Physiological changes in Biomphalaria glabrata Say, 1818 (Pulmonata: Planorbidae) caused by sub-lethal concentrations of the latex of Euphorbia splendens var. hislopii N.E.B (Euphorbiaceae)

Molluscides have been used as one of the strategies to control schistosomiasis. Many plant extracts with molluscidal effects have been tested, but the action of the latex of Euphorbia splendens var. hislopii is considered the most promising because it meets the recommendations of the World Health Organization (WHO). The objective of this study was to determine the lethal dose and identify the effects of the different doses of latex of E. splendens var. hislopii on the physiology of Biomphalaria glabrata submitted to treatment for 24 h. The concentrations of glucose, uric acid and total proteins in the hemolymph and of glycogen in the digestive gland and cephalopodal mass were determined. The LD50 value was 1 mg/l. The highest escape index was found to be at a concentration of 0.6 mg/l. The results showed that the latex of E. splendens var. hislopii caused a sharp reduction in the reserves of glycogen in the digestive gland and elevation of the protein content in the hemolymph of B. glabrata.     

The molluscicidal activity of the latex of Euphorbia splendens var. hislopii on Melanoides tuberculata (Thiaridae), a snail associated with habitats of Biomphalaria glabrata (Planorbidae)

The use of the latex of Euphorbia splendens var. hislopii was considered as an effective control method for Biomphalaria glabrata in Sumidouro, Rio de Janeiro. However, the appearance and expansion of the snail Melanoides tuberculata since August 1997, with the concomitant reduction of the population of B. glabrata suggest that competitive exclusion might be taking place. Depending on the susceptibility of the thiarid to the E. splendens toxin, the natural control that is occurring could be interrupted by the employment of the latex if the planorbid were less susceptible to the toxin. The aim of this study is to investigate the molluscicidal activity of the latex on M. tuberculata. We used 420 M. tuberculata, from Sumidouro. Fourteen different latex concentrations were tested using World Health Organization general methodology. Probit analysis was used for LD90 and LD50 determination. The LD50 was 3.57 mg/l and LD90 was 6.22 mg/l. At the highest concentration (10 mg/l) there was no survival. No significant differences among replicas (chi2 = 8.31; gl = 13; p > 0.05) were found. The LD90 dose for M. tuberculata was 13.8 times greater than that for B. glabrata, so that the molluscicide in the presence of the thiarid may have a synergic effect on reduction of Biomphalaria populations.     

Evaluation of the molluscicidal properties of Euphorbia splendens var. hislopii (N. E. B.) (Euphorbiaceae)--1. Experimental test in a lentic habitat.

The latex of Euphorbia splendens var. hislopii, at concentrations between 5 to 12 mg/l, kills 100% of the population of Biomphalaria glabrata in a lentic habitat, after 24 h. The lyophilized latex, stocked for 18 months, killed only 34.2% of the snails, at the concentration of 5 mg/l, and 96.0% at 12 mg/l. No lethal effect was observed among Pomacea haustrum exposed to the same concentrations of the molluscicide.     

Latex of "coroa de cristo" (Euphorbia splendens): an effective molluscicide

An aqueous solution of the latex of "coroa de cristo" (Euphorbia splendens var. hislopii) showed molluscicide action (LD90) at a concentration lower than 0.5 ppm on Biomphalaria glabrata and B. tenagophila reared in laboratory and at a concentration lower than 4.0 ppm for field B. tenagophila.     

Activity of Euphorbia splendens var. hislopii N.E.B. (Euphorbiaceae) latex against Lymnaea columella (Say, 1817) (Pulmonata: Lymnaeidae), intermediate host of Fasciola hepatica, Linnaeus, 1758 (Trematoda: Fasciolidae). 2: limited field-testing

The molluscicidal evaluation of Euphorbia splendens var. hislopii (Crown of thorns) against Lymnaea columella snails, intermediate host of Fasciola hepatica, in irrigation ditches of the Pisciculture Station at Universidade Federal Rural do Rio de Janeiro, was studied under limited field conditions. An aqueous solution of the latex at 5 mg/l was tested in two irrigation ditches (experimental and control ditches), after initial sampling of the snail population present. Twenty-four hours after application of the product, it was verified that 97.4% of free L. columella snails and 100% of snails of the same species captive in cages and used as sentinels at three points equidistant from the application site in the experimental ditch, died. For Biomphalaria tenagophila and Melanoides tuberculata snails, present in the experimental ditch, the mortality was 100%, for the species Pomacea spp. the mortality was 40%. No mortality was verified in the free mollusks, or in the sentinels in the ditch used as control. E. splendens var. hislopii latex is thus an efficient natural molluscicide, which may be used as an alternative control agent against L. columella.     

