
INTRODUCTION In pulmonate snails whose metabolism is carbohydrate orientated (Von Brand, 1931; Von Brand et al., 1957; Van der Horst and Zandee, 1973; Veld-
huijzen and Cuperus, 1976) the haemolymphatic glucose is the main precursor for the synthesis of the glycogen reserves stored in special cells (Hemminga et al., 198%). Nevertheless, the reproductive processes are more important in the removal of glucose from the haemolymph than the uptake in the storage regions (Veldhuijzen, 1975a,b; Veldhuijzen and Dogterom, 1975). The role of glucose as precursor in the polysaccharidic synthesis of the female part of the reproductive system was demonstrated in some snails and slugs ~Goudsmit and Aswell, 1965; Meenakshi and Scheer, 1969; Veldhuijzen, 197Sa). With respect to the study of secretory processes in the female accessory sex glands, it is primarily the albumen gland which produces the galactogen-rich perivitellin fluid which is deposited around fertilized oocytes (May, 1934; Goudsmit and Aswell, 1965). In the field, reproduction is strongly influenced by environmental conditions (Tompa, 1984 for review). In terrestrial snails, there are alternative periods of reproduction and inactivity or dormancy; hibernation at low temperatures in winter or estivation in summer at high temperatures and dryness. The dearth of edible snails in nature had required the development of commercial raising techniques which take into account the snails physiological characteristics (Gomot and Deray, 1987). In Helix aspersa maxima breeding methods, successive groups of newly hatched snails are reared throughout the year, from successive egg-
MATERIALSAND METHODS
In the week around 15 October, snails of the same age from a population raised under natural conditions were collected as soon as they became adult, having a fully formed reflected lip at the shell aperture. Snails to be studied in hibernation were placed in a cold chamber (6°C) in total obscurity and at a relative humidity of 50%. Those to be studied in estivatin~like conditions were kept in the laboratory at 18-20°C in a dark non-humidified container. Under these conditions, the snails remained inactive and withdrew into their shells. Tissues preparation Small groups of six snails were ~ndomly
chosen in
the stocks after 3 and 6 months of inactivity. After the animals had been weighed, haemolymph samples were taken with a hypodermic needle by puncturing the heart after perforating the shell with a scalpel. The haemolymph, flowing under its own pressure, 701
702
JACQUELINE BRIDEet al.
was collected in an Eppendorf tube for each snail and stored at -20°C. The albumen gland was removed and its relative wet weight, or maturation index, was estimated by the following: weight of albumen gland/weight of the animal x 100. A small piece of each gland was stored in an Eppendorf tube at -20°C for the determination of the amount of galactogen and glycogen. Polysaccharides determination Haemolymph glucose. Haemolymph samples were denatured (100 ~1) with 1.5 N perchloric acid (25 ~1). After neutralisation with 1.5 N KOH (25 pl) and centrifugation (7OOOg, 10 min), the glucose was spectrophotometrically determined in the supernatant using glucose dehydrogenase mutarotase (Boehringer) (Joosse and Van Elke, 1986). Albumen gland polysaccharides. After digestion of the glandular tissue in 1 ml of 2.5 N KOH at 80°C during 3 hr, the polysaccharides separation was carried out using the Van Handel procedure (1965). The polysaccharide pellets were hydrolyzed with 6 N H,SOI (3 hr, SO’C). The hydrolysate was neutralized for subsequent enzymatic determination of galactose (for galactogen) and glucose (for glycogen) using /?-galactose dehydrogenase and glucose dehydrogenase mutarotase (Boehringer), respectively (Joosse and Van Elke, 1986).
RESULTS
2
0
I
2
3
1
5
smonths
Fig. 2. Albumen gland relative wet weight (maturation index) in hibernation (A) and in estivation (B). Mean values k SEM (N = 6) sharing a common letter do not varying significantly (Mann-Witney U-test).
ing-like snails, the maturation index decreased by 37% after 3 months and by 67% after 6 months. Albumen gland polysaccharides (Figs 3, 4)
The amount of glycogen per gland (Fig. 3a) decreased until 3 months, by 35% in hibernating snails (A) and by 74% in estivating snails (B). The glycogen amount rose by 97% after 6 months in hibernating snails whereas it remained low in estivating snails (B). The concentration (Fig. 3b) varied in parallel to the quantity, except in estivation where no significant difference appeared on account of the strong decrease of the wet weight.
