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Amer. Maine. Bull. 22: 139-142 The concentration of calcium carbonate in shells of freshwater snails Meredith M. White1 Michael Chejlava1 Bernard Fried2 and Joseph Sherma1 , , , 1 Department ofChemistry, Lafayette College, Easton, Pennsylvania 18042,U.S.A. 2 Department ofBiology, Lafayette College, Easton, Pennsylvania, 18042, U.S.A., [email protected] Abstract: The range of concentration of calcium carbonate in the shells of various freshwater gastropods was determined using ion chromatography. Individuals ofHelisoma trivolvis (wild) were collected from three ponds in Pennsylvania and New Jersey; individuals of Physasp.werecollectedfromonepondinNewJersey;individualsofH. trivolvis (Coloradostrain) andBiomphalariaglabrata (NMRIstrain) were raised in thelaboratory; and individuals ofPomacea bridgesiiwere purchased commercially. The concentrations ofcalcium carbonate % (mean by dry weight) ofthe shells were as follows: H. trivolvis (wild), 97.0; Physa sp., 97.8; H. trivolvis (CO), 97.6; B. glabrata, 98.8; P. bridgesii, 98.2. Our data support and validate the previous claim that snail shells are comprised of95-99.9% calcium carbonate. Key words: Gastropoda, ion chromatography, Biomphalaria glabrata, Helisoma trivolvis, Physa sp., Pomacea bridgesii According to Hare and Abelson (1965) and Marxen et One of us (B. Fried) has maintained a continuous cul- al. (2003), molluscan shells consist of95-99.9% calcium car- ture ofHelisoma trivolvis (Colorado strain) for more than 15 bonate and 0.1-5% organic material by weight. However, years (Fried et al. 1987). Stock cultures ofBiompJialariagla- detailed analyses ofthe concentration ofcalcium carbonate brata (NMRI strain) were obtained from Dr. Fred Lewis, in the shells of individual species of pulmonate and proso- Schistosomiasis Laboratory, Biomedical Research Institute, branch snailsareforthemostpartnotavailable. Somevalues Rockville, Maryland, LISA. Individuals ofPomacea bridgesii ofcalcium carbonateintheshellsofseveral pulmonateswere were purchased from Carolina Biological Supply Company given incidental to a studythat examined the effects ofpara- (Burlington, North Carolina, USA). The specific identity of sitism by larval trematodes on the proportion of calcium these snails was confirmed by Dr. Robert H. Cowie, Univer- carbonate in the shells of selected gastropods (White et al. sityofHawaii, Center for Conservation Research and Train- 2005). To provide further confirmation of the 95-99.9% ing, Honolulu, Hawaii, LISA. Twenty individuals of H. tri- range ofcalcium carbonate reportedfor molluscan shells,we volvis (Colorado strain), 20 ol B. glabrata, or 2 to 3 of P. examined the calcium carbonate concentration ofindividu- bridgesii were maintained at 23 ± 1°C in aerated glass jars als from several species offield collected (wild) snails, Heli- containing 800 mL artificial springwater (ASW) as prepared soma trivolvis (Say, 1816) andPhysa sp., two species oflabo- by Ulmer (1970) under diffuse fluorescent light for a pho- ratory-reared snails, H. trivolvis and Biomphalaria glabrata toperiodof12 h perday. Thecultureswerefed adlibitum on (Say, 1816), and one snail species purchased from a com- boiled romaine lettuce leaves, and the water was changed mercial supplier, Pomacea bridgesii (Reeve, 1856). three times a week. Individuals of H. trivolvis (CO) and B. glabrataweremaintained in thelaboratoryforabout 6 weeks METHODS prior to use. At the time of analysis, they were sexually mm mature and exceeded 10 in shell diameter. Individuals Collection and maintenance of snails ofP. bridgesii were used within about 1 weekofreceipt from Individuals ofHelisoma trivolvis (Pennsylvania andNew the supplier. All ofthe snails were negative for larval trema- Jersey strain) were collected from Amwell Lake, Wildlife todes at necropsy. Management Area, East Amwell Township, Hunterdon County, New Jersey, USA (40°26'N, 74°49'W) on 15 July Calcium carbonate determination 2004 and from Hoch Pond, Northampton County, Pennsyl- Ten snailswererandomlyselected from each population vania, USA (40°47'20"N, 75°27'15"W) on 1 July 2004. Indi- for analysis. At necropsy, snails were measured with a ver- mm viduals of H. trivolvis and Physa sp. were collected from nier caliper to the nearest 0.1 tor Helisoma trivolvis, Delaware Pond, Knowlton Township, Columbia, New Jer- Biomphalaria glabrata, and Physa sp. and to the nearest 1 sey, USA (40°55'19.1"N, 75°03'49.5"W) on 1 July 2004. All mm for Pomacea bridgesii. Measurements were taken ofthe wild snails were analyzed within 1-2 days of collection. A maximum length ofindividuals ofPhysa sp. and P. bridgesii sample of water from each pond was also collected and and ofthe maximum diameterofH. trivolvisand B. glabrata. analyzed for calcium carbonate as well. The shell was dissected from the body and the body was 139 140 AMERICAN MALACOLOGICAL BULLETIN 22 • 1/2 • 2007 discarded. The operculum ofP. bridgesiiwas also discarded. carbonate ofthe shells ofvarious populations ofsnails. Ifa Each sample consistedofone snail shell, whichwasprepared significant difference (P < 0.05) was found, the data were and analyzed as described in White et al. (2005) using a subjected to the Bonferroni method to determine among Dionex DX-120 Ion Chromatograph (Dionex, Sunnyvale, which populations the difference occurred. When two California,USA) witha DionexAS40 automatedsampler, an means were compared, Student's f-test was used to deter- IonPac CG12A guard column (4 x 50 mm), an IonPac minewhetherthere was a significant difference (P< 0.05) in CS12A cation exchange analytical column (4 x 250 mm), the calcium carbonate concentration in the shells ofthe two and a conductivitydetector. The columnwas eluted isocrati- populations. SPSS version 12.0 softwarewas used for all data mM cally with 20 methanesulfonic acid at a flow rate of 1.0 analyses. mL/min. A Dionex cation self-regenerating suppressor ultra (100 mA) was utilized to suppress background conductivity. RESULTS Standard solutions ofthe calcium ion were prepared at 1.00 mg/L, 5.00 mg/L, 10.0 mg/L, 25.0 mg/L, 50.0 mg/L, 100 The percentage of calcium carbonate of the shells, the mg/L, and 200 mg/L and used for calibration. Each sample sizes of the snails used, and the concentrations of calcium was analyzed in triplicate with an injection volume of25 uL carbonate ofthe water in which the snails were maintained and the mean concentration of calcium ion (% by dry are presented in Table 1. The samples from Helisoma tri- weight) was calculated. The retention time for the calcium volvis from Hoch Pond showed a significantlylower concen- ion was 8.15 ± 0.50 min. Values ofconcentration ofcalcium tration ofcalcium carbonatethan anyother snail population carbonate ofthe test solutions were determined by PeakNet studied (ANOVA, P< 0.05). Shell size couldnotbe analyzed version 5.1 software as described in White et al. (2005). For as a factor when looking at the concentration of calcium each population, a blank was prepared in the same manner carbonate among all populations due to the natural size and was taken into account in calculating the final percent- differences between species. There was no significant differ- age ofcalcium carbonate ofeach snail shell. A single water ence in theconcentrations ofcalcium carbonate between the sample from each collection site and ASW were analyzed by two species oflaboratory-reared snails and the commercially ion chromatography following the same procedure. purchased snails (ANOVA, P > 0.05). The shells ofwild H. trivolvis showed no significant difference in the concentra- Statistical analysis tion of calcium carbonate from the laboratory-reared indi- For all experiments in which multiple sample means viduals of H. trivolvis (Student's f-test, P > 0.05); however, were compared, a single factor analysis