Molluscicidal activity of various solvent extracts from Solanum nigrum var. villosum L. aerial parts against Galba truncatula

Molluscicidal activity of Solanum nigrum var. villosum (morelle velue) extracts and their fractions were tested against the mollusca gastropoda Galba truncatula intermediate host of Fasciola hepatica. The results indicated that the hydro-methanol (MeOH-H2O) immature fruit extract possess the highest molluscicidal activity (LC50 = 3.96 mg/L) against Galba truncatula compared with other tested compounds. After acido-basic treatment, the methanolic extract fraction isolated from the immature fruits and the richest in alkaloids was the most toxic (LC50 = 1.65 mg/L). The fractions richest in saponosides obtained from the hydromethanolic and methanolic extracts of immature fruits showed interesting molluscicidal activities (LC50 = 6.15 mg/L and LC50 = 7.91 mg/L, respectively). The observed molluscicide activity could be attributed to the presence of alkaloids or saponosides. So, the immature fruits of Solanum nigrum var. villosum could be substrates of choice for molluscicide activity. In addition, total alkaloids and saponosides present in this plant deserve further investigations in order to identify the active principles and demonstrate their activities on mollusks in their natural habitat. According to the World Health Organization's guidelines on screening for plant molluscicides, use of these fractions may add to the arsenal of methods to control snail transmitting fasciolosis in tropical and Third World countries where fasciolosis is a common disease.     

Local adaptation of the trematode Fasciola hepatica to the snail Galba truncatula

Experimental infections of six riverbank populations of Galba truncatula with Fasciola hepatica were carried out to determine if the poor susceptibility of these populations to this digenean might be due to the scarcity or the absence of natural encounters between these snails and the parasite. The first three populations originated from banks frequented by cattle in the past (riverbank group) whereas the three others were living on islet banks without any known contact with local ruminants (islet group). After their exposure, all snails were placed in their natural habitats from the end of October up to their collection at the beginning of April. Compared to the riverbank group, snails, which died without cercarial shedding clearly predominated in the islet group, while the other infected snails were few in number. Most of these last snails released their cercariae during a single shedding wave. In islet snails dissected after their death, the redial and cercarial burdens were significantly lower than those noted in riverbank G. truncatula. Snails living on these islet banks are thus able to sustain larval development of F. hepatica. The modifications noted in the characteristics of snail infection suggest the existence of an incomplete adaptation between these G. truncatula and the parasite, probably due to the absence of natural contact between host and parasite.     

Fasciola hepatica: cercarial productivity of redial generations in long-surviving Galba truncatula

Bimiracidial infections of Galba truncatula with Fasciola hepatica were carried out to determine the effect of food quality on the frequency of 1- and 2-sporocyst infections, to analyse its impact on the developmental patterns (normal, or abnormal) of redial generations, and to verify its consequences on cercarial production. These investigations were performed in snails reared at 20 degrees C and provided with cos lettuce and commercial fish food (Tetraphyll) as a food source until their death. Double-sporocyst infections with normal development of redial generations were recorded in 43.9% of infected snails (out of 296). Single-sporocyst infections were noted in the other snails, with normal development of generations in 53.7% and abnormal development (the first mother redia early degenerated) in 2.4%. Four successive redial generations were found in long-surviving snails (more than 90 days). In both 1- and 2-sporocyst infections, showing normal development of generations, the daughter rediae, which exited from the first mother redia (R2a rediae), constituted the greater group of free rediae and produced the highest percentages of cercariae (46.2-48.2%). However, the development of these rediae inside the snail body was slower in 2-sporocyst infections than in 1-sporocyst infections. The numbers of rediae noted in subsequent generations (R2b/R3a and R3b/R4a rediae) were similar, whatever the number of full-grown sporocysts. The number of shed cercariae recorded in the 1- and 2-sporocyst infections did not significantly differ. When long-surviving snails died, 19.8-20.7% of cercariae produced by free rediae (essentially by R2b/R3a and R3b/R4a rediae) were still present in their bodies. The increased frequency of 2-sporocyst infections demonstrated that food quality had a significant effect on the redial burden of F. hepatica developing inside G. truncatula.     

