Introduction

Neutral lipids are involved in chemical communication of 37-collar spined Echinostoma, and most of the research into lipid function has involved the North American species E. trivolvis (referred to as E. revolutum in papers prior to 1988). The lipids appear to serve as pheromones, or as carriers of pheromones (Fried et al. 1980; Fried 1986; Haseeb and Fried 1988).

The neutral lipid excretory-secretory (ES) products are usually collected in test tubes following the incubation of worms in balanced salt solutions for various time periods (Fried et al. 1980). Little information is available on the optimal conditions needed to obtain these lipophilic products for behavioral studies or for chemical analysis. Information on tonicity, i.e., optimal salt concentrations of media used for in vitro maintenance of adult trematodes, is sparse, and most media that are used, i.e., Locke’s, Ringer’s, and Tyrode’s solutions, are based on the assumption that the worm tonicity is equivalent to conditions in the mammalian or avian gut.

Little information is available on the effects of media tonicity on the release of presumptive lipophilic pheromones in digeneans. Therefore, the main purpose of this study was to examine the release of neutral lipids in adults maintained at various tonicities.

Locke’s solutions were used at different concentrations, i.e., full strength (1×), half strength (0.5×), and double strength (2×), to study tonicity effects on the release of neutral lipid ES products. Adult digeneans are osmoconformers (Smyth and Halton 1983), and presumably the 1× and 0.5× Locke’s solutions are isotonic to the adults of E. caproni, whereas the 2× solution is hypertonic. We also incubated worms in 0.1% glucose in Locke’s solution to test the effects of a nutrient-enriched medium on the release of neutral lipid products from adult worms. Information on neutral lipid release was also obtained in a hypotonic medium, i.e., deionized water. In addition to studies on the lipophilic substances released into these media, observations were also made on the neutral lipid content of the worms. Previous observations on the neutral lipid content of E. caproni adults have been presented by Frazer et al. (1997) and Lee et al. (1998).

Marsit et al. (2000) examined lipids in encysted metacercariae of E. caproni. However, similar studies on lipids in excysted metacercariae of E. caproni are not available. Therefore, a final purpose of this study was to examine neutral lipids in excysted metacercariae and in their neutral lipid ES products.

Materials and methods

To obtain adult worms for this study, metacercarial cysts of E. caproni were removed from the kidney-pericardial cavity of experimentally infected Biomphalaria glabrata snails and were fed (approximately 50 cysts/host) to 20 outbred FVBN202 strain mice aged 6–8 weeks. These mice are transgenic for rat mammary carcinomas between 6 and 7 months of age (Kurt et al. 2000) and have not been used previously in echinostomiasis research. Mice were fed a rat-mouse-hamster 3000 diet (RHM) and water ad libitum.

Mice were necropsied at 18 days post-infection (PI). At necropsy, the small intestines were obtained from the pyloric sphincter to the ileocecal valve, and worms were removed from the intestines as described by Hosier and Fried (1986). Worms were gravid, released eggs, and were 2–4 mg in blotted wet weight, similar to what has been reported in other studies on E. caproni adults (Fried and Huffman 1996). Approximately 20±5 worms were recovered per host at necropsy, and worms were washed in several changes of Locke’s 1× solution (NaCl, 9.0 g/l; KCl, 0.4 g/l; CaCl2, 0.2 g/l, NaHCO3, 0.2 g/l)prior to use.

Previous studies on the physiology and biochemistry of E. caproni have used worms that were 14–21 days old (Fried and Huffman 1996). Such worms are ovigerous, 5–10 mm in length, and 2–4 mg in blotted wet weight. In the present study, neutral lipids released from E. caproni were used 18 days PI. A total of 120 worms were divided into six groups with 20 worms per group; five worms (10–20 mg) were used per sample, and four samples were used for each solution. The solutions used were Locke’s 0.5×, 1×, 2×, 0.1% glucose in Locke’s 1×, and deionized water. To obtain the release of neutral lipids into the medium, the worms were incubated in 1 ml of their respective solutions for 2 h at 37°C in 15 ml centrifuge tubes. A 1 ml aliquot of solution was removed with a Pasteur pipet and extracted in 2 ml of chloroform-methanol (2:1). Worms were homogenized in a glass homogenizer and extracted with 2 ml of chloroform-methanol (2:1). Each extract was filtered through glass wool, and to the filtrate was added 1.0 ml of Folch wash (0.88% aqueous KCl) to produce a biphasic mixture. The top hydrophilic layer was discarded, and the bottom lipophilic layer was evaporated under nitrogen and reconstituted in 25–60 μl of chloroform-methanol (2:1).

