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The effect of the growth hormone of the pond snail Lymnaea stagnalis on periostracum formation
Camp L3wi hem. t’hwol.. Vol 66A. pp. 687 10 690 Q Pergamon Press Ltd 1980 PrInted in Grra: Brilaln
THE EFFECT OF THE GROWTH HORMONE OF THE POND SNAIL LYMNAEA STAGNALZS ON PERIOSTRACUM FORMATION ARENDA. DOCTEROMand THEA JENTJENS Department
of Biology.
Free University.
(Recc+~d
30 Octohcr
Amsterdam.
The Netherlands
1979)
Abstract-l.
The effect of removal of the growth hormone producing neurosecretory Light Green Cells (LGC) on periostracum and shell matrix formation in the freshwater snail Lq’t~~~a stagnaliswas studied by determining the jH-tyrosine incorporation in these proteinaceous shell components. 2. LGC removal resulted in a considerable decrease of the periostracum formation at the shell edge. Shell matrix formation, however. was not affected. 3. It is concluded that the effect of the growth hormone on pcriostracum formation is crucial in the regulation of shell enlargement. 4. A summarizing model of shell growth (in length and in thickness) as affected by the growth hormone, is given.
INTRODUCTION Most gastropod shells have a persistent outer proteinaceous layer, the periostracum. On the inner surface of the newly formed edge of the periostracum the first calcium carbonate layer, the “outer crystalline layer”, is deposited. The greater part of the later formed shell material consists of the inner calcareous layers, which always have a proteinaceous matrix (cf. Wilbur, 1976). It is highly probable that special regions of the mantle underlying the shell successively produce (the precursors of) these components of the shell. The periostracum is formed by the mantle edge gland (Kniprath, 1972; Saleuddin, 1976). In a previous study (Dogterom rt al., 1979) it is argued that the whole edge, or a particular part of it-the “belt” (the thickened epithelium at the mantle edge; cf. Zylstra et al.. 1978), serves as the calcium donor for the outer crystalline layer. The remaining parts of the mantle epithelium provide the matrix and the calcium for the inner layers. In the freshwater snail, Lyrnnaea stagnalis, the neurosecretory Light Green Cells (LGC), which are located in the cerebral ganglia, produce a growth hormone (Geraerts, 1976), which stimulates the growth of all organs and of the shell. In the absence of growth hormone (after LGC extirpation) the formation of the inner calcareous layers continues, but the calcium deposition at the shell edge, i.e. the formation of the outer crystalline layer, ceases (Dogterom et al., 1979). The same was found for carbonate deposition (Dogterom & Van der Schors, 1980). The cessation of calcium deposition at the shell edge is due to a decreased calcium concentration in the mantle edge (Dogterom et al., 1979), and this is caused by a lowered concentration of a specific calcium binding protein in the mantle edge (Dogterom & Doderer, in prep.). The present study was undertaken to determine the role of the growth hormone of L. stagnalis in the formation of the two proteinaceous components of the shell: the periostracum and the matrix of the inner calcereous layers. 687
As in the previous experiments LGC exirpated animals were compared with sham-operated controls. To study the protein formation in the shell components the incorporation of injected 3H-tyrosine was determined. This amino acid was chosen, since it occurs in relatively large amounts in the periostracum of snails (Meenakshi et al., 1969; Waite, 1977). Moreover, preliminary experiments also showed that tyrosine is incorporated more rapidly than aspartic acid and equimolar amino acid mixtures. MATERIALSAND METHODS Breeding and experimental conditions, and the surgical and injection techniques, were as described by Dogterom et al. (I 979). The experiment included two groups of adult animals: sham operated controls and snails in which the LGC were cauterized. An aqueous solution of 3H-tyrosine (L-[2,3,15,6-~H]tyrosine: I mCi/ml. 82 Ci/mmol) was obtained from the Radiochemical Centre (Amersham. U.K.). Twenty-one days after the operations the snails were injected with 5 ~1 of this solution per gramme wet body weight. After the injection the animals were kept in polyethene beakers containing 300 ml tap water and some lettuce. After 30 or 120 min the snails were rinsed with tap water to remove possible adhering radioactivity. Under a dissection microscope the marginal shell edge was carefully removed with a piece of razor blade. Next it was decalcified (almost no calcium carbonate is present in the very edge) in 20~1 2 N HCI. To dissolve the proteinaceous parts 100 ~1 20% KOH was added. The samples were kept at 60°C for 18 hr. jH was counted in this solution. using Insta-Gel (Packard Instrument, Brussels). The animals could be easily removed from the remainder of the shells by freezing (in liquid nitrogen) and thawing. The shells were dried (18hr, 90°C) and weighed. Decalcification was performed in 5 ml 2 N HCI. Acid insoluble material was collected by filtration. The retentate was homogenized in 500~1 10% KOH and kept for 18 hr at 6O‘C. 3H was counted in the acid soluble and the acid insoluble fractions. The initial shell heights of both groups were about 24 mm. At the time of the injection, 21 days after the operation, the shams had shell heights of 28-29 mm, the LCG snails of 24-25 mm.
