Effects of increased salinity on survival and lipid composition of Helisoma trivolvis (Colorado Strain) and Biomphalaria glabrata in laboratory cultures

2007·2.7 MB·English

by  Danielle L Martin

THE VELIGER
TheVeliger49(2):101-104(July 2, 2007) © CMS, Inc., 2006
Effects of Increased Salinity on Survival and Lipid Composition of
Helisoma trivolvis (Colorado Strain) and Biomphalaria glabrata in
Laboratory Cultures
DANIELLE L. MARTIN,' BERNARD FRIED- * and JOSEPH SHERMA'
'Department ofChemistry and "Department of Biology, Lafayette College, Easton, Pennsylvania 18042, USA
Abstract. High performance thin layer chromatography (HPTLC) was used to study the effects of increased
salinity on the lipid composition of the digestive gland-gonad complex (DGG) of Biomphalaria glabrata (NMRI
strain) and Helisoma trivolvis (Colorado strain) snails. Both species ofsnails were maintained in laboratory cultures
containing various dilutions of artificial ocean water (AOW) for up to 2 weeks. Controls consisted of snails
maintained identically in deionized water (DI). Both species of snails maintained in AOW at a salinity of 20%
(196 mOsmol/kg) showed no significant changes in the concentrations ofthe major neutral lipid and phospholipid
classes compared to snails maintained in DI. Apparently, the ability to survive for 2 weeks at 20% salinity had no
DGG
noticeable effect on the concentrations of the depot or structural lipids associated with the of these snails.
Snails ofboth species were dead at 100, 50 and 25% sahnities within 90 min. At a salinity of20%, 3 of8 S. glabrata
and 4 of 8 //. trivolvis survived for 2 weeks; at 10% salinity, 6 of 8 i?. glabrata and 7of 8 H. trivolvis survived for
2 weeks. Most snails ofboth species survived for 2 weeks in DI. The ability ofplanorbid snails to survive relatively
high salinities has implications for the spread of larval trematodiases to endemic areas where salinities may be
elevated.
INTRODUCTION sparse, another purpose of this study was to examine
the effects of increased salinity on the survival of B.
Increased salinity in fresh water lakes and ponds is glabrata and H. trivolvis in the laboratory.
recognized as a serious environmental problem world-
wide (Williams, 1987). The question of how large an
increasein salinitycan be tolerated by most freshwater MATERIALS AND METHODS
snailshas not been wellexplored. In ourlaboratory, we Stock cultures of B. glabrata (NMRI strain) and H.
use two species of fresh water snails for various
trivolvis (Colorado strain) were maintained in the
NbiMolRogIical and chemical studies. These snails are an laboratory as described in Schneck & Fried (2005).
strain of the medically important planorbid ±1 mm
Adult snails (11 in shell diameter) of both
Biomphalaria glabrata (Say, 1816) and a Colorado
species were maintained, 4 or 5 snails per culture, in
sstormaain torfivotlhveisec(oSnaoy,mic1a8l1l6)y.imSpcohrnteacnkt &plaFnroirebdid(2H0e0l5i)- d8i.f5fecrmendtiacmoentceernftirnagteironbsowlosfeaacrhticfiocnitaalinioncgea1n00wmalteorf
recently described the growth of both of these snail (AOW) at 22 ± rC. The AOW was purchased as
species under the same laboratory conditions.
a prepared concentrate of artificial salts (Instant
Our laboratory has examined the effects of various Ocean) from Aquarium Systems (Mentor, OH). A
parameters on the lipid content of B. glabrata and H. stock solution of AOW was made using 43.13 g of
trivolvis, i.e., larval trematode parasitism, different Instant Ocean in 1 L of deionized (DI) water to
diets, snail age, starvation, and estivation. As reviewed represent full strength sea water with an osmolality of
in Bandstra et al. (2006a), alterations in lipids, i.e., 960 mOsmol/kg. Thefull strength seawaterwasdiluted
increase or decrease in the concentrations ofparticular usingDI water to obtain dilutions of50, 25, 20, 15, 10,
lipid classes, occur as a function of some of the and 5% sea water with osmolalities of 499, 251, 196,
aforementioned parameters. With this in mind, one 151, 100, and 40 mOsmol/kg, respectively. The osmol-
purpose of our study was to determine the effects of alities of the AOW solutions were measured using
increased salinity on the lipid content of B. glabrata a Wescor Inc. (Logan, UT) vapor pressure osmometer.