Haemolymphatic glucose (Fig. 1)
In estivating-like snails (B), the haemolymph glucose concentration remained rather constant with a gradual non-significant decrease of only 20% after 6 months. In hibernating snails (A), the glucose level exhibited a decrease of 55% after 3 months and 64% after 6 months. Albumen gland wet weight (Fig. 2)
After 3 months of hibernation, the albumen gland maturation index (relative wet weight) increased by 60% and then remained at the same level. In estivat-
b & 20
1;;:
a-1:
a I. Fig. 1. Haemolymph-glucose concentration (rg/ml) in hibernating snails (A) and in estivating snails (B). Mean values f SEM (N = 6) sharing a common letter do not differ significantly from each other but do differ from groups with another letter (Mann-Witney U-test).
, 0
I
2
3
1
5
6 months
Fig. 3. Albumen gland glycogen amount in mg per gland (a) and concentration in pg/mg wet weight (b) in hibernation (A) and estivation (B). Mean values f SEM (N = 6) sharing a common letter do not vary signilicantly (Mann-Witney U-test).
Temperature effect on Helix ~ly~~ha~des
:
.I
a M
20. 10. 0
1 0
t
2
2
‘
5
3
months
Fig. 4. Albumen gland galactogen amount (mg per gland) (a) and concentration (pg/mg wet weight) (b) in hibernation (A) and estivation (B). Mean values + SEM (N = 6) sharing a common letter do not differ significantly (Mann-Witney U-test).
Concerning the galactogen, no substantial variation in the amount per gland (Fig. 4a) occurred in hibernating snails. In contrast, after 3 months of estivation, the amount of galactogen strongly decreased (SO%, Fig 4a), the concentration also decreased (70%, Fig. 4b) and then remained at the same low level. DISCUSSiON The results indicated that in Helix aspersa maxima, the haemolymph glucose level and the female albumen gland of the genital tract were temperaturedependant during dormancy. Additionally, their evolution was related to the length of inactivity.
Haemolymphatic glucose and albumen gland evolution
In hi~mating snails, after an impo~ant decrease in the first 3 months, the haemolymph glucose rate remained low. The wet weight of the albumen gland and amount of glycogen rose after a decrease until the third month. Galactogen remained at the same level throughout the 6 months of the experiment. In estivating-like snails, in contrast, the haemolymph glucose rate did not change significantly but the albumen gland wet weight and polysaccharides (glycogen and galactogen) decreased dramatically during the first 3 months and then remained low. In pulmonates gastropods whose metabolism is carbohydrate orientated the rate of the haemolymphatic glucose and the storage of tissue glycogen as energetic reserves (Vedhuijzen and Cuperus, 1976; Hemminga et al., 198%) can be raised by feeding
703
carbohydrate-enriched food (Schwartz, 1934; Holtz and Von Brand, 1940; Scheerboom, 1978). During sta~ation or removal of glucose from the haemolymph by the high consuming synthesis of the female reproductive tract, the haemolymph glucose rate can be maintained by the control of a cerebral hyperglycemic factor which inhibits glycogen synthesis in storage tissues, stimulates glycogen breakdown and induces glucose release from this tissue (Hemminga et al., 1985a,b). Distinct regulatory mechanisms are operative for the control of the polysaccharide synthesis in the female accessory organs (Veldhuijzen and Dogterom, 1975). In the albumen gland, specific galactogen is synthesized by the endocrine control of either the dorsal bodies alone (Van Minnen and Sokolove, 1984), the brain (Goudsmit and Ram, 1982) or the dorsal bodies and the brain combined (Wijdenes et al., 1983; Miksys and Saleuddin, 1985). The gonad appears to have a direct or indirect effect (Berset De Vauileury et al., 1986; McCrone and Sokolove, 1986; Miksys and Saleuddin, 1985). Normally, the galactogen is not catabolized in the albumen gland but secreted during egg-laying to constitute the ~~vitelline fluid of the eggs. Nevertheless, during adverse conditions like starvation, low temperature or prolonged dormancy, the galactogen can be mobilized to he used as an energy reserve together with the glycogen (Veldhuijz~n and Cuperus, 1976) or when all glycogen has disappeared (Goddard and Martin, 1966; Barnett, 1971; Fantin and Gervaso, 1971). In experimental hibernation, the albumen gland glycogen decrease in the first 3 months suggested that polysaccharide regulation in the female genital tract is affected as in starving Lymnaea stagnalis (Veldhuijzen and Dogterom, 1975) and Biomp~a~ariu glabrata (De Jong-Brink, 1973; Vianey-Liaud, 1979). The simultaneous decrease of the albumen gland glycogen and the haemolymphatic glucose indicated that other tissues, probably the special reserves tissues, continue to remove glucose for their synthesis during the first 3 months of hibernation. After the third month, stimulation of the albumen gland glycogen synthesis would be controlled by gonadic factors because a stimulating effect of implantation of gonads from adult hibernating snails had been demonstrated on the glycogen of albumen glands of young snails, whereas the galactogen synthesis was stimulated by implantation of gonads from active snails (Berset De Vaufleury et al., 1986). The lack of a new synthesis of galactogen in hibernating snails would also he explained by a reduced activity of the endocrine dorsal bodies (Griffond and Vincent, 1985). The situation would be similar as in Lymnaea stagnalis maintained in starvation at low temperature (6°C) where the low level of dorsal bodies hormone does not stimulate the galactogen synthesis but prevents the catabolism of the secretory products of the albumen gland (Veldhuijzen and Cuperus, 1976). In contrast, in the absence of circulating dorsal bodies hormone, during
JACQUELINEBRIDE et al.
