Effects of diet on the lipid composition of the digestive gland-gonad complex of Biomphalaria Glabrata (Gastropoda) infected with larval Echinostoma Caproni (Trematoda)

This study examined the effects of a larval Echinostoma caproni infection on the neutral lipid composition of the digestive gland-gonad complex (DGG) of Biomphalaria glabrata snails fed hen's egg yolk supplemented with lettuce (Y-L) or lettuce supplemented with Tetramin (L-T). Snails were experimentally infected with the miracidial stage of this echinostome, and their DGGs containing daughter rediae were analyzed for neutral lipids five weeks post-infection by qualitative and quantitative thin-layer chromatography. Light microscopy using Oil Red O (ORO) staining and transmission electron microscopy (TEM) were used to localize neutral lipids in the rediae. The DGGs of infected snails maintained on the Y-L diet showed a significant increase in free sterols and a significant decrease in triacylglycerols compared to uninfected snails maintained on the Y-L diet. The DGGs of infected snails maintained on the L-T diet showed no significant difference in free sterols or triacylglycerols compared to uninfected snails maintained on the L-T diet. ORO staining and TEM showed the presence of lipid droplets in rediae from snails on the Y-L diet. The significant decrease in triacylglycerols in the DGGs of infected snails maintained on the Y-L diet suggests that triacylglycerols were utilized by the rediae.     

Effects of diet on the development of Schistosoma mansoni in Biomphalaria glabrata and on the neutral lipid content of the digestive gland-gonad complex of the snail

In a previous study, when the snail Biomphalaria glabrata was infected with Schistosoma mansoni and maintained on a diet of hen's egg yolk, it produced fully developed cercariae in about one-half the time taken by snails fed Romaine lettuce. Increased lipids were also noted in the snails fed the yolk diet. The purpose of the present study was to further investigate nutritional effects of a high-lipid diet on larval schistosome development and to reexamine the time to cercarial patency in infected snails maintained on either the yolk or lettuce diet and to use high-performance thin-layer chromatography (HPTLC) to analyze the neutral lipids in the digestive gland-gonad complex (DGG) of snails maintained on both diets. Infected snails maintained at 26 C and fed either diet produced fully developed cercariae by 4 wk postinfection (PI). Likewise, infected snails maintained at 23 C and fed either diet produced fully developed cercariae by 6 wk PI. The contention that the yolk diet enhanced the time to cercarial patency was not confirmed. The HPTLC analysis of neutral lipids showed that the DGG of infected snails fed the yolk diet contained significantly greater amounts of free sterols and cholesteryl esters but not triacylglycerols than that of the infected snails fed the lettuce diet.     

Schistosoma mansoni: Lysozyme activity in Biomphalaria glabrata during infection with two strains

Levels of lysozyme activity were determined in the hemolymph, digestive gland, and headfoot extracts of M-line stock of snails, Biomphalaria glabrata, during infection with the PR-1 and Lc-1 strains of the trematode, Schistosoma mansoni. At 3 hr postexposure there was a 10-fold increase in the levels of enzyme activity in the hemolymph of snails infected with the Lc-1 strain to which the snail is resistant. This increase was considerably higher when compared to the threefold increase in the PR-1-infected snails. The infection also induced a gradual depletion of lysozyme activity in the headfoot muscles of the two groups of infected snails. There were no changes in the levels of enzyme activity in the digestive gland extracts of the control and the two groups of infected snails. Similar changes in the levels of enzyme activity in the hemolymph and headfoot extracts of infected snails suggest a nonspecific response to a parasite infection and do not indicate that lysozyme is primarily responsible for the destruction of schistosome parasite in a resistant snail host.     

