Abstract:

Investigations of the early intramolluscan development of Echinostoma paraensei in juvenile M-line snails, Biomphalaria glabrata, have revealed the presence of a previously uncharacterized stage, a morphologically and behaviorally specialized mother redia, here termed the precocious mother redia (PMR). The PMR, which is produced in a sporocyst attached to the inner surface of the host's ventricle, develops before other mother rediae and is released as early as 6 days post-exposure (DPE). Unlike all other mother rediae later produced by the sporocyst, it attaches to the ventricle wall adjacent to the sporocyst and remains there for at least 31 days. It develops a long, sinuous body and a pharynx approximately twice as large as that of any other mother or daughter redia present. Developing rapidly, the PMR releases daughter rediae at 10 DPE, about 2 days before other mother rediae. When snails containing a single intraventricular sporocyst were challenged by exposure to additional miracidia, fewer challenge sporocysts were observed subsequently in the ventricle than in control snails; this effect was greater if the PMR of the original infection had emerged and was present in the ventricle. The proportion of challenge sporocysts that reached the ventricle but thensubsequently disappeared or failed to develop also increased significantly in snails in which a PMR was present. These results suggest that the PMR is specialized to protect the sporocyst and that it is capable of adversely affecting the development of challenge sporocysts. This mode of development was not observed in two other echinostome species (Echinostoma liei and E. trivolvis) examined.

Abstract:

Adults of the African ampullariid snail Pila ovata were examined for their ability to control laboratory populations of the pulmonate snail Biomphalaria pfeifferi, a widespread, intermediate host of the human pathogen Schistosoma mansoni in sub-Saharan Africa. In a 6-week experiment conducted in large (100 x 60 x 60 cm) outdoor tanks containing floating macrophytes (Nymphaea caerula) and initially set up with one adult ampullariid for every three adult pulmonates, the numbers of B. Pfeifferi egg masses were always about half those in similar tanks without P. ovata. Although, by week 6, the numbers of B. pfeifferi in the control tanks (without ampullariids) had increased 5-fold, from an initial mean of 30 snails/tank, there was no significant increase in the numbers of B. pfeifferi in the experimental tanks (containing ampullariids). Results of experiments conducted in indoor glass aquaria indicated that adult P. ovata rapidly attacked egg masses or neonates (< 2.5 mm shell diameter) of B. pfeifferi but had no effect on the adults. The adult ampullariids also significantly decreased cover by floating macrophytes over a 6-week period compared with that in similar but ampullariid-free aquaria. This decrease in plant cover is relevant to biological control of the schistosome vectors as macrophytes serve as food, shelter and oviposition sites for pulmonate snails. The present results indicate the ability of P. ovata to inhibit multiplication of B. pfeifferi populations, at least under laboratory conditions, both directly, through predation, and indirectly, by competition for resources. Pila ovata may therefore prove useful in the biological control of medically important, pulmonate snails.

Abstract:

Susceptibility of the snail Biomphalaria glabrata to infection with the digenetic trematode Echinostoma paraensei was correlated with the ability of secretory-excretory products (SEP) derived from sporocysts of this parasite to interfere with the spreading behavior of host hemocytes in an in vitro assay. In 15 separate experiments, the prevalence of infection achieved by miracidia was positively correlated (C.C. = 0.779, P < 0.001) with the ability of in vitro-transformed sporocysts derived from the same batches of miracidia to produce SEP that inhibited host hemocyte spreading. Under assay conditions reflecting differences in hemolymph volumes between juvenile snails (susceptible to infection) and adult snails (relatively refractory to infection), SEP had a significantly greater effect on hemocytes from juvenile snails. No significant differences in response to SEP were noted when equivalent numbers of adult and juvenile hemocytes were used in the assay. Snails of the RI line strain of B. glabrata are significantly more susceptible to infection with E. paraensei than 13-16-R1 strain snails. Exposure to SEP significantly increased the number of unspread hemocytes for both strains. However, significantly more 13-16-R1 than M line hemocytes remained spread following SEP treatment. Echinostoma paraensei sporocyst SEP effects on host hemocyte spreading mirror observed patterns in both age-and strain-related susceptibility of B. glabrata to this parasite. The results suggest that the number of hemocytes available to a particular snail influences its vulnerability to infection with E. paraensei. (C) 1998 Academic Press.