704
starvation at ambient temperature or after the dorsal bodies extirpation which does not prevent a normal eating activity in Lymnaea stagnaiis, the albumen gland galactogen is no longer protected against catabolism (Veldhuijzen and Cuperus, 1976). Thus, it can be suggested that the strong disappearance of the albumen gland galactogen in estivating-like Helix aspersa maxima would be caused by a total arrest of the dorsal bodies activity. In these snails, the polysaccharides mobilized from the albumen gland probably serve to maintain the haemolymph glucose rate. However, this does not preclude that glucose mobilized from the glycogen storage tissues by a cerebral hyperglycaemic factor (Hemminga et al., 1985b) may be implicated in maintaining the Constance of the haemolymphatic glucose. Repercussion of the thermal conditions during the dormancy on the ability of Helix aspersa maxima to reproduce
After a starvation period, the rise in haemolymph glucose concentration caused by the restart of feeding is the stimulus for the restarting of reproduction in suitable environmental conditions (Veldhuijzen, 1975a) probably by activation of the endocrine centers involved in the control of reproduction (Veldhuijzen, 1975b). Based on our above comparative observations, it appeared that the polysaccharide-rich well-developed albumen gland of 6-month hibernating snails have more capacity for synthetizing the important amount of galactogen for the eggs than the regressed albumen gland of estivating-like snails. As much as the hypoglycaemia of the hibernating snails would be rapidly balanced with the influx of glucose from the digestive system. The demonstration of the temperature dependence of the female accessory sex gland during the dormancy of Helix aspersa maxima is in agreement with results showing the temperature dependence of the gonadal activity. During hibernation, an evolution occurred in increasing the sensitivity of the spermatogonial multiplication to the stimulating effect of a rise in temperature (Gomot and Deray, 1990). The cerebral neuroendocrine system intervened as a relay between temperature and the spermatogonial multiplication (Gomot and Gomot, 1991). These results are important for the development of snail rearing methods because they show the need to use, as stock, adult animals which have hibernated. They support the theory that a short hibernation (less than 3 months) is correlated to a weak rate of reproduction whereas a longer hibernation, of at least 6 months, allows rapid reproduction and a high rate of egglaying (Bonnefoy-Claudet and Deray, 1984). SUMMARY
In this physiological study of the snail Helix asthe haemolymph glucose concen-
persa maxima,
tration, the albumen gland (female accessory sex gland), wet weight and polysaccharides (glycogen and galactogen) were determined during dormancy, as experimentally affected by low temperature (6”C, as hibernation) or high temperature (18-2O”C, as estivation) in the darkness. In hibernating snails, the haemolymphatic glucose decrease significantly by about 55% after 3 months and 64% after 6 months. The wet weight of the albumen gland increased of 50% after 3 months and of 60% after 6 months. Its glycogen contents decreased as far as 3 months but then raised of 97% after 6 months of inactivity. The galactogen had a rather constant level. At high temperature, the haemolymph glucose level did not change significantly. The albumen gland sustained a drastic decrease of its wet weight as far as 67% after 6 months. Since the third month, 74% of the glycogen and 70% of the galactogen were lost. These observations suggested that the temperature influenced the endocrine control of the polysaccharidic metabolism in inactive Helix aspersa maxima. The comparative study indicated that artificial hibernation appears to be the best method to keep a stock population of Helix aspersa maxima in order to preserve the female accessory sex gland which produce the perivitelline fluid of the eggs, thus increasing the probability of a rapid restart of reproduction after 6 months of inactivity. Acknowledgements-We
thank Brigitte Jolibois for the organization of the manuscript.