Encysted metacercariae of E. caproni were obtained from experimentally infected B. glabrata snails (Fried and Huffman 1996) at 8–10 weeks PI. Excysted metacercariae were obtained following treatment of cysts in an alkaline trypsin-bile salts medium (Fried and Roth 1974), and the excystment rate within 2 h at 41°C exceeded 80%. Excysted metacercariae were washed in three changes of Locke’s 0.5×, transferred to 2 ml Eppendorf tubes containing 0.5 ml of solution, and incubated at 37.5°C. In a single trial, excysted metacercariae were placed 500 per tube and incubated in Locke’s 0.5× for 2 h. Preliminary studies showed that the use of 300 or fewer excysted metacercariae failed to reveal neutral lipids using the high performance thin layer chromatography (HPTLC) methods described herein. The solution and excysted metacercariae were extracted, and neutral lipids were separated and detected as described below. E. caproni excysted metacercariae and rediae (as controls) were fixed in neutral buffered formalin for 2 days and then stained with oil red O (ORO) (Lillie 1944) for neutral fat.

Analysis by HPTLC was performed as described by Masterson et al. (1993) using Whatman (Clifton, N.J.) LHPKDF high-performance 10×20 cm, channeled, preadsorbent, silica gel plates. The standard used was Non-Polar Lipid Mixture-B (Matreya, Pleasant Gap, Pa.), which contained cholesteryl oleate, methyl oleate, triolein, oleic acid, and cholesterol at a concentration of 0.200 μg/μl each in chloroform. The compounds in the standard were used to identify the presence of cholesteryl esters, methyl esters, triacylglyerols, free fatty acids, and free sterols, respectively, and for quantification of sterols and triacylglycerols in the samples. Aliquots of the standard solution (2.00, 4.00, 8.00, 16.0 μl) and reconstituted sample solutions (2.00 and 8.00 μl) were spotted onto the preadsorbent areas of the plates using a 10- or 25-μl Drummond (Broomall, Pa.) digital microdispenser. Plates were developed using the Mangold (1969) mobile phase [petroleum ether (37.5–60°C)-diethyl ether-acetic acid, 80:20:2] to a distance of 9.0 cm past the preadsorbent-silica gel layer interface in a glass, paper-lined, twin-trough HPTLC chamber (Camag, Wimlington, N.C.). Development required 10 min.

The developed plates were dried using a hair dryer, and lipids were detected by spraying with 5% ethanolic phosphomolybdic acid (PMA) solution and heating for 15 min at 115°C on a Camag plate heater to produce blue zones on a light yellow background (see Masterson et al. 1993 for photographs of lipids separated in this HPTLC system). Sample and standard zones were scanned using a Camag TLC Scanner II, operating in the single-beam, single-wavelength mode with the tungsten light source set at 610 nm. Calculation of lipid percentages based on the scan areas of the four standards, used to produce a calibration curve, and the area of the single sample zone with a value closest to the two middle standards was performed as described by Higgs et al. (1990).

Results

Qualitative analysis of the extracts of the incubate samples in Locke’s 0.5×, 1×, 2×, 0.1% glucose in Locke’s 1×, and DI water showed the presence of free sterol (R f =0.21), free fatty acid (R f =0.25), and triacylglycerol (R f =0.55) zones. These sample zones migrated identically to the cholesterol, oleic acid, and triolein standard zones used as markers for the neutral lipid classes. Visual comparison of zone intensities indicated that Locke’s 0.5× solution had the greatest concentration of these neutral lipids, followed by Locke’s 1× and the 0.1% glucose in Locke’s 1×, followed by Locke’s 2×, and finally the DI water. A zone was present in the sample chromatograms with the same R f value as the cholesteryl oleate standard (0.71). Previous research indicates that this zone, which was more diffuse than the standard and had a blue-green color, probably contained steryl esters mixed with hydrocarbon contaminants (Cline et al. 2000). The Smith et al. (1995) solvent system (hexanes-petroleum ether-diethyl ether-glacial acetic acid, 50:25:5:1), which could have resolved the steryl ester/hydrocarbon sample zones and allowed quantification of the steryl esters, was not used because of a lack of sufficient sample for repeated analysis.

Quantitative analysis of the chromatograms by scanning densitometry showed the free sterol and free fatty acid sample zones were present below quantifiable levels. Triacylglycerols were the only neutral lipid present in high enough concentration for quantitative analysis. The mean concentration (ng/dl) and standard error values of triacylglycerols in all of the incubates are presented in Table 1. Using the ANOVA single factor comparison (P<0.05), it was determined that the amount of triacylglycerols in the extracted incubate solutions were significantly different from each other.

Table 1 Neutral lipid concentration in Echinostoma caproni adult worms incubated in various media for 2 h at 37.5°C and of ES products. n=4 except in the study on free sterols in deionized water where n=8
Full size table

Qualitative analysis of the homogenized worm samples showed the presence of free sterol, free fatty acid, and triacylglycerol standard and sample zones in the chromatograms. Zones in sample and standard chromatograms with R f =0.71 were present as described for the incubate solution extracts.