of variance the shells of the wild population of H. trivolvis as a group (ANOVA) was used to determine whether there was a sig- were significantly smaller in diameter than the shells ofthe nificant difference between the concentrations of calcium laboratory-reared H. trivolvis (Student's f-test, P < 0.05). Table 1. Percentage ofcalcium carbonate by dry weight ofsnail shell, shell size, and calcium carbonate content (mg/L ofwater. CaCO, content in shells (%) Size ofsnail (mm) CaCO, content Species Pond or ASW1 (mean ± SE) (mean ± SE) in water (mg/L)2 Wild snails Helisoma trivolvis3 Amwell Lake 97.6 ± 0.24 15.0±0.57 141.1 H. trivolvis5 Hoch Pond 95.2 ± 0.44 10.4±0.27 39.9 H. trivolis Delaware Pond 98.1 ± 0.6 10.6 ± 0.37 190.4 Physa sp.3 Delaware Pond 97.8 ± 0.5 8.4 ± O.l4,6 190.4 Laboratory-reared snails H. trivolvis (CO) ASW 97.6 ±0.44 13.2 ± 0.37 32.0 Biomphalaria glabrata3 ASW 98.8 ± 0.2 11.1 ± 0.37 32.0 Commercially purchased snails Pomacea bridgesii ASW 98.2 ± 0.4 36 ± 26 32.0 ' ASW = artificial spring water. 2Water from the collection sites for wild snails and ASW for all others. 3From White etal (2005). 4OnesamplewasdeterminedbytheQ-test(90%confidenceinterval)tobeanoutlierandwasexcludedfromtheresultsandallstatisticalanalyses,giving = 9. 5This sample had a mean concentration ofcalcium carbonate thatwas significantlylower than the othersamples (ANOVA, P < 0.05). 6Maximum length. 7Maximum diameter. CALCIUM CARBONATE IN SHELLS 141 When considering only shells from the wild populations of reared snails and the wild snails was that the laboratory- H. trivolvis, the group collected from Hoch Pond had a reared snails were fed ad libitum; we do not know how significantly lower concentration of calcium carbonate in adequate the food supply was for the snails obtained from their shells than did the groups collected from Amwell Lake the wild. Individuals ofLymnaeaperegra (Miiller, 1774) and and Delaware Pond. Phmorbariuscorneus (Linnaeus, 1758) reared in calcium-rich A correlation between the hardness ofthewater and the water, obtain calcium more from the water than from the concentration ofcalcium carbonate in the shells was found food. Individuals reared in calcium-poor water, however, only for the wild snails. Hoch Pond had the softest water obtain two to four times more calcium from the food than (although still abovethe 20 mg/LCaC03 minimum found to from the water (Young 1975). This relationship between limit the number ofspecies of snails that can survive; Boy- waterhardness andthesourceofcalcium maybe responsible cott 1936, Macan 1950). Shells ofsnails obtained from that in part for the results ot the present study; however, the pond had the lowest concentrations of calcium carbonate. relative role offood versus water as a source ofcalcium for Delaware Pond had the hardest water and the shells ofsnails the snails was undetermined in our study. collected from it had the highest concentrations ofcalcium The data showed no direct correlation between size of carbonate. Amwell Lake had a calcium content between that the shell andconcentrationofcalciumcarbonate. Withinthe ofHoch Pond and Delaware Pond. Shells ofsnails from that wild populations ofHelisoma trivolvis., the snails from Am- lake had concentrations ofcalcium carbonate between those well Lake were the largest; however, this did not correspond ofthe snails from Hoch Pond and the snails from Delaware to a lower or higher concentration ofcalcium carbonate in Pond. the shell. The shells from the wild population ofH. trivolvis The ASW had the lowest concentration ofcalcium car- showed no difference in concentration ofcalcium carbonate bonate, yet the shells ofthe laboratory-reared and commer- from those in the laboratoiy-reared strain, but the wild cially-purchased snails that were maintained in it had con- population was significantly smaller in diameter. centrations of calcium carbonate that were significantly The data