Lipid levels in Biomphalaria glabrata infected with different doses of Echinostoma paraensei miracidia

The effect of experimental exposure of Biomphalaria glabrata to different doses (5 and 50) of Echinostoma paraensei miracidia on the total levels of cholesterol and triglycerides circulating in the hemolymph and the neutral lipids in the digestive gland–gonad (DGG) complex were studied. The snails were dissected one, two, three and four weeks after infection to collect the hemolymph and DGG tissue, to measure the levels of cholesterol and triglycerides in the hemolymph and neutral lipids in the tissue. The results for the hemolymph showed a similar order of variation for both substrates tested in the first week after infection. The reduced levels of these lipids in the infected snails indicate intense use of these substrates both by the intermediate host and the parasite, suggesting its probable participation in the energy metabolism and structural construction of the developing larval stages. Alterations in the profile of neutral lipids in the DGG were also found. The results obtained indicate that in this model, the lipid metabolism depends on the miracidial dose used.     

Biomphalaria glabrata amoebocytes: Effect of Schistosoma mansoni infection on in vitro phagocytosis

The rate of phagocytosis by amoebocytes obtained from hemolymph of the pulmonate Biomphalaria glabrata infected with the trematode Schistosoma mansoni for 24 hr and 2, 4, and 6 weeks has been determined using the monolayer assay system. Amoebocyte preparations from snails infected for 4 and 6 weeks showed a gradual decrease in the phagocytic rates compared to those from uninfected controls. Snails harboring the parasite for 4 and 6 weeks also showed a significant increase in the number of amoebocytes in the hemolymph. No significant changes were detected in the rate of phagocytosis or number of amoebocytes in snails infected for 2 weeks or less. Alterations in the morphology and behavior of amoebocytes from infected snails were also noted.     

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).     

Distribution and variation of hemagglutinating activity in the hemolymph of Biomphalaria glabrata

A sensitive hemagglutination assay utilizing glutaraldehyde-fixed trypsinized calf erythrocytes (GTC) is described to test for agglutinin levels in hemolymph and albumen gland extracts from nine populations of Biomphalaria glabrata, and from B. straminea and B. obstructa. High levels of GTC-reactive hemagglutinin were found in all snail populations. There was no correlation between hemagglutinin titer and innate resistance of B. glabrata strains to Schistosoma mansoni. However, an increase in hemagglutinin titer occurs in B. glabrata M-RLc snails infected with Echinostoma lindoense and in snails sensitized and reexposed to this parasite.     

Characterization of the bacterial microbiota of Biomphalaria glabrata (Say, 1818) (Mollusca: Gastropoda) from Brazil

Roughly 200 000 000 people in 74 countries infected with schistosomes all share the fact that they came in contact freshwater harbouring infected snails. The aim of the study is to characterize the microbiota of wild and laboratory‐reared snails of Biomphalaria glabrata from Pernambuco, Brazil. The microbiota of these molluscs was identified biochemically by the VITEK 2 automated microbiological system. Antimicrobial susceptibility testing was carried out by the disc diffusion method with ß‐lactam antibiotics, aminoglycosides, quinolones, folate pathway inhibitors, fenicols and tetracyclines. The results showed that all bacteria identified were gram‐negative, including 11 bacterial genera: Aeromonas, Citrobacter, Enterobacter, Cupriavidus, Rhizobium, Stenotrophomonas, Pseudomonas, Klebsiella, Acinetobacter, Vibrio and Sphingomonas. Regarding the antimicrobial susceptibility, all the isolates exhibited resistance to amoxicillin and sensitivity to meropenem (beta‐lactam antimicrobials). The microbiota of the wild snails consisted predominantly of Enterobacter cloacae, while the laboratory‐reared snails predominantly showed Citrobacter freundii and Aeromonas sobria.

Significance and Impact of the Study

Biomphalaria glabrata is a Brazilian freshwater Planorbidae of great medical relevance as an intermediate host of Schistosoma mansoni. About a month after being infected by one or more miracidia larvae of a compatible schistosome, B. glabrata sheds thousands of cercariae into the water where they seek human skin and, if successful, penetrate to establish infection, eventually taking up residence and maturing in blood vessels of the small intestine. Results obtained from this study aim at targeting novel biological control strategies for schistosomiasis such as paratransgenesis. This is the first study on the microbiota of B. glabrata from Brazil.     