Quantitative analysis of the homogenized worm samples showed the presence of free sterols and triacylglycerols at quantifiable levels. Free fatty acid sample zones were present below the limit of quantification. The mean concentrations and standard errors of free sterols and triacylglycerols in the worm extracts are presented in Table 1. Using the ANOVA single factor comparison (P<0.05), it was determined that the amounts of free sterols and triacylglycerols were significantly different among all the worm samples. A zero hour control was extracted and tested immediately following worm removal from the gut, and the concentrations of free sterols and triacylglycerols in these worms were 0.385±0.046 and 1.28±0.11, respectively. The concentrations of free sterols and triacylglycerols in the zero hour worms were greater than the worms incubated in their respective solutions.

HPTLC analysis of a single pool of 500 excysted metacercariae revealed the presence of free sterols, free fatty acids, and triacylglycerols at quantifiable levels. The respective weights (ng) per organism were 16.2, 1.59, and 5.34 for excysted metacercariae incubated in 0.5 ml of Locke’s 0.5× for 2 h. The incubate media contained only free sterols, which were present in concentrations below the limit of quantification. Both the excysted metacercariae and the incubate media sample chromatograms had a steryl ester/hydrocarbon zone that migrated to the same R f value as in the incubate extracts.

Excysted metacercariae were determined to be ORO negative, and no evidence of lipid droplets was seen in the excretory system, as reported for excysted metacercariae of E. trivolvis (Butler and Fried 1977; Fried et al. 1998). Rediae treated identically to the excysted metacercariae of E. caproni were lipid positive in the redial gut, subtegument, and the zones between cercarial bodies (Marsit et. al 2000), indicating that the ORO test was working properly.

Discussion

Bailey and Fried (1977) showed that Locke’s 1× solution is isotonic to E. trivolvis, an allopatric species to E. caproni and one that also lives in the gut of avian and mammalian hosts. In the present study, E. caproni adults removed from the mammalian gut and placed in Locke’s 1×, 0.5×, or 0.1% glucose in Locke’s 1× were active during the 2 h incubation. The echinostomes placed in a hypotonic solution (deionized water) imbibed water, swelled, and were dead in this medium within 2 h. Worms placed in Locke’s 2× shrunk and were inactive by 2 h in the hypertonic medium. It appears that both 1× and 0.5× Locke’s are isotonic to adults of E. caproni.

The greatest release of triacylglycerols occurred in Locke’s 0.5×, suggesting that this medium is optimal for maximal release of this ES product. The release in Locke’s 0.5× was about 10× greater than in the hypertonic (Locke’s 2×) or the hypotonic medium (DI water). Behavioral studies in which the triacylglycerol fraction is to be tested as a pheromone or carrier of a pheromone should be collected from E. caproni adults in Locke’s 0.5×.

Fried and Appel (1977) reported the main neutral lipid fraction released by E. trivolvis adults (referred to as E. revolutum in that study) into Locke’s solution was free sterols, with lesser amounts of free fatty acids, triacylglycerols, steryl esters, and phospholipids released into the medium. The present study on E. caproni adults indicates that triacylglycerol is the main fraction released into the medium, with lesser amounts of free fatty acids and free sterols. Discrepancies in these studies on adults of these two allopatric species probably reflect species differences in neutral lipid content in both worm extracts and incubate products.

Lee et al. (1998) detected triacylglycerols and free sterols in E. caproni adults removed from the intestines of the ICR mouse and extracted immediately after removal. They reported values for free sterols and triacylglycerols of 0.38 and 3.0% wet weight, respectively. They also found significantly greater concentrations of triacylglycerols than free sterols in adult worm samples. Our findings are in accord with theirs in that triacylglycerols are a major fraction in E. caproni in contrast with E. trivolvis, in which the free sterol fraction is most abundant.

Butler and Fried (1977) and Fried et al. (1998) used ORO stain to show the presence of neutral lipids in the excretory system of the excysted metacercariae of E. trivolvis. The present histochemical study on E. caproni showed the absence of neutral lipid in excysted metacercariae. Marsit et al. (2000) also used ORO stain on encysted metacercariae and found that stage to be negative for neutral lipids. Based on our study, it is probable that E. caproni excysted metacercariae contain less neutral lipids than those of E. trivolvis. The function of neutral lipids in echinostome excysted metacercariae is not known. Excysted metacercariae of E. trivolvis do not pair in vitro (see Fried et al. 1980), and it is doubtful if neutral lipids serve as pheromones or carriers of pheromones in this larval stage of echinostome.

We used both metacercariae and adults of E. caproni in our study on the release of neutral lipids into incubation media. Our design was similar to that of Fried et al. (1980), who used incubation studies with both metacercariae and adults of E. trivolvis. Fried et al. (1980) found that the major neutral lipids released into incubation media by metacercariae and adults were free fatty acids and free sterols, respectively. Their results differed from what we found in the present study on E. caproni metacercariae and adults where free sterols and triacylglycerols were the major neutral lipids released into the incubation media, respectively.