Schistosoma mansoni: Pairing of male worms with artificial surrogate females

EXPERIMENTAL

PARASITOLOGY

Schistosoma

68,

202-207 (1989)

mansoni:

Pairing Surrogate

of Male Worms with Artificial Females

PAUL F. BASCH~AND CARMENCITANICOLAS Department

of Health

Research and Policy, Stanford, California

Stanford 94305,

University U.S.A.

School

of Medicine,

BASCH, P. F., AND NICOLAS, C. 1989. Schistosoma mansoni: Pairing of male worms with artificial surrogate females. Experimental Parasitology 68, 202-207. To determine whether male worms provide any specific polypeptides to females, we produced extruded alginate fibers that resembled the size and shape of mature female worms. Males clasped these surrogate “female worms” and remained “paired” with them for long periods. On polyacrylamide gel electrophoresis, fibers clasped for several days showed bands at approximately 40 and 46 kDa which were never found in fibers incubated in the same medium but not clasped by males. We believe that these may be substances transferred from male worms during normal pairing. Males biosynthetically labeled with [‘4C]leucine were permitted to pair with fibers, which took up a broad range of polypeptides visualized on long autoradiographic exposure to gek. 8 1989 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Schistosoma mansoni; Reproductive biology, Pairing behavior; Alginate; Artificial female; Polyacrylamide gel electrophoresis (PAGE); Ethylene diamino tetraacetic acid (EDTA); Sodium dodecyl sulfate (SDS).

It is well established that female Schistosoma mansoni worms grow and mature only after prolonged residence in the gynecophoral canal of males, but many questions concerning conjugal relations in this species remain unsettled. Some authors (e.g., Atkinson and Atkinson 1980; Shaw et al. 1977; Popiel and Basch 1984) have suggested a chemically based relationship, in which the male provides some hormone or nutrient material to the female. Alternatively, a physical-nutritional relationship, in which the male assists the female to feed, was proposed by Gupta and Basch (1987). Resolution of this question by biochemical means is difftcult. Male and female worms are fairly advanced organisms yielding similar complex patterns on gel electrophoresis. A substance exchanged between male to female in low concentration or transitory in nature could be easily overlooked in transfer experiments. r To whom correspondence should be addressed.

Many investigators have observed male schistosomes pairing with separated or previously unpaired female worms in vitro. We have noted that male schistosomes in culture will often “pair with” (i.e., clasp in the gynecophoral canal) cotton fibers from pipette plugs. Therefore it seemed reasonable that they may pair with appropriate materials introduced for the purpose. The idea arose of producing surrogate (artificial or imitation) “females” with the characteristics listed in Table I. Lacking the biochemical complexity of real female worms, these objects could absorb materials emitted by clasping males and provide a clearer indication of which, if any, chemicals are transferred from male to female schistosomes. After numerous experiments a suitable product was developed. MATERIALS AND METHODS Production of Surrogate Females A 1% solution of sodium alginate (alginic acid sodium salt, derived primarily from the kelp seaweed Macrocystis sp. and consisting of polymerized mannuronic acid residues) was made by slowly sprinkling the 202

0014-4894189 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ARTIFICIAL

TABLE I Desirable Characteristics of Materials for Use as Artificial (Surrogate) “Female” Schistosomes Approximate size, shape, and texture of real female worms Chemically inert; nontoxic Sterilizable, preferably by steam autoclave Readily absorb chemical materials Soluble: will release absorbed materials unaltered Visible in medium and in male worms Attractive to male worms and are clasped by them Easily produced in sufficient amounts; relatively inexpensive

