Homosexual male pairing in Schistosoma mansoni

and 1988. Homosexual male pairing in Schistosoma mansoni.International Journal for Parasitology 18: 1115–1117. To see whether male worms within the gynecophoral canal of another male worm would become feminized (i.e. express vestigial female-associated genes), we established homosexual pairs by twice exposing mice to male cercariae with a 4 or 6-week interval, and perfusing 3–5 weeks later. From 13 to 34% of these worms were found in pairs, compared with 0 to 7% in singly exposed controls. ‘Inner’ males in homosexual pairs showed no histological evidence of female reproductive structures, but were stunted, had poorly developed testes, and the high nuclear density characteristic of mature females. More vitelline follicles occurred in unpaired unisexual males than in homosexually paired males, fewest in bisexually paired males. Uptake of tyrosine, an indicator of vitelline development, occurred in the same relative order. The gynecophoral microenvironment often led to stunting, probably through starvation induced by the relative inaccessibility of host blood to homosexually clasped males.     

Effect of pairing in vitro on the glutathione level of male Schistosoma mansoni

The effect of in vitro incubation on the level of the intracellular nucleophile, glutathione (GSH), in adult Schistosoma mansoni was investigated. The GSH levels of freshly collected adult male and female parasites were 8.5 +/- 2.5 and 2.7 +/- 0.7 nmol/10 worms, respectively, as determined by an enzymatic assay. Twenty-four-hour incubation of unpaired males in RPMI-1640 medium at 37 C resulted in a 1.7-fold increase (P less than 0.001) in GSH level that remained elevated for at least 7 days. The increase was dependent on exogenous L-cystine, suggesting that it was due to biosynthesis of GSH. Biosynthesis in male S. mansoni was confirmed by isolating [3H] GSH from parasites incubated in medium containing L-[3H] cystine or [3H] glycine. In contrast to unpaired males, the GSH level of paired males as well as that of unpaired or paired females did not increase after 24 hr in vitro. When males that had been incubated unpaired for 24 hr were allowed to couple in vitro with freshly collected females, their GSH level fell to that of continuously paired males. These observations provide evidence that in vitro female schistosomes can influence the physiology of the male.     

Schistosoma mansoni: characterization of hemolytic activity from adult worms

Homogenates of adult Schistosoma mansoni worms contain a hemolytically active component(s). Centrifugation at 10,000 g shows the major activity is present in the pellet fraction. Red blood cell lysis with the schistosome hemolytic agent is optimal at acid pH (5.0) and highly temperature dependent. The hemolytic component is resistant to boiling (5 min) and stable for extended periods of time at 38 C (22 hr). The length of the lag phase prior to hemolysis and the rate of hemolysis are both concentration and temperature dependent. Following hemolysis, red blood cell ghosts remain.     

Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments

Maturation and maintenance of normal reproductive function in female Schistosoma mansoni require a permanent association with the male, but the nature of this relationship is not well understood. The regional localization of a stimulatory factor in the male and its target in the female were investigated. Unisexual female and mature male worms were transected into segments of various lengths. Various combinations of transected male and female segments and intact worms were transferred to the mesenteric veins of recipient hamsters and were also maintained in vitro. In hamsters and in vitro, pairing took place between intact worms of each sex and segments of the other, and between segments of both sexes. The majority of female worms and segments so paired showed some reproductive development, as assessed by vitelline gland differentiation. In intact unisexual females paired with small male segments, vitelline gland development was limited to that portion of the worm that had been held by the male. Worm segments continued to display normal body contractions throughout 24 days of in vitro maintenance and morphological integrity was retained. It is concluded that 1) in the absence of a functioning gut, worm segments can survive for prolonged periods on nutrients absorbed through the tegument; 2) worm pairing, male stimulation, and the female developmental response are independent of central nervous control by the cerebral ganglia; 3) males have no centralized localization for the female-stimulating factor; 4) vitelline gland differentiation in the female requires local stimulation through male contact, and this is not propagated throughout the worm.     

Schistosoma mansoni: cholesterol uptake by paired and unpaired worms

Mature males and females of Schistosoma mansoni were incubated for 24 hr in medium containing [3H]cholesterol. Worms thus labeled were paired for 24 hr with unlabeled partners, in vitro or in vivo by surgical implantation into hamsters. Controls consisted of additional unlabeled worms that did not pair. Scintillation counting of thin layer chromatographic separations of lipid extracts of schistosome tissues and of the culture medium indicated that [3H]cholesterol underwent no major metabolic changes during the course of the experiment. During the period of time allowed for pairing, labeled worms lost up to 65% of their [3H]cholesterol, which was detected in the pairing medium. In both unlabeled males and females which had paired with labeled partners, levels of [3H]cholesterol were higher than in unpaired controls. This suggests that normal cholesterol transfer in worm pairs is bidirectional and that it is facilitated by physical contact between juxtaposed membranes. Cholesterol exchange in schistosome worm pairs may be partly or wholly a consequence of normal tegumental turnover of the molecule.     

