Journal of Chemical Ecology, Vol. 12, No. 8, 1986

CHEMICAL COMMUNICATION IN A D U L T SCHISTOSOMES

M.A.

H A S E E B and L . K . E V E L A N D

Department of Microbiology and Immunology State University of New York, Downstate Medical Center Brooklyn, New York 11203 Department of Microbiology California State University Long Beach, California 90840 (Received October 1, 1985; accepted December 4, 1985) Abstraet--Lipids released by Schistosoma mansoni adult males attract females in vitro. Lipid release is modulated by the presence of other worms. Although S. mansoni males release lipid when paired with females, the release is enhanced when they are separated. S. japonicum adults release more free sterols when incubated individually than when incubated together. Similarly, individually incubated S. haematobium males release more free sterols than when incubated in groups. However, S. haematobium females incubated in groups release more free fatty acids than do equal numbers of males or pairs incubated in groups. There is evidence that S. mansoni adult females concomitantly accumulate and release cholesterol in the absence of an exogenous supply, although de novo synthesis of cholesterol in schistosomes has not yet been demonstrated. Schistosomula and adult schistosomes incorporate exogenous lipids. Lipids are incorporated chiefly through the tegument. Cholesterol is transferred between males and females. Key Words--Trematoda, Digenea, Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, behavior, chemoattraction, chemical communication, pheromones, lipids, receptors, histochemistry, electron microscopy, thin-layer chromatography.

INTRODUCTION H o w m a l e and f e m a l e s c h i s t o s o m e s first locate e a c h o t h e r in v i v o is still a m a t t e r o f conjecture, but there is n o w e v i d e n c e f r o m in vitro studies that pairing is c h e m i c a l l y m e d i a t e d . Interest in interactions b e t w e e n m a l e s and f e m a l e s date back to the 1920s w h e n S e v e r i n g h a u s (1928) and S a g a w a et al. (1928) inde1699 0098-0331/86/0800-1699505.00/0 9 1986 Plenum Publishing Corporation

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pendently postulated hormonal dependence of females upon males for maturation. Later studies by Vogel (1941, 1942, 1947), Standen (1953), and Moore et al. (1954) provided further evidence for male-induced female maturation. Several studies have indicated that pairing with males is a prerequisite not only for female maturation but also for migration of paired worms to mesenteric and bladder veins and for continued egg laying (Standen, 1953; Moore et al., 1954; Armstrong, 1965; Michaels, 1969; Shaw, 1977). MATE LOCATION AND MIGRATION IN THE MAMMALIAN HOST

Because he observed homosexual male pairs, Armstrong (1965) believed that in vivo mating of Schistosoma mansoni occurs by trial and error and that pairing results from thigmotaxis rather than chemotaxis. He also concluded that a pheromone from males is responsible for maturation of females in heterosexual pairs and retards the development of one male in homosexual pairs. We now know that S. mansoni females require continuous male contact for reproduction. When mature females from bisexual infections are deprived of males, they are reduced in size and their vitellaria and ovaries degenerate. These regressive changes are reversed if pairing is resumed with males (Clough, 1981; Popiel et al., 1984). Males in the absence of females produce normal sperm and show no degenerative changes (Floyd and Nollen, 1977; Kolzow and Nollen, 1978; see Nollen, 1983, for review). The route of schistosome migration in the mammalian host following cercarial penetration has been studied by several workers and several hypotheses have been proposed (see Miller and Wilson, 1978, 1980; Wheater and Wilson, 1979, for review). However, none of these studies addresses the issue of initial pairing. Our information is limited to the fact that males are required to escort females to the small veins of mesentery or bladder wall (Standen, 1953). Recent studies have demonstrated that adult S. mansoni attract each other in vitro, and the attraction is chemically mediated (Imperia et al., 1980; Eveland et al., 1982). Imperia et al. (1980) stated that adult males emit pheromone(s) which attract females. The in vitro studies by Eveland et al. (1983) and Shirazian and Schiller (1982) suggest worm-finding and pairing mechanisms which may operate in vivo. Autoradiographic tracking of schistosomula can also elucidate events which occur in vivo prior to, during, and following mating. These techniques have been profitably used in the schistosome-mouse model for purposes other than identified here (Georgi, t982; Mangold and Dean, 1983). Several studies have indicated that young schistosomes pair on arrival in the liver. No serious effort has been made to elucidate mechanisms of site selection in the mammalian host except an in vitro study by Awwad and Bell (1978) which indicated that S. mansoni pairs were attracted more strongly than

