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.     

Tegumental glucose permeability in male and female Schistosoma mansoni

Tegumental hexose transporters have been kinetically characterized in mated and separated male and female Schistosoma mansoni 8-12 wk postinfection. Significant gender-specific differences in Km and Vmax were observed. In mated males, the estimated constants (mean +/- SE) were: Km = 0.63 +/- 0.31 mM, Vmax = 0.93 +/- 0.44 nmol/mg worm water/min, and the Kd = 0.25 +/- 0.09 microliter/mg worm water/min. In mated females the kinetics were: Km = 0.99 +/- 0.40 mM, Vmax = 1.22 +/- 0.42 nmol/mg worm water/min, and Kd = 0.60 +/- 0.14 microliter/mg worm water/min. The influx of 2-deoxy-D-glucose and 3-O-methylglucose has been similarly characterized; these analogs share the same glucose transporter in male and female schistosomes. 2-Deoxy-D-glucose has a higher affinity, and 3-O-methylglucose a lower affinity, than does glucose. Because mated male schistosomes supply glucose to female partners, similarities between the free glucose concentration of the male and the affinity of the transporter determined for mated female schistosomes suggest that male-to-female transfer may be a potentially rate-limiting step in glucose utilization by the female. Permeability x surface are (PS) products and Vmax/Km ratios were significantly elevated in mated schistosomes, suggesting that the transporter is primarily localized to the dorsal surface of the male. Gender- and mating-specific analyses of PS products indicate that tegumental permeability to glucose is significantly increased in mated schistosomes, and compares very favorably to that of the host liver.     

Schistosoma mansoni: a comparison of male and female muscle physiology

Surface electrical activity and membrane potentials recorded from male and female Schistosoma mansoni are similar. Surface electrical activity and responses to electrical stimulation are slightly higher in females. Contractures induced by praziquantel, 60 mM K+, ouabain or 5 degrees C are slightly less in females but responses to putative neurotransmitters (5-HT, dopamine and carbachol) are the same in both sexes. Females are more susceptible to the removal of Ca2+ and to the increase of Mg2+ in the medium. These differences may be due to anatomical differences in the sexes or to the recording methods used.     

Schistosoma mansoni: glutathione S-transferase-catalyzed detoxication of dichlorvos

Dialyzed cytosol of adult Schistosoma mansoni worm pairs catalyzed the glutathione-dependent O-demethylation of dichlorvos (2,2-dichlorovinyl dimethylphosphate), the active form of the antischistosomal drug metrifonate, to form a thioether conjugate, S-methylglutathione, and desmethyl dichlorvos. The reaction rate was dependent on both time and protein concentration, and no product was formed when either dichlorvos or glutathione was omitted from the reaction mixture. Female worm cytosols were about 2.5-fold more active per milligram of protein that those of males. Partial purification of glutathione S-transferases from male worms by affinity chromatography on glutathione-agarose showed that the reaction could be catalyzed by a preparation containing the three major isoenzymes, but that the unbound fraction, which contains at least one additional form of the enzyme that is particularly active with epoxide substrates, was 16-fold more active toward dichlorvos than the bound fraction. S-Methylglutathione also was formed by S. mansoni worm pairs incubated in the presence but not in the absence of dichlorvos. Because GSH S-transferase-catalyzed metabolism of dichlorvos results in the formation of desmethyldichlorvos, which unlike the parent compound is not an effective acetylcholinesterase inhibitor, the reaction represents a pathway of detoxication in schistosomes. It is the first example of a clinically used schistosomicide shown to be detoxicated by a conjugation pathway. These results raise the possibility that dichlorvos detoxication by S. mansoni may help explain why this species is normally refractory to metrifonate.     

Transintegumental uptake of metabolic substrates in male and female Schistosoma mansoni.