AREND A. D~GTEROM and THEA JENTJENS
688
This procedure could not bc used in the present experiments, because always small shell fragments remain in the clay. Therefore, Fig. IB was used as a standard graph for the determination of the circumference of the openings of the shells (of known heights) of the experimental animals.
RESl L.TS
Table I shows that in the shell edge of the - LGC snails significantly less 3H-tyrosine has been incorporated than in that of the shams. In the other fractions no differences between the two groups were found. After 120min the quantities incorporated in the shell edge were higher than after 30min, but in the other fractions the quantities did not increase during this period.
DlSCUSSIOIV
20
22 24 26 mm shell height la)
26
Fig. 1. The relation between shell height (a) and circumference of the shell opening (b). Shells of different heights have shell openings of different size i.e. the circumferences of the shell edges are different. The 3H-tyrosine incorporation occurs in the newly formed margin of the periostracum at the shell edge. Therefore, if the incorporation in shells of different size has to be compared, the incorporation has to be expressed per unit of length of the margin. It is difficult to determine the circumference of the shell edge (Fig. IA) directly. Therefore prints of the shell openings were made in clay, and their circumference were measured using a thin iron wire. Figure 1B gives the results obtained with 20 shells of different heights. It is clear that there is a linear relation between the circumference of the shell opening and shell height.
Table
I. Incorporation
Shell part
Shell edge
Acid soluble shell fraction Acid
insoluble
shell fraction
In the present study the shell was divided into two parts: the shell edge and the rest of the shell. The shell edge consisted of the newly formed margin of the periostracum and a small part of the outer crystalline layer. The rest of the shell consisted of the old periostracum, the old outer crystalline layer and the old and recently formed inner layers with matrix. Therefore, the data on 3H-tyrosine incorporation in the shell edge and in the rest of the shell are measures for periostracum and for matrix formation, respectively. The quantity of tyrosine incorporated in the periostracum did increase from 30 to 120 min after the injection, whereas that in the matrix did not. A similar difference was found between calcium and carbonate depositions in the shell edge and those in the rest of the shell (Dogterom t’t ul.. 1979: Dogterom & Van der Schors, 1980). This strongly suggests that the formations of the periostracum and of the outer crystalline layer are coupled. and that the same applies to those of the matrix and the inner calcareous layers. LGC removal strongly diminishes periostracum formation, whereas matrix formation continues, This completely agrees with the effects of LGC removal on the calcium and carbonate depositions in the shell
of 3H-tyrosine into the proteinaceous L. stagnalis, 21 days after the operations Incubation time (min)
30
of the shell of
-LGC
SHAM
47 +
parts
53 (7)
14 +
8 (9) t
32 +
21 (8) *
120
632 + 671
(8)
30
899 + 644
(7)
1173 + 624
(9)
120
519 + 330
(8)
762 L 675
(8)
149 + 220
(7)
153 +
88
(9)
75 (8)
120 -+
48
(8)
30 120
153 +
Values (k SD) in dpm/mm (shell edge) or in dpm!mg dry shell (otbcr fractions); in brackets: number of observations; *: - LGC differ significantly from shams (1 < 0.05; test of Wilcoxon).
