The effect of fresh toad bile on the induction of encystation in Opalina sudafricana parasitic in Bufo regularis

Opalina sudafricana reacted by encystation to small doses (0.04 ml) of fresh toad bile injected subcutaneously into its host Bufo regularis. It is speculated that androgens present in the injected bile of male toads, and oestrogens found in injected bile of female hosts reach the parasites in the recta of the treated animals and induce them to encyst. High doses of bile killed the parasites and their hosts. Small doses of bile (0·04 ml) added to in vitro cultures induced encystation in the opalinids. High doses killed the opalinids in vitro. There is also the possibility that bufodeoxycholic acid found in the toad bile may play a role in inducing the parasites to divide and encyst.     

Induction of sexual reproduction in Opalina sudafricana by injecting its host Bufo regularis with gibberellic acid

Induction of sexual reproduction in Opalina sudafricana by injecting its host Bufo regularis with gibberellic acid. International Journal for Parasitology4, 203–206. Opalina sudafricana parasitic in the rectum of Bufo regularis was induced to reproduce sexually when its host was injected subcutaneously with 0·3 mg of gibberellin-A₃. This plant growth substance had no effect on the induction of encystation in the parasites in vitro. Urine of toads injected with gibberellin-A₃ induced sexual reproduction (encystation) in the opalinids in vitro. It is speculated that the plant hormone must either be broken down into an active substance by the toad or cause the toad to excrete its own gonadal hormones (or other hormones) into the urine. This active substance or the excreted hormones may induce division in the parasites resulting in the formation of small forms which encyst.     

A new biological assay, utilizing parasitic protozoa, for screening carcinogenic substances which induce bladder cancer in man and other mammals

Injections of aromatic amines (β-naphthylamine, benzidine, O-dianisidine or N-2-fluorenyl acetamide), tryptophan metabolites (3-hydroxyanthranilic acid, xanthurenic acid or LD-kynurenine sulphate), oestrone, and nicotine, which are known bladder carcinogens in man and some other mammals induced sexual reproduction (encystation) in Opalina sudafricana when injected into its host Bufo regularis. This may be used as a new biological assay for screening substances which induce bladder cancer in man and some other mammals. It is speculated that the metabolites of the injected carcinogenic substances used in this work are excreted in the urine of the host, hydrolysed by the hydrolytic enzymes and become carcinogenic. These carcinogenic metabolites reach the parasites in the rectum of the toads and induce them to divide mitotically to form small forms which eventually encyst. It is speculated that the presence of cysts in the rectum of the injected toads is indicative that a carcinogenic effect took place in the parasites. Oestrone is the only carcinogenic substance which induced encystation in the opalinids in vitro. Urine of toads injected with β-naphthylamine, benzidine, O-dianisidine, N-2-fluorenyl acetamide, 3-hydroxyanthranilic acid, xanthurenic acid, DL-kynurenine sulphate, oestrone and nicotine induced cyst formation in the parasites in vitro.     

The use of Opalina sudafricana in a biological test for diagnosing disordered metabolism of tryptophan in human subjects

Injections of urine of patients with bladder cancer linked with bilharziasis, simple urinary bilharziasis, ascariasis or ancylostomiasis, induced cyst formation found in Opalina sudafricana when injected into its host Bufo regularis. It is suggested that the carcinogenic tryptophan metabolites present in the injected urine reach the parasites in the recta of the experimental toads and stimulate them to divide mitotically to form small forms which eventually encyst. This test may be of a diagnostic help in detecting any abnormality in tryptophan metabolism in some human patients.     

Ecdysterone induced sexual reproduction in Opalina sudafricana parasitic in Bufo regularis

Ecdysterone induced sexual reproduction in Opalina sudafricana parasitic in Bufo regularis. International Journal for Parasitology 3: 863–868. Ecdysterone injections (0·5 mg/ml) induced sexual reproduction in Opalina sudafricana parasitic in male or female Bufo regularis after 12 days. Hypophysectomy or gonadectomy did not prevent the hormone from producing its effect on the parasites. Ecdysterone did not induce sexual reproduction in the parasites in vitro. Urine of toads injected with ecdysterone induced the opalinids to encyst in vitro.     

