Background: Two aspects need to be considered for schistosomiasis control: morbidity and transmission. In this context, many soluble substances have been tested and Euphorbia <i>milii</i> latex is one of the most promising Brazilian molluscicides. Phytochemical studies involving simulation of the applicability of this latex on all aquatic forms of the <i>S. mansoni</i> life cycle are rare in the literature. The aim of this work was to evaluate the effect of <i>E. milii</i> latex on <i>S. mansoni</i> in the egg, miracidium and different developmental stages in <i>Biomphalaria glabrata</i>. Methods: The laboratory study was designed to simulate the different forms of exposure of the life cycle stages of <i>S. mansoni</i> to the LC<sub>50</sub> of <i>E. milii</i> latex; we tested the exposition from four situations of <i>S. mansoni</i> contact with the latex and observed the exposure on different snails’ infection stage too. All snails were analyzed weekly for cercarial shedding and reproductive biology. Results: The results showed that contact of <i>S. mansoni</i> eggs and miracidia with the LC<sub>50</sub> of <i>E. milii</i> negatively influenced the development of the parasite life cycle in the intermediate host, with consequent reduction of cercarial shedding. The exposure of infected snails affected the reproductive biology and cercarial shedding in all intra-mollusk development stages of <i>S. mansoni</i>, but the reduction was greater in the first, fourth, fifth, sixth, seventh and eighth weeks of infection. The LC<sub>50</sub> of <i>E. milii</i> latex had toxic action on eggs and miracidia, and the number of cercariae shed by snails during the study period declined by about 80%. Conclusions: We can conclude that the use of natural biodegradable compounds containing low concentrations of substances already characterized as having eco-toxico- logical potential can be an important tool to reduce the transmission of Schistosomiasis.
For schistosomiasis control, two aspects need to be considered: morbidity and transmission. For morbidity control, chemotherapy using praziquantel is generally successful in reducing the number of severe cases, but chemotherapy is not enough for transmission control and it is also necessary to use water-soluble substances [
Several studies have shown different activities of E. milii latex on helminths. The molluscicidal effects of the crude latex of E. milii were a good tool against schistosomiasis dynamics [
The E. milii latex samples were collected in the Ilha do Governador district (22˚48'09"S/43˚12'35"W) of the city of Rio de Janeiro, Brazil, as described by Vasconcellos & Amorin (2003). The calculation of the lethal concentration (LC) of the aqueous extract of the latex was carried out by probit analysis [
The laboratory study was designed to simulate the different forms of exposure of the life cycle stages of S. mansoni to the LC50 of E. milii latex. The situations analyzed were:
Infection of snails with eggs and/ormiracidia exposed to the LC50 of the aqueous extract of E. milli: four situations of S. mansoni contact with the latex LC50 were analyzed: Group 1―control (eggs and miracidia were maintained and the snails were infected in distilled water), Group 2―hatching of miracidia in distilled water, but the snails were infected in the presence of the latex LC50; Group 3―hatching of miracidia in the presence of the LC50 and infection of snails in distilled water; and Group 4―hatching of miracidia and infection of snails in the presence of the latex LC50. All the groups were composed of 30 snails. After infection, the snails were kept individually in 50 mL beakers with distilled water and the reproductive parameters were recorded weekly. The snails were feds ad libitum with lettuce leaves three times a week.
Infected snails: The exposure to the latex LC50 was varied according to the snails’ infection stage ( [
All snails were analyzed weekly for cercarial shedding and reproductive biology. The results were expressed as mean ± standard deviation and analyzed by analysis of variance (ANOVA), the Tukey-Kramer test and Student’s t-test for unpaired data to compare means (α = 5) (Instat, GraphPad, v.4.00, Prism, GraphPad, v.3.02, Prism Inc.).
The survival of the snails in the control group (Group 1) was 45.3% greater at the end of the experiment than observed in Group 4, in which the process of hatching and infection in the laboratory occurred in contact with the LC50 of the E. milii latex. The latex contact at the time of hatching of the miracidia or infection caused reductions of 27.3% and 18.2% in the survival of the snails, respectively. The infection rate of the snails was 30% higher in Group 1 than in Group 4, where the hatching and infection occurred in contact with the latex. The reproductive parameters of B. glabrata infected with eggs and miracidia of S. mansoni exposed to LC50 were observed during the entire experiment, but there were no significant differences (α = 5%).