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unpaired worms to a dialysate of human feces. Chemical substances providing site-finding cues to schistosomes may originate in the human diet and from metabolic products of normal enteric flora. Whether S. haematobium pairs migrate to veins of the urinary bladder because of substances absorbed from the bladder, and whether they would respond to urine in vitro, both remain to be determined. CHEMICAL INTERACTIONS IN VITRO

Although studies on hermaphroditic trematodes have yielded considerable information on pairing behavior (Fried and Leiby, 1982; Fried and Jacobs, 1980), these digeneans are not suitable models for sex-dependent behavior studies. Schistosomes are ideally suited for studies on both heterosexual and homosexual interactions between worms. Shaw et al. (1977) showed that acetone and ether extracts of male S. mansoni induced development of vitelline cells with a corresponding increase in body length of females from unisexual infections. Results of another study also suggest involvement of lipids in the development of females. Unisexually developed females showed increased [3H]tyrosine uptake when they were exposed to males or chloroform extracts of males (Popiel and Erasmus, 1981). These observations suggest that factors produced by males induce maturation in females and that the developmental changes are mediated by lipids. Alternatively, the active substance(s) may be factors for which lipids serve as carriers or solvents. The pheromone of the hard ticks has been shown to be dissolved in neutral lipid (Sonenshine et al., 1981). LIPID RELEASE IN SCHISTOSOMES

Free sterols and triacylglycerols are the major neutral lipid fractions in extracts of both male and female S. mansoni (Smith and Brooks, 1969; Fried et al., 1981), and free sterols constitute the major lipid fraction in male excretorysecretory (ES) products (Fried et al., 1981). Cholesterol is the most abundant lipid and the major free sterol in S. mansoni adults (Smith and Brooks, 1969; Meyer et al., 1970). Since free sterol fraction is the major neutral lipid fraction in worm ES products (Fried et al., 1981), Fried et al. (1983) quantitated cholesterol in adult males and females and their ES products to study cholesterol accumulation and release over time (Table 1). They found that males tend to maintain their cholesterol for at least 0.5 hr following removal from the host. At recovery, males contained 1.2-1.5 /xg cholesterol/worm and at 0.5 hr they had 0.8-1.5 t~g. However, during 0.5 hr incubation, males released 0.0120.028 t~g cholesterol/worm into the incubation medium. Females initially contained 0.13 #g cholesterol/worm and by 0.5 hr their content had risen to 0.42 #g/worm. Females had, however, released 0.008-0.013 ~g cholesterol/worm

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TABLE 1. AMOUNTS OF CHOLESTEROL (/zg) IN Schistosoma mansoni ADULTS AND THEIR WORM-FREE INCUBATES a

Extracts Worms

0 hr

0.5 hr

Pairs Males Females

1.9-2.8 1.2-1.5 0.13

1.2-1.5 0.8-1.5 0.42

Incubate 0.003-0.013 0.012-0.028 0.008-0.013

~Data from Fried et al. (1983).