Dolichol phosphate mannose synthase is differentially expressed in male and female worms of Schistosoma mansoni

The cDNA encoding the Schistosoma mansoni dolichol phosphate mannose synthase was completely sequenced, displaying the highest homology with Cricetulus griseus and Saccharomyces pombe genes. The Schistosome enzyme had a K(m) of 0.127 microM, a value that is within the range of those reported for several other species. Thin-layer chromatography of the radiolabelled schistosome lipid intermediate showed it was identical to dolichol-phosphate (C80-C105). Expression of dolichol phosphate mannose synthase of S. mansoni (SmDPMS) was analysed by Northern blot and quantified by semi-quantitative RT-PCR with cDNA from mature and immature male and female worms. Northern blot analysis revealed a single 1-kb band. Both approaches confirmed a higher level of expression in mature female worms, as compared to immature and male worms.     

Effect of immature Schistosoma mansoni worms on hamsters' plasma enzymes

1. The pattern and activity of isocitrate, lactate and malate dehydrogenases and malic enzyme were studied in plasma of normal hamsters and hamsters at the 26th day of infection with S. mansoni. 2. Although the electrophoretic patterns of these enzymes were similar in normal and infected hamsters, their activities were higher in the latter than the former group of animals. The elevation in the enzymic activity indicates that there is tissue damage caused by the larvae at this stage.     

Schistosoma mansoni: effects of antimony on immature and adult worms.

Immature Schistosoma mansoni in mice are less susceptible to antimony therapy than adult worms. KSb tartrate inhibited phosphofructokinase (PFK) (EC 2.7.1.11) to a greater extent in extracts of 3-week-old worms than adults, and inhibited production of lactate in both immature and adult worms in vitro. In vivo, KSb tartrate was accumulated similarly by 3-week-old worms and by adults: measurements of hexosephosphate following drug treatment suggested similar inhibition of PFK in the two worm stages. If antimony acts by inhibition of PFK it is not clear why the young worms are more resistant to chemotherapy than adults.     

Dolichol phosphate is a rate-limiting factor in mannosyl transferase activity of adult male worms of Schistosoma mansoni

The formation of mannolipid through catalysis by mannosyl transferase of adult females of Schistosoma mansoni was found to be 2-3 fold higher than male worms. In contrast, mannosyl transferase in immature females generated approximately the same amount of mannolipid as male worms, immature or not. Exogenous dolichol phosphate added to homogenates of male worms produced a stoichiometric increase in mannolipid formation. Saturating amounts of dolichol phosphate generated similar mannosyl transferase activities in male and female homogenates, showing that in S. mansoni, dolichol phosphate is the lipid intermediate in the glycosylation reaction and that this mannolipid is a rate-limiting substrate. Thin layer chromatography revealed that the mannolipid was identical in male and female worms. Adult males incubated with ¹⁴C-acetate synthesised several apolar compounds, one of which displayed a R_f identical to that of the mannolipid. When exposed to ¹⁴C-acetate treated males in vitro, untreated females were able to incorporate a compound, which partitioned in the same way as the mannolipid. The increased mannosyl transferase-dependent mannolipid formation in adult females may reflect a higher energy demand by these parasites, which is probably associated with oogenesis.     

Schistosoma mansoni: nitrothiazolines and the male tegument

Within 24 hr of treatment of the mouse host with BW484C, 2-[5-nitro-2-(pivaloylimino)-4-thiazoline-3-yl]diacetamide, pairs of Schistosoma mansoni exhibited "hepatic shift" and began to leave the mesenteric veins. The tegument of the males was altered, both morphologically and physiologically, while that of females was unaffected. This morphological damage to males correlated well with therapeutic efficacy against both sexes in a range of analogues of BW484C. However, parasites removed from mice after treatment but before the hepatic shift and then maintained in vitro were far from moribund as treated males could be maintained for 8 days in vitro, although this was 5 days less than males from untreated mice. Females survived as well as control worms. In contrast, male and female S. mansoni remaining in their host after therapy were invaded by host cells in the liver after 2 days. The morphological effects and reduction of the in vitro survival of males treated in the mouse and removed after 24 hr could be simulated by in vitro exposure for 24 hr to 10(-5) M BW484C. Females were not susceptible to this regime. It was concluded that worm pairs were swept to the liver as a result of drug dependent damage to the tegument of the male and that phagocytic invasion of male and female schistosomes by host cells within the liver was an important factor in the efficacy of BW484C. The biochemical events underlying the effects on the tegument of male worms remain unknown.     

