and Joseph Sherma2| Parasitology Research |
| vol. 88, nr. 12, p. 1080 - 1082 |
| © Springer-Verlag 2002 |
| DOI 10.1007/s00436-002-0718-0 |
Seth W. Kaufer2, Michael Chejlava2, Bernard Fried1, and Joseph Sherma2
| (1) | Department of Biology, Lafayette College, Easton, PA 18042, USA |
| (2) | Department of Chemistry, Lafayette College, Easton, PA 18042, USA |
| E-mail: friedb@lafayette.edu Phone: +1-610-3305463 Fax: +1-610-3305705 |
Received: 29 May 2002 / Accepted: 11 July 2002 / Published online: 15 August 2002
Abstract. Graphite furnace atomic absorption spectrometry and ion chromatography were used to study the metallic ions in the digestive gland-gonad complex (DGG) of Cerithidea californica snails infected with the daughter rediae and cercariae of Euhaplorchis californiensis and in uninfected DGGs. Seven metals (calcium, copper, iron, magnesium, potassium, sodium, zinc) were found to be present in infected and uninfected DGGs at concentrations above the minimum levels required for detection. Of these, calcium was present in significantly higher amounts (Student's t-test, confidence level of 95%) in the infected versus uninfected DGGs; magnesium occurred in significantly lower amounts in the infected DGGs. Our results were compared with a previous study that analyzed metallic ions in the DGG of Helisoma trivolvis naturally infected with Echinostoma trivolvis. That study reported a significant elevation of sodium but a reduction of magnesium and manganese in the DGG of infected snails. Variations in the results of the two studies reflect intrinsic differences in the larval trematode-snail systems used.
There is little information on the effects of larval trematode parasitism on the metallic ion content of marine gastropod first intermediate hosts. Evans et al. (2001) examined the effects of undetermined larval trematode infections in the marine snail Littorina littorea. They found that infected littorines had significantly lower levels of iron, copper, and nickel than uninfected snails. Our laboratory was concerned with the pathobiochemical effects of larval trematode parasitism on the marine snail Cerithidea californica. This snail is infected with at least 18 species of larval trematodes (Martin 1972). In our studies with this larval trematode-snail system, we examined the effects of larval parasitism on the neutral lipid and phospholipid composition of the snail (Cline et al. 2000), effects of infection on lutein and beta-carotene concentrations in the snails (Marsit et al. 2000a), and effects of infection on the carbohydrate content of the snail (Marsit et al. 2000b). In the aforementioned studies, the larval trematodes were associated with pathobiochemical effects on the snails; there were qualitative and quantitative differences in the analytes in the infected versus uninfected snails.
We have not examined the effects of larval trematode infection on the metallic ion content of C. californica. We were able to obtain a population of snails infected only with the intramolluscan stages of the heterophyid Euhaplorchis californiensis (see Martin 1972 for a description of these stages) and uninfected cohorts from the same collection site. We were interested in this study because metallic ions are important indicators of biopollution in gastropod molluscs. They accumulate in the tissues of gastropods, particularly in the digestive gland. Because of the bio-accumulative properties of gastropods, they are useful monitors of metal pollution. Furthermore, many gastropods are infected with larval digeneans and there is scant information on the effects of metal bio-accumulation in uninfected snails versus those infected with larval digeneans. The purpose of this study was to determine the concentrations of certain metal ions in uninfected C. californica and those infected with the intramolluscan stages of E. californiensis, using graphite furnace atomic absorption spectrometry (GFAAS) and ion chromatography (IC). Because the digestive gland-gonad complex (DGG) of C. californica is the main site of infection with the rediae and cercariae of E. californiensis and because it is a major site for the storage of metal ions, it was this complex that was examined in our study.