REFERENCES
Barnett J. E. G. (1971) An acid a-galactosidase from the albumen gland of Helix aspersa. Comp. Biochem. Physiol. 4OB, 585-592. Berset De Vatieury J. P., Bride J. and Gomot L. (1986) Mise en Cvidence du r6le endocrine de la gonade dans le dkveloppement de l’appareil gknital de l’escargot He1i.v aspersa. C.R. Sot. Biol. 180, 196196. Bonnefoy-Claudet R. and Deray A. (1984) Influence de la dun% d’hibemation sur l’activitt reproductrice de l’escargot Helix aspersa Miiller. C.R. Sot. Biol. 178, 442-449.
Brand T. von (1931) Der Jahreszyklus im StolTbestand der Weinberg-schnecke (Helix pomaiia). 2. Vergleich. Physiol. 14, 200-264.
Brand T. von. McMahon P. and Nolan M. 0. (1957) Physiological observations on starvation and desiccation of the snail Australorbis elabratus. Biol. Bull. 113.89-102. Fantin A. M. B. and Gervaso M. V. (1971) A hktoenzymatic investigation of galactogen synthesis in the albumen gland of Helix pomatia and Lymnaea stagnalis. Hisrochemie 28, 88-94.
Goddard C. K. and Martin A. W. (1966) Carbohydrate metabolism. In Physiology of Mollusca (Edited by Wilbur K. M. and Yonge C. M.), Vol. II, pp. 275-308. Academic Press, London. Gomot L. and Deray A. (1987) Les Escargots. La Recherche 18, 302-3 11. Gomot P. and Deray A. (1990) The length of hibernation affects temperature-induced (25°C) spermatogenic multiplication in Helix aspersa Miill. Experientia 46, 684-686.
Temperature effect on Helix polysaccharides Gomot P. and Gomot L. (1991) Length of hibernation and the brain’s influence on temperature-induced spermatogenie DNA synthesis in Helix aspersa. Comp. Biochem. Physiol. lOOA, 689492.
Goudsmit E. M. and Aswell G. (1965) Enzymatic synthesis of galactogen in the snail Helix pomatia. Biochem. Res. Comm. 19, 417-422.
Goudsmit E. M. and Ram J. L. (1982) Stimulation of Helix pomatia albumen gland galactogen synthesis by putative neurohonnone and by cyclic AMP analogues. Camp. Biochem. Physiol. 71B; 4i7422.
Griffond B. and Vincent C. (1985) Etude de I’activid des corps dorsaux de l’escargot Helix aspersa Mtiller au tours des phases physiologiques de la vie adulte et sous differentes vhotooeriodes. ht. J. Inv. Reprod. Devel. 8. 27-37.
_
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Handel H. van (1965) Estimation of glycogen in small amount of tissue. Ann. Biochem. 11, 256265. Hemminga M. A., Koomen W., Maaskant J. J. and Joosse J. (1985a) Effect of photoperiod and temperature on the glycogen stores in the mantle and the head-foot muscle of the freshwater pulmonate snail Lymnaea stagnalis. Comp. Biochem. Physiol. 8OB, 139-143. Hemminga M. A., Maaskant J. J., Koomen W. and Joosse J. (1985b) Neuroendocrine control of glycogen mobilization in the freshwater Lymnaea stagnalis. Gen. camp. Endocrinol. 57, 117-123.
Hemminga M. A., Maaskant J. J., Jager J. C. and Joosse J. (1985~) Glycogen metabolism in isolated glycogen cells of the freshwater snail Lymnaea stagnalis, as influenced by external glucose concentration. Comp. Biochem. Physiol. 82A, 239-246.
Holtz F. and Brand T. von (1940) Quantitative studies upon some blood constituants of Helix pomatia. Biol. Bull. 79, 42343 1. Horst D. J. van der and Zandee D. I. (1973) Invariability of the composition of fatty acids and other lipids in the pulmonate land snail Cepaea nemoralis (L.) during an annual cycle. J. camp. Physiol. 85, 317-3% Jot-in-Brink M. de (1973) The effect of desiccation and starvation upon weight, histology and ultrastructure of the reproductive tract of Biomphalaria glabrata, intermediate host of Schistosoma mansoni, Z. Zellforsch. 136, 229-262. Joosse J. and Elk R. Van (1986) Trichobilharzia ocellata physiological characterization of giant growth, glycogen depletion and absence of reproductive activity in the intermediate snail Lymnaea stagnalis. Exp. Parasitol. 62, 1-3.
