Complex interactions among a nematode parasite (Daubaylia potomaca), a commensalistic annelid (Chaetogaster limnaei limnaei), and trematode parasites in a snail host (Helisoma anceps)

Many biotic interactions can affect the prevalence and intensity of parasite infections in aquatic snails. Historically, these studies have centered on interactions between trematode parasites or between trematodes and other organisms. The present investigation focuses on the nematode parasite Daubaylia potomaca and its interactions with a commensal, Chaetogaster limnaei limnaei , and a variety of trematode species. It was found that the presence of C. l. limnaei indirectly increased the mean intensity of D. potomaca infections, apparently by acting as a restraint for various trematode parasites, particularly the rediae of Echinostoma sp. In turn, Echinostoma sp. rediae adversely affected the mean intensity of D. potomaca by their consumption of both juvenile and adult nematodes present in tissues of the snail. These organisms not only belong to 3 different phyla but occupy distinct trophic levels as well. The complex interactions among these 3 organisms in the snail host provide an excellent example of biotic interactions influencing the infection dynamics of parasites in aquatic snails.     

The role of Chaetogaster limnaei in the dynamics of trematode transmission in natural populations of freshwater snails

Chaetogaster limnaei, an oligochaete was always found infesting Bulinus (Phy.) globosus, B.(B.) forskalii and Lymnaea natalensis in Bo, Sierra Leone, living below the shell or embedded in the mucus of the foot. The worms had no preference for snails with trematode infection. The population was highest in the dry season when fewer snails were infected by trematodes. This is suggested to be a result of the protection the worms give to snails against invading miracidia.     

Malacological assessment and natural infestation of Biomphalaria straminea (Dunker, 1848) by Schistosoma mansoni (Sambon, 1907) And Chaetogaster limnaei (K. Von Baer, 1827) in an urban eutrophic watershed.

The objective of this study was to perform a malacological assessment at the Ibirité reservoir watershed in the metropolitan region of Belo Horizonte (Minas Gerais) and to evaluate the natural infestation rate of Biomphalaria straminea (Gastropoda: Planorbidae) by Schistosoma mansoni (Platyhelminthes: Trematoda) and Chaetogaster limnaei (Oligochaeta: Naididae). The samples were collected from July to August 2002. The B. straminea individuals collected were kept in the laboratory; the natural infestation rate by S. mansoni and C. limnaei was assessed weekly. The malacological assessment identified five mollusk species present in the Ibirité reservoir watershed: B. straminea, Physa marmorata, Lymnea sp., Melanoides tuberculatus, and Pomacea austrum. Laboratory observations showed that the B. straminea individuals were infected by C. limnaei rather than S. mansoni. Although there was no infection of B. straminea by S. mansoni, presence of B. straminea in itself merits close attention due to possible risk of human schistosomiasis by the local population.     

The association of Zygocotyle lunata and Echinostoma trivolvis with Chaetogaster limnaei, an ectosymbiont of Helisoma trivolvis

Helisoma trivolvis snails collected from a lake in Warren County, New Jersey, in June 2007 possessed the ectosymbiont Chaetogaster limnaei (Annelida). Some of these snails were also infected with larval stages of Zygocotyle lunata and Echinostoma trivolvis. Chaetogaster limnaei associated with the infected snails fed on the cercariae of both digeneans. Zygocotylae lunata cercariae were observed in the stomach of C. limnaei and whole cercariae were loosely attached to the ventral surface of the chaetogasters. Cercariae in the stomachs were digested within 48 hr and probably served as a source of nutrient for the annelids. Whole cercariae and 1 nonviable metacercaria of E. trivolvis were seen in the stomachs of the chaetogasters. The protective action of the chaetogasters on the transmission of the cercariae of E. trivolvis and Z. lunata to second intermediate hosts in the wild awaits further study.     