Abstract:

After infection with the digenetic trematode Echinostoma paraensei, hemolymph of the snail Biomphalaria. glabrata contains lectins comprised of 65-kDa subunits that precipitate polypeptides secreted by E. paraensei intramolluscan larvae, Comparable activity is lacking in hemolymph of uninfected snails. Three different cDNAs with sequence similarities to peptides derived from the 65-kDa lectins were obtained and unexpectedly found to encode fibrinogen-related proteins (FREPs). These FREPs also contained regions with sequence similarity to Ig superfamily members, B. glabrata has at least five FREP genes, three of which are expressed at increased levels after infection, Elucidation of components of the defense system of B. glabrata is relevant because this snail is an intermediate host for Schistosoma mansoni, the most widely distributed causative agent of human schistosomiasis. These results are novel in suggesting a role for invertebrate FREPs in recognition of parasite-derived molecules and also provide a model for investigating the diversity of molecules functioning in nonself-recognition in an invertebrate.

Abstract:

A nested polymerase chain reaction (PCR) protocol was developed for detecting the presence of Schistosoma mansoni sporocysts in intermediate host snails of the genus Biomphalaria. To accomplish this, rDNA genes encoding the 18S rRNA of S. mansoni and Biomphalaria alexandrina from Egypt were sequenced, as were 18S-encoding genes of the 13-16-R1 and Salvador strains of Biomphalaria glabrata. Based on a comparison of host and parasite sequences, a nested set of PCR primers was designed to allow specific amplification of portions of S. mansoni 18S rDNA. These primers allowed detection of as little as 10 fg of S. mansoni DNA diluted in 100 ng of snail DNA and did not allow amplification of snail 18S sequences. Using nested PCR, the presence of a single S. mansoni sporocyst within an adult snail could be detected at 1 day postexposure. In DNA samples extracted from each of 74 snails of the M-line strain of B. glabrata exposed to from 1 to 10 S. mansoni miracidia for intervals ranging from 1 to 44 days, use of the outside primer pair alone detected the parasite's presence in 51% of the snails, whereas the sequential use of outside and nested primer pairs detected parasites in 92% of the snails. This approach has utility in determining if snails in endemic areas bear prepatent or inactive infections and in assessing the degree of compatibility between local snail and schistosome populations. It will also facilitate studies of resistance of snails to infection.

Abstract:

The Orthonectida is a small, poorly known phylum of parasites of marine invertebrates. Their phylogenetic placement is obscure; they have been considered to be multicellular protozoans, primitive animals at a ''mesozoan'' grade of organization, or secondarily simplified flatworm-like organisms. The best known species in the phylum, Rhopalura ophiocomae, was collected on San Juan Island, Wash. and a complete 18S rDNA sequence was obtained. Using the models of minimum evolution and parsimony, phylogenetic analyses were undertaken and the results lend support to the following hypotheses about orthonectids: (1) orthonectids are more closely aligned with triploblastic metazoan taxa than with the protist or diploblastic metazoan taxa considered in this analysis; (2) orthonectids are not derived members of the phylum Platyhelminthes; and (3) orthonectids and rhombozoans are not each other's closest relatives, thus casting further doubt on the validity of the phylum Mesozoa previously used to encompass both groups.

Abstract:

Freshwater snails in the genus Biomphalaria transmit Schistosoma mansoni in Africa, South America and the Caribbean region. Although considerable attention has been given to the identification of species, little is known of evolutionary relationships among the species. A phylogenetic analysis of 25 populations representing 11 species was performed on 25 enzyme loci examined using starch gel electrophoresis. A phylogenetic analysis of the individual populations produced 60 trees of equal length. The 60 trees have a consistency index value of 75.9% and a retention index value of 76.5%. The phylogenetic analysis provided strong support for the monophyly of Biomphalaria with either 14 or 15 synapomorphies uniting all of the species included and separating them from the outgroup, two species of Helisoma. Four nominal species represented by multiple populations formed monophyletic groups. Populations of B. sudanica, B. choanomphala, and B. alexandrina were interspersed. Ten arrangements were obtained for the populations of these three species. A variety of ingroup taxa were used to root the trees, and all provided support for the use of Helisoma species as an outgroup. In all of the trees obtained, the African species together formed a monophyletic group. In none of the trees obtained did the neotropical species form a monophyletic group. A constrained analysis requiring the monophyly of the neotropical species as well as the African species resulted in 90 trees just two steps longer than the shortest trees. Analysis of the species from either hemisphere alone resulted in decreased resolution, as measured by an increase in the number of trees obtained. This finding suggests that further comparisons of species from the two hemispheres will be of considerable value. Finally, two species which are resistant to infection with S. mansoni were included among the eleven studied. Neither of these species formed the sister group to all of the other species included, indicating that susceptibility is the plesiomorphic state, and that resistance is derived. Similarly, in none of the trees obtained did the two resistant species fall out as sister taxa, indicating that resistance arose independently twice.

Abstract:

Freshwater snails in the genus Biomphalaria transmit Schistosoma mansoni, a causative agent of human schistosomiasis in Africa and the Neotropical region. Twenty-five populations representing 11 species of Biomphalaria from both Africa and the neotropical region were examined using starch gel electrophoresis for 22 loci. The percentage of polymorphic loci observed per population varied from 4.5 to 63.6%, with a mean of 28.7%; observed heterozygosity levels varied from 0.002 to 0.126, with an average of 0.052. Observed heterozygosity and polymorphism values fell within the range of values reported in previous studies of the genus, but observed heterozygosity in most cases was less than expected under Hardy-Weinberg equilibrium. The genetic variance was partitioned among species, populations and individuals using hierarchical G-statistics. For all of the populations considered together, the variance among species was 58.4%, among populations within species 24.9%, and among individuals within a population 16.7%. When only the Neotropical species were considered, the percentages of the total variance attributable to variation among species, among populations within species, and among individuals were 18.1%, 29.2% and 52.7%, respectively. For the African species, 27.8% of the variance was among species, 42.3% among populations, and 29.9% among individuals. Intraspecific Roger's genetic distances ranged from 0.01 to 0.38, and interspecific distances from 0.1 to 0.84. Nei's unbiased distances ranged from 0.00 to 0.44 within a nominal species, and from 0.02 to 1.82 between species. Non-metric multidimensional scaling of Rogers' distances revealed a clear separation of African species from Neotropical species and of the Neotropical species from each other. The African species formed overlapping clusters.

Abstract:

The digenetic trematodes Schistosoma mansoni and Echinostoma paraensei have a common snail host Biomphalaria glabrata. in this article, Eric Loker and Coen Adema discuss how these parasite species evade the host immune response in fundamentally different ways.

Abstract:

Production of elevated haemolymph agglutination titres by Biomphalaria glabrata following exposure to Echinostoma paraensei miracidia was investigated, to characterize this parasite-induced response and to understand its functional relevance. Both the dose of infection (1, 10 or 100 miracidia per snail) or the number of separate exposures to infection (between one and three, over a 4 or 8 day interval) were varied, and assuming a threshold dosage (10 miracidia per snail or higher) was exceeded, titres of juvenile snails peaked at 8-16 times the values for unexposed control snails, regardless of the exposure regimen. Adult snails, which are relatively refractory to infection, have slightly higher resting titres than juveniles, but exhibit only a 2- to 4-fold increase in titre following exposure. Juveniles exposed to infection but lacking demonstrable infection had lower titres than snails with confirmed infections. Exposure to infection increased heterogeneity of plasma agglutinins and provoked production of unique specificities not found in unexposed snails. However, the overall pattern of agglutination responses for snails with successfully developed parasites did not differ from those in which parasite development was unsuccessful. Agglutinating activity was inhibitable by several different monosaccharides, although plasma from infected snails was relatively unaffected by nr-acetyl-glucosamine or N-acetyl-galactosamine. Wounding of snails provoked no change in plasma agglutination activity. As the highest agglutination titres were produced in snails with successfully developing parasites and agglutinin composition did not differ between snails with successful or unsuccessful parasites, the functional relevance of the response remains enigmatic. The production of unique agglutinins following exposure deserves additional study.