and H. trivolvis. Because information on the tolerance For both the survival and lipid-high performance thin
of planorbid snails to increased salinities is relatively layer chromatography (HPTLC) studies, a group of4
snails of each species was maintained in separate
'Corresponding author, e-mail: [email protected] cultures at most salinities for up to 2 weeks. Survival
Page 102 The Veliger, Vol. 49, No. 2
data determined the salinity that was used in the rate lanes in aliquots of 1.00. 2.00, 4.00, 8.00. and
HPTLC-Upid studies. Cukures were fed romaine 16.0 |aL, and reconstituted samples were applied in
lettuce leaves ad libitum and the water was changed 2.00 |a^L-16.0 jiL aliquots. Applied aliquots were al-
every two days. Snails were examined daily for 1 to lowed to dry for 30 s beforedevelopment oftheplatein
14 days after the cultures were set up. Dead snails were a rectangular Camag (Wilmington, NC, USA) HPTLC
removed from the culture on the same day they died. twin-trough chamber that was lined with a saturation
Snails that did not react to mechanical probing were pad (Analtech, Newark, DE, USA) and allowed to
considered dead. Controls for the salinity studies equilibrate with the mobile phase for at least 15 min
consisted ofsnails maintained identically but in DI. before insertingthe plate. Themobile phase, petroleum
For HPTLC analysis, snail digestive gland-gonad ether-diethyl ether-glacial acetic acid (80:20:1), was
complexes (DGGs) were obtained and used as de- allowed to reach a level 9 cm above the preadsorbent-
scribed in Ong et al. (2006). Three DGGs ofeach snail silica gel interface, which required approximately
species (n = 3) maintained in 20% AOW and DI water 10 min. Developed plates were dried in a fume hood
were prepared for the neutral lipid analysis, and three for 1 min using cool air from a hairdryer and sprayed
snail DGGs ofeach species (n = 3) for analysis ofthe with 5% ethanolic phosphomolybdic acid (PMA)
AOW
phospholipids. The use ofthe 20% was based on solution. Theplatewasheatedforat 115'C onaCamag
the relatively good survival of both snail species for plate heater until blue neutral lipid bands appeared on
2 weeks at this salinity (see Results for further in- a yellow background.
formation). The blotted wet weight of the H. trivolvis The standard for polar lipid analysis. Polar Lipid
snails ranged from 9.8-19.7 mg arid the B. glahrata Mix (Matreya, Inc., Pleasant Gap, PA, USA), con-
ranged from 8.0-23.9 mg. Each sample consisted of tained 25.0% each of CH, phosphatidylethanolamine
DGG
a single and was prVepWarRed in a tissue grinder (PE), phosphatidylcholine (PC), and lysophosphatidyl-
(15 mL, KT885300-0015. International Inc.. choline (LPC) with a total of 25.0 mg of lipid. The
West Chester, PA, USA) using chloroform-methanol standard was placed in a 25 mL volumetric flask and
(2:1) with a volume that was twenty times the weight of diluted with chloroform-methanol (2:1) to prepare
the sample. After homogenization, the supernatant was
a solution containing 0.0250 |ig/|iL of each lipid.
filtered through cotton, and a volume of Folch wash HPTLC analysis ofpolar lipids was done on the same
(0.88% KCl) was added that was Va that of chloro- plates and with the same standard and sample volumes
form-methanol (2:1) used for lipid extraction. The
as described previously for the neutral lipids, but the
upper hydrophilic layer was removed using a Pasteur
mobile phasewaschloroform-methanol-water(65:25:4)
pipet and discarded; thelowerlipophiliclayerwas dried with a development time ofapproximately 20 min. The
under nitrogen gas in a waterbath (40-60°C). Residues dried plates were sprayed with a 10% cupric sulfate
were reconstituted with 80-200 )iL of chloroform-
solution and then heated at 140'C until brown bands
methanol (2:1), the exact volume chosen based upon on a white background were formed.
the amount ofthe residue. To quantify the amount oflipid in samples, a Camag
For neutral lipid analysis, the standard was Non- TLC Scanner II was used with the following settings:
Polar Lipid Mixture-B (Materya Inc., Pleasant Gap, tungsten light source at 610 nm for neutral lipids and
tPeAr,ol U(SCAH)),, wohlieicch accoindta(iOnAe)d,2t0r.i0ol%eineac(hTO)o,f cmheotlheysl- deuterium light source at 370 nm for polar lipids, slit
oleate (MO), and cholesteryl oleate (CO) with a total width 4, slit length 4, and scanning speed 4 mm/sec.