Associations between two trematode parasites, an ectosymbiotic annelid, and Thiara (Tarebia) granifera (Gastropoda) in Jamaica

This work describes associations of Thiara (Tarebia) granifera, its larval trematode community, and Chaetogaster limnaei limnaei at a freshwater reservoir in Jamaica. Larvae of 2 trematodes were present, i.e., a notocotylid (15.3%) and Philophthalmus sp. (1.3%), in 3,575 T. granifera examined. The prevalence of both infections increased with snail shell length (H = 56, P < 0.01, H = 23.1, P < 0.01, respectively). Only 3.0% (n = 595) of infected snails possessed reproductive stages, compared with 90.3% (n = 2,980) of uninfected snails (χ(2) = 2,059.8, df = 1, P < 0.001); both trematodes negatively impacted snail reproduction. Chaetogaster l. limnaei occurred within the mantle cavity of T. granifera with a prevalence of 2.3% (n = 3,575); intensity ranged from 1 to 6 annelids. Notocotylid larvae occurred in 32.5% (n = 83) of snails also harboring C. l. limnaei, compared with 14.9% (n = 3,492) of snails lacking the annelid (χ(2) = 18.127; P < 0.001). Chaetogaster l. limnaei appears not to influence the recruitment of egg-transmitted, notocotylid infections to snails. Ingestion of emergent cercariae by the annelid was observed; this may impact transmission of the parasite. The article presents the first report of a notocotylid and C. l. limnaei in T. granifera, and of Philophthalmus sp. in Jamaica.     

Effects of Plagiorchis elegans (Digenea: Plagiorchiidae) infection of Biomphalaria glabrata (Pulmonata: Planorbidae) on a challenge infection with Schistosoma mansoni (Digenea: Schistosomatidae)

Prior exposure of Biomphalaria glabrata to the eggs of an incompatible digenean, Plagiorchis elegans, rendered this snail host less suitable to a compatible species, Schistosoma mansoni. Although P. elegans failed to develop patent infections in B. glabrata, it reduced the production of S. mansoni cercariae by 88%. Concomitantly, host attributes such as reproduction, growth, and survival were compromised. The effect of P. elegans infection was most severe among snails that, in addition, had developed patent schistosome infections. Although few S. mansoni cercariae were produced, egg production by B. glabrata was only 4% of control values. Furthermore, no doubly infected snails survived for more than 3 wk after patency, whereas controls experienced no mortality during the same time period. The above effects were attributable to the establishment and persistence of P. elegans sporocysts in the tissues of the incompatible snail host. Their indirect antagonistic interaction with thelarval stages of S. mansoni may be mediated, in part, through their long-term stimulation of the host's internal defense mechanisms. These findings are discussed with a view to use P. elegans and other plagiorchiid digeneans as agents in the biological control of snails and snail-borne diseases.     

Experimental double infection of Biomphalaria glabrata snails with Angiostrongylus cantonensis and Schistosoma mansoni

Two groups of Biomphalaria glabrata snails primarily infected with Angiostrongylus contonensis were secondarily exposed to infection with Schistosoma mansoni. To investigate any anatagonistic effect of the first infection on a superimposed one and to compare to singly and non-infected snails, a series of experiments was undertaken in which snails were individually exposed, variously, to 1,000 and 2,000 first-stage larvae of A. cantonensis and then to 5 and 10 miracidia of S. mansoni 1 day and 3 weeks later. Snails became infected with S. mansoni in both groups of snails with double infections and shed cercariae after the same incubation period as in the singly infected groups. The number of snails shedding cercariae simultaneously was similar in single and double infection groups during the first two weeks of shedding, after which this number decreased somewhat in doubly infected groups. Snails with double infection showed higher cumulative mortality rates than in snail groups with single infection with either A. cantonensis or S. mansoni. Therefore, initial infection of B. glabrata with A. cantonensis produced no inhibitory or retarding effect on subsequent infection of snails with S. mansoni.     