Abstract

A chelating anti-clumping (alpha-C) buffer allowed blood cells (hemocytes) of a gastropod, Lymnaea stagnalis to be separated by discontinuous Percoll density gradient centrifugation. The hemocytes of L. stagnalis were separated into five fractions, having a density lower than 10, 20, 30, 40, and 50% Percoll, respectively. Trypan blue exclusion assays showed viability of separated hemocytes to be between 81 and 89%. Cytospin preparations of these hemocytes were examined. Small cells were mainly observed at high densities; at lower densities medium and large hemocytes were also present. No absolute separation was achieved. Some density fractions were enriched for hemocytes with regard to the distributions of two endogenous lysosomal enzymes (alpha-naphthyl acetate esterase and acid phosphatase).

Abstract:

Polyclonal antibodies were raised in rabbits to two groups of diffusely staining M line Biomphalaria glabrata plasma polypeptides, of 150-210 and 70-120 kDa, designated as Group 1 molecules (G1M) and group 2 molecules (G2M), respectively. G1M and G2M are known to increase in abundance and to become more diverse following infection of B. glabrata with the digenetic trematode Echinostoma paraensei. These antibodies were used in conjunction with immunoblotting and slot blotting procedures to document binding of G1M/G2M from plasma of unexposed control snails (C plasma) or plasma from snails with 8-day infections of E. paraensei (I plasma) to various targets. Binding of G1M/G2M to both gram-positive and gram-negative bacteria, human A, B, and O and rabbit erythrocytes, and to sporocysts and rediae of E. paraensei was documented. Immunoblots revealed that erythrocytes are bound by a particular G2M band of 100-120 kDa present in I plasma that is in low concentration or lacking in C plasma. This explains previous results indicating that I plasma agglutinates all 4 erythrocytes examined whereas C plasma agglutinates only rabbit erythrocytes. More G1M/G2M binding to sporocysts occurred if I plasma rather than C plasma was used for incubations. Also, monosaccharide inhibition of G1M/G2M binding to sporocysts was observed in I plasma but not in C plasma. The results indicate that infection with E. paraensei induces production by B. glabrata of unique plasma polypeptides and that molecules present in I plasma can bind to the surfaces of non-self objects including E. paraensei larvae in a lectin like fashion. (C) 1994 Academic Press, Inc.

Abstract:

Many parasites develop in invertebrate hosts that possess internal defense systems (IDS) that vigorously defend self-integrity. Invertebrates apparently do not produce a large diversity of finely tuned immunorecognition molecules but rather rely on recognition of patterns. As a consequence, requirements for immune evasion are likely to be fundamentally different in such hosts. Although parasites of invertebrates certainly employ diverse tactics to evade host IDS, this review focuses on parasite-mediated interference with the structural and functional integrity of host hemocytes and argues that this is a common strategy of immune evasion. Parasites mediating such effects on host hemocytes are termed suppressors. In some cases, interference is mediated by mutualistic symbionts carried by the suppressors. Hemocytes from infected hosts exhibit diminished adherence to substrates, impaired spreading ability, and reduced ability to participate in phagocytosis or encapsulation reactions. As a result of the action of suppressors, the host's vulnerability to opportunistic parasites is increased, a phenomenon termed acquired susceptibility. A strategy of interference is therefore risky, particularly for suppressors with relatively long development times. As a result, suppressors may provoke either a partial generalized interference or a selective interference with host IDS function, actively contribute to protection of the host to discourage growth of opportunists (termed parasite-mediated internal defense), or induce compensatory host responses that protect the host but that do not jeopardize their own development. Some parasites consistently colonize previously infected hosts and seem to be specialized opportunists.