powder, with stirring, into boiling distilled water. The solution was filtered with suction through Whatman No. 2 filter paper and distributed in lo-ml aliquots in screw-capped glass tubes, which were autoclaved to inhibit possible microbial growth. To make the fibers, sodium alginate solution was drawn into a 5-ml glass syringe fitted into a footswitch-controlled plunger-type infusion pump (Harvard Apparatus Co. Model 975) adjusted to an output of approximately 1.4 ml/min. A blunted 20-gauge syringe needle was fitted into about a meter of polyethylene tubing (Intramedic 7426, Clay Adams, Parsippany, NJ, U.S.A.) whose other end led to a SO-mm length of finely drawn-out small-bore glass capillary tubing. (Syringe, needle, and tubing sizes are not critical, but all connections must be strong.) The viscous alginate solution was extruded with steady pressure through the glass capillary tubing held under the surface of a 2% solution of calcium chloride in distilled water. The sodium alginate solution was converted immediately to a continuous strand of flexible, insoluble calcium alginate. This process was carried out under a binocular dissecting microscope. Control of pumping rate and orifice size yielded fibers of varying diameters. Those around 80- to 12O+m thick were selected, washed several times by centrifugation in distilled water, cut into approximately lO-mm lengths with a razor blade in a Petri dish, and autoclaved. Because calcium alginate fibers are transparent and often difficult to see within the male worm, visibility was improved by incorporating tine insoluble opaque particles. This was done by grinding charcoal granules (Darco; Atlas Chemical Corp., Wilmington, DE, U.S.A.) in a mortar and pestle to produce a dust, which was mixed with an excess of distilled water. After gravity sedimentation for 5 to 10 min, the sediment was discarded and the supematant containing suspended carbon particles was used to dissolve the sodium alginate, care being taken that the capillary

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tube orifice remained open. The resultant fibers met all the requirements listed in Table I.

Pairing of Male Worms with Fibers Worms of a Puerto Rican strain of S. mansoni originally obtained from the laboratory of Dr. David Erasmus (Cardiff, Wales, UK) were recovered from 5- to IO-week unisexual or bisexual infections in mice by the perfusion method of Duvall and Dewitt (1967). Paired males were separated gently from their female partners, and males from unisexual infections were used directly. After washing several times in medium containing antibiotics (100 units of penicillin and 100 mcg streptomycin sulfate/ml), about three to four male worms/ml of Medium 169 (Basch, 1981) containing about 10 fiber segments/male were maintained at 37 C in flowing 5% CO,. We tried a variety of containers, including polystyrene Petri dishes of 35-mm diameter with 2 ml of medium, glass Leighton tubes with 1.5 ml of medium, and glass screw-capped tubes of approximately 50-ml capacity containing 10 ml of medium. Worms were examined daily by transmitted light under the dissecting microscope for evidence of pairing with the fiber segments. Fibers were removed from the gynecophoral canal with fine watchmakers forceps and pooled in the smallest possible drop of serum-free medium to reduce leaching of any absorbed substances.

Bisected Worms To determine whether the instinct to clasp an inanimate object required an intact worm, some males were bisected transversely with a sterile scalpel blade as described by Basch (1988) before being placed with fibers.

Signal Detection 1. PAGE. Paired and unpaired fibers were solubilized in a minimal volume of 0.1 M EDTA tetrasodium salt. An equal amount of double-strength SDS-PAGE sample buffer (Takacs 1979) was added. The drop of medium in which paired fibers were collected (or other samples of medium) was mixed with an equal volume of double-strength sample buffer. Tubes containing samples were immersed in boiling water for 5 min. Samples were applied to wells in a 4.7% polyacrylamide stacking gel and were run on 10% polyacrylamide separating gels in a Bio-Rad Protean slab cell by standard methods. Homogenized male worms solubilized in sample buffer were also electrophoresed on gels. Dried gels were stained with Coomassie blue or with silver stain (Bio-Rad, Richmond, CA, U.S.A.) by standard methods. 2. Transfer of radiolabel. To see whether newly synthesized polypeptide materials were transferred from males to fibers, males were incubated 24 hr in medium containing 10 @i/ml of [i4C]leucine, then rinsed well

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and placed in cultures with fibers as above. Paired fibers were removed and electrophoresed as described. Gels were stained with Coomassie blue, destained in methanol-acetic acid, impregnated for 1 hr in En3Hance (New England Nuclear, Boston, MA, U.S.A.), rinsed in water for 30 min, and dried onto Whatman 3-mm filter paper. Gels were autoradiographed at - 70 C by exposure to Kodak X-Omat film, with enhancing screens, for periods from 2 days to 2 months to look for specific transfers of labeled bands and to compare patterns among male worms, paired, and unpaired fibers. We presumed that materials transferred in small amounts would be more easily detected by long autoradiographic exposures than by direct staining of gels. To see if the male makes some specific material and exports it to the female (surrogate) when specifically stimulated by pairing, we labeled the males strongly (20 &i/ml overnight) and rinsed them in cold medium. A gel was made from the labeled males immediately,

and remaining worms were incubated in unlabeled medium. Some males were placed with fibers under optimal conditions for pairing and were permitted to pair for 24 hr. Gels were made from the males and from the fibers, and also from unpaired males and fibers. We looked for extra bands in paired males, with transfer to the fibers. In another experiment we maintained worms in labeled medium throughout and prepared gels of the same types. RESULTS

Pairing

of Male Worms with Fibers

In general, pairing (Fig. 1)occurred more readily in round-bottomed 50-ml culture tubes than in Leighton tubes or Petri dishes. We noted that male worms from 5week infections paired more readily with

FIG. 1. Male Schistosoma mansoni clasping calcium alginate fibers (arrows) in vitro. All are of intact worms except C, which shows the cut end of a bisected male. A, B, and E show the posterior end of the intact male. Note the carbon particles added for visibility. Scale bars = 200 pm.