In vitro development of the strobilar stage of Mesocestoides corti

Thompson R. C. A., Jue Sue L. P. and Buckley S. J. 1982. In vitro development of the strobilar stage of Mesocestoides corti. International Journal for Parasitology12: 303–314. Sexually mature strobilated adults of Mesocestoides corti were grown consistently from undifferentiated tetrathyridia in vitro using a conventional diphasic culture system. Development (growth, strobilisation and maturation) was compared in vitro and in vivo. Although growth and strobilisation were comparable in vitro and in vivo, during the first 18 days, total length and numbers of proglottids decreased in vivo but continued to increase in vitro after day 18. Both male and female reproductive systems appeared to develop normally in vitro and self copulation was frequently observed in cultured worms. However, fully developed oncospheres were not produced in vitro.     

Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro

Haseeb M. A., Eveland L. K. and Fried B. 1984. Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro. International Journal for Parasitology14: 83–88. Schistosoma mansoni male and female adults were incubated at 37°C for 0.5 and 1.0 h in Earle's balanced salt solution containing 0.1% glucose and 0.5% lactalbumin hydrolysate, then examined by histochemistry and scanning electron microscopy. Histochemical analysis of cryostat sections stained with Oil Red O showed that males contain neutral lipid mainly in the parenchyma and tubercles, while females contain neutral lipid in the vitellaria. Neutral lipids are released from the tubercles of both paired and unpaired males maintained in vitro. There is evidence of in situ lipid transfer from males to blood vessel walls. Neutral lipid was not seen in females from unisexual infections. Sudan Black B staining fo total lipids is positive in tubercles, parenchyma, and vitellaria. Nile Blue Sulphate stains acidic lipids in male caecal walls. Scanning electron microscopy reveals no tegumental damage.     

Spontaneous sexual differentiation of Mesocestoides corti tetrathyridia in vitro

Barrett N. J., Smyth J. D. and Ong S. J. 1982. Spontaneous sexual differentiation of Mesocestoides corti tetrathyridia in vitro. International Journal for Parasitology12: 315–322. Tetrathyridia of Mesocestoides corti, from the body cavity of mice, maintained in the laboratory by intraperitoneal infection, were used for in vitro culture. In an initial experiment, after 50 days asexual multiplication in vitro one tetrathyridium spontaneously segmented and developed into a sexually mature adult. Further experiments were carried out in an attempt to determine the conditions favouring segmentation and sexual differentiation. A combination of 5 or 10 ml liquid medium S1OE.H (basically composed of CMRL 1066 and foetal calf serum with supplements) changed every 3 days, in a Leighton tube (19 × 105 mm), rotated at 38°C and gassed with 10 or 20% CO₂, containing between 100 and 200 tetrathyridia, has proved to be most suitable so far. Numerous adult worms with normal male and female genitalia have been obtained in this system. However, segmentation is sporadic, rather than consistent and only a few shelled eggs with hooked oncospheres have so far been obtained, suggesting that impregnation and fertilization in vitro is not fully comparable with that in vivo.     

Nippostrongylus brasiliensis: effect of host hormones on helminth ingestion in vivo

Nippostrongylus brasiliensis: effect of host hormones on helminth ingestion in vivo. International Journal for Parasitology16: 77–80. Ingestion of dye in vivo by both sexes of Nippostrongylus brasiliensis was increased in gonadectomized male, but not female, mice that were implanted with testosterone-filled capsules. The nematodes increased their ingestion of dye in both sexes of gonadectomized hosts that were implanted with 17 β-estradiol and decreased their uptake of dye in progesterone-implanted animals. Implantation of intact male mice with capsules of hydrocortisone and cholesterol suggested that ingestion by N. brasiliensis was decreased and increased, respectively, in these animals. Ligation of the bile duct in gonadectomized male mice that were implanted with testosterone capsules reduced the ingestion of dye by both sexes of nematode.     