Schistosoma mansoni does not and cannot oxidise fatty acids,but these are used for biosynthetic purposes instead

Adult schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, have always been considered to be homolactic fermenters and, in their energy metabolism, strictly dependent on carbohydrates. However, more recent studies suggested that fatty acid β-oxidation is essential for egg production by adult female Schistosoma mansoni. To address this conundrum, we performed a comprehensive study on the lipid metabolism of S. mansoni. Incubations with [¹⁴C]-labelled fatty acids demonstrated that adults, eggs and miracidia of S. mansoni did not oxidise fatty acids, as no ¹⁴CO₂ production could be detected. We then re-examined the S. mansoni genome using the genes known to be involved in fatty acid oxidation in six eukaryotic model reference species. This showed that the earlier automatically annotated genes for fatty acid oxidation were in fact incorrectly annotated. In a further analysis we could not detect any genes encoding β-oxidation enzymes, which demonstrates that S. mansoni cannot use this pathway in any of its lifecycle stages. The same was true for Schistosoma japonicum and all other schistosome species that have been sequenced. Absence of β-oxidation, however, does not imply that fatty acids from the host are not metabolised by schistosomes. Adult schistosomes can use and modify fatty acids from their host for biosynthetic purposes and incorporate those in phospholipids and neutral lipids. Female worms deposit large amounts of these lipids in the eggs they produce, which explains why interference with the lipid metabolism in females will disturb egg formation, even though fatty acid β-oxidation does not occur in schistosomes. Our analyses of S. mansoni further revealed that during the development and maturation of the miracidium inside the egg, changes in lipid composition occur which indicate that fatty acids deposited in the egg by the female worm are used for phospholipid biosynthesis required for membrane formation in the developing miracidium.     

Effects of diet on the carotenoid pigment and lipid content of Pomacea bridgesii as determined by quantitative high performance thin layer chromatography

High performance thin layer chromatography was used to quantify the concentrations of beta-carotene and lutein in the whole bodies and digestive gland-gonad complexes of Pomacea bridgesii fed on a lettuce diet and a hen's egg yolk diet, and snails starved for one week. beta-carotene and lutein concentrations were determined in the fecal matter of cultures on the lettuce and yolk diets as well. Significantly higher amounts of beta-carotene were found in the whole bodies of snails fed lettuce compared with those starved for a week. beta-carotene was not detected in the whole bodies and digestive gland-gonad complexes of snails fed hen's egg yolk. Neutral and polar lipid levels were also quantified in P. bridgesii fed lettuce and yolk. Significantly higher amounts of triacylglycerols were detected in the digestive gland-gonad complexes of snails maintained on the egg yolk diet.     

The hemolymph of Biomphalaria snail vectors of schistosomiasis supports a diverse microbiome

The microbiome - the microorganism community that is found on or within an organism's body - is increasingly recognized to shape many aspects of its host biology and is a key determinant of health and disease. Microbiomes modulate the capacity of insect disease vectors (mosquitoes, tsetse flies, sandflies) to transmit parasites and disease. We investigate the diversity and abundance of microorganisms within the hemolymph (i.e. blood) of Biomphalaria snails, the intermediate host for Schistosoma mansoni, using Illumina MiSeq sequencing of the bacterial 16S V4 rDNA. We sampled hemolymph from five snails from six different laboratory populations of B. glabrata and one population of B. alexandrina. We observed 279.84 ± 0.79 amplicon sequence variants per snail. There were significant differences in microbiome composition at the level of individual snails, snail populations and species. Snail microbiomes were dominated by Proteobacteria and Bacteroidetes while water microbiomes from snail tank were dominated by Actinobacteria. We investigated the absolute bacterial load using qPCR: hemolymph samples contained 2784 ± 339 bacteria/μl. We speculate that the microbiome may represent a critical, but unexplored intermediary in the snail–schistosome interaction as hemolymph is in very close contact with the parasite at each step of its development.     

Purification and binding properties of hemagglutinin from Biomphalaria glabrata.