May F. (1934) Der Jahreszyklus im Galaktogen und Glykogenbestand der Weinbergschnecke. Zeit. Biol. 95, 401430.
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McCrone E. J. and Sokolove P. G. (1986) Photoperiodic activation of brains in castrate and the role of the gonad in reproductive maturation of Limax maximus. J. Comp. Physiol. MA, 151-158. Meenakshi V. R. and Scheer B. T. (1969) Regulation of the galactogen synthesis in the slug.. Ariolimax colombianus. Coma. Biochem. Phvsiol. 29. 841-845.
Miksys S. and Saleuddin A. S. M. (1985) Effects of castration on growth and reproduction of Helisoma duryi (Mollusca, Pulmonata). Int. J. Inv. Reprod. Devel. 12, 145-160.
Minnen J. van and Sokolove P. G. (1984) Galactogenstimulating factor in the slug Limax maximus: cellular localization and partial purification. Gen. camp. Endocrinol. 49, 114-122.
Scheerboom J. E. M. (1978) The influence of food quantity and food quality on assimilation, body growth and egg production in the pond snail Lymnaea stagnalis (L.), with particular reference to the hemolymph-glucose concentration. Proc. Kon. Ned. Akad. Wet. 81C, 173-183.
Schwartz K. (1934) Uber den Blutzucker der Weinbergschnecke. Biochem. Z. 275, 262-269. Tompa A. S. (1984) Land snails (Stylommatophora). In The Mollusca, Vol. 7, Reproduction (Edited by Tompa A. S., Verdonk N. H. and Van den Biggelaar J. A. M.), pp. 47-140. Academic Press, New York. Veldhuijzen J. P. (1975a) Effects of different kinds of food, starvation and restart of feeding on the haemolymphglucose of the pond snail Lymnaea stagnalis. Neth. J. Zool. 25, 89-102.
Veldhuijzen J. P. (1975b) Glucose-tolerance in the pond snail Lymnaea stagnalis as affected by temperature and starvation. Neth. J. Zool. 25, 206-218. Veldhuijzen J. P. and Dogterom G. E. (1975) Incorporation of 14C glucose in the polysaccharides of various body parts of the pond snail Lymnaea stagnalis as affected by starvation. Neth. J. Zool. 25, 247-260. Veldhuijzen J. P. and Cuperus R. (1976) Effects of starvation, low temperature and the dorsal body hormone on the in vitro synthesis of galactogen and glycogen in the albumen gland and the mantle of the pond snail Lymnaea stagnalis. Neth. J. Zool. 26, 119-135. Vianey-Liaud M. (1979) Influence du jeiine et de la renutrition sur l’oviposition et les gametogeneses chez le planorbe Biomphalaria glabrata (Gasteropode Pulmont Basommatophore). Malacologia 18, 40-406. Wijdenes J., Elk R. van and Joosse J. (1983) Effects of two gonadotropic hormones on polysaccharide synthesis on the albumen gland of Lymnaea stagnalis, studies with the organ culture technique. Gen. camp. Endocrinol. 51, 263-27
1.
0300-9629/93$6.00+ 0.00 0 1993Pergamon Press Ltd
1993
Printed in Great Britain
EFFECT OF TEMPERATURE ON HAEMOLYMPHAT~C GLUCOSE AND ALBUMEN GLAND POLYSACCHARIDES DURING DORMANCY IN THE SNAIL HELLY ASPERSA MAXIA4A JACQURLINEBRIDE, &MY ~NNE~Y-CLAUDET Laboratoire
and LUCIRN C&MOT
de Zoologie et Embryologic, U.F.R. Sciences et Techniques, place Markhal 25030 Besancon Cedex, France
Leclerc,
(Received 22 January 1993; accepted 26 February 1993) Abstract-l. In artificial hibernation at 6”C, a rapid decrease in haemol~ph glucose concentration occurred up to the third month and thereafter decreased slowly. The wet weight of the female albumen gland increased and its glycogen content increased after the third month whereas galactogen levels remained constant. 2. At 18-20°C in dryness (artificial estivation), haemolymph glucose remained constant. The albumen gland sustained a drastic decrease in wet weight. Its glycogen and galactogen were lost after the third month. 3. The results suggest that a low temperature during a &months artificial dormancy of Helix asperse rnuxima would promote ~pr~uction.
laying. Thus, it is necessary to have at one’s disposal a field-collected stock population of adult snails stored in dormancy, to have available egg-laying snails according to the needs. In order to determine whether low or high temperature during the dormancy of the stock population of Helix aspersa maxima maximizes reproductive success, we examined comparatively the haemolymph glucose level and the female albumen gland.