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fibers than did worms from 7-week or older infections. In a typical experiment with 5week-old males, approximately 40% would be found clasping fibers after 2 to 3 days in culture. Using 7-week or older worms, about 20 to 25% were more commonly found paired. The vigorous movement of the worms often caused fibers to break, particularly at the anterior end of the male worm. After a few days the fibers within the gynecophoral canal were frequently found in several pieces because of stretching and flexing movements of the worms. No differences in pairing behavior were noted between males grown unisexually in mice and those formerly paired. Bisected Worms

Both anterior and posterior halves of bisected males survived well and were able to clasp fibers with apparently equal ease, and in proportions similar to intact worms (Fig. 1). Transfer of Materials

Unincubated control fibers in distilled water or simple medium without serum left no trace on gels. The alginate fibers absorbed polypeptides over a wide range of molecular weights, and dissolved readily in EDTA, releasing all of their chemical contents. The drop of unused serum-free medium to which clasped fibers were removed reflected their polypeptide profile within a few minutes, indicating that the alginate material leached its contents quickly. The fibers absorbed materials from the complete medium, which, presumably for undelined physical-chemical reasons, did not yield exactly the same pattern as the medium itself (Fig. 2, lanes 2 and 3). Figure 2 shows typical PAGE lanes of paired and unpaired fibers. The gels strongly suggested that there were differences between fibers maintained in complete medium with or without worms, and those actually clasped by male worms for 24 hr or more. Unpaired fibers or those incubated in me-

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FIG. 2. Silver-stained polyacrylamide gel electrophoretic patterns of polypeptides from solubilized alginate fibers. All lanes represent a single gel. Lane 1, fibers that had been clasped by male worms for 48 to 72 hr. Lane 2, fibers that were in the same cultures but not clasped. Each lane represents a pool of approximately 100 fiber segments. Note the additional bands present in the clasped (“paired”) fibers (arrows). Lane 3 is fresh Medium 169 containing 8% human serum, and lane 4 is the same after 2 days of incubation with male worms; essentially all bands in these lanes originate in the serum. Numbers to the left are approximate molecular weight markers, in kilodaltons.

dium without worms freely absorbed materials from the serum component of the medium (Fig. 2, lane 2). On most gels of paired fibers, more or less prominent bands were seen at approximately 40 and 46 kDa (Fig. 2, lane 1). These bands were detected by silver staining of paired fibers and of the drop of serum-free medium in which they were collected, but not in unpaired fibers or in the general culture medium. Gels and autoradiographs of male worms (Fig. 3, lanes 4 and 5) showed such a large number of polypeptides that it was not possible to identify the corresponding bands with contidence . Autoradiographs of the major polypeptides present in male worms were exposed sufficiently in 2-3 days (Fig. 3). Autoradiographs of paired fibers, whose polypeptide pattern in general reflected that of male

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FIG. 3. Autoradiographs of polyacrylamide gel electrophoretic patterns. All lanes represent a single gel. Lane 1, fibers that had been clasped for 48 to % hr by male worms previously incubated in medium containing [14C]leucine. Lane 2, fibers that had been in the same cultures, but not clasped, failed to take up any detectable label. Lane 3, the drop of medium in which “paired” fibers (used in lane 1) had been collected after separation from males and prior to solubilization. Note the rapid leaching of all components. Lanes 4 and 5, two different [r4C]leucine-labeled homogenized whole male worms that had “paired” with the fibers. Exposures of film to gels were 8.5 weeks (lanes l-3) and 2 days (lanes 4 and 5).

worms, usually required months of exposure for adequate demonstration of banding. Even after such lengthy periods, fibers not in contact with male worms failed to register on the photographic film (Fig. 3, lane 2). No differences were detected in autoradiographic patterns of male worms that had or had not paired. DISCUSSION