Molluscicidal action of the latex of Euphorbia splendens var. hislopii N.E.B. ("Christ's Crown") (Euphorbiaceae) against Lymnaea columella (Say, 1817) (Pulmonata: Lymnaeidae), intermediate host of Fasciola hepatica Linnaeus, 1758 (Trematode: Fasciolidae): 1- test in laboratory

The latex action of Euphorbia splendens var. hislopii (Christ's Crown) against snails Lymnaea columella, intermediate host of Fasciola hepatica, derived from irrigation ditches of the Station of Pisciculture at Universidade Federal Rural do Rio de Janeiro, was studied in the laboratory. Lab bioassays, using aqueous solutions of the latex, varying between 0.1 and 10 mg/l, have proven molluscicidal activity of the product collected on the same day the tests were performed, during the four seasons of the year, finding the following lethal concentrations (LC90): 1.51 mg/l in the spring; 0.55 mg/l in the summer; 0.74 mg/l in the fall and 0.93 mg/l in winter, after 24 h exposure of the snails, showing significant differences among the seasons of the year (ANOVA test, F = 11.01, G.L.= 3/33, p < 0.05), as well as among the concentrations (ANOVA test, F = 27.38, G.L.= 11/33, p < 0.05). In the summer, mortality reached 100% from concentration at 0.6 mg/l, the same during fall and in winter as of 1 mg/l, while in spring it only reached 100% mortality as of 2 mg/l. Mortality in the controls was low, reaching 5% in the summer and winter and 10% in the fall and spring. None of the samples died. During the assay, with an aqueous solution of the latex at a concentration of 5 mg/l, in order to check the time of duration of the product effect, in the laboratory, it was observed that the molluscicidal activity remained stable up to the 15th day after the beginning of the test with 100% mortality of L. columella, gradually losing its effect until the 23rd day, when we no longer observed animal mortality. In the control group, there was a random daily variation in mortality rate ranging 0-50% after 48 h of observation for 30 days.     

The molluscicidal activity of niclosamide (Bayluscide WP70(R)) on Melanoides tuberculata (Thiaridae), a snail associated with habitats of Biomphalaria glabrata (Planorbidae)

The aim of this study was to determine the toxicity of niclosamide (Bayluscide (R)) on Melanoides tuberculata and Biomphalaria glabrata under laboratory conditions. The latter species is the intermediate host of Schistosoma mansoni (Sambon 1917). M. tuberculata was successfully used as competitor of B. glabrata in biological control programs in French West Indies. Both molluscicide and biological control using M. tuberculata have proved to be successful in reducing the population density of B. glabrata. The associated use of molluscicide in this area would be an effective measure if M. tuberculata were less susceptibility to the molluscicide than B. glabrata. Three hundreds individuals each of B. glabrata and of M. tuberculata, collected in Sumidouro, State of Rio de Janeiro, were used in the experiment. The molluscs were exposed to 14 different concentrations of niclosamide as recommended by the World Health Organization. Probit analysis was used to determine the LC 50 and LC 90. The LC 50 and LC 90 values for B. glabrata were 0.077 mg/l and 0.175 mg/l, respectively and the LC 50 and LC 90 values for M. tuberculata were 0.082 mg/l and 0.221 mg/l respectively. As the lethal concentrations of niclosamide were approximately the same to both species, this could be a disadvantage when controlling B. glabrata with niclosamide in an area of M. tuberculata occurrence. It might therefore be preferable to utilize the latex extracted from the Euphorbia splendens, which presented a much higher efficiency for B. glabrata than to M. tuberculata.     