into the incubation medium during 0.5 hr incubation. Heterosexual worm pairs contained 1.9-2.8/~g cholesterol/worm pair and at 0.5 hr incubation, they contained 1.2-1.5 #g cholesterol/worm pair. Their incubation medium contained 0.003-0.013 /zg cholesterol/worm pair at 0.5 hr. These data indicate that the two sexes handle lipid differently and that pairing influences lipid release. However, separate lipid analyses of males and females following incubation of pairs would more clearly delineate the influence of the sexes on each other. Earlier studies on S. mansoni showed that in males lipids are localized in the parenchyma and tubercles (Grnnert, 1955; Smith et al., 1969; Fried et al., 1981) and in females they are found primarily in the vitellaria (Fried et al., 1981). Even though neutral lipid release was reported from S. mansoni (Fried et al., 1981, 1983), no specific structure was associated with the release. Recent histochemical and electron microscopic studies indicated that neutral lipids are released from the tegument (Haseeb et al., 1984, 1985b). These studies confirmed previous observations on lipid distribution in adult schistosomes and demonstrated marked differences between males and females in lipid release. In S. mansoni males which were separated from females immediately following their recovery from the host, lipid was usually seen in the parenchyma but rarely in tubercles. However, when incubated in vitro for 0.51.0 hr, males accumulated lipid in both the parenchyma and tubercles with release from the latter. These studies demonstrated lipid accumulation and release from the dorsal tegument of unpaired males but not from females. Senft et al. (1978) believed that the gynecophoral canal tegument is a site of active lipid secretion, but our studies do not confirm their observations. Males which had not been separated from females after recovery from the host contained lipid both in the parenchyma and tubercles with release from the latter. At 0.5 hr incubation in vitro, these males were not observed to release lipid, although their tubercles and parenchyma were lipid-positive. But at 1.0 hr of incubation, they were again releasing lipid and lipid droplet shift from parenchyma to the tubercles was obvious. No prominent differences could be noticed in females which had been incubated with males and those which were

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TABLE 2. SUMMARYOF HISTOCHEMICALDETECTION OF LIPIDSIN TEGUMENTOF Schistosoma japonicum ADULTSMAINTAINEDIN VITROa Separated worms

Paired worms 0 hr

0 hr

m ORO NBS

(A) (N)

f

0.5 hr

m

f

1.0 hr

m

f

m

f

+ +r

-

+r

-

+ +r

-

+|

-

+ +r

+ +

+ +r

+ -

+

-

+ +

+ -

~Data from Haseeb et al. (1986). + +, moderately positive; +, weakly positive; - , negative; ORO, Oil red O; NBS, Nile blue sulfate; A, acidic lipid; N, neutral lipid; r0 lipid droplet release; | occasional release. Worms pairs were either fixed at 4~ in 10% neutral buffered formalin immediately following recovery from hosts or mechanically separated and fixed without incubation (0 hr) or after 0.5 and 1.0 hr incubation in Earle's balanced salt solution containing 0.1% glucose and 0.5 % lactalbumin hydrolystate. incubated singly. F e m a l e s contained lipids in the vitellaria and at times in the lumen of intestinal ceca. However, females from unisexual infections were lipidnegative (Haseeb et al., 1984). Similar studies on S. japonicum revealed a similar pattern o f lipid distribution as in S. mansoni adults, although, patterns o f lipid release are different in the two species. Although neutral lipid release was restricted to the dorsal tegument in S. mansoni males, in S. japonicum males release occurred from both the dorsal and ventral teguments. Haseeb et al. (1984) reported acidic lipid release into the ceca o f S. mansoni males, but it was not observed in S. japonicum. S. japonicum males also released neutral lipid from the intestinal ceca. Females released neutral lipid into the intestinal ceca but not from the tegument. Acidic lipid release was not observed from worms o f either sex (Haseeb et al., 1986). Results o f histochemical lipid studies on S. japonicum are summarized in Tables 2 - 4 . These differences in lipid release patterns between the two schistosome species cannot be explained simply on the basis o f absence o f tubercles in S. japonicum. Although it was conceived earlier that S. mansoni males release lipids from their tubercles, electron microscopic studies showed that the whole dorsal tegument is involved in this process (Haseeb et al., 1985b). These studies further indicated differentiation o f dorsal and ventral teguments in males o f these species. Lipid distribution in S. haematobium remains to be determined. Even though we have demonstrated neutral lipid release in S. mansoni and S. japonicum by both histochemical and biochemical methods, information on lipid secretion at the cellular level is needed. Further studies are also required to determine if released lipid droplets are membrane bound. There is some evidence that schistosomes release lipid in vivo. Cryostat

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TABLE 3. SUMMARYOF HISTOCHEMICALDETECTIONOF LIPIDS IN PARENCHYMAa OF Schistosoma japonicum ADULTS MAINTAINEDIN VITROb Paired worms