The number of cercariae eliminated/snail in all treatment groups (Groups 2, 3 and 4) was significantly less than in the control group (Group 1) (
All infected snails used in the experiment shed S. mansoni cercariae. There was a significant decrease in the survival rate in all weeks of infection compared to the control group (unexposed and uninfected snails), but the survival was lower in snails infected and exposed to the latex, where survival ranged from 40% to 0% in the first and eighth week of infection, respectively.
The above results show a clear reduction in the cercarial shedding per snail with longer time of infection. The mean cercarial shedding rates observed during 35 days demonstrated that exposure decreased the elimination of cercariae in all infected groups (
The present work demonstrated that contact with the LC50 of E. milli latex by eggs, miracidia and infected snails can change the parasite dynamics of S. mansoni. The results show that contact of S. mansoni eggs and miracidia with the sublethal concentration of E. milii negatively influenced the development of the parasite’slife cycle in the intermediate host, with consequent reduction of cercarial shedding.
De Jong-Brink et al. (1988) elucidated the neuroendocrine mechanism of sporocysts in the snail. The development of sporocystsis responsible for increasing the concentration of the neuropeptide schistosomin in the snail’s hemolymph, and the increase of this peptide is antagonistic to calfluxin, a peptide responsible for the
Groups | 3rd week of infection | 4th week of infection | 5th week of infection | 6th week of infection |
---|---|---|---|---|
1) Control | 1.59 ± 1.17a | 3.33 ± 0.21a | 3.37 ± 0.2a | 3.2 ± 0.09a |
2) Infection + latex | 1.74 ± 0.96a | 2.82 ± 0.38a,b | 3.27 ± 0.15a | 3.11 ± 0.5a |
3) Hatching + latex | 1.34 ± 0.97a | 1.87 ± 1.08c | 2.77 ± 0.3b | 2.86 ± 0.42a |
4) Hatching + infection + latex | 1.11 ± 0.95a | 1.98 ± 0.77b,c | 2.74 ± 0.51b | 3.06 ± 0.29a |
Eggs/snail | Egg masses/snail | Eggs/egg mass | ||
---|---|---|---|---|
Uninfected and unexposed | 27.6 ± 13.1a | 5 ± 2.3a | 7.8 ± 6.5a | |
Uninfected and exposed | 23.6 ± 14.5a | 4 ± 2.3a,c | 6.2 ± 1.8a | |
1st week of infection | Infected and unexposed | 6.3 ± 6.1a | 0.5 ± 0.4b | 19.9 ± 6.5a |
Infected and exposed | 2.8 ± 6.3b | 0.1 ± 0.3b | 10 ± 14a | |
2nd week of infection | Infected and unexposed | 6.6 ± 6.1a | 0.5 ± 0.4b | 11.1 ± 5.4a |
Infected and exposed | 1 ± 2.4b | 0.06 ± 0.1b | 3.6 ± 8a | |
3rd week of infection | Infected and unexposed | 7.5 ± 5.1a | 0.5 ± 0.4b | 14 ± 2.2a |
Infected and exposed | 0.92 ± 0.89b | 0.1 ± 0.1b | 6.5 ± 6.6a | |
4th week of infection | Infected and unexposed | 6.4 ± 3a | 0.5 ± 0.3b | 18.2 ± 13.2a |
Infected and exposed | 0.0 ± 0.0* | 0.0 ± 0.0* | 0.0 ± 0.0* | |
5th week of infection | Infected and unexposed | 12 ± 4a | 0.9 ± 0.2b | 13.2 ± 6.2a |
Infected and exposed | 0.0 ± 0.0* | 0.0 ± 0.0* | 0.0 ± 0.0* | |
6th week of infection | Infected and unexposed | 17.16a | 0.9 ± 0.2b | 21.8 ± 10.2a |
Infected and exposed | 13 ± 3a | 1 ± 0.1b | 13.6 ± 6.2a | |
7th week of infection | Infected and unexposed | 22.5 ± 23.7a | 1.7 ± 1.4b,c | 10.6 ± 3.6a |
Infected and exposed | 7.2 ± 4.3a | 1.2 ± 1.4b | 10.2 ± 6.8a | |
8th week of infection | Infected and unexposed | 28.8 ± 2a | 1.5 ± 0.7b | 20.8 ± 17.9a |
Infected and exposed | 0.0 ± 0.0* | 0.0 ± 0.0* | 0.0 ± 0.0* |
Control | Exposed | Reduction of cercariae shed | |||
---|---|---|---|---|---|
Number of cercariae | Number of snails at the end | Number of cercariae | Number of snails at the end | (%) | |
1st week of infection | 239,157 | 4 | 35,705 | 0 | 85.07 |
2nd week of infection | 12,410 | 5 | 11,430 | 4 | 7.89 |