Control of periostracum formation in Lymnaea
689
Haemoiymph
‘-----~I------,------------CO"
r--coca, I inner co/core0us
HCO,
f
> motncfn
toyers
>
Mantle
I
matrix
Shell
Fig. 2. The role of the growth hormone of L. stagnalis in shell formation (for explanation, see text). CA: carbonic anhydrase, Ph. ox.: phenol oxidase. edge and in the rest of the shell (Dogterom et al., 1979; Dogterom & Van der &hors (1980). The present study completes our analysis of the effects of the growth hormone on the formation of the various shell components. Figure 2 summarizes the results. The experiments have demonstrated that the growth hormone of L. stagnalis has two clear effects. It stimulates the formation of the periostracum and of the outer crystalline layer. The first process consists of two components: 1. the production of the precursor periostracin (Waite et al., 197(j), and 2. the tanning of periostracin by phenoloxidase (Waite & Wilbur, 1976). Further investigations are needed to elucidate the details of this first effect of the growth hormone. Secondly, the effect on the formation of the outer crystalline layer is achieved by facilitating cells in the mantle edge (probably the “belt”) to maintain a relatively high calcium concentration (higher than in other tissues) by means of a specific calcium-binding protein (Dogterom & Doderer, in prep.). The bicarbonate movements, however, are not affected by the growth hormone: carbonate deposition in the shell edge is lowered after LGC removal, but 2 I days after the operation carbonic anhydrase is still present in the “belt” as well as in the other parts of the mantle epithelium. As has been discussed previously, carbonate deposition is secondarily affected via the calcium movements (Dogterom & Van der Schors, 1980). Finally, neither the formation of the matrix (from the hypothetical precursor matricin) nor that of the
inner calcareous layers is affected by the growth hormone (Dogterom et al., 1979; Dogterom & Van der Schors, 1980). Acknowledgements-The authors thank Professor J. Joosse for stimulating discussions during the course of the work and the preparation of the manuscript, Professor J. Lever for critically reading the manuscript, Mr G. W. H. van den Berg for drawing the figures, and Helen van Hekelen for typing the manuscript.
REFERENCES
DOGTEROM A. A., LOENHOUT H. VAN& SCHORSR. C. VAN DER(1979) The effect of the growth hormone of Lymnaea stagnalis 63-68.
on shell calcification.
Gen. camp. Endocr.
39,
D~GTEROM A. A. & SCHORSR. C. VANDER(I 980) The effect of the growth hormone of Lymnaea stagnalis on (bi) carcarbonate movements, especially with regard to shell formation. Gen. camp. Endocr. In press. GARAERTSW. P. M. (1976) Control of growth by the neurosecretory hormone of the Light Green Cells in the freshwater snail Lymnaea stagnalis. Gen. camp. Endocr. 29, 61-71.
KNIPRATHE. (1972) Formation and structure of the periostracum in Lymnaea stagnalis. Calc$ 7% Res. 9. 26&27
I.
MEENAKSHI V. R., HAREP. E., WATABEN. & WILBURK. M. (1969) The chemical composition of the periostracum of the molluscan shell. Comp. Biochem. Physiol. 29, 611620.
690
AKEI;U A. DOGTEKOM and
THEA
JENTJENS
SALtitiotxh A. S. M. (1976) Ultrastructural studrcs on the WAITE J. H.. SALEUDDIN A. S. M. & ANDERSEN S. 0. (1979) structure and formation of the periostracum in Hdisorna Periostracin --A soluble precursor of sclerotized periostracum in M,rfi/u.s r&lis L. J. cofnp. PlzJxiol. 130, 301 307. (Mollusca). In T/w A4cchu~~iam.s of Miwru/ixtion irr t/w Inwrr&rorcs trntl Pltrnts. (Edited by WATABI- N. & WILWILBCK K. M. (1976) Recent studies of invertebrate mtneralization In The Mcc~lkrrrisnrs f,f Mi,ic,,-trli,-clrio,l in t/w BC’K K. M.). pp. 309-337. Unwersity of South Carolina fntwwh,utes trntl PIunrs. (Edrted by WA~ARC N. & WnPress. ntx K. M. pp. 97- IOX. University of South Carolina WAITE J. H. (1977) Evidence for the mode of sclerotization Press. m a molluscan periostracum. Co~rn. Biochcw. P/r>,siol. S8B, IS7 162. ZkLSl’KA U., BOI.R H. H. & SMINIA T. (1978) Ultrastructure, WAITI J. H. & WILBIIK K. M. (1976) Phenoloxidase in the histology and innervation of the mantle edge of the periostracum of the marine bivalve &lodinlrrs ~L’I~I~ssL~.~ freshwater pulmonate snails Lrnrrturcl sfcquulis and Dillwyn. .1. C.Yp.Zoo/. 195, 359-36X. Bionrphcrluriu pftiiflcri. CLI/CI/: Ti\s. Rex 26, 27 I -2X?.