Separated worms

0 hr

0 hr

m ORO NBS (A) (N)

++ + ++

0.5 hr

1.0 hr

f

m

f

m

f

m

f

+

+

+

++

+

+++

+

++ +

++ +

++ -

+ ++

++ +

++ ++

++ +

Vitellaria in females. bData from Haseeb et al. (1986). + + +, strongly positive; + +, moderately positive; +, weakly positive; - , negative; ORO, Oil red O; NBS, Nile blue sulfate; A, acidic lipid; N, neutral lipid. See legend to Table 2. sections o f m o u s e m e s e n t e r i c veins harboring adult w o r m s r e v e a l e d neutral lipid droplets in parts o f b l o o d v e s s e l walls w h i c h w e r e in contact with m a l e w o r m s . T h e s e droplets had staining properties similar to those o b s e r v e d in schistosomes. O n l y parts o f the m a l e g y n e c o p h o r a l fold e x p o s e d to f e m a l e contain lipid, suggesting an influence o f females on m a l e lipid secretion. A l t h o u g h males o f w o r m pairs released neutral lipid b o t h in vitro and in situ ( H a s e e b et al., 1984), the release was greater in m a l e s after they w e r e separated f r o m f e m a l e s (Haseeb et al., 1985b). L i p i d droplets released f r o m the ventral t e g u m e n t o f S. japonicum males w e r e o b s e r v e d in the g y n e c o p h o r a l canal and o c c a s i o n a l l y in the adjacent fem a l e t e g u m e n t , suggesting possible lipid transfer f r o m m a l e to female. A c e t o n e and ether extracts ( p r e s u m a b l y lipids) o f S. mansoni m a l e s induce maturation in f e m a l e s (Shaw et al., 1977), and f e m a l e s s h o w vitelline gland differentiation only in regions w h i c h contact m a l e s (Popiel and Basch, 1984a). TABLE 4. SUMMARYOF HISTOCHEMICALDETECTION OF LIPIDS IN CECAL WALLS OF Schistosoma japonicum ADULTS MAINTAINEDIN VITROa Separated worms

Paired worms 0 hr

0 hr

ORO NBS (A) (N)

m

f

++r

++r

++ ++

+ +

0,5 hr

m

f

m

-

-

++ +

++ -

1.0 hr f

m

f

+r

-

++r

+

+ +

++ -

++ +r

++ -

~Data from Haseeb et al. (1986). + +, moderately positive; +, weakly positive; - , negative; ORO, Oil red O; NBS, Nile blue sulfate; A, acidic lipid; N, neutral lipid; r, lipid droplet release. See legend to Table 2.

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Although S. japonicum females released neutral lipid into the intestinal ceca only when paired with males, lipids were detected in worm-free incubates of separated females (Haseeb et al., 1986). Densitometric thin-layer chromatographic data on neutral lipid release in S. japonicum and S. haematobium are also available. Pooled worm-free incubates of 10 singly incubated S. japonicum males contained 0.30 /zg of free sterols, whereas 10 males incubated together failed to release enough to be detected by densitometry. Similarly, females incubated separately released more free sterols than those incubated together (Haseeb et al., 1986). S. haematobium males also released more free sterols when incubated singly than a similar number incubated in groups. Ten females incubated together released more free fatty acids than 10 males or 10 pairs incubated in groups (Haseeb et al., 1985c). Our histochemical and TLC studies clearly indicate that lipid release is affected by the presence of other worms. These observations are in accord with the findings of Eveland et al. (1983), who reported that worms of either sex do not show attraction to worm pairs, two males or two females, although heterosexual attraction to single worms occurred. They proposed that either the "window effect" (Kemp and Devine, 1982) or a "shut-down" mechanism is involved in diminishing attraction. Demonstration of a dose-response relationship would help clarify this issue. Lipid release and its modulation by the presence of other worms is one aspect of the complex chemical communication system in schistosomes. We do not know how this modulation occurs or what chemical messages are involved. Whether the messages are different from those which mediate attraction is also unknown. We do know that lipids are involved in worm attraction. Excretorysecretory products of worms incubated in vitro and their lipophilic fractions prepared in chloroform-methanol attract worms of the opposite sex (Eveland et al., 1984). Our recent studies indicate that lipophilic fractions of worm-free incubates prepared in n-hexane attract worms of the opposite sex better than those prepared in chloroform-methanol (Eveland and Haseeb, 1986). Because all animal pheromones do not belong to a single group of chemical substances (Law and Regnier, 1971; Silverstein, 1981), we must consider that chemicals used in separation procedures may interfere with biologic activities of potential chemoattractants. For example, lipid and protein moieties of most lipoproteins are separated in procedures using chloroform-methanol since methanol precipitates proteins. In separation procedures employing n-hexane, such precipitates are not observed. METABOLIC DEPENDENCE