INTRODUCTION In pulmonate snails whose metabolism is carbohydrate orientated (Von Brand, 1931; Von Brand et al., 1957; Van der Horst and Zandee, 1973; Veld-
huijzen and Cuperus, 1976) the haemolymphatic glucose is the main precursor for the synthesis of the glycogen reserves stored in special cells (Hemminga et al., 198%). Nevertheless, the reproductive processes are more important in the removal of glucose from the haemolymph than the uptake in the storage regions (Veldhuijzen, 1975a,b; Veldhuijzen and Dogterom, 1975). The role of glucose as precursor in the polysaccharidic synthesis of the female part of the reproductive system was demonstrated in some snails and slugs ~Goudsmit and Aswell, 1965; Meenakshi and Scheer, 1969; Veldhuijzen, 197Sa). With respect to the study of secretory processes in the female accessory sex glands, it is primarily the albumen gland which produces the galactogen-rich perivitellin fluid which is deposited around fertilized oocytes (May, 1934; Goudsmit and Aswell, 1965). In the field, reproduction is strongly influenced by environmental conditions (Tompa, 1984 for review). In terrestrial snails, there are alternative periods of reproduction and inactivity or dormancy; hibernation at low temperatures in winter or estivation in summer at high temperatures and dryness. The dearth of edible snails in nature had required the development of commercial raising techniques which take into account the snails physiological characteristics (Gomot and Deray, 1987). In Helix aspersa maxima breeding methods, successive groups of newly hatched snails are reared throughout the year, from successive egg-
MATERIALSAND METHODS
In the week around 15 October, snails of the same age from a population raised under natural conditions were collected as soon as they became adult, having a fully formed reflected lip at the shell aperture. Snails to be studied in hibernation were placed in a cold chamber (6°C) in total obscurity and at a relative humidity of 50%. Those to be studied in estivatin~like conditions were kept in the laboratory at 18-20°C in a dark non-humidified container. Under these conditions, the snails remained inactive and withdrew into their shells. Tissues preparation Small groups of six snails were ~ndomly
chosen in
the stocks after 3 and 6 months of inactivity. After the animals had been weighed, haemolymph samples were taken with a hypodermic needle by puncturing the heart after perforating the shell with a scalpel. The haemolymph, flowing under its own pressure, 701
702
JACQUELINE BRIDEet al.
was collected in an Eppendorf tube for each snail and stored at -20°C. The albumen gland was removed and its relative wet weight, or maturation index, was estimated by the following: weight of albumen gland/weight of the animal x 100. A small piece of each gland was stored in an Eppendorf tube at -20°C for the determination of the amount of galactogen and glycogen. Polysaccharides determination Haemolymph glucose. Haemolymph samples were denatured (100 ~1) with 1.5 N perchloric acid (25 ~1). After neutralisation with 1.5 N KOH (25 pl) and centrifugation (7OOOg, 10 min), the glucose was spectrophotometrically determined in the supernatant using glucose dehydrogenase mutarotase (Boehringer) (Joosse and Van Elke, 1986). Albumen gland polysaccharides. After digestion of the glandular tissue in 1 ml of 2.5 N KOH at 80°C during 3 hr, the polysaccharides separation was carried out using the Van Handel procedure (1965). The polysaccharide pellets were hydrolyzed with 6 N H,SOI (3 hr, SO’C). The hydrolysate was neutralized for subsequent enzymatic determination of galactose (for galactogen) and glucose (for glycogen) using /?-galactose dehydrogenase and glucose dehydrogenase mutarotase (Boehringer), respectively (Joosse and Van Elke, 1986).
RESULTS
2
0
I
2
3
1
5
smonths
Fig. 2. Albumen gland relative wet weight (maturation index) in hibernation (A) and in estivation (B). Mean values k SEM (N = 6) sharing a common letter do not varying significantly (Mann-Witney U-test).
ing-like snails, the maturation index decreased by 37% after 3 months and by 67% after 6 months. Albumen gland polysaccharides (Figs 3, 4)
The amount of glycogen per gland (Fig. 3a) decreased until 3 months, by 35% in hibernating snails (A) and by 74% in estivating snails (B). The glycogen amount rose by 97% after 6 months in hibernating snails whereas it remained low in estivating snails (B). The concentration (Fig. 3b) varied in parallel to the quantity, except in estivation where no significant difference appeared on account of the strong decrease of the wet weight.