THE EFFECT OF THE GROWTH HORMONE OF THE POND SNAIL LYMNAEA STAGNALZS ON PERIOSTRACUM FORMATION ARENDA. DOCTEROMand THEA JENTJENS Department
of Biology.
Free University.
(Recc+~d
30 Octohcr
Amsterdam.
The Netherlands
1979)
Abstract-l.
The effect of removal of the growth hormone producing neurosecretory Light Green Cells (LGC) on periostracum and shell matrix formation in the freshwater snail Lq’t~~~a stagnaliswas studied by determining the jH-tyrosine incorporation in these proteinaceous shell components. 2. LGC removal resulted in a considerable decrease of the periostracum formation at the shell edge. Shell matrix formation, however. was not affected. 3. It is concluded that the effect of the growth hormone on pcriostracum formation is crucial in the regulation of shell enlargement. 4. A summarizing model of shell growth (in length and in thickness) as affected by the growth hormone, is given.
INTRODUCTION Most gastropod shells have a persistent outer proteinaceous layer, the periostracum. On the inner surface of the newly formed edge of the periostracum the first calcium carbonate layer, the “outer crystalline layer”, is deposited. The greater part of the later formed shell material consists of the inner calcareous layers, which always have a proteinaceous matrix (cf. Wilbur, 1976). It is highly probable that special regions of the mantle underlying the shell successively produce (the precursors of) these components of the shell. The periostracum is formed by the mantle edge gland (Kniprath, 1972; Saleuddin, 1976). In a previous study (Dogterom rt al., 1979) it is argued that the whole edge, or a particular part of it-the “belt” (the thickened epithelium at the mantle edge; cf. Zylstra et al.. 1978), serves as the calcium donor for the outer crystalline layer. The remaining parts of the mantle epithelium provide the matrix and the calcium for the inner layers. In the freshwater snail, Lyrnnaea stagnalis, the neurosecretory Light Green Cells (LGC), which are located in the cerebral ganglia, produce a growth hormone (Geraerts, 1976), which stimulates the growth of all organs and of the shell. In the absence of growth hormone (after LGC extirpation) the formation of the inner calcareous layers continues, but the calcium deposition at the shell edge, i.e. the formation of the outer crystalline layer, ceases (Dogterom et al., 1979). The same was found for carbonate deposition (Dogterom & Van der Schors, 1980). The cessation of calcium deposition at the shell edge is due to a decreased calcium concentration in the mantle edge (Dogterom et al., 1979), and this is caused by a lowered concentration of a specific calcium binding protein in the mantle edge (Dogterom & Doderer, in prep.). The present study was undertaken to determine the role of the growth hormone of L. stagnalis in the formation of the two proteinaceous components of the shell: the periostracum and the matrix of the inner calcereous layers. 687
As in the previous experiments LGC exirpated animals were compared with sham-operated controls. To study the protein formation in the shell components the incorporation of injected 3H-tyrosine was determined. This amino acid was chosen, since it occurs in relatively large amounts in the periostracum of snails (Meenakshi et al., 1969; Waite, 1977). Moreover, preliminary experiments also showed that tyrosine is incorporated more rapidly than aspartic acid and equimolar amino acid mixtures. MATERIALSAND METHODS Breeding and experimental conditions, and the surgical and injection techniques, were as described by Dogterom et al. (I 979). The experiment included two groups of adult animals: sham operated controls and snails in which the LGC were cauterized. An aqueous solution of 3H-tyrosine (L-[2,3,15,6-~H]tyrosine: I mCi/ml. 82 Ci/mmol) was obtained from the Radiochemical Centre (Amersham. U.K.). Twenty-one days after the operations the snails were injected with 5 ~1 of this solution per gramme wet body weight. After the injection the animals were kept in polyethene beakers containing 300 ml tap water and some lettuce. After 30 or 120 min the snails were rinsed with tap water to remove possible adhering radioactivity. Under a dissection microscope the marginal shell edge was carefully removed with a piece of razor blade. Next it was decalcified (almost no calcium carbonate is present in the very edge) in 20~1 2 N HCI. To dissolve the proteinaceous parts 100 ~1 20% KOH was added. The samples were kept at 60°C for 18 hr. jH was counted in this solution. using Insta-Gel (Packard Instrument, Brussels). The animals could be easily removed from the remainder of the shells by freezing (in liquid nitrogen) and thawing. The shells were dried (18hr, 90°C) and weighed. Decalcification was performed in 5 ml 2 N HCI. Acid insoluble material was collected by filtration. The retentate was homogenized in 500~1 10% KOH and kept for 18 hr at 6O‘C. 3H was counted in the acid soluble and the acid insoluble fractions. The initial shell heights of both groups were about 24 mm. At the time of the injection, 21 days after the operation, the shams had shell heights of 28-29 mm, the LCG snails of 24-25 mm.