Tegumental Ca-stimulated adenosine triphosphatase activity in adult Schistosoma mansoni worms

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized within the tegument of fixed worms on the cytoplasmic leaflet of both the double surface membrane and the basement membrane; this reaction was inhibited by 1 microM chlorpromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosomes are discussed.     

Schistosoma mansoni: activation of hemolytic activity in homogenates from live adult worms

We have previously described hemolytic activity in extracts of lyophilized Schistosoma mansoni adult worms. In contrast, freshly homogenized live worms have little hemolytic activity. However, preincubation of the homogenate at 38 C, pH 5.1 for 22 hr resulted in an 18 fold increase in specific activity (Hb released/mg protein) due to dramatic increases in hemolytic activity and decreases in protein concentration. No activation occurred when the homogenate was boiled prior to preincubation or when preincubation was performed at 4 C. In addition, a thermostable inhibitor of hemolytic activity is present in freshly homogenized live adult S. mansoni. Hydrolysis of the inhibitor and activation of the hemolytic agent appear to be due in part to hydrolytic activity of one or more cysteinyl proteinases.     

Schistosoma mansoni: purification and characterization of the major acidic proteinase from adult worms

We report purification of the major digestive proteinase from adult worms of Schistosoma mansoni. This enzyme is a thiol proteinase with a pH optimum of 5 and is activated by thiol reagents. It was purified 300-fold using a combination of gel chromatography and chromatofocusing. It readily hydrolyzed hemoglobin with an apparent Km of 0.29 microM and a specific activity of 27 micrograms degraded/min/mg enzyme at 37 C. Peptides with positively charged amino acids were preferentially cleaved. The enzyme degraded Boc-Arg-Arg-7-amino-4-methyl coumarin with a kcat/Km of 9083 M-1 sec-1. Lengthening the peptide chain to 3 amino acids or substituting glycine for the amino terminal arginine resulted in decreased activity. The enzyme was inhibited by chloromethylketone-derivatized peptides of similar sequence and by leupeptin. The purified proteinase exhibits microheterogeneity in different preparations with forms ranging in molecular weight from 30,000 to 35,000, and pI 5.7-6.0.     

Transfer of [14C] cholesterol and its metabolites between adult male and female worms of Schistosoma mansoni

Adult male and female worms of Schistosoma mansoni are able to incorporate [14C]cholesterol and convert it into several metabolites. Schistosomula on the other hand cannot convert the incorporated cholesterol. Male worms are able to transfer the [14C]cholesterol and labelled metabolites to female worms but labelled female worms can transfer cholesterol but are unable to transfer the conversion products of cholesterol to male worms. Uptake by female worms of labelled products shed by male worms seems to occur more efficiently in the presence of serum than in its absence.     

Morphometric study of Schistosoma mansoni adult worms recovered from Undernourished infected mice

Some unfavourable effects of malnutrition of the host on Schistosoma mansoni worm biology and structure have been reported based upon brigthfield microscopy. This paper aims to study by morphometric techniques, some morphological parameters in male and female adult worms recovered from undernourished albino mice in comparison with parasites recovered from well-fed infected mice. Undernourished animals were fed a multideficient and essentially low protein diet (RBD diet) and compared to well-fed control mice fed with the commercial diet NUVILAB. Seventy-five days post-infection with 80 cercarie (BL strain) animals were sacrificed. All adult worms were fixed in 10% formalin and stained with carmine chloride. One hundred male and 60 female specimens from each group (undernourished and control) were examined using an image system analysis Leica Quantimet 500C and the Sigma Scan Measurement System. The following morphometrical parameters were studied: body length and width, oral and ventral suckers, number and area of testicular lobes, length and width of ovary and uterine egg. For statistical analysis, the Student's t test for unpaired samples was applied. Significant differences (p < 0.05) were detected in body length and width, in parameters of suckers, uterine egg width, ovary length and area of testicular lobes, with lower values for specimens from undernourished mice. The nutritional status of the host has negative influence on S. mansoni adult worms, probably through unavailability of essential nutrients to the parasites.