The effect of social isolation on androgen and corticosteroid levels in a cichlid fish (Haplochromis burtoni) and in swordtails (Xiphophorus helleri)

The blood androgen and glucocorticoid levels of male Haplochromis burtoni and Xiphophorus helleri were measured in socially isolated individuals (8 and 4 weeks, respectively) and nonisolated controls. In both species social isolation leads to significant reductions of mean concentrations and variances, both for androgens and corticosteroids. It is known for H. burtoni—and new evidence for X. helleri is presented—that social isolation reduces aggressivity in nonescalating situations. It is proposed that social stimuli in both species stimulate the testes to produce more androgenic hormones, and that this can lead to higher levels of aggression. At least in tropical fish species the androgenic optimum for defending a territory or a social status may be much more controlled by social stimuli than previously realized.     

New bufadienolides and C23 steroids from the venom of Bufo bufo gargarizans

Six new bufadienolides (1-6) and two new C₂₃ steroids (7 and 8), together with three known compounds (9-11) were isolated from the venom of Bufo bufo gargarizans. Their structures were elucidated by spectroscopic analysis and single-crystal X-ray diffraction. In vitro cytotoxicities of all compounds were evaluated in A549 cancer cell line. Compounds 2, 3 and 10 showed significant cytotoxic activities.     

Effect of host sex on passive immunity in mice infected with Nematospiroides dubius

Dobson C. & Owen M. E. 1978. Effect of host sex on passive immunity in mice infected with Nematospiroides dubius. International Journal for Parasitology8: 359–364. Female C₃H but not Quackenbush (Q) mice harboured fewer Nematospiroides dubius than male C₃H and Q mice. Both strains lost worms 21 days after infection. C₃H and Q mice became progressively immune to infection following 4 sequential doses of N. dubius larvae and showed a sex resistance to infection. Hypothymic nu/nu CBA Balb/c mice did not show these effects on N. dubius infection. The reciprocal transfer of male and female immune mesenteric lymph node cells (IMLNC) to syngeneic male and female recipients showed that the female environment enhanced the protective qualities of both male and female IMLNC but the male environment suppressed these effects. Gonadectomized male and female recipients of male and female IMLNC had levels of infection similar to the entire female recipients. Serum from immune female donor mice protected both male and female recipients better than immune serum from male donors, but female mice in each treatment group were better protected than male mice. Immune serum transferred greater levels of protection then IMLNC to recipient mice against N. dubius infections. These data are consistent with the conclusion that the male environment suppresses lymphocyte activity and the production of protective antibodies and additionally may depress the effectiveness of sensitized lymphocytes and antibodies in ejecting N. dubius. On the other hand the female environment does not appear to adversely affect the mobilization of the protective immune response and may enhance immune effector mechanisms in protecting mice against N. dubius infections.     

Schistosoma mansoni: One- and two-dimensional electrophoresis of proteins synthesized in vitro by males,females, and juveniles

Polyacrylamide gel electrophoresis coupled with fluorography is a sensitive method for visualizing individual gene products synthesized in vitro by Schistosoma mansoni (K. Atkinson and B. G. Atkinson 1980, Nature (London)283, 478–479). In vitro labelling with radioactive amino acids ensures that the proteins are of parasite origin and fluorography permits detection of minute amounts of newly synthesized, electrophoretically separated gene products. One-dimensional electrophoretic separation in polyacrylamide gels with sodium dodecyl sulphate and fluorography of juvenile and adult proteins reveal that juveniles produce most adult proteins. Although similar studies with proteins from sexed adults imply that analogous gene products are elaborated by both sexes, a number of sex-specific gene products are resolvable by more rigorous two-dimensional electrophoretic separations. The homogametic male produces 5 polypeptides not produced by the heterogametic female. Three outstanding male-specific gene products include a polypeptide with a molecular weight (MW) of 88 kilodaltons (kd) and an isoelectric point (pI) of 5.65, one with an MW of 66 kd and a pI of 5.25, and one with an MW of 58 kd and a pI of 5.25. Other, readily detectable male-specific polypeptides include one which coelectrophoreses with β-actin and one which coelectrophoreses with β-tropomyosin. The female synthesizes 4 specific polypeptides which have isoelectric points between 4.3 and 4.7, are of low molecular weight, and are resolvable only with 12% acrylamide gels. Two-dimensional electrophoresis resolves 74 major polypeptides synthesized by adult worms, and a code is presented which identifies each polypeptide by sex specificity, isoelectric point, and molecular weight.     