Parasite development and visceral pathology in Galba truncatula co-infected with Fasciola hepatica and Paramphistomum daubneyi

Histological investigations in Galba truncatula naturally or experimentally co-infected with Fasciola hepatica and Paramphistomum daubneyi were carried out to study parasite development and the responses of the digestive gland and kidney of snails, as larval forms of these digeneans often use these two sites for their growth within the snail's body. The number of live rediae per snail ranged from 2.4 to 4.2 for the dominating parasite (it developed in the digestive gland) and was less than 2.0 for the other species. When the dominating species was F. hepatica, most snails harboured cercariae-containing rediae; if this parasite was P. daubneyi, procercariae-containing rediae with or without free procercariae were observed in most snails. In contrast, most rediae of the other species were immature. The pathology caused by the dominating species in the digestive gland was greater than that recorded in the kidney, where the other parasite was generally located. The most frequent tissue lesions in the digestive gland were generalized epithelial necrosis and epithelial reconstitution. In the kidney, multifocal epithelial necrosis was frequently observed, particularly when P. daubneyi was the dominating species. The frequencies of lesions in the digestive gland agreed with percentages reported by our team in other snails mono-infected with F. hepatica or P. daubneyi. In contrast, multifocal necrosis in the kidney was clearly greater in the present study and this finding might be explained by assuming that a sufficient number of free larvae within the snail would be necessary for the development of epithelial necrosis in the whole kidney.     

The experimental production of Fasciola hepatica metacercariae from three aquatic populations of Galba truncatula

Laboratory investigations on three aquatic populations of Galba truncatula, originating from the Peruvian Altiplano and French Massif Central, were carried out during three successive snail generations to determine if these populations might be successfully used for the metacercarial production of Fasciola hepatica under experimental conditions. High numbers of surviving snails at day 30 post-exposure (>70%), high prevalences of F. hepatica infections (>60%), and prolonged productions of cercariae for a mean period of 35 to 47 days were observed in the three populations, whatever the snail generation. In the Peruvian population, metacercariae of F. hepatica significantly decreased in numbers from a mean of 251 in the parent snails to 124 per snail in the F2 generation, whereas no significant variation was observed in the two French populations. As these aquatic snails rarely emerged out of water, the use of these populations for the commercial production of F. hepatica metacercariae was of great interest, because the daily time spent watching the breeding boxes of snails was clearly shorter, thereby reducing the cost of producing metacercariae compared with using amphibious snails reared with romaine lettuce.     

The activity of dispiro peroxides against Fasciola hepatica

Dispiro 1,2,4-trioxanes and 1,2,4,5-tetraoxanes had superior efficacy against Fasciola hepatica than the corresponding ozonides (1,2,4-trioxolanes). For highest efficacy, spiroadamantane and carboxymethyl substructures were required. Three compounds completely cured F. hepatica-infected mice at single oral doses of 50mg/kg and two were partially curative at single doses of 25mg/kg.     

Fasciola hepatica: the growth and larval productivity of redial generations in Galba truncatula subjected to miracidia differing in their mammalian origin

Experimental infections of Galba truncatula with 4 isolates of Fasciola hepatica miracidia differing by their mammalian origin (cattle, nutrias, rabbits, or sheep) were carried out to determine if parasite origin had an effect on the number of free rediae, their growth, and their larval productivity in each redia category. The mammalian origin of miracidia had a significant influence on the numbers of free rediae (they were higher in cattle-group snails) and the lengths of rediae (they were lower in rabbit groups). The redia category had also a significant effect on body and pharyngeal measurements. In all groups, the majority of cercariae (55.8-63.2%) were produced by the daughter rediae (R2a rediae) originating from the first mother redia. Compared with the other groups, the mean number of cercariae at day 49 postexposure was twice as high in cattle groups. In contrast, the mean number of daughter rediae produced by each second-appearing mother redia or each R2a redia was higher in the nutria, rabbit, and sheep groups. The mammalian origin of F. hepatica miracidia had an effect on the number of live rediae, their length, and their redial and cercarial productivity.     

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.     