supported the claim that shells of freshwater % higher than those of the wild snails (Student's f-test, snails are comprised of 95-99.9 calcium carbonate by P<0.05). According to the standard classification for water weight. hardness of the U.S. Geological Survey (2006), the water from Hoch Pond and the ASW were soft, the water from Amwell Lake was hard, and the water from Delaware Pond ACKNOWLEDGEMENTS was very hard. We are grateful to Dr. Fred A. Lewis, Head, Schistoso- miasis Laboratory, Biomedical Research Institute, Rockville, DISCUSSION Maryland, USA. for supplying individuals of Biomphalaria glabrata used in this work through NIH-NIAID contract In spite ofconsiderable variation in the calcium content N01-AI-55270. We also thank Dr. John R. Stonesifer, Lafay- (mg/L) ofthe water in which the snails were maintained, all ette College Mathematics Department, Easton, Pennsylva- ofthe snails showed concentrations o%fcalcium carbonate in nia, USA, for his help in the statistical analysis. White's work their shells in the range of95.2-98.8 by weight, similar to was supported by a Camille and Henry Dreyfus Foundation therangereportedbyHare andAbelson (1965). Thus, under Senior Scientist Mentor Initiative Grant awarded to J. conditions of variable concentrations of calcium carbonate Sherma. in the water, freshwater snails are able to maintain a high concentration ofcalcium carbonate in their shells. No clear trend between the concentrations of calcium carbonate of LITERATURE CITED the external media and the concentrations ofthe snail shells was found when both wild and laboratory-reared populations wereconsidered. Freshwatersnailsobtaintheircalcium Bevelpaonsdietri,onG.of19C5a2.45CaalncdifPicatiionnrienlatmioolnlutsokss.heIlIlI.fIonrtmaakteioann.dBdieo-- from the surrounding water and their food source (van der logical Bulletin 102: 9-15. Borght and van Puymbroeck 1966, Young 1975), first local- Borght, O. van derand S. van Puymhroek. 1966. Calcium metabo- izing the calcium in the mantle before depositing it in the lism in a freshwater mollusk. Quantitative importance ofwa- shell (Bevelander 1952). It is surprising that the laboratory- terand foodassupplyforcalcium duringgrowth. Nature210: reared snails, which were raised in the softest water, had 791-793. relatively high concentrations ofcalcium carbonate in their Boycott,A. E. 1936.Thehabitatsoffresh-waterMolluscain Britain. shells. One important difference between the laboratory- Journal ofAnimal Ecology 5: 116-186. 142 AMERICAN MALACOLOGICAL BULLETIN 22 • 1/2 • 2007 Fried, B., S. Scheuermann, and }. Moore. 1987. InfectivityofEchi- nostoma revohitum miracidia for laboratory raised pulmonate snails, journal ofParasitology 73: 1047-1048. Hare, P. E. and P. H. Abelson. 1965. Amino acid composition of some calcified proteins. Carnegie Institution of Washington Yearbook 64: 223-232. Macan, T. T. 1950. Ecology offreshwater Mollusca in the English Lake District. Journal ofAnimal Ecology 19: 124-146. Marxen, J. C, W. Becker, D. Finke, B. Hasse, and M. Epple. 2003. Early mineralization in Biomphalaria glabrata: Microscopic and structural results. Journal of Molluscan Studies 69: L13- 121. Ulmer, M. J. 1970. Notes on rearing ofsnails in the laboratory. In: A. ]. Maclnnis and M.Voge, eds., Experimentsand Techniques in Parasitology. W. H. Freeman, San Francisco. Pp.143-144. U.S. GeologicalSurvey. 2006. Explanationofhardness.Availableat: http://water.usgs.gov/owq/Explanation.html 7 November 2006. White, M.M., M. Chejlava, B. Fried, and I. Sherma. 2005. Effects of various larval digeneans on the calcium carbonate content of the shells of Helisoma trivolvis, Biomphalaria glabrata, and Physa sp. Parasitology Research 95: 252-255. Young, J.O. 1975. A laboratory study, using 45Ca tracer, on the source ofcalcium during growth in two freshwater species of Gastropoda. Proceedings oftheMalacologicalSocietyofLondon 41: 439-445. Accepted: 7 November 2006