Cerithidea californica snails naturally infected with the intramolluscan stages of Euhaplorchis californiensis and uninfected snails were collected in the Colorado Lagoon, Long Beach, California, USA, by a commercial supplier (Jones Biological Laboratory, Long Beach, Calif.). Snails were shipped overnight on sphagnum moss by express mail to Easton, Pennsylvania and maintained at 12 °C in 18-cm diameter finger bowls half-filled with artificial sea water (Carolina Biological Supply Co., Elon, N.C.) until they were used (within 3 days of receipt). Snails were isolated individually in Stender dishes containing 5 ml of sea water to determine larval infection with E. californiensis. Infected snails released large numbers of heterophyid cercariae (cercarial body with characteristic eye spots and cercarial tail with characteristic fin folds containing tegumentary spines) identified as E. californiensis. Snails determined to be infected by isolation were subsequently crushed in one-half strength Locke's solution (containing, per liter:4.5 g NaCl, 0.2 g KCl, 0.1 g CaCl2, 0.1 g NaHCO3) to obtain the infected DGG, i.e., the gland complex plus the rediae. DGGs were pooled to achieve a blotted wet weight of about 100 mg (usually 1-2 DGGs/sample). Likewise, samples of uninfected DGGs were pooled to obtain a similar blotted wet weight (usually 2 uninfected DGGs). Most of the snails that were negative upon isolation were negative when the DGGs were examined after the snails were crushed. Snails were dissected and examined very carefully to exclude infection with other helminths. Prior to use in an analysis, each sample was rinsed several times in deionized water and then digested in 5 ml of boiling concentrated nitric acid in a 10-ml beaker. Each digested sample was transferred and diluted to 10 ml in a volumetric flask with 1% nitric acid and stored in plastic bottles to prevent sodium from leaching into solution from glass. Aliquots from samples to be analyzed by GFAAS and IC were diluted 1:100 with 1% nitric acid to bring them into the range of the calibration curves. Samples to be analyzed by IC were additionally treated as follows: 1 ml was removed from diluted samples, boiled down on a hot plate, and reconstituted in 1 ml of 0.001 M nitric acid. A total of about 30 snails was used for the entire study; samples were used for several different measurements of metal ion concentrations.
The samples were quantitatively analyzed for seven metallic ions: iron, copper, zinc, sodium, potassium, calcium, and magnesium. Five of the metals were quantified by GFAAS: iron, copper, zinc, sodium, and magnesium. The GFAAS instrument was a GBC 932 (GBC, Arlington Heights, Il.) plus atomic absorption spectrometer with GBC GF3000 graphite furnace system, separate hollow cathode lamps (Varian Techtron, Australia) for each element determined, GBC PAL3000 auto-sampler, and GBC Advanta version 1.33 software. The instrument has a double-beam design and a deuterium background-correction system. Absorbance values of the test solutions were measured by the instrument in the form of peak areas. All standard and sample volumes were 20 µl. Bulk solutions of each metallic ion in 1% HNO3 were made and auto-diluted with 1% HNO3 into multiple standards by the instrument. These ranges and other instrument parameters varied by metal. Ten infected and ten uninfected samples were analyzed in triplicate to obtain absorbance values. The instrument provided the measured concentration of each test solution by interpolation from the calibration curve. The metal concentration in the diluted snail samples was converted to a snail-mass basis using the calculation: final metal concentration (mg/g) = (i×v×d)/106 m, where i is the test solution concentration from the instrument in parts per billion (ppb; ng/ml), v is the original volume of the samples (10 ml), d is the dilution factor (100), and m is the mass of the snail DGG in grams. The infected and uninfected sets of ten samples were then averaged and compared for statistical difference using Student's t-test at the 95% confidence interval.
Quantification of two metals that could not be analyzed by GFAAS with our instrumentation was performed by IC: potassium and calcium. The instrument was a DX-120 ion chromatograph (Dionex, Sunnyvale, Calif.) with Dionex AS40 automated sampler. A Dionex IonPac CS12A (4×250 mm) cation exchange column functionalized with weak phosphonic and carboxylic acids was used. The column was eluted isocratically with 20 mM methanesulfonic acid at a flow rate of 1 ml/min. A Dionex cation self-regenerating suppressor ultra (100 mA) was utilized to remove the eluent ions during analyte detection with the conductivity detector. Mixed standard solutions of the two metal cations were prepared at 100 ppb, 1 parts per million (ppm), 10 ppm, and 100 ppm and used for calibration. Ten infected and ten uninfected samples were analyzed in duplicate with an injection volume of 25 µl at a flow rate of 1.0 ml/min. The retention times for potassium and calcium ions were 4.67 min and 8.37 min, respectively. Concentration values of test solutions were determined by the instrument; these were converted to concentrations in the snail samples using the calculation: final metal concentration (mg/g) = (i×v×d)/1,000 m, where i is the test solution concentration from the instrument in ppm (µg/ml), v is the original volume of the samples (10 ml), d is the dilution factor (100), and m is the mass of the snail DGG in grams.
GFAAS experimentation provided calibration curves for each of the tested metals with a representative nonlinear least squares correlation coefficient of approximately 0.995. The triplicate test solution concentration measurements all had relative standard deviation values less than 10%. The mass-adjusted snail concentrations (Table 1) fell in the parts per thousand to parts per million range and had much larger uncertainty than the instrument values of the test solutions. Sodium and magnesium were the metals in highest concentration in uninfected snails, with lower levels of iron, zinc, and copper. Sodium and magnesium were also present in the highest concentrations in infected snails, with less iron, zinc, and copper. The concentration level of magnesium was significantly lower in infected compared to uninfected snails (Student's t-test, 95% confidence level). No significant differences were detected between the concentrations in infected and uninfected samples of the other metals.