A somatically diversified defense factor, FREP3, is a determinant of snail resistance to schistosome infection

Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Fibrinogen-related protein 3 (FREP3) has been previously associated with snail defense against digenetic trematode infection. It is a member of a large family of immune molecules with a unique structure consisting of one or two immunoglobulin superfamily domains connected to a fibrinogen domain; to date fibrinogen containing proteins with this arrangement are found only in gastropod molluscs. Furthermore, specific gastropod FREPs have been shown to undergo somatic diversification. Here we demonstrate that siRNA mediated knockdown of FREP3 results in a phenotypic loss of resistance to Schistosoma mansoni infection in 15 of 70 (21.4%) snails of the resistant BS-90 strain of Biomphalaria glabrata. In contrast, none of the 64 control BS-90 snails receiving a GFP siRNA construct and then exposed to S. mansoni became infected. Furthermore, resistance to S. mansoni was overcome in 22 of 48 snails (46%) by pre-exposure to another digenetic trematode, Echinostoma paraensei. Loss of resistance in this case was shown by microarray analysis to be associated with strong down-regulation of FREP3, and other candidate immune molecules. Although many factors are certainly involved in snail defense from trematode infection, this study identifies for the first time the involvement of a specific snail gene, FREP3, in the phenotype of resistance to the medically important parasite, S. mansoni. The results have implications for revealing the underlying mechanisms involved in dictating the range of snail strains used by S. mansoni, and, more generally, for better understanding the phenomena of host specificity and host switching. It also highlights the role of a diversified invertebrate immune molecule in defense against a human pathogen. It suggests new lines of investigation for understanding how susceptibility of snails in areas endemic for S. mansoni could be manipulated and diminished.     

Echinostoma paraensei: hemocytes of Biomphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni

Earlier in vivo work by Lie et al. (1977) indicated that the innate resistance of the 10R2 strain of Biomphalaria glabrata to PR1 Schistosoma mansoni could be interfered with if the snails were infected previously with another trematode, Echinostoma paraensei. We have studied this interference phenomenon using in vitro methods in an attempt to understand its mechanistic basis. Hemolymph, derived from 10R2 snails infected with E. paraensei for 14-28 days, killed 25% of S. mansoni sporocysts in vitro, significantly less (P less than 0.001) than the 90% killing rate observed with hemolymph from uninfected, control 10R2 snails. Hemolymph from the infected 10R2 snails and from schistosome susceptible M line snails did not differ significantly (P greater than 0.1) in their relative inability to kill S. mansoni sporocysts in vitro. The defect in sporocyst killing exhibited by echinostome infected 10R2 snails was traced to the cellular, rather than the humoral, component of the hemolymph. Preparations containing uninfected 10R2 snail hemolymph and echinostome daughter rediae exhibited significantly less (P less than 0.001) killing of S. mansoni sporocysts than did controls containing only 10R2 hemolymph and S. mansoni sporocysts. Our results suggest that echinostome larvae release factors that interfere with the ability of B. glabrata hemocytes to kill S. mansoni sporocysts.     

Multiplex PCR for both identification of Brazilian Biomphalaria species (Gastropoda: Planorbidae) and diagnosis of infection by Schistosoma mansoni (Trematoda: Schistosomatidae)

A simple and single-step technique based on multiplex PCR (multiplex polymerase chain reaction) has been developed for simultaneous identification of Brazilian Biomphalaria species, the intermediate hosts of Schistosoma mansoni, and their diagnosis of infection by the trematode. We used species-specific primers directed both to the internal transcribed spacer 2 (ITS2) of ribosomal DNA from 3 of the S. mansoni host species and to the mitochondrial DNA (mtDNA) from the trematode. Those primers were used simultaneously in a single multiplex-PCR reaction, and template DNA was obtained from S. mansoni-infected and noninfected snails. The results were visualized in silver stained polyacrylamide gels, revealing the presence of specific bands. The methodology has shown to be efficient, fast, and reproducible for Biomphalaria species identification and diagnosis of snails infected by S. mansoni during prepatent periods.     