Opalinidae in African Anura I. Genus Opalina

During 1988 and 1989, 409 specimens of southern African Anura comprising 50 species in nine families were checked for opalinids in the cloaca. Opalina natalensis Metcalf, 1923 was found in four of 12 Phrynobatrachus natalensis (Ranidae) and O. duquesnei n. sp. in four of five Chiromantis xerampelina (Rhacophoridae). Opalina amygdala (Boisson, 1965) and O. foliacea (Boisson, 1959) are proposed as new combinations. It is suggested that the Ranidae (in particular the genera Phrynobatrachus and Ptychadena), the Hyperoliidae (in particular the genus Leptopelis) and the Rhacophoridae (with the genus Chiromantis) are among the major carriers of Opalina in the Afrotropical Region.     

Cultivation in vitro of Schistosomatium douthitti (trematoda: Schistosomatidae)

Cultivation in vitro of Schistosomatium douthitti (Trematoda : Schistosomatidae). International Journal for Parasitology12: 541–545. We grew S. douthitti in vitro from the cercaria to pairing ovigerous adults, using methods and media previously reported for cultivation of Schistosoma mansoni. Growth of S. douthitti in vitro was much more rapid than that of S. mansoni, but the cultures achieved a similar stage of maximal development. Cultured S. douthitti worms were smaller than those from animals and eggs were inviable. We determined that females of this species, known to produce eggs in unisexual animal infections, will also do so in culture in the absence of males.     

Bioactive polyhydroxylated sterols from the marine sponge Haliclona crassiloba

Four new polyhydroxylated sterols, named halicrasterols A–D (1–4), together with six known analogs (5–10) were isolated from the marine sponge Haliclona crassiloba. Compounds 1 and 2 represented rare examples of steroids featuring 17(20)E-double bonds. The structures of 1–10 were elucidated by spectroscopic analysis and comparison with reported data. This is the first report of a steroid profile for this species. The antimicrobial activities of 1–10 were evaluated against a panel of bacterial and fungal strains in vitro, and compounds 4 and 9 showed moderate activity against some of the Gram-positive strains with MICs ranging from 4 to 32 μg/mL.     

Sesquiterpene lactones from Vernonia scorpioides and their in vitro cytotoxicity

Fresh leaves of Vernonia scorpioides are widely used in Brazil to treat a variety of skin disorders. Previous in vivo studies with extracts of this species had also demonstrated a high antitumor potential. This paper reports isolation of four sesquiterpene lactones (hirsutinolides and glaucolides), together with diacetylpiptocarphol, 8-acetyl-13-etoxypiptocarphol, luteolin, apigenin, and ethyl caffeate from fresh leaves and flowers of Vernonia scorpioides. The hypothesis that hirsutinolide 3 is formed during extraction was verified theoretically using Density Functional Theory. The effects of isolated compounds on in vitro tumor cells were investigated, as well as their genotoxicity by means of an in vitro comet assay. The results indicate that glaucolide 2 and hirsutinolide 4 are toxic to HeLa cells. These compounds were genotoxic in vitro, a property that appears to be related to the presence of their epoxy groups, which has been a more reliable indication of toxicity than substitution on C-13 or the presence of α,β-unsaturated keto-groups. These results need to be replicated in vivo in order to ascertain their toxicity.     

An olfactory basis for maternal discrimination of sex of offspring in rats (rattus norvegicus)

Lactating Long-Evans rats (Rattus norvegicus) were presented with three-day-old male and female pups that were either untreated, coated with collodion in the anogenital region or on the neck, or perfumed; or with female pups odorized with either male or female pup urine. As measured by related t-tests of maternal anogenital licking, male and female pups were discriminated in the untreated condition, but not when masking stimuli were present. Female pups treated with male urine elicited more licking than female pups treated with female urine. Overall, maternal licking was decreased by olfactory masking agents and increased by pup urine. It was concluded that olfactory stimuli from pups both stimulate maternal licking and serve as a basis for discriminating male and female offspring.