AREND A. D~GTEROM and THEA JENTJENS
688
This procedure could not bc used in the present experiments, because always small shell fragments remain in the clay. Therefore, Fig. IB was used as a standard graph for the determination of the circumference of the openings of the shells (of known heights) of the experimental animals.
RESl L.TS
Table I shows that in the shell edge of the - LGC snails significantly less 3H-tyrosine has been incorporated than in that of the shams. In the other fractions no differences between the two groups were found. After 120min the quantities incorporated in the shell edge were higher than after 30min, but in the other fractions the quantities did not increase during this period.
DlSCUSSIOIV
20
22 24 26 mm shell height la)
26
Fig. 1. The relation between shell height (a) and circumference of the shell opening (b). Shells of different heights have shell openings of different size i.e. the circumferences of the shell edges are different. The 3H-tyrosine incorporation occurs in the newly formed margin of the periostracum at the shell edge. Therefore, if the incorporation in shells of different size has to be compared, the incorporation has to be expressed per unit of length of the margin. It is difficult to determine the circumference of the shell edge (Fig. IA) directly. Therefore prints of the shell openings were made in clay, and their circumference were measured using a thin iron wire. Figure 1B gives the results obtained with 20 shells of different heights. It is clear that there is a linear relation between the circumference of the shell opening and shell height.
Table
I. Incorporation
Shell part
Shell edge
Acid soluble shell fraction Acid
insoluble
shell fraction
In the present study the shell was divided into two parts: the shell edge and the rest of the shell. The shell edge consisted of the newly formed margin of the periostracum and a small part of the outer crystalline layer. The rest of the shell consisted of the old periostracum, the old outer crystalline layer and the old and recently formed inner layers with matrix. Therefore, the data on 3H-tyrosine incorporation in the shell edge and in the rest of the shell are measures for periostracum and for matrix formation, respectively. The quantity of tyrosine incorporated in the periostracum did increase from 30 to 120 min after the injection, whereas that in the matrix did not. A similar difference was found between calcium and carbonate depositions in the shell edge and those in the rest of the shell (Dogterom t’t ul.. 1979: Dogterom & Van der Schors, 1980). This strongly suggests that the formations of the periostracum and of the outer crystalline layer are coupled. and that the same applies to those of the matrix and the inner calcareous layers. LGC removal strongly diminishes periostracum formation, whereas matrix formation continues, This completely agrees with the effects of LGC removal on the calcium and carbonate depositions in the shell
of 3H-tyrosine into the proteinaceous L. stagnalis, 21 days after the operations Incubation time (min)
30
of the shell of
-LGC
SHAM
47 +
parts
53 (7)
14 +
8 (9) t
32 +
21 (8) *
120
632 + 671
(8)
30
899 + 644
(7)
1173 + 624
(9)
120
519 + 330
(8)
762 L 675
(8)
149 + 220
(7)
153 +
88
(9)
75 (8)
120 -+
48
(8)
30 120
153 +
Values (k SD) in dpm/mm (shell edge) or in dpm!mg dry shell (otbcr fractions); in brackets: number of observations; *: - LGC differ significantly from shams (1 < 0.05; test of Wilcoxon).