Effects of snail size on encystment of Echinostoma caproni in juvenile Biomphalaria glabrata (NMRI strain) and observations on the survival of infected snails

The effects of snail size on encystment of Echinostoma caproni cercariae in neonatal and juvenile Biomphalaria glabrata (NMRI strain) snails were studied. Encystment in neonatal (0.7-1.1 mm shell diameter) and juvenile (2-3 mm shell diameter) snails was compared 24 h post-infection (PI) following individual exposure of snails of each size to 1, 5, 10, 25 and 50 cercariae. Significantly more cysts were recovered from juveniles exposed to 1, 5, 10 and 50 cercariae than from neonatals with comparable exposure. Size of B. glabrata was a major factor in determining cyst burden in this planorbid. Survival of infected versus uninfected neonatals and juveniles was also examined for 7 days. Neonatals exposed to 10 cercariae showed a significant decrease in survival at 3, 6 and 7 days PI when compared to the uninfected controls. There was no significant decrease in the survival of juveniles exposed to 10 cercariae compared to uninfected controls at any time point. Snail size was a factor in mortality associated with echinostome cercarial penetration and encystment.     

Studies of the relationship between Schistosoma and their intermediate hosts. III. The genus Biomphalaria and Schistosoma mansoni from Egypt, Kenya, Sudan, Uganda, West Indies (St. Lucia) and Zaire (two different strains: Katanga and Kinshasa)

The compatibility between strains of Schistosoma mansoni from Egypt, Kenya, Sudan, Uganda, the West Indies, and Zaire (two strains which came from Katanga and from Kinshasa), and various species and strains of Biomphalaria, i.e. Biomphalaria pfeifferi, B. alexandrina, B. glabrata and B. camerunensis was investigated. Data as mortality, rate of infection of the surviving snails, duration of infection, cercarial production per day per positive snail, etc., were observed. The main emphasis was placed on determining the total cercarial production per 100 exposed snails for each snail population. It was possible to infect all the tested populations of B pfeifferi with the various strains of S. mansoni, but the observation as e.g. TCP/100 exposed snails varied greatly according to the population of snail and the strain of S. mansoni. The results for the remaining species of Biomphalaria varied greatly, depending on the combination, e.g. B. alexandrina was only susceptible to the local S. mansoni from Egypt. The highest TCP/100 exposed snails was more than 1 million for the strains of S. mansoni from Egypt, Kenya and the West Indies in B. alexandrina, B. pfeifferi and B. glabrata, respectively. The next group, with a TCP/100 exposed snails on 7--800 000 consists of S. mansoni from Sudan, Uganda and Zaire (Katanga) all in B. pfeifferi. The last tested strain of S. mansoni, Zaire (Kinshasa) yielded a cercarial production on 500 000 per 100 exposed snails in B. pfeifferi and B. camerunensis. The shortest prepatent period, 19 days, was observed for S. mansoni from Kinshasa, Zaire, in B. camerunensis, and the longest prepatent period, 25 days, was found for strains from Egypt and from the West Indies in B. alexandrina and B. glabrata, respectively. In general, a very long duration of infection, lasting up to 200 days, was observed.     

Molluscicide activity of a butanol extract from Phytolacca dodecandra (endod) on Biomphalaria glabrata

A buthanol extract of Phytolacca dodecandra (type 44) obtained from Ethiopia berries, was tested as molluscicide in our laboratory and in the field. The lethal dose (LD90) for adult snails, newly hatched and egg-masses of Biomphalaria glabrata, in 24 hours exposure, were of 4.5, 23.0 and 102.0 ppm respectively. The LD90 for the fish Lebistes reticulatus was of 2.0 ppm. These results are similar to those of Lemma (1984) in Ethiopia. In two water ponds treated with 10 ppm of the buthanol extract or 3 ppm of niclosamide the mortality rates of B. glabrata were of 84.6 and 100.0%, respectively. Both treatments were toxic for L. reticulatus in the field trials. The possibility of using molluscicides derived from plants is discussed as an alternative for treatment of schistosomiasis foci in Brazil.     

Resistance of Biomphalaria occidentalis from Varzea das Flores dam, Minas Gerais, to Schistosoma mansoni infection detected by low stringency polymerase chain reaction

Biomphalaria occidentalis Paraense, 1981 from Varzea das Flores dam, MG, Brazil, was exposed to infection with Schistosoma mansoni. Individual infection was performed with 140 B. occidentalis and 100 B. glabrata snails using LE and SJ strains. Two groups of B. occidentalis were killed after seven day-miracidia exposure to detect S. mansoni DNA, through the low stringency polymerase chain reaction (LS-PCR), and were negative. The infection rates were 69.2% (LE strain) and 96.7% (SJ strain) for B. glabrata and 0% for B. occidentalis. LS-PCR enabled early resistance diagnosis.     