A hemagglutinin has been purified from Biomphalaria glabrata (PR-B) hemolymph, albumin glands, and egg masses using affinity chromatography with Sephadex gels. The purified material from any of the sources above demonstrated identical immunological properties during immunoelectrophoresis or immunodiffusion, and similar serological specificity for human A₁ erythrocytes and to a lesser extent A₂ erythrocytes. Hemagglutinin was able to bind in vitro to the tegumental surface of cultured Schistosoma mansoni sporocysts, cercariae, and miracidia. Sporocysts dissected from infected snails and shed cercariae were already found to have hemagglutinin on their tegumental surface as demonstrated by immunofluorescence. It is postulated that hemagglutinin binding to the surface of larval helminths may “mask” them from being recognized by the snail host's cellular defense system.     

Chronobiological aspects of the host-parasite relationships between Biomphalaria glabrata and Schistosoma mansoni: cercarial production and infectivity, and growth kinetics of the host

In the Biomphalaria glabrata-Schistosoma mansoni association, variations in cercarial production, in cercarial infectivity, and in the growth of infected snails are rhythmic. These chronobiological aspects are correlated with the dynamics of the intramolluscan larval stages of the parasite during the course of the infection. Rhythmic variations in the growth kinetics of infected snails are interpreted in terms of host-parasite metabolic exchanges.     

Studies on resistance in snails: Interference by nonirradiated echinostome larvae with natural resistance to Schistosoma mansoni in Biomphalaria glabrata

Most of the genetically selected juvenile Biomphalaria glabrata snails, normally strongly resistant to Schistosoma mansoni, lost their juvenile resistance to this parasite when other trematodes were concurrently present in the snail. Three echinostome species all were able to reduce this genetically controlled juvenile resistance: Echinostoma lindoense, E. paraensei, and e. liei. Subsequently, adult resistance to S. mansoni, clearly present in control snails of the same age and strain that were not doubly infected, failed to develop in most of the snails that also harbored echinostomes. Other snails, selected for resistance as adults to S. mansoni, also usually became susceptible to this parasite following infection with E. paraensei. The capacity of E. paraensei to interfere with the snails' resistance to S. mansoni was greater than that of E. lindoense. Destruction by predation of primary sporocysts of S. mansoni by echinostome rediae prevented completion of development of the S. mansoni infections. In a number of snails all primary S. mansoni sporocysts were consumed before secondary sporocysts could be formed. In most experimental snails, however, some of the schistosomes survived, often as a small number of degenerated secondary S. mansoni sporocysts. The capability of flukes to interfere with the natural defense of snails may be an important phenomenon whereby trematode species survive in their snail hosts.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.     

Alterations in total serum proteins and protein fractions in Biomphalaria glabrata parasitized by Schistosoma mansoni

Samples of serum from healthy Biomphalaria glabrata and from those infected with the larval stages of Schistosoma mansoni were subjected to quantitative electrophoretic analysis and total protein determinations. In the case of the infected snails, samples of serum were taken at 14 time intervals post-exposure to 5 miracidia ranging from 1 hr to 70 days.A total of 34 serum protein fractions have been identified in the hemolymph of uninfacted B. glabrata, although all these fractions usually do not occur in every snail. In infected snails, there is a gradual reduction in the staining intensity of the serum protein fractions until day 35 post-exposure to miracidia, and this is correlated with a reduction in the total serum protein concentration. By day 70 post-exposure to the parasite, the total protein concentration had declined to one-third of that in uninfected snails.Although the decrease in the amount of serum protein fractions as a result of parasitization by S. mansoni is generally nonselective; i.e., almost all the fractions are reduced, three fractions, 7, 9, and 10, do not appear to be reduced in quantity. These remain essentially unaltered in their intensity to staining for up to 70 days, the duration of this experiment.It is postulated that the reduction of serum protein is due to utilization by sporocysts of S. mansoni.     