lipid concentration of 25.0 mg/mL. These compounds The CATS-3 software was used to generate a linear
regressioncalibrationplot byrelatingtheweights ofthe
were used to represent free sterols, free fatty acids,
standard zones to their peak areas. From the calibra-
triacylglycerols, methyl esters, and steryl esters, re-
spectively. The standard was dissolved in a 25.0 mL tion plot, the weights of the sample zones were
automatically interpolated based on their measured
volumetric flask in chloroform-methanol (2:1) to yield
a final concentration of 0.200 |ig/|iL of each com- peak areas. After the data was collected, if more than
pound. one ahquot ofa singlesamplefellwithinthe calibration
Analysis was performed on HPTLC silica gel 60 curve, the weight corresponding to the sample area
CF254 channeled silica gel plates (EMD Chemicals, closest to the mean of the two middle standard areas
Inc., Gibbstown, NJ, an affiliate of MerckKGaA, was used for the calculation of lipid percent. The
Darmstart,Germany), 10 X 20 cm,withapreadsorbent weight percentages of neutral and polar lipids in the
zone and 19 scored lanes. Plates were prewashed by whole snail DGGs were calculated using the following
development to the top with methanol and dried in equation:
a fume hood. Standards of neutral lipids were applied
with a 10.0 fiL Drummond (Broomall, PA, USA) % Lipid (w)(R)(F)(100)
digital microdispenser onto the preadsorbent of sepa- Hg sample
D. L. Martin et al., 2006 Page 103
Table
1
Weight percent (mean SE) ofneutral and polar lipids in the DGGs ofB. glabrata (Bg) andH. trivolvis (Ht) in 20%
artificial ocean water (AOW) and deionized water (DI).
Snail FS TG PC PE
Ht in AOW 0.0766 ± 0.0243 0.239 ± 0.0407 0.6846 ±0.135 0.201 ± 0.0343
Ht in DI 0.0742 ± 0.00504 0.153 ± 0.101 0.5973 ± 0.059 0.352 ± 0.099
Bg in AOW 0.0753 ± 0.0048 0.1697 ± 0.0621 0.8023 ±0.311 0.4235 ± 0.0172
Bg in DI 0.0679 ± 0.0172 0.2214 ± 0.0776 1.288 ±0.139 0.32234 ±0.153
FS = Free sterols.
TG = Triacylglycerols.
PC = phosphatidylcholine.
PE = phosphatidylethanolamine.
n = 3 samples for each analysis.
where w = |ag interpolated from the plot and R = samples, and since their identities were uncertain, they
[reconstituted volume (|aL)]/[spotted volume (\xL)\. For were not further characterized.
quantification ofsome samples, dilution or concentra- Samples analyzed for polar lipids showed zones with
tion was necessary in order to have scan area ofat least comparablemigrationtothe standardwith Rfvalues of
one sample aliquot bracketed within the calibration 0.28 (PC) and 0.47 (PE). LPC was not able to be
plot. Inthesecases, a suitable factor(F)wasincludedin detected with the cupric sulfate reagent at the
the calculations. concentrations applied.
Table 1 shows the lipid percent ± standard error
RESULTS data ofFS, TG, PC, and PE fractions ofthe DGGs of
H. trivolvis andB. glabrata DGGs ofsnails maintained
Survival data was as follow: all snails (n = 4 for each in 20% saline and DI water. The greatest concentra-
species at each salinity) were dead at salinities of 100, tions in both species ofsnails were the phospholipids.
50, and 25% S within 90 min. A marked difference In spite of apparent differences in the means of
occurred at 20% salinity where 3 of 8 fi. glabrata certain values of some lipids in snails maintained in
(37.5%) and 4 of 8 //. trivolvis (50%) survived the 20% saline versus DI water (for instance triacylglycer-
2 week experimental period. Because of the relatively ols from H. trivolvis snails , PC from B. glabrata snails
high survival in 20% salinity, it was this salt and PE from //. trivolvis snails), Student's r-test (P >
concentration that was selected for the lipid-HPTLC 0.05) showed no intraspecific differences in the
studies. Additional survival data showed that at 10% concentration ofany lipids in snails maintained in the
salinity, 6 of 8 (75%) B. glabrata and 7 of 8 (87.5%) 20% saline solution versus those in DI water for the
H.trivolvis survived for 2 weeks. Controls in deionized 2 week period.
water showed the following survival data at 2 weeks: 8
DISCUSSION
of8 B. glabrata (100%) and 7 of8 77. trivolvis (87.5%).