Influence of saccharose on the development of cercariae from Schistosoma mansoni strains BH and SJ.

The development of cercariae from Schistosoma mansoni strains BH and SJ in Biomphalaria glabrata and Biomphalaria tenagophila treated with saccharose was studied. The molluscs were maintained in dechlorinated tap water containing 0.01% saccharose. After one week of treatment with saccharose, B. glabrata and B. tenagophila were exposed to ten S. mansoni miracidia, from BH and SJ strains respectively. Control snails of both species were maintained in dechlorinated tap water without saccharose and exposed to the same number of miracidia. There was no significant difference between the infection rates of snails treated or not with saccharose. However, the two groups of B. glabrata had significantly greater infection rates than the corresponding B. tenagophila groups. Molluscs treated with saccharose had a lower survival rate, with the greatest mortality occurring immediately before and at the beginning of cercariae release. Treatment with saccharose did not result in the release of more cercariae, but larvae from molluscs so treated showed a greater capacity to penetrate mouse skin, which was attributed to the greater energy supply during larval development in the mollusc.     

Experimental exposure of Helisoma trivolvis and Biomphalaria glabrata (Gastropoda) to Ribeiroia ondatrae (Trematoda)

Experimental infections provide an important foundation for understanding host responses to parasites. While infections with Ribeiroia ondatrae cause mortality and malformations in a wide range of amphibian second intermediate host species, little is known about how the parasite affects its snail first intermediate hosts or even what species can support infection. In this study, we experimentally exposed Helisoma trivolvis, a commonly reported host of R. ondatrae, and Biomphalaria glabrata, a confamilial snail known to host Ribeiroia marini, to increasing concentrations of embryonated eggs of R. ondatrae obtained from surrogate definitive hosts. Over the course of 8 wk, we examined the effect of parasite exposure on infection status, time-to-cercariae release, host size, and mortality of both snail species. Helisoma trivolvis was a highly competent host for R. ondatrae infection, with over 93% infection in all exposed snails, regardless of egg exposure level. However, no infections were detected among exposed B. glabrata, despite previous accounts of this snail hosting a congener parasite. Among exposed H. trivolvis, high parasite exposure reduced growth, decreased time-to-cercariae release, and caused marginally significant increases in mortality. Interestingly, while B. glabrata snails did not become infected with R. ondatrae, individuals exposed to 650 R. ondatrae eggs grew less rapidly than unexposed snails, suggesting a sub-lethal energetic cost associated with parasite exposure. Our results highlight the importance of using experimental infections to understand the effects of parasite exposure on host- and non-host species, each of which can be affected by exposure.     

Circadian Rhythms in the Cercarial Emergence of Schistosoma mansoni by Biomphalaria tenagophila at Outdoors: A Comparative Study with Biomphalaria glabrata

Circadian rhythms in the emergence of S. mansoni cercariae from Biomphalaria tenagophila and B. glabrata, snail hosts of schistosomosis in Brazil, were investigated. A total of 35 specimens of B. tenagophila (São Paulo, Brazil) and 12 B. glabrata (Minas Gerais, Brazil) exposed individually to five miracidia of Schistosoma mansoni originated from the same biotope as their snail hosts, were tested. Observations were carried out at outdoors, with the quantification of cercarial emergence at 3h intervals during three consecutive days in November 1989 and in May 1990. Cercarial emergence was essentially diurnal (from 06.00-18.00h) in both species. Circadian rhythms were detected by the Single Cosinor Method among 74.3% of B. tenagophila and 91.7% of B. glabrata snails. The acrophases corresponding to individual snails were between 11.37 e 17.54h in B. tenagophila and between 14.15 and 16.29h in B. glabrata. These findings confirm our preliminary observations in B. tenagophila and are in accordance to those of other authors in regard to B. glabrata. The acrophases of individual snails were similar within each species, thus indicating that at populacional level cercarial emergence was concentrated in particular times of the day. Group acrophases for each species varied from 13.22 to 15.22h and were not significantly different between B. tenagophila and B. glabrata. Cercariae emerging from B. tenagophila snails seemed to be more sensitive to environmental temperature than those emerging from B. glabrata, at least in the temperature range prevailing along the tests. Further chronobiological studies on host-parasite interactions are encouraged to improve our knowledge about temporal aspects of schistosomosis transmission.     