The pattern of mortality in mice experimentally infected with Fasciola hepatica

The extent and pattern of mortality of mice exposed to low levels of F. hepatica were determined for a 56-day period. Mice were exposed to 1, 2, 3, 4 or 6 metacercariae per mouse with emphasis placed on one cyst infections. Eighty per cent of all deaths occurred between days 27 and 35 post exposure with the mean death day for all being from 31 to 34 days postexposure. The mortality followed a statistically predictable pattern with the use of one-cyst infections as an extrapolation point. As the dose increased, the percentage mortality increased as a simple multiple of probabilities indicating the absence of synergism or antagonism in producing death when more than one parasite was present. Relatively low levels of infection in mice reliably produced high mortality within a limited time period and, thus, mortality could be used as a parameter of infection for studies with a variety of aims.     

Larval development of Fasciola hepatica in experimental infections: variations with populations of Lymnaea truncatula

A retrospective study was undertaken on 70 French populations of Lymnaea truncatula experimentally infected with Fasciola hepatica to determine whether or not susceptibility of snails to infection influenced redial and cercarial production. Results were compared with those obtained from two control populations, known for prevalences higher than 60% when experimentally infected with F. hepatica. In the 70 other populations examined, the prevalences ranged from 2 to 75%. In 55 of these populations, where the prevalence was more than 20%, a high proportion (50.1-56.8%) of snails died after cercarial shedding, whereas in the other groups (non-shedding snails with the most differentiated larvae being free cercariae, rediae containing cercariae, immature rediae, or sporocysts, respectively), snail death was significantly less. In 11 populations, where the prevalence values were 5-19%, only 14% of snails died after cercarial shedding, whereas snails with free cercariae, rediae with cercariae, or immature rediae showed significant increases in snail mortality. In the remaining four snail populations, with prevalences of less than 5%, the most differentiated larval forms were only immature rediae and/or sporocysts. Overall, the number of rediae containing cercariae significantly decreased with decreasing prevalence values. The low prevalence of experimental infection in several populations of snails might be explained by the occurrence of natural infections with miracidia originating from a mammalian host other than cattle, and/or by genetic variability in the susceptibility of snails to infection.     

Allopatric combination of Fasciola hepatica and Lymnaea truncatula is more efficient than sympatric ones

Parasites are capable of rapid evolutionary changes relative to their hosts, due to short life cycle, short generation time, and high fecundity. The direction of the evolution of parasite virulence can be studied in cross-transfer experiments, combining hosts and parasites from different localities, and comparing the outcome of established (sympatric and potentially locally adapted) and novel (allopatric) combinations of hosts and parasites. We aimed to compare the compatibility with snails hosts, the infectivity of metacercariae in rabbits and rats, and the fitness among different combinations (French-FF and Spanish-SS sympatries and allopatry-FS). The first isolate of Fasciola hepatica and its corresponding intermediate host, Lymnaea truncatula originated from Lugo's northwestern Spain. The second isolate of parasite and snail was collected in the Limoges area in central France. The Spanish snails were more susceptible to their sympatric trematode than the French snails. The Spanish flukes were more infective to intermediate hosts (snails) than the French flukes, but subsequent definitive hosts (rats or rabbits) infections remained similar. The estimated fitness was low in sympatric infections and highly similar (from 4.7 to 5.3). The fitness similarity corresponds, however, to different variations in life-history traits that could represent different strategies among the host-parasite local combinations. The infection rate in snails, metacercarial productivity, metacercarial infectivity, and the estimated fitness were better for allopatric combination (FS). The susceptibility data showed a higher efficiency of flukes in the allopatric snail population than in their local snail population. However, our results were obtained after one generation and from a single isolate and it remains to be determined if all allopatric fluke-snail isolates may present a better fitness. Nevertheless our results indicate that introduction of liver fluke-infected cattle should be monitored carefully, as it could result in the introduction of more efficient parasites.     

