Scanning electron microscopical studies on the tegument of adult worms of Schistosoma mansoni originating from ultraviolet-irradiated and non-irradiated cercariae.

The surface topography and ultrastructural changes of Schistosoma mansoni worms developed from ultraviolet-irradiated cercariae were compared with those from non-irradiated cercariae. The tegument of worms developed from irradiated cercariae showed a variety of changes including the occurrence of oedemata and most of the tubercles being torn at the tip and having lost their spines on the dorsal surface of some male worms. In females, parts of the tegument were devoid of spines and surface lesions were present. The range and extent of these changes differed not only among individual worms but also between different regions of individual worms resulting in generalized deformities in those worms originating from irradiated cercariae.     

Schistosoma mansoni: male-biased sex ratios in snails and mice

Adult sex ratios of Schistosoma mansoni, in mice, were shown to be biased toward males (3:1) despite the finding that sex ratios of miracidia were 50:50. The adult male bias was caused by greater male infectivity of miracidia for snails and cercariae for mice. A significantly higher percentage of male miracidia developed to cercarial production in unimiracidial infections (57 male, 34 female), and a significantly higher percentage of male cercariae developed to adulthood in mice (143 male, 79 female worms resulted from 900 male and 900 female cercariae). No significant differences were found between male and female parasites for longevity of miracidia (both sexes, 10 hr) and cercariae (males 21.3 +/- 5.75 hr, females 25.0 +/- 7.02 hr), prepatent periods in snail hosts (male 34 +/- 2.92 days, females 33 +/- 2.36 days), longevity of snail infections (males 96.6 +/- 25.15 days, females 115.2 +/- 82.43 days), and the numbers of cercariae produced per snail lifetime (males 30,751.44 +/- 18,064.33, females 34,083.00 +/- 33,732.82). Present results provide a better understanding of the life cycle of S. mansoni, are of theoretical significance for theories of biased sex ratios (which at present cannot account for the male-biased ratio of S. mansoni), and also suggest that schistosomiasis transmission models assuming a 50:50 sex ratio at all stages of the life cycle should be reassessed.     

<Emphasis Type="Italic">Trichobilharzia regenti</Emphasis>: Antigenic structures of intravertebrate stages

Like several other bird schistosomes, neurotropic schistosome of Trichobilharzia regenti can invade also mammals, including humans. Repeated infections cause cercarial dermatitis, a skin inflammatory reaction leading to parasite elimination in non-specific mammalian hosts. However, in experimentally primo-infected mice, the worms escape from the skin and migrate to the central nervous system. In order to evade host immune reactions, schistosomes undergo cercaria/schistosomulum transformation accompanied with changes of surface antigens. The present study is focused on localization of the main antigens of T. regenti; cercariae, schistosomula developed under different conditions and adults were compared. Antigens were localized by immunofluorescence and ultrastructural immunocytochemistry using sera of mice repeatedly infected with T. regenti. Detected antibody targets were located in glycocalyx and penetration glands of cercariae and in tegument of cercariae, schistosomula and adults. Shedding of cercarial glycocalyx significantly reduced surface reactivity; further decrease was reported during ongoing development of schistosomula. Spherical bodies, probably transported from subtegumental cell bodies to worm surface, were identified as the most reactive tegumental structures. Based on similar results for schistosomula developed in specific, non-specific hosts and in vitro, it seems that the ability of T. regenti to decrease the surface immunoreactivity during ontogenesis is independent on the host type.     

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.     

Androgen receptor immunoreactivity in the male and female Syrian hamster brain.

