EFFECTS OF COEXPOSURE WITH ECHINOSTOMA CAPRONI AND SCHISTOSOMA MANSONI MIRACIDIA ON NEUTRAL AND POLAR LIPIDS OF BIOMPHALARIA GLABRATA AS DETERMINED BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY-DENSITOMETRY AND OBSERVATIONS ON SNAIL SURVIVAL AND FECUNDITY

Abstract

High performance thin-layer chromatography (HPTLC)-densitometry was used to characterize and quantify neutral and polar lipids in Biomphalaria glabrata snails subjected to either Echinostoma caproni and Schistosoma mansoni miracidia coexposure, or single exposure to each of these trematode parasites. Observations on survival and fecundity were made on an unexposed population (2 cultures of 25 snails each); a population exposed exclusively to S. mansoni(2 cultures of 25 snails, each exposed to 6 miracidia per snail); a population exposed exclusively to E. caproni (2 cultures of 25 snails, each exposed to 10 miracidia per snail); and a population exposed to S. mansoni and one week later exposed to E. caproni (2 cultures of 25, each exposed to 10 E. caproni miracidia and 6 S. mansonimiracidia per snail). Each culture contained 800 mL of artificial spring water; snails were maintained at 25 ± 1°C and fed boiled romaine lettuce ad libitum. A sample of each population was necropsied at 2, 4, 6, and 8 weeks post exposure to E. caproni. The digestive-gland gonad complex (DGG) of each snail was extracted in chloroform-methanol for lipid analysis. Lipids were separated on 10 × 20 cm Analtech channeled HPTLC-HLF silica gel plates with a preadsorbent zone. For neutral lipids, the mobile phase and detection reagent used were petroleum ether-diethyl ether-glacial acetic acid (80:20:1) and 5% ethanolic phosphomolybdic acid, respectively. For polar lipids, a chloroform-methanol-deionized water (65:25:4) mobile phase and a 10% cupric sulfate in 8% phosphoric acid detection reagent were used. Quantitative densitometric analysis was performed using a CAMAG TLC Scanner 3 with the tungsten light source set at 610 nm for neutral lipids and the deuterium light source set to 370 nm for polar lipids. Significant differences in lipid concentrations were only observed for the free fatty acids (ANOVA, P<0.0125). Survival of snails declined mainly in the group exposed only to E. caproni miracidia and in the coexposed group. Fecundity (based on egg laying) increased in the group exposed only to E. caproni miracidia, but declined in the other groups.

Keywords:

• Biomphalaria glabrata
• coinfection
• densitometry
• Echinostoma caproni
• high-performance thin-layer chromatography
• HPTLC
• miracidia
• neutral lipids
• polar lipids
• Schistosoma mansoni
• snail fecundity
• snail survival

ACKNOWLEDGMENT

We are indebted to Dr. Fred A. Lewis for supplying the B. glabrata snails used in this study through NIH-NIAID contract HHSN2711010000051. The authors thank Steven Miles of Analtech Inc. for providing the HPTLC plates. Daniel Beideman was supported by a Camille and Henry Dreyfus Foundation Senior Scientist Mentor Program award to Professor Joseph Sherma and the Lafayette College EXCEL Scholars Program.

Notes

^a A, S. mansoni exposed; B, coexposed (S. mansoni and E. caproni); C, E. caproni exposed; D, control (unexposed snails).

^a A, S. mansoni exposed; B, coexposed (S. mansoni and E. caproni); C, E. caproni exposed; D, control (unexposed snails).

^a A, S. mansoni exposed; B, coexposed (S. mansoni and E. caproni exposed); C, E. caproni exposed; D, control (unexposed control).

^b Sample size of DGGs used: A = 6, B = 6, C = 4, D = 6.