Host reproductive fitness: the influence of increasing parasite pressure in a Biomphalaria glabrata/Schistosoma mansoni system

Summary

Host life history traits are often shaped by trade-offs between the current and potential future costs of parasitism. Reproductive tissues are not normally essential for host survival and diversion of resources elsewhere is a common effect of parasitic infection. Variations in reproductive output may therefore indicate overall fitness correlated to the host response to parasite pressure. Here, we investigated reproductive fitness in a Biomphalaria glabrata—Schistosoma mansoni system, a laboratory model for schistosomiasis. Five matched groups of unselected B. glabrata snails were individually exposed to doses of 1, 2, 5, 10 or 20 S. mansoni miracidia, respectively. A sixth group remained unexposed providing a control. Fertility (defined as actual reproductive performance, measured as the number of offspring produced) and fecundity (defined as potential reproductive capacity, measured as number of eggs and embryos formed) were monitored for each group at weekly intervals. Our results revealed that both parasite dose and infection status had a significant effect on the potential reproductive capacity of the host, but this was not always reflected in the actual reproductive success. Egg mass production showed a negative association with increasing parasite dose in patently infected snails. In contrast, snails exposed to the parasite, but within which infection did not establish, demonstrated a positive association between egg mass production and parasite dose. This suggests the existence of a fecundity compensation mechanism occurring within the post-patent period of infection. This is, to our knowledge, the first report of such an effect in a snail-trematode system and, indeed, in any host-parasite association.     

Ribeiroia marini: pathogenesis and larval trematode antagonism in the snail, Biomphalaria glabrata

Larval trematode antagonism between Ribeiroia marini and Schistosoma mansoni was studied in the snail Biomphalaria glabrata. A laboratory-raised Puerto Rican strain of B. glabrata was exposed to single and double infections with given numbers of: (1) embryonated eggs of R. marini from laboratory rats, and (2) miracidia of S. mansoni from mice. Snails were maintained in outside environmental tanks in San Juan, Puerto Rico and larval trematode interactions were examined in a series of five experiments. Snails of all sizes were highly susceptible to single infections with R. marini. Rediae and cercariae caused extensive damage to the digestive gland and ovotestis resulting in premature death of snails. Heavily infected snails were castrated and stopped laying eggs. Snails infected first with S. mansoni were only partly susceptible to superinfection with R. marini given on Day 23. In a reverse experiment, snails infected first with R. marini were only partly susceptible to a second infection with S. mansoni given on Day 23. In simultaneous exposures, snails developed double infections (22%) with R. marini dominant and S. mansoni sporocyst and cercaria production reduced. While R. marini is not a strong direct antagonist against established S. mansoni infections, it has several attributes as a possible biological control agent: hardy eggs easily produced in rats; high infectivity to snails of all ages; and ability to castrate and prematurely kill B. glabrata. The R. marini-rat system described here provides a convenient laboratory and field model for the study of intrasnail trematode antagonism and biological control.     

Biomphalaria species in Alexandria water channels

Of the several species of Biomphalaria snails worldwide that serve as the intermediate host for Schistosoma mansoni, Biomphalaria alexandrina is a species that is indigenous to Egypt. Recently, there has been much debate concerning the presence of Biomphalaria glabrata and the hybrid of the species with Biomphalaria alexandrina. Due to this debate, the absence of a clear explanation for the presence of B. glabrata in Egyptian water channels and the probability that they may be reintroduced, we conducted this field study to identify Biomphalaria species present in Alexandria water channels. Laboratory-adapted susceptible snails to Schistosoma mansoni of the following species were used as a reference; Biomphalaria alexandrina, Biomphalaria glabrata and their hybrid. These snails were used to perpetuate the Schistosoma life cycle at the Theodor Bilharz Research Institute (TBRI), Cairo, Egypt. Morphological and molecular studies were conducted on these reference snails as well as on the first generation of Biomphalaria snails from two areas in the Alexandria governorate. The morphological study included both external shell morphology and internal anatomy of the renal ridge. The molecular study used a species-specific PCR technique.The results demonstrated that there was an absence of Biomphalaria glabrata and the hybrid from Alexandria water channels. Moreover, the susceptibility patterns of these reference snails were studied by measuring the different parasitological parameters. It was found that Biomphalaria glabrata and the hybrid were significantly more susceptible than Biomphalaria alexandrina to the Egyptian strain of Schistosoma mansoni. The results demonstrated that if Biomphalaria glabrata was reintroduced and adapted to the local environment in Egypt, it would have important epidemiologic impacts that would have a serious effect on the health of Egyptian people.