To investigate potential mechanisms for sex differences in the physiologic response to androgens, the present study compared the hormonal regulation of intracellular androgen receptor partitioning and the distribution of androgen receptor immunoreactivity in select brain regions from male and female hamsters. Androgen receptors were visualized on coronal brain sections. Two weeks after castration, androgen receptor immunoreactivity filled the neuronal nuclei and cytoplasm in males and females. In gonad-intact males and females, androgen receptor immunoreactivity was limited to the cell nucleus. Whereas exogenous dihydrotestosterone prevented cytoplasmic immunoreactivity, estrogen at physiologic levels did not. These results suggest that nuclear androgen receptor immunoreactivity in gonad-intact females is maintained by endogenous androgens, and that androgens have the potential to influence neuronal activity in either sex. However, sex differences in the number and staining intensity of androgen-responsive neurons were apparent in select brain regions. In the ventral premammillary nucleus, ventromedial nucleus of the hypothalamus, and medial amygdaloid nucleus, androgen receptor staining was similar in gonadectomized males and females. In the lateral septum, posteromedial bed nucleus of the stria terminalis (BNSTpm), and medial preoptic nucleus, the number of androgen receptor-immunoreactive neurons was significantly lower in females (p < .05). Moreover, the integrated optical density/cell in BNSTpm was significantly less in females (1.28+/-0.3 units) than in males (2.21+/-0.2 units; p < .05). These sex differences in the number and staining intensity of androgen-responsive neurons may contribute to sex differences in the behavioral and neuroendocrine responses to androgens.     

Androgen receptor immunoreactivity in the male and female Syrian hamster brain

To investigate potential mechanisms for sex differences in the physiologic response to androgens, the present study compared the hormonal regulation of intracellular androgen receptor partitioning and the distribution of androgen receptor immunoreactivity in select brain regions from male and female hamsters. Androgen receptors were visualized on coronal brain sections. Two weeks after castration, androgen receptor immunoreactivity filled the neuronal nuclei and cytoplasm in males and females. In gonad‐intact males and females, androgen receptor immunoreactivity was limited to the cell nucleus. Whereas exogenous dihydrotestosterone prevented cytoplasmic immunoreactivity, estrogen at physiologic levels did not. These results suggest that nuclear androgen receptor immunoreactivity in gonad‐intact females is maintained by endogenous androgens, and that androgens have the potential to influence neuronal activity in either sex. However, sex differences in the number and staining intensity of androgen‐responsive neurons were apparent in select brain regions. In the ventral premammillary nucleus, ventromedial nucleus of the hypothalamus, and medial amygdaloid nucleus, androgen receptor staining was similar in gonadectomized males and females. In the lateral septum, posteromedial bed nucleus of the stria terminalis (BNSTpm), and medial preoptic nucleus, the number of androgen receptor–immunoreactive neurons was significantly lower in females (p < .05). Moreover, the integrated optical density/cell in BNSTpm was significantly less in females (1.28 ± 0.3 units) than in males (2.21 ± 0.2 units; p < .05). These sex differences in the number and staining intensity of androgen‐responsive neurons may contribute to sex differences in the behavioral and neuroendocrine responses to androgens. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 359–370, 1999     

Sex and estrous cycle-dependent differences in glial fibrillary acidic protein immunoreactivity in the adult rat hippocampus

Sex differences in the morphology and function of the hippocampus have been reported in several species, but it is unknown whether a sexual dimorphism exists in glial fibrillary acidic protein (GFAP) expression in the rat hippocampus. We analyzed GFAP immunoreactivity in the hippocampus of intact adult male rats as well as in females during diestrus and proestrus phases of the estrous cycle. We found that in CA1, CA3, and dentate gyrus, GFAP immunoreactivity was higher in proestrus females as compared with males and diestrus females. In CA1, a similar GFAP immunoreactivity was found in males and in diestrus females, but in dentate gyrus, males presented the lowest GFAP content. Interestingly, differences in astrocyte morphology were also found. Rounded cells with numerous and short processes were mainly observed in the hippocampus during proestrus whereas cells with stellate shape with few and long processes were present in the hippocampus of males and diestrus females. The marked sex and estrous cycle-dependent differences in GFAP immunoreactivity density and in astrocyte number and morphology found in the rat hippocampus, suggest the involvement of sex steroid hormones in the sexually dimorphic functions of the hippocampus, and in the change in its activity during the estrous cycle.     