Certain substances such as cholesterol (Popiel and Basch 1986), glycoprotein (Gupta and Basch 1987a), and glucose (Cornford and Huot 1981) are known to be exchanged between male and female schistosomes, but these ubiquitous substances are found in both sexes and appear to lack maturational stimulation. Atkinson and Atkinson (1980) described the transfer of large amounts of a polypeptide of approximately 65 kD from male to female Schistosoma

NICOLAS

munsoni, but we were unable to duplicate those observations (Popiel and Basch 1984). The present study demonstrated that alginate fibers readily absorb materials from their surroundings and release materials into new media. We studied the polypeptide profiles of hundreds of fibers incubated in medium with and without serum, with and without the presence of male worms, and clasped or not clasped by males. Fibers paired with males for an extended period absorbed some polypeptides (at about 40 and 46 kDa) that were not found in unpaired fibers in the same medium. Films from gels of fibers paired with radiolabeled worms showed a more complex pattern after prolonged exposure (8.5 weeks) than did silver-stained gels, reflecting the greater sensitivity of the autoradiographic method. It was difficult to correlate the patterns obtained by these two methods, particularly to identify the 40 and 46 kDa bands on autoradiographic films. Autoradiographs do not express banding in an identical manner to stained gels from which they were made. The concentration of radioactivity may not be proportional to the protein content because of differences in biosynthetic incorporation into the various polypeptides, due to different turnover rates or varying proportions of the particular labeled amino acid. For these reasons we could not determine with certainty which bands on the autoradiograph represented those pointed out on Fig. 2, lane 1. The use of inert artificial females provides a strategy for capture and release of chemical substances emitted by males. Assuming that the synthetic alginate fibers are treated by the male worms in the same way as females, the current study suggests that some materials, synthesized by the male, are transferred to their partners through the gynecophoral surface. The size of these molecules, about 40 and 46 kDa, makes it unlikely that they are nonspecific or waste products. As their composition and func-

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tion remain unknown, it is not clear whether these materials play a specific role in growth or maturation of female worms. Further studies of this system appear warranted. ACKNOWLEDGMENTS

This work was supported by Grant AI-18792 from the National Institutes of Health, U.S. Public Health Service. We thank Natalicia Basch for expert technical assistance. REFERENCES

ATKINSON, K. H., AND ATKINSON, B. G. 1980. Biochemical basis for the continuous copulation of female Schistosoma mansoni. Nature (London) 283, 47W79. BASCH, P. F. 1981. Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing. Journal of Parasitology 67, 179-185. BASCH, P. F. 1988. Schistosoma mansoni: Nucleic acid synthesis in immature females from single-sex infections, paired in vitro with intact males or male segments. Comparative Biochemistry and Physiology B 90, 389-392.

CORNFORD, E. M., AND HUOT, M. E. 1981. Glucose transfer from male to female schistosomes. Science 213, 1169-l

171.

DUVALL, R. H., AND DEWITT, W. B. 1967. An im-

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proved perfusion technique for recovering adult schistosomes from laboratory animals. American Journal of Tropical Medicine and Hygiene, 16,483486. GUPTA, B. C., AND BASCH, P. F. 1987. The role of Schistosoma mansoni males in feeding and development of females. Journal of Parasitology 73, 481486. GUPTA, B. C., AND BASCH, P. F. 1987a. Evidence for transfer of a glycoprotein from male to female Schistosoma mansoni during pairing. Journal of Parasitology

73, 674-675.

PAYARES, G., AND SIMPSON, A. J. G. 1985. Schistosoma mansoni surface glycoproteins analysis of their expression and antigenicity. European Journal of Biochemistry 153, 195-201. POPIEL, I., AND BASCH, P. F. 1984. Putative polypeptide transfer from male to female Schistosoma mansoni. Molecular and Biochemical Parasitology 11, 179-188. POPIEL, I., AND BASCH, P. F. 1986. Schistosoma mansoni: Cholesterol uptake by paired and unpaired worms. Experimental Parasitology 61, 343-347. SHAW, J. R., MARSHALL, I., AND ERASMUS, D. A. 1977. Schistosoma mansoni: In vitro stimulation of vitelline gland development by extracts of male worms. Experimental Parasitology 42, M-20. TAKACS, B. 1979. Electrophoresis of proteins in polya&amide slab gels. In “Immunological Methods” (I. Letkovits and B. Pernis, Eds.), Vol. 1, pp. 81105. Academic Press, New York. Received 15 August 1988; accepted with revision 20 October 1988