Abstract:

In vitro interactions between intramolluscan stages (sporocyst, daughter rediae, and metacercariae) of the trematode parasite Echinostoma paraensei and adherent hemocytes from the gastropods Biomphalaria glabrata (intermediate host) and Helix aspersa (non-host) were visualized by time-lapse videomicroscopy. Hemocytes of either species not exposed to E. paraensei displayed extensive mobility and activity of cellular extensions. Image analysis disclosed no significant change in the total surface area occupied by hemocytes in a selected field of view over 2 hr. Echinostoma paraensei exerted life stage-specific effects on the behavior of B. glabrata hemocytes; the cells moved away from sporocysts and daughter rediae but not encysted metacercariae. In the presence of sporocysts, hemocytes rounded up, whereas hemocytes adjacent to rediae assumed a stringy, beady appearance. Hemocytes close to the parasite were affected more rapidly than more distant cells. In 2 hr, a hemocyte-free ''halo'' formed around the parasite larvae, significantly reducing the hemocyte-occupied surface area (to 43% by sporocysts and to 70% by rediae). The changes induced by sporocysts and rediae are similar to those noted in both in vivo and in vitro studies of the B. glabrata-E. paraensei model system and are interpreted as manifestations of parasite-mediated interference with host hemocyte function. Helix aspersa (non-host) hemocytes were not affected, suggesting that E. paraensei-mediated effects on hemocytes exhibit a degree of specificity.

Abstract:

Hemolymph lectins may play an important role in the internal defense responses of gastropods to parasites. Two groups of known carbohydrate-binding polypeptides, of 150-220 kDa (designated as group 1 molecules, or G1M) and of 75-130 kDa (group 2 molecules, or G2M), were harvested from pooled plasma samples of Biomphlaria glabrata using affinity chromatography and examined using 2-dimensional electrophoresis. Plasma samples were derived from control snails or snails exposed 8 days earlier lo the trematodes Echinostoma paraensei or Schistosoma mansoni. Plasma of control and S. mansoni-exposed snails contained little or no G1M, whereas plasma from E. paraensei-infected snails contained G1M covering a broad pI spectrum. G2M resolved as 1-2 isoforms in control plasma and up to 4 relatively faint isoforms in plasma from S. mansoni-exposed snails. and as 5-6 resolvable isoforms in plasma from E. paraensei-infected snails. Plasma from individual snails contained as many as 5 G2M polypeptides following exposure to E. paraensei. Exposure to trematode larvae stimulated production by B. glabrata of increased abundance and diversity of carbohydrate-binding proteins. The 2 trematode species provoked different responses, and 2 B. glabrata strains studied (M line and 13-16-R1 strains) differed from one another in their responses.

Abstract:

The propensity of molluscan haemocytes to clump irreversibly upon mixing in suspension, together with their strong adherence to many substrates, compromises many efforts to obtain plasma-free or treated cells for subsequent use in experimental protocols. An anti-clumping buffer developed for use with haemocytes from the gastropod Lymnaea stagnalis was tested for its effectiveness with cells from Biomphalaria glabrata. While we were unable to obtain sufficient numbers of viable haemocytes for subsequent experimentation after centrifugation, this divalent cation-chelating buffer induced rounding of adherent cells monolayered on glass. Thus, cells washed in physiological buffers could subsequently be retrieved as monodispersed suspensions. Such haemocytes retained some phagocytic activity, with levels of uptake improved when cells were held in buffeT with excess divalent cations before addition of target particles.

Abstract:

The internal defence system (immune system) of the pond snail Lymnaea stagnalis is reviewed. Humoral defence activities are agglutination, opsonisation and inhibition of bacterial growth. Cellular defence is exerted by antigen trapping endothelial cells, foreign protein engulfing pore cells, phagocytic reticulum cells and mobile haemocytes. The haemocytes contribute most to defence and are therefore treated in more detail. There is one type of haemocyte, morphological heterogeneity of the haemocyte population is due to varying states of differentiation of the cells. Haemocytopoiesis is through mitosis of haemocytes, both in the pool of tissue-dwelling cells and in circulation. Haemocytes resemble monocytes/macrophages, they are typical phagocytes equipped with recognition factors (lectins), lysosomal enzymes and a cytotoxicity mechanism using reative oxygen intermediates. A comparison is made of the internal defence systems of the three main metazoan taxa, insects, molluscs and vertebrates.