Upregulation of astrocytic basic fibroblast growth factor in the cingulate cortex of lactating rats: time course and role of suckling stimulation

Previous work from our laboratory has shown that there is a much higher level of bFGF and GFAP immunoreactivity in area 2 of the cingulate cortex (Cg2) of rats on day 16 of lactation than in cycling or late pregnant females. To examine the time course of this change, in the first of the current studies, we compared bFGF and GFAP immunoreactivity in the brains of lactating females on postpartum day 4 (PP4), day 10 (PP10), day 16 (PP16), and day 24 (PP24) with that of cycling and ovariectomized (OVX) females. In the second study, we investigated whether the maintenance of these changes in bFGF and GFAP depended on suckling stimulation by removing litters on day 1 or day 16 postpartum and examining the brains of the dams on day 4 (Pr4) or day 24 (Pr24) postpartum, respectively. bFGF and GFAP immunoreactivity within Cg2 and the medial preoptic area (MPOA) were measured. In both experiments astrocytic bFGF and GFAP surface density in the Cg2 varied significantly across groups. All postpartum rats, regardless of stage of lactation or presence of the litter, had significantly higher levels of bFGF and GFAP immunoreactivity than cycling animals. Thus, the maintenance of this upregulation in bFGF and GFAP immunoreactivity does not depend on suckling stimulation. Consistent with our previous report, astrocytic bFGF was also elevated in the MPOA of PP16 animals. These data suggest a robust, long-lasting, postpartum change in bFGF and GFAP immunoreactivity in Cg2 and a role for this area of the cortex in the physiological and behavioral adaptations that accompany reproductive experience.     

Modulation of galanin and neuromedin U-like immunoreactivity in rat corticotropes after alteration of endocrine status

The localization of galanin in rat lactotropes and human corticotropes is well established. Neuromedin U immunoreactivity is present in rat corticotropes but radioimmunoassay of thyroid-manipulated rat pituitaries has also linked it to the thyroid axis. We found galanin immunoreactivity in some rat corticotropes, so we have re-examined rat anterior pituitary galanin- and neuromedin U-like immunoreactivity by use of immunocytochemistry and electron microscopy in rats in the normal state and after estrogen administration or adrenalectomy. In normal rats galanin immunoreactivity was present in a few corticotropes and lactotropes, females showing more than males; neuromedin U-like immunoreactivity was present in some thyrotropes and most corticotropes, in both sexes. Where galanin, neuromedin U and ACTH immunoreactivities were colocalized in corticotropes they were present in the same granules. Estrogen administration caused an increase in number of galanin immunoreactive lactotropes, as previously shown. The proportion of neuromedin U-positive corticotropes was not affected. After adrenalectomy, only females showed a significant increase in the proportion of galanin-positive corticotropes. Neuromedin U immunoreactivity was significantly increased in both sexes, as previously shown. Thus, in rat, as in man, galanin can be present in corticotropes and its expression appears to be sexrelated. This finding, and the demonstration of thyrotrope neuromedin U (only examined in normal females), provide correlation with previous experiments. The influence of endocrine status on the expression of these novel peptides underlines the inherent plasticity of pituitary endocrine cells.Visiting Senior Research Fellow from the Institute of Human Anatomy, University of Naples (Italy), and is in receipt of a grant from the EEC (n.SC1 282)     

Differences in Brazilian strains of Schistosoma mansoni evaluated by means of morphometric analysis of cercariae of both sexes

Morphometrics of Brazilian strains (BH, SJ and CMO) of Schistosoma mansoni cercariae were obtained with a computerized image analyzer (IMAGE PRO PLUS, MEDIA CYBERNETICS), considering the following characters: body area, tail, furcae, oral and ventral suckers and distance between them. For statistical analysis, the variance test (one-way Anova) was applied and significant differences of p< 0.05 were considered. All morphometric values in the BH strain were significantly higher (p< 0.05) than in the others. Lower values were obtained in females of SJ strain for all characters, excepting the body area. Only this character showed to be significantly different in males and females of the three strains. Specimens of both sexes in the BH and SJ strains showed significant differences regarding all characters. It was observed that this morphometric analysis permits the characterization of strains and also the sex identification in S. mansoni cercariae. Due to its feasibility, this method can be applied as a tool in laboratories devoid of more complex equipment.     