Haemolymphatic glucose (Fig. 1)
In estivating-like snails (B), the haemolymph glucose concentration remained rather constant with a gradual non-significant decrease of only 20% after 6 months. In hibernating snails (A), the glucose level exhibited a decrease of 55% after 3 months and 64% after 6 months. Albumen gland wet weight (Fig. 2)
After 3 months of hibernation, the albumen gland maturation index (relative wet weight) increased by 60% and then remained at the same level. In estivat-
b & 20
1;;:
a-1:
a I. Fig. 1. Haemolymph-glucose concentration (rg/ml) in hibernating snails (A) and in estivating snails (B). Mean values f SEM (N = 6) sharing a common letter do not differ significantly from each other but do differ from groups with another letter (Mann-Witney U-test).
, 0
I
2
3
1
5
6 months
Fig. 3. Albumen gland glycogen amount in mg per gland (a) and concentration in pg/mg wet weight (b) in hibernation (A) and estivation (B). Mean values f SEM (N = 6) sharing a common letter do not vary signilicantly (Mann-Witney U-test).
Temperature effect on Helix ~ly~~ha~des
:
.I
a M
20. 10. 0
1 0
t
2
2
‘
5
3
months
Fig. 4. Albumen gland galactogen amount (mg per gland) (a) and concentration (pg/mg wet weight) (b) in hibernation (A) and estivation (B). Mean values + SEM (N = 6) sharing a common letter do not differ significantly (Mann-Witney U-test).
Concerning the galactogen, no substantial variation in the amount per gland (Fig. 4a) occurred in hibernating snails. In contrast, after 3 months of estivation, the amount of galactogen strongly decreased (SO%, Fig 4a), the concentration also decreased (70%, Fig. 4b) and then remained at the same low level. DISCUSSiON The results indicated that in Helix aspersa maxima, the haemolymph glucose level and the female albumen gland of the genital tract were temperaturedependant during dormancy. Additionally, their evolution was related to the length of inactivity.
Haemolymphatic glucose and albumen gland evolution
In hi~mating snails, after an impo~ant decrease in the first 3 months, the haemolymph glucose rate remained low. The wet weight of the albumen gland and amount of glycogen rose after a decrease until the third month. Galactogen remained at the same level throughout the 6 months of the experiment. In estivating-like snails, in contrast, the haemolymph glucose rate did not change significantly but the albumen gland wet weight and polysaccharides (glycogen and galactogen) decreased dramatically during the first 3 months and then remained low. In pulmonates gastropods whose metabolism is carbohydrate orientated the rate of the haemolymphatic glucose and the storage of tissue glycogen as energetic reserves (Vedhuijzen and Cuperus, 1976; Hemminga et al., 198%) can be raised by feeding
703
carbohydrate-enriched food (Schwartz, 1934; Holtz and Von Brand, 1940; Scheerboom, 1978). During sta~ation or removal of glucose from the haemolymph by the high consuming synthesis of the female reproductive tract, the haemolymph glucose rate can be maintained by the control of a cerebral hyperglycemic factor which inhibits glycogen synthesis in storage tissues, stimulates glycogen breakdown and induces glucose release from this tissue (Hemminga et al., 1985a,b). Distinct regulatory mechanisms are operative for the control of the polysaccharide synthesis in the female accessory organs (Veldhuijzen and Dogterom, 1975). In the albumen gland, specific galactogen is synthesized by the endocrine control of either the dorsal bodies alone (Van Minnen and Sokolove, 1984), the brain (Goudsmit and Ram, 1982) or the dorsal bodies and the brain combined (Wijdenes et al., 1983; Miksys and Saleuddin, 1985). The gonad appears to have a direct or indirect effect (Berset De Vauileury et al., 1986; McCrone and Sokolove, 1986; Miksys and Saleuddin, 1985). Normally, the galactogen is not catabolized in the albumen gland but secreted during egg-laying to constitute the ~~vitelline fluid of the eggs. Nevertheless, during adverse conditions like starvation, low temperature or prolonged dormancy, the galactogen can be mobilized to he used as an energy reserve together with the glycogen (Veldhuijz~n and Cuperus, 1976) or when all glycogen has disappeared (Goddard and Martin, 1966; Barnett, 1971; Fantin and Gervaso, 1971). In experimental hibernation, the albumen gland glycogen decrease in the first 3 months suggested that polysaccharide regulation in the female genital tract is affected as in starving Lymnaea stagnalis (Veldhuijzen and Dogterom, 1975) and Biomp~a~ariu glabrata (De Jong-Brink, 1973; Vianey-Liaud, 1979). The simultaneous decrease of the albumen gland glycogen and the haemolymphatic glucose indicated that other tissues, probably the special reserves tissues, continue to remove glucose for their synthesis during the first 3 months of hibernation. After the third month, stimulation of the albumen gland glycogen synthesis would be controlled by gonadic factors because a stimulating effect of implantation of gonads from adult hibernating snails had been demonstrated on the glycogen of albumen glands of young snails, whereas the galactogen synthesis was stimulated by implantation of gonads from active snails (Berset De Vaufleury et al., 1986). The lack of a new synthesis of galactogen in hibernating snails would also he explained by a reduced activity of the endocrine dorsal bodies (Griffond and Vincent, 1985). The situation would be similar as in Lymnaea stagnalis maintained in starvation at low temperature (6°C) where the low level of dorsal bodies hormone does not stimulate the galactogen synthesis but prevents the catabolism of the secretory products of the albumen gland (Veldhuijzen and Cuperus, 1976). In contrast, in the absence of circulating dorsal bodies hormone, during
JACQUELINEBRIDE et al.