In the HPTLC studies, the major lipids quantified Relatively few studies are available on the survival of
were free sterols (FSs), (TGs), PC and PE. The linear fresh water pulmonates at various salinities. Of the
regression calibration plots relating the scan areas to pulmonale snails previously studied, Lymnaea peregra
weights ofneutral lipid standard zones (0.400-3.20 )4g) (Miiller, 1774) wasable to survive upto 10-11 partsper
andpolarlipids (0.500-4.00 |dg)consistently gavelinear thousand (ppt) and Physa fontinalis (Linnaeus, 1758)
regression correlation (r) values of 0.98 and 0.99, survived up to 6 ppt (Hyman, 1967). Ourstudy showed
respectively. good survival of H. trivolvis and B. glabrata at 1 ppt
Samples analyzed for neutral lipids showed zones and moderate survival of both species at 20 ppt. A
that comigrated with standards with Rf values of 0.19 marked reduction in survival was seen in both species
(CH) and0.57 (TO) ineverysample. Some sampleshad at 25 ppt, compared to 20 ppt. We can offer no
low amounts of free fatty acids that comigrated with explanation at this time for such a dramatic difference
the oleic acid standard at R, 0.38. The faster moving in survival at 25 versus 20 ppt salinities.
neutral lipid sample zones, tentatively thought to be The results of our HPTLC-lipid studies showed no
steryl esters and methyl esters, did not comigrate with significant changes in the concentrations of phospho-
CO and MO, respectively in the neutral lipid solvent lipids and neutral lipid classes in the DGGs of B.
system. These zones were relatively sparse in the snail glabrata and H. trivolvis maintained in DI versus 20%
Page 104 The Veliger, Vol. 49, No. 2
saline. Our results on the effects ofsalinity on the lipid Acknowledgments. We would like to thank Dr. Charles
class composition in the DGGs ofplanorbids contrast HoUiday for assistance in using the osmometer and Meredith
noticeably to other studies, i.e., snail aging (Schneck et White for assistance with the statistical analysis.
al., 2004), larval trematode parasitism (Bandstra et al., LITERATURE CITED
2006b), and estivation and starvation (White et al.,
2006), which showed significant increases or decreases Bandstra, S. R., B. Fried & J. Sherma. 2006a. Effects of
in the concentration of one or more lipid classes in B. diet and larval trematode parasitism on lipids in snails as
glabrata and H trivolvis snails as a function of the PdleatnearrmiCnhedrombyatothgirn-alpahyeyr—McohdreomrantoTgLrCaph1y9.:18J0o-u1r8n6a.l of
vWaeriacbolnecsltuuddeiedth(asteeaBdaanpdtsattriaonetoalf.,t2h0e0s6easfnoarilasretvoiewt)h.e Bandpsetrrfaor,maSn.ceR.,thBi.n-lFaryieredc&hroJm.atSohgerrampah.ic20a0n6abl.ysiHsigho-f
different salinities used in our study did not interfere neutral lipids and phospholipids in Biomphalariaglabrata
with the biochemistry of the lipid depot storage patently infected with Echinostotna caproni. Parasitology
involvingneutral lipids or the integrity ofthe structural Research 99:414-418.
lipids involving phospholipids. DA Silva,P. B., C. S. Barbosa, O. Pieri,A.Travassos&L.
Florencio. 2006. Physico-chemical and biological as-
Survival of B. glabrata in our study at 10 and even pectsrelatedtotheoccurrenceofBiomphalariaglabratain
20 ppt is of interest because de (da Silva et al., 2006) foci of schistosomiasis in coastal areas of the state of
showed survival ofthis medically important snail from Pernambuco, Brazil. Quimica Nova 29:901-906.
aquatic environments in coastal areas of Brazil at Hyman, L. H. 1967. Pulmonata: physiology. P. 645 in The
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masoni, at such a relatively high salinity constitutes an Effects of a hen's egg yolk diet on certain inorganic
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Although we did not monitor food intake or Schneck,J. L., B. Fried&J. Sherma. 2004. Effectsofaging
fecundity of our snails, we made incidental observa- on the neutral and polar lipid composition of Biompha-
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experimental conditions for 2 weeks in DI. We have oftheeffects ofestivation and starvation onneutrallipids
always used ASW as described by Ulmer (1970) to and phospholipids in Biomphalaria glabrata (NMRI
culture our planorbids (see Schneck & Fried, 2005 for strain) and Helisoma trivolvis (Colorado strain) snails by
quantitative high performance thin layer chromatogra-
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