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.     

Interspecific antagonism and virulence in hosts exposed to two parasite species

Co-infection of host organisms by multiple parasite species has evolutionary consequences for all participants in the symbiosis. In this study, we co-exposed aquatic-snails (Biomphalaria glabrata) to two of their trematode parasites, Schistosoma mansoni and Echinostoma caproni. In co-exposed snails, E. caproni prevalence was 63% compared to only 23% for S. mansoni. Co-exposed E. caproni-infected snails exhibited reduced fecundity, higher mortality, and higher parasite reproduction (higher virulence) compared to hosts exposed to echinostomes alone. Conversely, co-exposed S. mansoni-infected snails released fewer parasites and produced greater numbers of eggs compared to hosts exposed to S. mansoni alone. These results suggest that co-exposure not only influences the establishment (presence or absence) of particular parasite species, but also impacts host life history, parasite reproduction, and the virulence of the interaction.     

Echinostoma paraensei and Schistosoma mansoni: adherence of unaltered or modified latex beads to hemocytes of the host snail Biomphalaria glabrata.

Hemocytes derived from a strain (13-16-R1) of Biomphalaria glabrata resistant to Schistosoma mansoni were significantly more likely to bind untreated latex beads than hemocytes from the schistosome-susceptible M line strain. Beads preincubated in 13-16-R1 plasma were more readily bound by both 13-16-R1 and M line hemocytes than beads preincubated in M line plasma. Beads preincubated in plasma derived from snails of either strain infected with the trematode Echinostoma paraensei were more readily bound by hemocytes than beads preincubated in plasma from control snails of the corresponding strain. Plasma from snails exposed to S. mansoni did not have a similar effect. Throughout these experiments, beads receiving a particular treatment were consistently bound at higher rates by 13-16-R1 than M line hemocytes. SDS-PAGE of plasma components eluted from beads revealed differences between treatments, particularly in diffuse bands falling into two groups, of 75-130 and 150-220 kDa. The results indicate that both hemocytes and plasma components from the two host strains differ and identify plasma molecules deserving of additional study as possible modulators of hemocyte effector functions. Also, S. mansoni and E. paraensei provoked different responses in the same host snail.     

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.     

Dynamics of the leukocytic response of Biomphalaria glabrata during the larval development of Schistosoma mansoni and Echinostoma liei

The daily evolution of the number of hemocytes in Biomphalaria glabrata was ascertained under three conditions: uninfected snails, snails infected with Schistosoma mansoni, and snails infected with Echinostoma liei. The Results show differences between the three experiments as well as in the average hemocyte density over the whole experimental period, as in the temporal dynamics of circulating hemocyte number. Specifically, it appears that the development of E. liei in B. glabrata induces a density of circulating hemocytes greater than that in uninfected B. glabrata or in snails infected with S. mansoni. The hemocyte dynamics observed in both experimental groups might best be interpreted by taking into account differences in the immunogenic stimulating capacity of the two trematodes and different physiological functions of the hemocytes brought into play during the infection: wound repair, nutrient digestion and transport, and excretion.     

Diagnostic of Biomphalaria snails and Schistosoma mansoni: DNA obtained from traces of shell organic materials

Freshwater snails belonging to the genus Biomphalaria act as intermediate hosts for the parasite trematode Schistosoma mansoni in Africa and in the neotropical region. Identification of such molluscs is carried out based on morphological characters and the presence of cercariae is verified through squeezing snails between two glass slides or by exposing them to artificial light. However, sometimes, the material collected includes molluscs with decomposed bodies or, yet, only empty shells, which precludes their identification and S. mansoni detection. Due to these difficulties, we have developed a methodology in which DNA may be extracted from traces of organic material from inside shells in order to identify molluscs through polymerase chain reaction and restriction fragment length polymorphism and to detect S. mansoni into these snails, by using low stringency polymerase chain reaction. Species-specific profiles obtained from B. glabrata, B. straminea, and B. tenagophila snails and their shells, maintained in laboratory for ten years, showed the same profiles. S. mansoni profiles showed to be present in shell specimens as far as the eighth week after being removed from aquarium.     