Effects of infection with Angiostrongylus cantonensis on the circulating haemocyte population and the haematopoietic organ of the host snail M-line Biomphalaria glabrata

The number of circulating haemocytes, the size of the haematopoietic organ, and the size of haemocyte capsules around the parasite were studied in M-line Biomphalaria glabrata snails exposed to 100 or 400 first-stage larvae of Angiostrongylus cantonensis. The number of haemocytes in exposed snails increased significantly at 1 day post-exposure, decreased to control value, and then increased again. The decrease in number of circulating haemocytes is probably due to the removal of cells from the circulation to participate in encapsulation of larvae. The majority of circulating haemocytes in M-line B. glabrata are fully-spread granulocytes, which increase significantly in number in snails following exposure to A. cantonensis larvae. However, populations of partially-spread granulocytes, round cells, hyalinocytes and miscellaneous haemocytes were relatively constant. The size of capsules around the parasite increased during the 42-day interval of the experiment. The haematopoietic organ increased in size in response to infection.     

Experimental infection of Physa heterostropha, Helisoma trivolvis, and Biomphalaria glabrata (Gastropoda) with Echinostoma revolutum (Trematoda) Cercariae

Gross and histologic studies were done on laboratory-raised Physa heterostropha, Helisoma trivolvis, and Biomphalaria glabrata snails exposed individually to 100 cercariae of Echinostoma revolutum. Cercariae showed a predisposition for the kidneys of snails. They entered the nephridiopore, migrated up the tubular kidney, and encysted in the saccular kidney within 2 hr. Considerably more cysts were in the kidney of B. glabrata at 24 hr than in H. trivolvis or P. heterostropha kidneys. Encysted metacercariae were not infective to domestic chicks at 2 hr, but were infective by 4 hr. Biomphalaria glabrata exposed to about 1,000 cercariae/snail and necropsied either 10 or 16 wk postexposure contained 300-500 cysts/kidney; about one-half the cysts were viable and infective to chicks. Biomphalaria glabrata is an excellent second intermediate host for the laboratory propagation of E. revolutum.     

Dose-dependent schistosome-induced mortality and morbidity risk elevates host reproductive effort

Parasitism changes the host environment and may influence resource allocation between reproductive effort and somatic maintenance. We characterized the impact of dose-dependent schistosome exposure and/or infection establishment on intermediate host survival and reproduction. Four matched groups of Biomphalaria glabrata snails were individually exposed to increasing doses of Schistosoma mansoni parasites, with a fifth control group remaining unexposed. Increased mortality was observed amongst both snails infected and also those snails exposed to the parasite but within which infection did not establish, although only exposed but uninfected snails showed a dose-dependent increase in mortality. Snails also facultatively altered their reproductive output in response to parasite exposure: egg mass production decreased with increasing parasite dose in patently infected snails, whilst, in contrast, exposed but uninfected snails demonstrated a positive association between egg mass production and parasite dose in the post-patent period. These results uniquely suggest an exposure-dose-dependent post-patent fecundity compensation occurring in relation to the risk of future parasite-associated mortality.     

Rescue of sporocysts of Schistosoma mansoni in nonsusceptible Biomphalaria by head-foot transplantation into susceptible snails

To measure the longevity of sporocysts of Schistosoma mansoni in nonsusceptible snails (13-16-R1 and Salvador strains of Biomphalaria glabrata, and Biomphalaria obstructa), the head-foot (HF) of miracidia-exposed snails was transplanted into the hemocoel of a susceptible NIH albino recipient at 1-36 days postexposure (DPE). Recipient snails which were not exposed to miracidia then were monitored for infection transferred by the implant, and infection prevalences in recipients of HF transplants from nonsusceptible donors were compared to those in snails implanted with an HF from NIH albino donors. Transplants from NIH albino snails between 1 to 15 DPE infected 98% of recipients. Similarly, at 1 DPE, 69-85% of transplants from nonsusceptible snails contained viable sporocysts, as shown by resulting patent infections in the recipients. Recipient infection prevalence, and presumably numbers of transplants containing viable sporocysts, declined as a function of DPE, and by 5-9 DPE this decrease was significant for all 3 types of nonsusceptible donors. However, viable sporocysts still occurred in B. obstructa and 13-16-R1 B. glabrata as late as 19 and 20 DPE, respectively, and in Salvador B. glabrata as late as 33 DPE. Thus, sporocysts persist in nonsusceptible snails considerably longer than suggested by results of previous histological studies.     