Antibody profiles by EITB and ELISA of cattle and sheep infected with Fasciola hepatica

In evaluating potential mechanisms of immunity in fascioliasis we compared the time-course analysis of the antibody responses between a resistant (cattle) and a susceptible model (sheep). Sera from sheep and cows experimentally infected with F. hepatica were reacted with both somatic (FhWWE) and excretory-secretory (ES) antigens in order to evaluate the antibody repertoires in the 2 different hosts. Analysis of these sera by ELISA showed a significant increase in antibody levels by 2 wk in most infected cattle using both somatic and ES antigens, whereas with most infected sheep antibodies are not clearly detected until week 4. By EITB, both infected sheep and cows recognize major somatic polypeptides in a molecular weight range of 30-38 kDa by 8 wk. Cattle recognized 3 additional major antigens of 56, 64, and 69 kDa as early as 6 wk. Various polypeptides of 20-25 kDa are prominently detected by most sheep and very faintly, if at all, by the cow sera. The sera from both sheep and cows also identify ES polypeptides of 20-28 kDa. The patterns of polypeptides recognized by sheep infected with S. mansoni and challenged with F. hepatica in EITB are almost identical to those with a simple F. hepatica primary infection. No significant differences were detected in the antibody kinetics in ELISA between these 2 groups. Differences and similarities between these models could eventually help determine which antibodies may be predictive of resistance or susceptibility in fascioliasis.     

Molluscicidal activity of the Puerto Rican Weed,Solatium nodiflorum,against snail hosts ofFasciola hepatica

After screening 200 species of Puerto Rican plants, Solanum nodiflorumwas selected for comprehensive laboratory evaluation as a source of natural molluscicide. All parts of this plant were found toxic to the snails Fossaria (Lymnaea) cubensis, Lymnaea columella, Biomphalaria glabrata,and Physa cubensis.No toxicity was found against Marisa cornuarietisand Tarebia granifera. The toxicant was not affected by pH and dry storage. There was a direct correlation between temperature and molluscicidal activity. The plant is easy to find locally and could be developed into cheap, safe means for controlling both Schistosomaand Fasciolasnail hosts in Puerto Rico.     

Host plant defenses of black (Solanum nigrum L.) and red nightshade (Solanum villosum Mill.) against specialist Solanaceae herbivore Leptinotarsa decemlineata (Say)

Black nightshade (Solanum nigrum, S. nigrum L.) and red nightshade ( Solanum villosum, S. villosum Mill.) are medicinal plants from the Solanaceae family that synthesize glycoalkaloids and other secondary metabolites. To recognize the potential insecticide activity of these compounds, leaf extracts (containing glycoalkaloid and methanol fractions) were tested for enzyme inhibition, antifeedant activity and toxicity. For in‐vitro glutathione S‐transferase (GST) inhibition activity, we used insecticide‐resistant Colorado potato beetle, Leptinotarsa decemlineata ( L. decemlineata; Say) midgut and fat‐body homogenate. In‐vivo toxicity and the antifeedant activity were performed using larval bioassays. The methanol extracts had greater GST inhibitory activity compared to the glycoalkaloids, as well as greater 2nd instar larvae mortality and antifeedant activity. Furthermore, the green leaf volatile compound, cis‐hex‐3‐enyl acetate, at the concentration of 5 ppm, caused 50% mortality of 2nd instar larvae. Our findings suggest the potential usefulness of S. nigrum and S. villosum extracts to control L. decemlineata.     

The stress of Lymnaea truncatula just before miracidial exposure with Fasciola hepatica increased the prevalence of infection.

Single-miracidium infections of Lymnaea truncatula with Fasciola hepatica were carried out under laboratory conditions to determine whether the stress of snails just before miracidial exposure had any influence on the prevalence of Fasciola infection, redial burden, and cercarial shedding. Three methods, i.e., the fasting of L. truncatula for 3 days in water filtered through a Millipore membrane, the effect of 6-8 degrees C water for 15 min, or the immersion of L. truncatula in a detergent solution at low concentration for 15 min, were used to stress snails. Enhanced susceptibility of snails to F. hepatica infection was noted in stressed groups (93-96% vs 48-50% in controls). The number of free rediae did not show any variation in controls as well as in stressed groups, except for fasted snails in which free rediae were significantly fewer. No differences in cercarial production between controls and the cold group were noted. Fasting, cold shock, or detergent exposure prior to exposure to F. hepatica miracidia might have weakened the snails so that they were not as efficient in avoiding miracidial penetration, thus leading to higher infection rates.