Metabolic interdependence of male and female schistosomes has been the subject of several studies. Trarisfer of [laC]glucose from males to females has been demonstrated in S. mansoni, S. haematobium, and S. japonicum in vitro (Comford and Huot, 1981). These workers further demonstrated that the rate

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TABLE 5. [4-14C]CHOLESTEROL (rig/WoRM + S E M ) TRANSFER BETWEEN Schistosoma

mansoni ADULTS 0 hr

3.0 hr

Males

Males

Females

0.722 + 0.083-0.792 _ 0.063

0.346 + 0.025

0.090 + 0.003

Females

Females

Males

0.245 + 0.020-0.298 + 0.031

0.167 _+ 0.012

0.086 + 0.004

aData from Haseeb et al. (1985a).

of glucose assimilation by worms was significantly greater in paired than in separated worms and that unpaired males contained more glycogen than did paired males. Atkinson and Atkinson (1980) reported that " T h e male worm retains little of the protein it produces in greatest abundance, and this protein is electrophoretically identical to the most abundant protein found in, but not synthesized by, the female." This 66-kd polypeptide occurs naturally in males and females from both unisexual and bisexual infections and, when such worms are exposed to [14C]leucine, it is incorporated into 66-kd bands of mature and unisexually developed females as well as mature males (Popiel and Basch, 1984b). Our recent studies have shown that cholesterol is transferred in both directions between males and females (Table 5). In these experiments single worms removed from pairs were incubated for 3.0 hr in RPMI 1640 containing 0.16 /zCi [4-14C]cholesterol/ml. After initial labeling, these worms were incubated with unlabeled, freshly recovered and separated worms of the opposite sex for another 3.0 hr. Females incorporated 0.090 + 0.003 ng cholesterol/worm w aen incubated with males containing 0.722 _+ 0.083 ng cholesterol/worm. Males contained 0.346 + 0.025 ng cholesterol/worm following incubation. Males incorporated 0.086 _ 0.004 ng cholesterol/worm when incubated with females containing 0.245 + 0.02 ng cholesterol/worm. At the end of the experiment, females contained 0.167 + 0.012 ng cholesterol/worm (Haseeb et al., 1985a). Light-level autoradiography revealed the tegument to be the major site of cholesterol uptake. In males, more transtegumental uptake occurred on the dorsal than the ventral surface and label was heavier in the tubercles than the rest of the dorsal surface. The tubercles on the mid-dorsal surface were more heavily tagged than those near the periphery. The cecal walls were tagged in both males and females. Dorsal subtegumental parenchyma was more heavily tagged than the ventral. Vitellaria accumulated most of the label in females and the tegument was not as heavily labeled as in males (Haseeb et al., 1985a). S. mansoni females accumulate labeled cholesterol in the vitellaria but radioproline is rather sparingly incorporated into the vitellaria (Senft, 1968). S.