Cercarial chaetotaxy and sex differentiation of Schistosoma mansoni deriving from humans and Nectomys squamipes (Muridae: Sigmondontinae) in Brazil

A comparative study was made between sympatric isolates of Schistosoma mansoni: one from a wild rodent (R) Nectomys squamipes and another one from humans (H) isolated from a low endemic schistosomiasis transmission area in Brazil. Our purpose was to detect differences between them concerning chaetotaxy (number and pattern of distribution of the argentophilic papillae) of the cercariae by means of silver impregnation. No significant difference (x > 0.05) between isolates was noted. Nevertheless, a significant difference (x < 0.05) was observed in the cercarial index (ratio of the distance between the first and the second preacetabular papillane and the distance between the first and the second dorsal preacetabular papillae) of male and female cercariae in both isolates. Males presented a greater cercarial index than females. By means of multivariate analysis, male cercariae were distinguished from female cercariae through the following characteristics: average number of dorsal papillae on the right quadrant, average number of ventral middle papillae on the right quadrant (H isolate) and average number of dorsal middle papillae on the left quadrant (R isolate). The results suggest that R and H isolates belong to the same population that could complete its life cycle in rodent-snail-rodent fashion.     

Infectivity of Diplostomum spp. in Arctic charr: aspects of exposure duration and cercariae morphology

The diplostomid flukes, Diplostomum spp., infect fish and cause cataract opacities in the eye lens. The effect of exposure dose on abundance of Diplostomum spp. eye flukes in fish is known, but the effect of the duration of cercariae exposure has not been studied. However, under natural conditions, the temporal window for a successful cercaria attachment on fish is very short and, consequently, differences in infectivity of eye fluke cercariae, in the short-exposure durations of a few seconds, are probably biologically the most meaningful. We investigated infectivity of Diplostomum spp. cercariae originating from snail hosts in 3 lakes (3 Lymnaea stagnalis populations and 1 Radix balthica population) in 6 exposure times, ranging from 5 sec to 15 min, in young-of-the-year Arctic charr Salvelinus alpinus. In addition, we compared the infectivity to the cross-morphology of the cercariae. In the long-exposure duration, i.e., > or = 5 min, infectivity of Diplostomum spp. did not vary between the snail host species (L. stagnalis and R. balthica) of the same lake or across the L. stagnalis populations of 3 different lakes. However, in the short-exposure duration, i.e., < or = 60 sec, Diplostomum spp. cercariae shed from L. stagnalis had higher infectivity than did cercariae from R. balthica of the same lake. This indicates that that there is an interaction between length of cercariae exposure and origin of Diplostomum spp., and that the duration of exposure may influence the results when fish are experimentally infected. Within a lake, cercariae shed from L. stagnalis were also smaller than cercariae shed from R. balthica.     

Small estuarine fishes feed on large trematode cercariae: lab and field investigations

In aquatic ecosystems, dense populations of snails can shed millions of digenean trematode cercariae every day. These short-lived, free-living larvae are rich in energy and present a potential resource for consumers. We investigated whether estuarine fishes eat cercariae shed by trematodes of the estuarine snail Cerithidea californica. In aquaria we presented cercariae from 10 native trematode species to 6 species of native estuarine fishes. Many of these fishes readily engorged on cercariae. To determine if fishes ate cercariae in the field, we collected the most common fish species, Fundulus parvipinnis (California killifish), from shallow water on rising tides when snails shed cercariae. Of 61 killifish, 3 had recognizable cercariae in their gut. Because cercariae are common in this estuary, they could be frequent sources of energy for small fishes. In turn, predation on cercariae by fishes (and other predators) could also reduce the transmission success of trematodes.     