Host parasite relationship among 3 Schistosoma mansoni and 6 Biomphalaria glabrata strains from epidemic and no epidemic Venezuelan areas is studied

Six lots of 18 B. glabrata from: La Victoria, Turmero, Cagua in Aragua state; Caserío El 25 in Carabobo state, Chabasquén in Portuguesa state and Humocaro Bajo in Lara state, were experimentally infected with miracidia of SM, C5 and C6 strains of Schistosoma mansoni (18 snails/Schistosoma mansoni strain). The averages of the intramolluscal period (IMP) obtained for the S. mansoni strains were very similar and comprised between 35.4 and 36.1 days. No significative statistical differences in the IMP were found according to the S. mansoni strain and the size of snails: < 7 mm and > 7 mm. However, significative statistical differences in the IMP were found, in relation to the B. glabrata strain and between the snails classified in two groups according to the S. mansoni dose (5 miracidia/snail and 10 miracidia/snail). The higher percentages of infection (PI) were found for the following parasite-snail combinations: C6-Cas. El 25 (80.7%), SM-La Victoria (73.1%) and C5-Cagua (62%). No significative statistical differences were found for the PI a) between the snail classified in two groups according to the size (< 7 mm and > 7 mm), b) in relation to the miracidium dosification (5 and 10 miracidia/snail and c) in accord to the S. mansoni strain. However, significative statistical differences were found for the PI obtained with different strains of the snail.     

Respiratory burst of Biomphalaria glabrata hemocytes: Schistosoma mansoni-resistant snails produce more extracellular H2O2 than susceptible snails

The production of reactive oxygen species by hemocytes from the gastropod Biomphalaria glabrata has been linked to their ability to kill the trematode parasite Schistosoma mansoni. For 2 laboratory strains of the snail, 1 resistant (13-16-R1) and 1 susceptible (MO) to the PR1 strain of S. mansoni, we compared hemocyte production of extracellular hydrogen peroxide when stimulated with the protein kinase C agonist phorbol myristate acetate (PMA). The time course of the PMA-induced response is similar in both strains with respect to onset, peak production, and termination of the respiratory burst. However, the magnitude of the response differs between strains, in that hemocytes from resistant snails generate significantly more hydrogen peroxide. These findings suggest that the capacity to produce hydrogen peroxide could be critical in determining susceptibility or resistance to S. mansoni.     

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.     

Effects of aestivation and starvation on the neutral lipid and phospholipid content of Biomphalaria glabrata infected with Schistosoma mansoni

The effects of aestivation or starvation on the neutral lipid and phospholipid content of Biomphalaria glabrata patently infected with Schistosoma mansoni were determined by high-performance thin-layer chromatography-densitometry. Infected-aestivated snails were maintained in a moist chamber at 24 +/- 1 C and a relative humidity of 98 +/- 1%. Infected-starved snails were maintained in artificial spring water (ASW) at 23 +/- 1 C without exogenous food. Infected snails (the controls) were maintained in ASW at 23 +/- 1 C and fed lettuce ad libitum. The 3 groups were maintained in the laboratory for 7 days, and then the lipids from the digestive gland-gonad complex (DGG) were extracted and analyzed by class. Infected-aestivated snails exhibited greater mortality rate and weight loss after 7 days than did the infected-starved snails. The steryl ester concentration in the infected-starved snails was significantly increased (P = 0.010) compared with the controls but not compared with infected-aestivated snails; the concentration of phosphatidylcholine in infected-aestivated snails was significantly decreased (P = 0.007) compared with the controls but not when compared with the infected-starved snails. Aestivation or starvation had a significant effect on the concentration of certain lipid classes in the DGG of B. glabrata infected with S. mansoni.