The lethality of Euphorbia conspicua to adults of Biomphalaria glabrata, cercaria of Schistosoma mansoni and larvae of Artemia salina

Leaf extracts of Euphorbia conspicua (Euphorbiaceae), together with the latex and fractions derived therefrom, were evaluated for their molluscicidal and cercaricidal activities and their toxicities to brine shrimps. Whilst the leaf extracts were inactive against Biomphalaria glabrata, the latex, its triterpenic fraction and irritant fractions I and II exhibited high activities against adult snails with LC90 values of 4.87, 10.55, 0.64 and 0.10 microg/mL, respectively. The latex and its derived fractions were considered lethal to the cercaria of Schistosoma mansoni at concentrations of 100 microg/mL. The toxicities of the latex and the irritant fractions, but not of the triterpenic fraction, against Artemia salina were high with LC50 values < 10 microg/mL. The possible application of the latex of E. conspicua as an alternative natural molluscicide is considered.     

Effects of copper sulfate toxicity on cercariae and metacercariae of Echinostoma caproni and Echinostoma trivolvis and on the survival of Biomphalaria glabrata snails

Copper in the form of copper sulfate (CuSO4) decreases the survival of Biomphalaria glabrata snails, but the effects of this molluscicide on Echinostoma caproni and Echinostoma trivolvis, 2 species of digeneans that use B. glabrata as intermediate hosts, are not known. Studies were done on the effects of various concentrations of CuSO4 in artificial spring water (ASW) on the survival and infectivity of E. caproni and E. trivolvis cercariae. Solutions containing 1.0, 0.1, and 0.01% CuSO4 were 100% lethal within 2 hr of exposure for both species. Time to 50% mortality in 0.001% CuSO4 was 8 hr for E. caproni and 16 hr for E. trivolvis; at 24 hr, the controls showed 50 and 65% mortality, respectively. Treatment of cercariae of both species for 0.5 hr in 0.001% CuSO4 had no effect on the ability of cercariae to form normal cysts in juvenile B. glabrata snails. However, treatment with 0.01% CuSO4 for 0.5 hr caused a significant reduction in the ability of cercariae of both species to encyst in snails. Treatment of encysted metacercariae of both species in 0.001% CuSO4 for I hr had no effect on subsequent excystation of these echinostomes in a trypsin-bile salts medium, whereas concentrations of 1.0, 0.1, and 0.01% CuSO4 and 1.0 and 0.1% CuSO4 decreased chemical excystation of E. caproni and E. trivolvis cysts, respectively. Survival studies on the effects of CuSO4 in Locke's solution on chemically excysted metacercariae of both species were also done. Excysted metacercariae of both species were killed by 2 hr in either 0.1 or 0.01% CuSO4 in Locke's solution. However, time to 50% mortality for both species of excysted metacercariae in 0.001% CuSO4 was approximately 5 hr. Time to 50% mortality for the controls was about 12 hr. Survival of juvenile B. glabrata snails was also examined. All B. glabrata snails were dead by 6 hr in 1 and 0.1% CuSO4 in ASW. Biomphalaria glabrata snails showed 50% mortality by about 6 hr in 0.01% CuSO4 and about 80% were still alive at 24 hr in 0.001% CuSO4. All controls were alive at 24 hr, at which time the experiment was terminated. Concentrations greater than 0.001% CuSO4 increased snail mortality, as well as that of the cercariae and excysted metacercariae of E. caproni and E. trivolvis. Our findings suggest that concentrations of copper sufficient to eliminate juvenile B. glabratta snails are also sufficient to kill the cercariae and excysted metacercariae of these digeneans but not the encysted metacercariae, which may be protected by their cyst walls.