The influence of anhydrobiosis on the infestation of Biomphalaria glabrata by Schistosoma mansoni miracidium

410 Biomphalaria glabrata (Caribbean strain of Guadeloupe) have been infected with one miracidium of Schistosoma mansoni, 110 snails, used as controls have been kept into water; the survival rate was 96.4% after 4 weeks and 25.4% produced cercariae. 300 snails were kept on wet soil, and submitted for 6 weeks to progressive desiccation. The survival rate was 23.4% but only 9 of them produced cercariae. Periodic variations of the production of male and female larvae have been shown by the weekly test of the cercariae productions. In previously desiccated snails, the production of male and female cercariae is similar while in controls the production of female larvae is more important. In experimental snails, the larval development seems to be stopped during anhydrobiosis. The production of cercariae is just delayed for the length of the dry keeping.     

Histopathology of experimental Schistosoma incognitum infection in mice following exposure to normal and irradiated cercariae.

The tissue reactions in mice, experimentally infected with normal and irradiated cercariae of S. incognitum were studied. The lesions observed in the skin, liver, lungs and the intestine of mice infected with normal cercariae are briefly described, and compared with those observed with cercariae irradiated at 3000 r of gamma rays. In general, the reactions in mice exposed to normal cercariae were more intense than in those infected with irradiated cercariae. The severity of the reactions appeared largely due to the deposition of eggs in the tissues of the mice infected with normal cercariae. The experimental evidence suggested that most of the flukes from the irradiated cercariae are destroyed in the liver by tissue reaction.     

Cercarial productivity of redial generations in single-miracidium infections of Lymnaea truncatula with Paramphistomum daubneyi or Fasciola hepatica

Single-miracidium infections of Lymnaea truncatula with Paramphistomum daubneyi or with Fasciola hepatica were carried out under laboratory conditions to count free rediae, their germinal embryos, and to determine the cercarial productivity of each redial generation. In snails infected by P. daubneyi, the cercariae were produced by the first (8.7 cercariae per redia) and second (8.9 per redia) generations. At day 63 post-exposure, they corresponded, respectively, to 53.9% and 46.1% of cercariae produced by all rediae. In snails infected by F. hepatica, the majority of cercariae were produced by the R2a group (18.2 cercariae per redia) and corresponded to 66.0% of cercariae produced all rediae. The cercariae produced by the other redial groups were more limited in number: 17.5 per redia in the R1b group (28.7%) and 2.0 per redia in the R2b/R3a group (5.3%). Cercarial productivity of P. daubneyi until day 63 post-exposure was more limited in number than that of F. hepatica: a total of 145 cercariae per snail versus 427 per snail.     

Ribeiroia ondatrae cercariae are consumed by aquatic invertebrate predators

Trematodes amplify asexually in their snail intermediate hosts, resulting in the potential release of hundreds to thousands of free-living cercariae per day for the life of the snail. The high number of cercariae released into the environment undoubtedly increases the probability of transmission. Although many individual cercariae successfully infect another host in their life cycle, most fail. Factors that prevent successful transmission of cercariae are poorly understood. Microcrustaceans and fish have been observed to eat cercariae of some species, although the possibility that predation represents a significant source of mortality for cercariae has been largely unexplored. We tested the cercariophagic activity of several freshwater invertebrates on Ribeiroia ondatrae, a trematode that causes limb deformities in amphibians. Individuals of potential predators were placed into wells of multiwell plates with 10-15 cercariae, and numbers of cercariae remaining over time were recorded and compared with numbers in control wells that contained no predators. Of the species tested, Hydra sp., damselfly (Odonata, Coenagrionidae) larvae, dragonfly (Odonata, Libellulidae), larvae, and copepods (Cyclopoida) consumed cercariae. In some cases, 80-90% of the cercariae offered to damselfly and dragonfly larvae were consumed within 10 min. In most cases, predators continued to consume cercariae at the same average rates when offered cercariae together with individuals of an alternate prey item. Hydra sp. ate fewer cercariae in these trials. Our findings suggest the need for field and laboratory studies to further explore the effects of predators on transmission of R. ondatrae to amphibian larvae. In addition, the results suggest that conservation of the biodiversity and numbers of aquatic predators may limit adverse impacts of trematode infections in vertebrate hosts.     