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mansoni adults incorporate proline through the gut and tegument (Senft, 1968), but Fripp (1967) did not detect any radioactivity in S. haematobium gut after providing [1-14C]glucose in vitro. In our experiments in which the worms were labeled individually, dense labeling was on the dorsal surface. Therefore, it appears that receptors for different biologically active molecules are located at different sites in schistosomes. If the worms were incorporating cholesterol through the cecal route, heavier tagging might be expected in the anterior than the posterior half of the gut. Since the tagging in cecal walls was uniform throughout the length of worms of both sexes, it seems unlikely that cholesterol uptake occurs through the ceca, but suggests that excess cholesterol is released into the ceca following transtegumental uptake. Catabolism of lipids as an alternative source of energy probably does not occur in schistosomes since they do not consume 02 for their energy-yielding metabolic reactions (Bueding, 1950). Moreover, lipids absorbed by schistosomes are mainly utilized as constituents of parasite membranes (Rumjanek and Simpson, t980). Cholesterol is incorporated into the outer half of the outer lipid bilayer (Torpier and Capron, 1980). A recent study has shown de novo synthesis of cholesterol in Fasciola hepatica (Gerasimova and Leutskaya, 1983). In contrast, schistosomes do not appear to synthesize cholesterol de novo (Smith et al., 1970; Meyer et al., 1970). Cholesterol metabolism in schistosomes appears to be in a state of dynamic equilibrium. Both the incorporation (Rumjanek and Simpson, 1980; Haseeb et al., 1985a) and release (Fried et al., 1983) of cholesterol are obviously governed by the physiologic state of the worm, but the precise regulatory mechanism is not known. Lipid incorporation in schistosomula is modulated by the amount of lipid present in the incubation medium (Rumjanek and McLaren, 1981). Such data are not available for adult worms in which the presence or absence of worms of the opposite sex may affect lipid uptake. As mentioned above, the presence of worms of the opposite sex affects lipid release (Haseeb et al., 1985c, 1986). Biologically active ecdysteroids are known to occur in certain helminths, such as Dirofilaria immitis (Mendis et al., 1983) and Moniezia expansa (Mendis et al., 1984). Ecdysone and 20-hydroxyecdysone have also been detected in S. mansoni (Torpier et al., 1982; Nirde et al., 1983). These molecules have been shown to be produced by the worms and have been detected in serum and urine of patients (Nirde et al., 1984). Their role in the biology of the parasite is unclear. The function of ecdysteroids in insects has been reviewed by Loof et al. (1984). RECEPTORS

From her studies on the mating of transected worms in vitro, Michaels (1969) postulated that the mating position is determined by linear receptors on

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S. mansoni males and females. She used several enzymes, including ribonucleases and deoxyribonucleases, and snake venoms in an attempt to determine the nature of postulated receptors, but these attempts remained unsuccessful. Results of her studies are difficult to interpret since normal and abnormal mating positions of transected worms are not clearly defined. She fiarther reported that only one in 10 males mated with frozen and thawed females, which argues against the occurrence of receptors that determine mating position. Lectin-binding sites have been demonstrated on both schistosomula and adult schistosomes (Stein and Lumsden, 1973; Bennett and Seed, 1977; Murrell et al., 1978). Several oligosaccharide residues, notably alpha-methyl-o-mannoside, D-galactose, D-mannose and/or o-glucose, N-acetyl-D-glucosamine, Nacetyl-D-galactosamine and sialic acid occur on the schistosome surface (Simpson and Smithers, 1980; Simpson and McLaren, 1982). In these studies, however, distinction between male and female worms was not made. Bennett and Seed (1977) believed that these lectin-binding carbohydrates occur as glycoproteins. Results of other studies did not support this notion (Mun-ell et al., 1978), A recent study of the interaction of Salmonella and schistosomes suggest that at least the mannose-containing receptors are glycolipids (Melhem and LoVerde, 1984). Although these studies have provided information about lectin-binding receptors in schistosomes, there has been no attempt to determine the role of receptors in chemoattraction and/or pairing and mating. Such information is available for other systems, e.g., lectin-mediated functional impairment and inhibition of chemotaxis have been demonstrated in Caenorhabditis elegans by Jeyaprakash et al. (1985). Enzyme-mediated inhibition of chemotaxis in this worm has also been demonstrated by Jansson et al. (1984). These workers reported that mannosidase and sialidase caused 100 % inhibition and trypsin caused a 50 % reduction of chemotactic behavior in C. elegans. Host immunoglobulin binding to schistosomes has been shown to be mediated by Fc receptors present on the worm surface. In addition, receptors for C3 and Clq have also been demonstrated on schistosomula (Smithers and Doenhoff, 1982) but differences between males and females were not examined. At present, there is no evidence of involvement of these receptors in interactions between male and female worms. Although electron microscopy has revealed structures on schistosome surfaces which may serve as sensory receptors, there is still no information on their function (Hockley, 1973; McLaren, 1980). ULTRASTRUCTURAL INTEGRITY OF WORMS MAINTAINED IN VITRO