Table 1. Graphite furnace atomic absorption spectrometry (GFAAS) and ion chromatography (IC) data for seven metals in infected versus uninfected snails (mean ± SD).Ten infected and ten uninfected samples were analyzed. The t-test was performed at the 95% confidence level with ttable = 2.26. If tcalc > ttable, then the numbers are significantly different. An asterisk indicates a significant difference between infected and uninfected samples
| Method | Metal | Infected snail (mg/g) | Uninfected snail (mg/g) | tcalc |
| GFAAS | Fe | 0.15±0.03 | 0.14±0.05 | 0.110 |
| GFAAS | Cu | 0.04±0.02 | 0.04±0.03 | 0.0733 |
| GFAAS | Zn | 0.2±0.1 | 0.2±0.1 | 1.31 |
| GFAAS | Na | 2.7±0.5 | 2.2±0.8 | 1.40 |
| GFAAS | Mg* | 0.5±0.1 | 1.0±0.1 | 9.18 |
| IC | K | 0.7±0.4 | 1.2±0.6 | 1.71 |
| IC | Ca* | 0.6±0.2 | 0.3±0.2 | 2.73 |
IC determinations provided calibration curves for potassium and calcium with linear least squares correlation coefficients of approximately 0.985. The mass-corrected snail concentrations of the metals were determined in the same samples as analyzed by GFAAS. The data resulted in the parts per thousand to parts per million range and are shown in Table 1. The concentration of calcium was significantly higher in the infected snails (Student's t-test, 95% confidence level). The potassium concentrations were not significantly different.
Invasion of the DGG of Cerithidea californica by larval Euhaplorchis californiensis causes disruption of the architecture of this complex (see Yoshino 1976 for a review). Concomitant with gross and histological changes in the DGG are changes in the concentrations of neutral lipids, phospholipids, and lipophilic pigments in the infected complex (Cline et al. 2000; Marsit et al. 2000a).
The results of our study show that there are also significant changes in certain metallic ions as a result of larval E. californiensis infection in the DGG of C. californica. Thus, we found significantly higher amounts (at the 95% confidence level) of calcium but significantly lower amounts of magnesium in this complex in infected snails. In a similar study using the freshwater snail Helisoma trivolvis naturally infected with the ubiquitous digenean Echinostoma trivolvis, Layman et al. (1996) found significantly higher amounts of sodium and lower amounts of magnesium and manganese in the DGGs of infected snails. Variations in the results of the two studies reflect intrinsic differences in the larval trematode-snail systems used. In the only other published study using a marine snail infected with larval trematodes, Evans et al. (2001) reported significant decreases in iron, copper, and nickel concentrations in whole snail bodies of Littorina littorea infected with unidentified larval trematodes, compared with uninfected snails. Evans et al. (2001) used atomic absorption spectrometry to analyze heavy metals (lead, iron, copper, nickel) in the whole snail bodies of their infected and uninfected littorines. With the exception of iron and copper, our analyses were made on various other metallic ions not examined by Evans et al. (2001). Moreover, the two studies are not comparable since their analyses were made on whole snail bodies, whereas we examined only the target site of the larval trematode, the DGG. Nevertheless, their study and ours showed that larval trematode parasites are associated with significant changes in the metallic ion content of first intermediate snail hosts.
We do not know why the concentrations of certain metals are significantly increased, whereas others are significantly decreased in infected versus uninfected snails. As discussed by Evans et al. (2001) for their L. littorea-larval trematode model, a major reason for the difference is the pathologic effect of the larval trematodes on the digestive gland cells of the infected snails. A major function of digestive gland cells is the storage of various metals (see Bebianno and Langston 1995 for details), holding them in membrane-insoluble granules in the cells. Destruction of the digestive gland cells by larval trematodes probably reduces storage volume and holding capacity of metals in the infected snails.
Our study has important implications for future work that attempts to analyze metallic ions in C. californica and other marine gastropods associated with coastal habitats. Such snails are usually heavily parasitized with larval trematodes, which could alter baseline values for certain metals in the snails. Workers who attempt to analyze metallic ions in these snails should be aware of the influence of parasitism on their studies and should make efforts to distinguish snails infected with larval trematodes versus those uninfected prior to any analysis of metallic ions.
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