3rd week of infection | 45,130 | 7 | 30,680 | 3 | 32.01 |
4th week of infection | 87,090 | 4 | 27,900 | 2 | 67.96 |
5th week of infection | 57,130 | 4 | 7,300 | 1 | 87.22 |
6th week of infection | 63,920 | 6 | 6,372 | 1 | 90.03 |
7th week of infection | 79,290 | 5 | 11,040 | 4 | 86.07 |
8th week of infection | 29,360 | 7 | 2,930 | 0 | 90.02 |
Total | 613.487 | 42 | 133.357 | 15 | 78.26 |
reproductive activity of gastropods [
The exposure of infected snails to the E. milii latex affected the reproductive biology in all intra-mollusk development stages of S. mansoni. The reduction in reproductive activity of snails infected with larval trematodes is due to metabolic changes in glucose and glycogen concentrations in the hemolymph, as described by Mello-Silva et al. (2010). During development of the parasite in the digestive gland (primary sporocyst → secondary sporocyst), glucose levels in the hemolymph decline, causing increased activity of glycogen at catalytic sites. Mello-Silva et al. (2010) compared the carbohydrate metabolism between snails infected or not by S. mansoni and exposed to this latex and observed that in infected snails exposed to the molluscicide, the reduction in glucose levels in the hemolymph occurs on the first day after exposure while the glycogen level in the digestive gland only declines after 1 week of exposure. We observed significant reductions in reproductive activity of snails exposed to LC50in the first week of infection (55.6%), second week (84.8%) and third week (87.7%), after which the reduction was 100%. The increase and development of sporocysts pose energy needs and the increased activity of carbohydrate catalytic sites affects other events that require the same energy reserves, such as the snail’s reproductive biology.
From an epidemiological standpoint, we observed snails infected and not infected with S. mansoni, but snails can be infected at different weeks of the parasite’s development inside the intermediate host. In this sense, analyzing the effects of exposure to phytochemical products on snail population is very important to better understand the transmission dynamics during control actions
Over the 35 days, a population of 80 infected snails produced more than 613 thousand cercariae and the mortality rate was approximately 58% (
We can conclude that the use of natural biodegradable compounds containing low concentrations of substances already characterized as having eco-toxicological potential can be an important tool to reduce the transmission of schistosomiasis. The LC50 of the E. milii latex had toxic action on eggs and miracidia, and the number of cercariae shed by infected snails was approximately 80% less during the study period. Considering the parameters analyzed (survival, reproductive activity and number of cercariae shed), the latex was more toxic to infected snails, regardless of the week of infection, corroborating the hypothesis of selective action of this product.
We are grateful to Dr. Patricia Pinto Machado from Laboratório de Esquistossomose Experimental (IOC/Fiocruz) for the technical and scientific support. This study was funded by the Fundação Oswaldo Cruz and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
The author(s) declare that they have no competing interests.
RCA: Responsible to conception, design, acquisition and analysis of data and the construction of the manuscript.
GF: Have made substantial contributions to acquisition of data.
MCV: Have made substantial contributions to interpretation of data.
MLAR: Have made substantial contributions to interpretation of data.
CCMello-Silva: Have made substantial contributions of design, interpretation and construction of the manuscript.