704
starvation at ambient temperature or after the dorsal bodies extirpation which does not prevent a normal eating activity in Lymnaea stagnaiis, the albumen gland galactogen is no longer protected against catabolism (Veldhuijzen and Cuperus, 1976). Thus, it can be suggested that the strong disappearance of the albumen gland galactogen in estivating-like Helix aspersa maxima would be caused by a total arrest of the dorsal bodies activity. In these snails, the polysaccharides mobilized from the albumen gland probably serve to maintain the haemolymph glucose rate. However, this does not preclude that glucose mobilized from the glycogen storage tissues by a cerebral hyperglycaemic factor (Hemminga et al., 1985b) may be implicated in maintaining the Constance of the haemolymphatic glucose. Repercussion of the thermal conditions during the dormancy on the ability of Helix aspersa maxima to reproduce
After a starvation period, the rise in haemolymph glucose concentration caused by the restart of feeding is the stimulus for the restarting of reproduction in suitable environmental conditions (Veldhuijzen, 1975a) probably by activation of the endocrine centers involved in the control of reproduction (Veldhuijzen, 1975b). Based on our above comparative observations, it appeared that the polysaccharide-rich well-developed albumen gland of 6-month hibernating snails have more capacity for synthetizing the important amount of galactogen for the eggs than the regressed albumen gland of estivating-like snails. As much as the hypoglycaemia of the hibernating snails would be rapidly balanced with the influx of glucose from the digestive system. The demonstration of the temperature dependence of the female accessory sex gland during the dormancy of Helix aspersa maxima is in agreement with results showing the temperature dependence of the gonadal activity. During hibernation, an evolution occurred in increasing the sensitivity of the spermatogonial multiplication to the stimulating effect of a rise in temperature (Gomot and Deray, 1990). The cerebral neuroendocrine system intervened as a relay between temperature and the spermatogonial multiplication (Gomot and Gomot, 1991). These results are important for the development of snail rearing methods because they show the need to use, as stock, adult animals which have hibernated. They support the theory that a short hibernation (less than 3 months) is correlated to a weak rate of reproduction whereas a longer hibernation, of at least 6 months, allows rapid reproduction and a high rate of egglaying (Bonnefoy-Claudet and Deray, 1984). SUMMARY
In this physiological study of the snail Helix asthe haemolymph glucose concen-
persa maxima,
tration, the albumen gland (female accessory sex gland), wet weight and polysaccharides (glycogen and galactogen) were determined during dormancy, as experimentally affected by low temperature (6”C, as hibernation) or high temperature (18-2O”C, as estivation) in the darkness. In hibernating snails, the haemolymphatic glucose decrease significantly by about 55% after 3 months and 64% after 6 months. The wet weight of the albumen gland increased of 50% after 3 months and of 60% after 6 months. Its glycogen contents decreased as far as 3 months but then raised of 97% after 6 months of inactivity. The galactogen had a rather constant level. At high temperature, the haemolymph glucose level did not change significantly. The albumen gland sustained a drastic decrease of its wet weight as far as 67% after 6 months. Since the third month, 74% of the glycogen and 70% of the galactogen were lost. These observations suggested that the temperature influenced the endocrine control of the polysaccharidic metabolism in inactive Helix aspersa maxima. The comparative study indicated that artificial hibernation appears to be the best method to keep a stock population of Helix aspersa maxima in order to preserve the female accessory sex gland which produce the perivitelline fluid of the eggs, thus increasing the probability of a rapid restart of reproduction after 6 months of inactivity. Acknowledgements-We
thank Brigitte Jolibois for the organization of the manuscript.
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