Schistosoma mansoni: Long-term maintenance of clones by microsurgical transplantation of sporocysts

Schistosoma mansoni sporocysts originally derived from monomiracidially infected Biomphalaria glabrata snails were serially transplanted into the cephalopedal sinus of anesthetized snails by the microsurgical implantation of fragments of parasitized hepato-pancreas and ovotestis. Three to six passages each of five male and five female clones were maintained for as long as 2.0 years. Of the recipient snails which survived surgery, 87% released cercariae, usually beginning 5–7 weeks after surgery. The percentage of snails which released cercariae increased with successive passages. The mean survival time of surgically infected snails after cercarial emergence began was 9.2 ± 0.5 weeks, nearly the same as that of miracidially infected snails. Longevities of snails infected with male or female clones were similar. Recipient snail size and age did not influence cloning success. Beginning 5 weeks from the onset of cercarial emergence large numbers of cercariae (a mean of 3900/snail from male clones and 1300/snail from females) were obtained during each shedding period. These results clearly demonstrate that the microsurgical transplantation of sporocysts is a practical means of maintaining and expanding populations of genetically homogeneous schistosomes (clones).     

In vivo and in vitro encystment of Echinochasmus liliputanus cercariae and biological activity of the metacercariae

In vivo and in vitro encystment of the cercariae of Echinochasmus liliputanus and biological activity of the metacercariae were studied. In vivo encystment of cercariae occurred in the gills of goldfish, the second intermediate host. However, the cercariae also encysted in vitro in Locke solution (0.6x to 1.2x strength), 0.7-1.2% NaCI, artificial gastric juice, and human gastric juice. Locke or NaCI solutions were shown to be appropriate for in vitro encystment to occur within 24 hr; however, full-strength Locke solution was shown to be optimal. The 1-day-old metacercariae formed in vivo and treated in 0.1% sodium deoxycholate excystation medium at 37 C for 1 hr showed 88.5% excystation. The metacercariae formed in vitro, however, showed 88.6% and 85.0% excystation for normal and abnormal ones, respectively. Abnormal cysts at room temperature usually die within 10 days. About 70% of the normal cysts, both in vivo and in vitro, can still excyst after being stored in Locke 0.5x solution at 4 C for 3 mo. Cysts formed in vivo and in vitro were equally infective. The encystment of the cercariae in vitro could be inhibited when the cercariae were treated with 1 micromol silver nitrate. Because silver nitrate binds to the papillae, especially to the ciliated papillae, on the cercaria surface, it is suggested that papillary chemoreceptors may be involved in encystment of the cercariae. The finding of E. liliputanus cercariae encysting in vitro, especially in human gastric juice, might be helpful in elucidating mechanisms of the definitive hosts that are directly infected by the cercariae.     

Localization of Methionine-Enkephalin-Like Peptides in the Nervous System and Ovaries of the Calliphora vomitoria During the First Reproductive Cycle

Summary

Using the peroxidase antiperoxidase immunocytochemical method (PAP) Met-enkephalin immunoreactivity has been demonstrated in brains (males and females) and ovaries of Calliphora vomitoria. The Met-enkephalir immunoreactive neuronal structures are present in the cerebrum, sub- oesophageal ganglion and optic lobes. A similar distribution pattern of Met- enkephalin immunoreactivity is observed in both brains and ovaries of severa stages from 0.30 hr to 48 hr after copulation. In the ovaries, the distributior of the Met-enkephalin-like peptide is dependent in the stage of development o: the ovarian follicles. In trophocytes and oocytes, the immunoreactivity firsl appears around the nuclei, and is then dispersed in the cytoplasm. Later, al the end of vitellogenesis it is only present in the periphery of the cells and finally, it disappears. No Met-enkephalin immunoreactivity is found in tht follicle cells.