Smith et al. (1969) described degenerative changes in adults following 90 min incubation in either isotonic phosphate-buffered saline, normal human serum, hyperimmune serum, or horseradish peroxidase. Hocldey (1970) reported de-

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generative changes in worms maintained in hypotonic and hypertonic media (Hockley, 1973). Simpson and McLaren (1982) observed more degenerative changes in worms maintained in RPMI 1640 than in those maintained in Earle's medium supplemented with lactalbumin hydrolysate (LAH). Other studies have reported morphologic changes in worms maintained in vitro in a variety of media (Carlisle et al., 1983; Weisberg et al., 1983). In our laboratory, scanning and transmission electron microscopy following in vitro incubation for 1.0 hr did not reveal any morphologic damage (Haseeb et al., 1984, 1985b). Since Carlisle et al. (1983) and Weisberg et al. (1983) used suction to collect worms, it is possible that they damaged worm surfaces. They also reported that LAH, a constituent of EBSS, appeared to adversely affect the morphology of adult worms. We have not observed damage in the presence of LAH. In other studies LAH has been reported to stimulate egg maturation and production in S. mansoni females maintained in vitro over a period of 14 days (Newport and Weller, 1982), which argues against deleterious effects. Acknowledgments--This work was supported in part by NIH grant AI-17540. Materials used in this study were provided by an NIAID supply contract AI 02656. REFERENCES

ARMSTRONG,J.C. 1965. Mating behavior and development of schistosomes in the mouse. J. Parasitol. 51:605-616. ATKINSON, K.H., and ATKINSON, B.G. 1980. Biochemical basis for the continuous copulation of female Schistosoma mansoni. Nature 283:478-479. AWWAD, M., and BELL, D.R. 1978. Faecal extract attracts copulating schistosomes. Ann. Trop. Med. Parasitol. 72:389-390. BENNETT, J.L., and SEED, J.L. 1977. Characterization and isolation of concanavalin A binding sites from the epidermis of S. mansoni. J. Parasitol. 63:250-258. BUEDING, E. 1950. Carbohydrate metabolism of Schistosoma mansoni. J. Gen. Physiol. 33:475495. CARLISLE,S., WEISBERG,L.S., and BENTLEY, A.G. 1983. Schistosoma mansoni: Morphologic changes induced by maintenance in vitro. J. Parasitol. 69:319-334. CLOUGH, E.R. 1981. Morphology of reproductive organs and oogenesis in bisexual and unisexual transplants of mature Schistosoma mansoni females. J. Parasitol. 67:535-539. CORNFORD, E.M., and HUOT, M.E. 1981. Glucose transfer from male to female schistosomes. Science 213:1169-1171. EVELAND,L.K. and HASEEB,M.A. 1986. Sehistosome behavior in vitro. J. Chem. Ecol. 12:16871698. EVELAND, L.K., FRIED, B., and COHEN, L.M. 1982. Schistosoma mansoni: Adult worm chemoattraction with and without barriers. Exp. Parasitol. 54:271-276. EVELAND, L.K., FRIED, B., and COHEN, L.M. 1983. Schistosoma mansoni: Adult worm chemoattraction with barriers of specific molecular weight exclusions. Exp. Parasitol. 56:255258. EVELAND,L.K., FRIED, B., and HASEEB,M.A. 1984. Schistosoma mansoni: Chemoattraction studies with excretory-secretory, lipophilic and hydrophilic worm products. Program and Abstracts. 59th Annual Meeting. American Society of Parasitology, Snowbird, Utah, p. 35. FLOYD, R.D., and NOLLEN, P.M. 1977. Effects of stressful conditions on the development and movement of reproductive cells in Schistosoma mansoni. J. Parasitol. 63:87-90.

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