A new method for measuring transintegumental uptake in living schistosomes in vitro has been applied to the study of individual males and females. Uptake of a 14-C labeled test metabolite was compared to that of tritiated water (a highly diffusible reference substance). Use of the short half-life (T 1/2 = 100 min) isotope 113m-Indium, bound to EDTA (ethylene diamine tetra-acetic acid, a nondiffusible reference substance) permitted quantification of the relative amount of 14-C test substance passively adhering to the schistosoma surface. Substraction of this amount provided an estimate of net uptake. D-glucose uptake, as measured by this method, increased with time, approaching equilibrium by two min; a positive correlation between temperature and glucose uptake was also observed. Nondialyzable components in rat, human, horse and fetal calf sera did not enhance glucose uptake. In both male and female schistosomes, minimal uptakes were seen for the nonmetabolizable sugar alcohol mannitol (MW = 182). L-glucose uptake was similarly low, but high uptakes were observed in both sexes for D-glucose. In addition to confirming the stereospecificity of hexose uptake, these studies suggested our technique provides a sensitive method for measurement of both high and low uptake compounds. The uptakes of D-glucose and the L-amino acids--arginine, ornithine, lysine, histidine, phenylalanine and serine--were comparatively higher in female than male schistosomes. Slight elevations in uptake by females were observed for threonine, valine and glycine, but aspartate uptake was slightly higher in males. No dramatic male-female differences were immediately apparent for the uptakes of proline, leucine, isoleucine, tyrosine and glutamate. Schistosomal uptake of L-amino acids that are essential for vertebrates was generally higher than uptake of the nonessential amino acids.     

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.     

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.     

Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.     

Origin and diversification of the human parasite Schistosoma mansoni

Schistosoma mansoni is the most widespread of the human-infecting schistosomes, present in 54 countries, predominantly in Africa, but also in Madagascar, the Arabian Peninsula, and the Neotropics. Adult-stage parasites that infect humans are also occasionally recovered from baboons, rodents, and other mammals. Larval stages of the parasite are dependent upon certain species of freshwater snails in the genus Biomphalaria, which largely determine the parasite's geographical range. How S. mansoni genetic diversity is distributed geographically and among isolates using different hosts has never been examined with DNA sequence data. Here we describe the global phylogeography of S. mansoni using more than 2500 bp of mitochondrial DNA (mtDNA) from 143 parasites collected in 53 geographically widespread localities. Considerable within-species mtDNA diversity was found, with 85 unique haplotypes grouping into five distinct lineages. Geographical separation, and not host use, appears to be the most important factor in the diversification of the parasite. East African specimens showed a remarkable amount of variation, comprising three clades and basal members of a fourth, strongly suggesting an East African origin for the parasite 0.30-0.43 million years ago, a time frame that follows the arrival of its snail host. Less but still substantial variation was found in the rest of Africa. A recent colonization of the New World is supported by finding only seven closely related New World haplotypes which have West African affinities. All Brazilian isolates have nearly identical mtDNA haplotypes, suggesting a founder effect from the establishment and spread of the parasite in this large country.     

Host reproductive fitness: the influence of increasing parasite pressure in a Biomphalaria glabrata/Schistosoma mansoni system

Summary

Host life history traits are often shaped by trade-offs between the current and potential future costs of parasitism. Reproductive tissues are not normally essential for host survival and diversion of resources elsewhere is a common effect of parasitic infection. Variations in reproductive output may therefore indicate overall fitness correlated to the host response to parasite pressure. Here, we investigated reproductive fitness in a Biomphalaria glabrata—Schistosoma mansoni system, a laboratory model for schistosomiasis. Five matched groups of unselected B. glabrata snails were individually exposed to doses of 1, 2, 5, 10 or 20 S. mansoni miracidia, respectively. A sixth group remained unexposed providing a control. Fertility (defined as actual reproductive performance, measured as the number of offspring produced) and fecundity (defined as potential reproductive capacity, measured as number of eggs and embryos formed) were monitored for each group at weekly intervals. Our results revealed that both parasite dose and infection status had a significant effect on the potential reproductive capacity of the host, but this was not always reflected in the actual reproductive success. Egg mass production showed a negative association with increasing parasite dose in patently infected snails. In contrast, snails exposed to the parasite, but within which infection did not establish, demonstrated a positive association between egg mass production and parasite dose. This suggests the existence of a fecundity compensation mechanism occurring within the post-patent period of infection. This is, to our knowledge, the first report of such an effect in a snail-trematode system and, indeed, in any host-parasite association.     

Schistosoma mansoni: mortality, pathophysiology, and susceptibility differences in male and female mice.

In parallel studies of Schistosoma mansoni infections in male and female CBA/J mice, major sex-related differences are seen in the development of infection and disease. Upon equal subcutaneous exposures to 45 cercariae female mice present a more severe clinical course with consequent higher mortality than male mice. By 12 weeks of infection, more than 80% of female mice die, while less than 20% of infected males succumb to infection. This greater index of mortality is apparently due to the higher susceptibility of female mice to the development of adult worms. Exposed to 45 cercariae, virtually all females develop patent infections, but 8-34% of male mice do not do so. Also, the recovery rate of adult worms per cercariae from female mice is much higher than that from males, indicating that schistosomula are more successful in developing into adult worms in female mice. Additional studies indicate that this dichotomy of schistosomiasis in the sexes is not restricted to mice of the CBA/J strain, but also occurs in C57BL/6 and outbred CF1 strain mice.     