Control of periostracum formation in Lymnaea
689
Haemoiymph
‘-----~I------,------------CO"
r--coca, I inner co/core0us
HCO,
f
> motncfn
toyers
>
Mantle
I
matrix
Shell
Fig. 2. The role of the growth hormone of L. stagnalis in shell formation (for explanation, see text). CA: carbonic anhydrase, Ph. ox.: phenol oxidase. edge and in the rest of the shell (Dogterom et al., 1979; Dogterom & Van der &hors (1980). The present study completes our analysis of the effects of the growth hormone on the formation of the various shell components. Figure 2 summarizes the results. The experiments have demonstrated that the growth hormone of L. stagnalis has two clear effects. It stimulates the formation of the periostracum and of the outer crystalline layer. The first process consists of two components: 1. the production of the precursor periostracin (Waite et al., 197(j), and 2. the tanning of periostracin by phenoloxidase (Waite & Wilbur, 1976). Further investigations are needed to elucidate the details of this first effect of the growth hormone. Secondly, the effect on the formation of the outer crystalline layer is achieved by facilitating cells in the mantle edge (probably the “belt”) to maintain a relatively high calcium concentration (higher than in other tissues) by means of a specific calcium-binding protein (Dogterom & Doderer, in prep.). The bicarbonate movements, however, are not affected by the growth hormone: carbonate deposition in the shell edge is lowered after LGC removal, but 2 I days after the operation carbonic anhydrase is still present in the “belt” as well as in the other parts of the mantle epithelium. As has been discussed previously, carbonate deposition is secondarily affected via the calcium movements (Dogterom & Van der Schors, 1980). Finally, neither the formation of the matrix (from the hypothetical precursor matricin) nor that of the
inner calcareous layers is affected by the growth hormone (Dogterom et al., 1979; Dogterom & Van der Schors, 1980). Acknowledgements-The authors thank Professor J. Joosse for stimulating discussions during the course of the work and the preparation of the manuscript, Professor J. Lever for critically reading the manuscript, Mr G. W. H. van den Berg for drawing the figures, and Helen van Hekelen for typing the manuscript.
REFERENCES
DOGTEROM A. A., LOENHOUT H. VAN& SCHORSR. C. VAN DER(1979) The effect of the growth hormone of Lymnaea stagnalis 63-68.
on shell calcification.
Gen. camp. Endocr.
39,
D~GTEROM A. A. & SCHORSR. C. VANDER(I 980) The effect of the growth hormone of Lymnaea stagnalis on (bi) carcarbonate movements, especially with regard to shell formation. Gen. camp. Endocr. In press. GARAERTSW. P. M. (1976) Control of growth by the neurosecretory hormone of the Light Green Cells in the freshwater snail Lymnaea stagnalis. Gen. camp. Endocr. 29, 61-71.
KNIPRATHE. (1972) Formation and structure of the periostracum in Lymnaea stagnalis. Calc$ 7% Res. 9. 26&27
I.
MEENAKSHI V. R., HAREP. E., WATABEN. & WILBURK. M. (1969) The chemical composition of the periostracum of the molluscan shell. Comp. Biochem. Physiol. 29, 611620.
690
AKEI;U A. DOGTEKOM and
THEA
JENTJENS
SALtitiotxh A. S. M. (1976) Ultrastructural studrcs on the WAITE J. H.. SALEUDDIN A. S. M. & ANDERSEN S. 0. (1979) structure and formation of the periostracum in Hdisorna Periostracin --A soluble precursor of sclerotized periostracum in M,rfi/u.s r&lis L. J. cofnp. PlzJxiol. 130, 301 307. (Mollusca). In T/w A4cchu~~iam.s of Miwru/ixtion irr t/w Inwrr&rorcs trntl Pltrnts. (Edited by WATABI- N. & WILWILBCK K. M. (1976) Recent studies of invertebrate mtneralization In The Mcc~lkrrrisnrs f,f Mi,ic,,-trli,-clrio,l in t/w BC’K K. M.). pp. 309-337. Unwersity of South Carolina fntwwh,utes trntl PIunrs. (Edrted by WA~ARC N. & WnPress. ntx K. M. pp. 97- IOX. University of South Carolina WAITE J. H. (1977) Evidence for the mode of sclerotization Press. m a molluscan periostracum. Co~rn. Biochcw. P/r>,siol. S8B, IS7 162. ZkLSl’KA U., BOI.R H. H. & SMINIA T. (1978) Ultrastructure, WAITI J. H. & WILBIIK K. M. (1976) Phenoloxidase in the histology and innervation of the mantle edge of the periostracum of the marine bivalve &lodinlrrs ~L’I~I~ssL~.~ freshwater pulmonate snails Lrnrrturcl sfcquulis and Dillwyn. .1. C.Yp.Zoo/. 195, 359-36X. Bionrphcrluriu pftiiflcri. CLI/CI/: Ti\s. Rex 26, 27 I -2X?.