Abstract:

No abstract

Abstract:

An enzyme cytochemical, light microscopy study was undertaken to evaluate the effect of haemolymph extraction on distribution of acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and peroxidase in haemocytes of Lymnaea stagnalis. The study revealed that discrete acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and peroxidase-staining subpopulations of haemocytes do exist in L. stagnalis, and that there are differences in distribution. A high percentage, about 96%, of haemocytes showed positive reaction for peroxidase, the percentage of haemocytes showing activity for alpha-naphthyl acetate esterase, acid phosphatase, and alkaline phosphatase, was about 54, 42, and 34, respectively. Haemolymph extraction by foot retraction, significantly affected enzyme distribution. Whereas distribution of acid phosphatase increased significantly from day 1 to 5 after extraction of haemolymph, that of all other enzymes showed significant decrease from day 1 to 7 depending upon the enzyme. The distribution of all enzymes stabilized by day 8 after extraction of haemolymph. Probable reasons for the observed fluctuations in the distribution of enzymes subsequent to haemolymph extraction are discussed.

Abstract:

Haemocytes taken from six different gastropod snail species, Achatina achatina, A. fulica, Biomphalaria glabrata, Bulinus natalensis 272, Helix aspersa and Lymnaea stagnalis, were compared for surface staining with lectins and with monoclonal antibodies raised against haemocytes of Biomphalaria glabrata and L. stagnalis respectively. The lectin staining did not serve to differentiate haemocyte populations of any species, with haemocytes of all but L. stagnalis staining with concanavalin A, and haemocytes of this species being stained with wheatgerm agglutinin. It was apparent that monoclonal antibodies (Mabs) raised to haemocyte markers of one species of snail were capable of recognizing determinants on haemocytes of other snail species. The degree, location and subpopulation specificity of staining differed for the Mabs employed. In particular, Mab Ls8 stained cytoplasmic granules of L. stagnalis haemocytes, surface structures on haemocytes of Biomphalaria glabrata, Bulinus natalensis 272, A. achatina and H. aspersa, whereas haemocytes of A. fulica did not stain. The two most-related snail species, the Achatina spp. showed differential staining patterns with both lectin and antibodies.

Abstract:

The kinetics of oxygen radical production by phagocytosing hemocytes of the pond snail Lymnaea stagnalis were investigated. After contact had been established between zymosan and hemocytes in monolayers at 0-degrees-C, phagocytosis was initiated by a shift to room temperature. Until the internalization phase of phagocytosis was completed, oxidative activity was detected mainly extracellularly (superoxide dismutase inhibitable cytochrome C reduction and peroxidase-catalyzed phenol red oxidation were used for the detection of superoxide and hydrogen peroxide, respectively). Thereafter, extracellular oxygen radical production by phagocytosing hemocytes decreased. Luminol-dependent chemiluminescence activity grew and, after the internalization phase of phagocytosis, remained at a high level, suggesting continued oxygen radical activity intracellularly. These results indicate that contact between zymosan and the hemocyte's plasma membrane stimulates a membrane-bound system to generate and release oxygen radicals. After internalization, this system appears to continue oxygen radical production inside the phagosome. So far, oxygen radical production in snail hemocytes shows many similarities to the mechanism in mammalian leucocytes.

Abstract:

Molluscan internal defense or immune systems have a cellular and a humoral limb. The hemocyte is the most important of the four defense cell types. The humoral part of the system is comprised of diverse factors, mainly produced by hemocytes. Target recognition and effector activation are essential for elimination of the target. Humoral, i.e., cell-independent, killing of foreign organisms by soluble factors occurs, and there is hemocyte-mediated cytotoxicity. This is the major effector mechanism against non-self. Cytotoxicity is exerted by lytic enzymes which degrade foreign organisms, extracellularly or in phagosomes. Moreover, evidence is mounting that reactive oxygen intermediates play an important part in hemocyte-mediated cytotoxicity. Hemocyte-target contact activates an enzyme located in the hemocyte's plasma membrane to generate oxygen radicals (superoxide, hydrogen peroxide, and probably others). These are potent killers of encapsulated metazoans and phagocytosed monocellular organisms. This system of oxidative killing is the main subject of this paper.