Schistosoma mansoni: a complement-dependent receptor on adult male parasites

The dorsal surface of adult male Schistosoma mansoni exhibits an affinity for Salmonella typhi when these bacteria are preincubated in normal serum from mice, guinea pigs, or humans. The complement (C) system was shown to be responsible for the bacterial binding. Bacteria not preincubated with normal serum or preincubated in normal serum which had been treated with ethylenediamine tetraacetic acid (EDTA), or cobra venom factor (CoF), or heated at 56 C for 60 min did not bind to the parasite's surface. Further experiments utilizing ethylene glycol tetraacetic acid (EGTA) plus Mg²⁺, heat inactivation at 50 C for 30 min, and zymosan treatment of the serum indicated the C fixation and deposition on the bacteria occurs via the alternative C pathway. These observations indicate the presence of a complement-dependent receptor on the dorsal tegumental surface of the adult male parasite.     

Schistosoma mansoni: praziquantel-induced changes to the female reproductive system

The long-term in vivo effects of a single subcurative dose (200 mg/kg body wt of mouse) of praziquantel on the ultrastructure of the female reproductive system of Schistosoma mansoni were investigated. Morphological changes in the structure of both the vitelline gland and the ovary were apparent within 24 hr post-treatment, and lead to a partial or complete regression of both organ systems. Associated with this regression was a cessation of egg production. In surviving, paired females, irrespective of the initial severity of the drug-induced damage, both the vitelline gland and the ovary completely redeveloped and lead eventually to a resumption of egg production. In contrast, in unpaired, previously mature females the reproductive system also regressed but did not redevelop. In these cases, although the initial changes in the reproductive system were the result of drug action, the long-term regressive changes were due to discontinued male stimulation.     

Diversification of the insulin receptor family in the helminth parasite Schistosoma mansoni

Insulin signalling is a very ancient and well conserved pathway in metazoan cells, dependent on insulin receptors (IR) which are transmembrane proteins with tyrosine kinase activity. A unique IR is usually present in invertebrates whereas two IR members are found with different functions in vertebrates. This work demonstrates the existence of two distinct IR homologs (SmIR-1 and SmIR-2) in the parasite trematode Schistosoma mansoni. These two receptors display differences in several structural motifs essential for signalling and are differentially expressed in parasite tissues, suggesting that they could have distinct functions. The gene organization of SmIR-1 and SmIR-2 is similar to that of the human IR and to that of the IR homolog from Echinococcus multilocularis (EmIR), another parasitic platyhelminth. SmIR-1 and SmIR-2 were shown to interact with human pro-insulin but not with pro-insulin-like growth factor-1 in two-hybrid assays. Phylogenetic results indicated that SmIR-2 and EmIR might be functional orthologs whereas SmIR-1 would have emerged to fulfil specific functions in schistosomes.     

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.     

Immunocytochemical localization of the major glutathione S-transferases in adult Schistosoma mansoni

Indirect immunofluorescence was used to investigate the tissue distribution of the major isoenzymes of Schistosoma mansoni glutathione S-transferase (GSH S-transferase). When polyclonal rabbit antisera against GSH S-transferase isoenzymes SmGST-1, -02, and -3 were applied to cryostat or plastic-embedded sections of fixed adult worms, a punctate pattern of enzyme distribution was observed that was restricted to the parenchyma. Labeling was much more pronounced in males than females, consistent with the biochemically determined distribution of these enzymes between the sexes. Intense immunolabeling was noted within the subectocytoplasmic core tissue of the tubercles of the male that appeared to be connected to deep parenchymal cells by immunoreactive cell processes. Immunofluorescence could be blocked completely by prior incubation of antisera with affinity-purified enzyme. Although schistosome GSH S-transferases have been reported to be protective antigens, no immunoreactivity was detected within or on the tegument, including the dorsal spines of the male. The lack of tegumental immunoreactivity was confirmed by immunoblotting of tegumental membrane preparations following SDS-PAGE. Muscle fibers, vitelline cells, and cecal epithelium also failed to react. The fact that the GSH S-transferases were not uniformly distributed among all parenchymal cells suggests the existence of subpopulations of parenchymal cells that are preferentially involved in the conjugation of electrophiles with glutathione.