Timothy P. Yoshino

Schistosoma mansoni: Passive transfer of resistance by serum in the vector snail, Biomphalaria glabrata

Passive transfer of natural resistance to Schistosoma mansoni (PR-1 strain) has been successfully accomplished in the snail intermediate host, Biomphalaria glabrata (PR albino, M-line strain). Injection of serum (cell-free hemolymph) from a naturally schistosome-resistant strain of B. glabrata (10-R2) into PR albino snails induced a complete protection from a primary infection with the parasite in 29 of 48 snails (60.4%). In comparison, inoculation of homologous PR albino serum or heterologous proteins (fetal calf serum) had no effect. Moreover, this protection could be induced 24 hr prior to, or 24 hr after, exposure to the parasite, although heating of 10-R2 serum to 70 C for 30 min destroyed its protective ability. When in vitro transformed sporocysts were preincubated in 10-R2 or PR albino serum and then were injected into susceptible snails, a high level of infection (88.5 and 83.3%, respectively) was produced in both groups. Thus, the 10-R2 serum factor does not appear to be mediating specific parasite recognition by host hemocytes. Alternatively, our results suggest that 10-R2 serum possesses a heat-labile factor which specifically activate B. glabrata hemocytes to encapsulate and destroy sporocysts whereas PR albino serum lacks this factor.

Schistosoma mansoni: effect of infection on reproduction and gonadal growth in Biomphalaria glabrata

Sexually mature Biomphalaria glabrata were exposed to 12 miracidia of Schistosoma mansoni, and egg production of snails was monitored over a period of 5 weeks. During the study period, exposed snails grew at approximately the same rate as unexposed controls. Castration, as measured by a reduction in the mean number of eggs laid per snail occurred between 14 and 21 days postexposure (PE). The reduction in fecundity in infected snails coincided with the migration and establishment of daughter sporocysts in the digestive gland and gonad. Enumeration of individual oocytes in longitudinal sections of the ovotestis revealed that uninfected snails contained significantly more oocytes per section than infected snails at 27, 31, and 40 days PE. In addition, the mean area of gonadal sections of control snails increased over the 40-day experimental period, whereas there was no such increase in gonadal area of infected snails. These data suggest that there is an inhibition in gonadal growth in infected snails. When oocyte data were expressed in terms of mean gonadal area, the mean number of oocytes per mm2 of gonad of uninfected and infected snails did not differ significantly over the study period, except at Day 14 PE, when infected snails contained a significantly greater number of oocytes per mm2 of gonad than did uninfected controls. It is hypothesized that daughter sporocysts of S. mansoni are primarily responsible for the inhibition of host reproductive activity, and may be mediating their effects through mechanisms involved in the regulation of gonadal growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of concanavalin a-reactive determinants on hemocytes of two Biomphalaria glabrata snail stocks: receptor binding and redistribution

Fluorescein-labeled concanavalin A (F-Con A) was employed as a membrane probe to compare the surface glycosyl components associated with glass-adherent circulating hemocytes from a schistosome-susceptible (PR albino) and schistosome-refractory (10-R2) stock of snails Biomphalaria glabrata. Under hemolymph-free conditions, granular hemocytes (granulocytes), composed of two morphologically-distinct cell populations (rounded and spread hemocytes) bound F-Con A via specific carbohydrate surface receptors. Con A-binding induced in rounded hemocytes a redistribution (patching and capping) of lectin-receptor complexes. Receptor redistribution in both snail stocks was characterized by a high capping frequency (80–90% of adherent rounded cells) and temperature dependency. Differences in the response of albino and 10-R2 snail hemocytes to differing F-Con A concentrations, however, suggest that cells may possess subtle differences in physiology or function.

Surface antigens of Biomphalaria glabrata (Gastropoda) hemocytes: Functional heterogeneity in cell subpopulations recognized by a monoclonal antibody

Abstract A hemocyte surface membrane marker (BGH 1 ) has been identified using hemocyte-specific monoclonal antibodies (mABs) generated by somatic cell fusion methods. The BGH 1 epitope was expressed on a subpopulation of circulating, glass-adherent blood cells from two strains of the snail, Biomphalaria glabrata . Approximately 40% of the circulating hemocytes from the PR albino (M-line) B. glabrata strain were BGH 1 − , compared to a prevalence of 10% BGH 1 + cells in the 10-R2 snail strain. When hemocytes were firmly attached and spread on a glass surface, BGH 1 + cells were morphologically distinguishable from BGH 1 − cells by their ovoid shape and the presence of short, thin filopodial projections along the ectoplasmic border. In contrast, BGH 1 − hemocytes were more pleomorphic and possessed long, spike-like filopodia. Moreover, the BGH 1 epitope was trypsin-resistant and retained its antigenic reactivity with probe mABs following fixation with paraformaldehyde or paraformaldehyde/MeOH. Fixation with glutaraldehyde, however, significantly reduced mAB binding to the BGH 1 surface epitope. There was no apparent age-dependent expression of the BGH 1 determinant since circulating hemocyte populations in very young (1–2 mm) to adult (10–12 mm) snails were composed of both BGH 1 + and BGH 1 − subpopulations. Quantitative shifts in the prevalence of epitope-bearing hemocytes between the smallest snail size class (1–2 mm) and the larger snails (3–4 and 10–12 mm) are believed to be due to a differential production and/or release of BGH 1 − hemocytes within the blood circulation rather than a gradual age-related change in the expression of surface antigens on individual cells. Experiments designed to assess the in vitro phagocytic capability and lysosomal acid phosphatase (APase) activity of mAB-reactive hemocytes revealed that BGH 1 + cells, when compared to those lacking the surface marker, were significantly reduced in both their phagocytic and APase-producing activities. Since the PR albino strain of B. glabrata possesses a higher proportion of BGH 1 − hemocytes and a lower total concentration of circulating cells than do snails of the 10-R2 strain, PR albino snails are thus potentially reduced in their natural capacity to mount cellular reactions against foreign materials.

Identification of antigenically distinct hemocyte subpopulations in Biomphalaria glabrata (Gastropoda) using monoclonal antibodies to surface membrane markers.

SummaryFive monoclonal antibodies (mABs) against surface antigens on circulating, glass-adherent hemocytes of the snail, Biomphalaria glabrata, were produced by somatic cell hybridization methods. Two mABs (IID2.6-Bg and IID4.8-Bg) are pan-hemocytic, reacting uniformly with epitopes shared by all adherent hemocytes. Determinants recognized by these mABs also are present in soluble form and appear to be associated with a hemoglobin-depleted ultracentrifuged fraction of snail hemolymph. Hybridoma-derived mABs IIC6.8-Bg and VB10.3-Bg recognize hemocyte surface epitopes expressed by only 50–60% of the adherent cell population. These mABs also are reactive with soluble hemolymph antigens but apparently recognize determinants which are different from the IID2.6-Bg and IID4.8-Bg reactive sites. Another antigenically distinct hemocyte subpopulation is recognized by mAB IID7.1-Bg. Epitopes that are reactive with this mAB differ from the previously described determinants by their asymmetrical distribution on the surface of positive cells and the absence of soluble antigenic components in hemolymph. Furthermore, unlike the other mABs, the prevalence of hemocytes staining with IID7.1-Bg antibodies differed between two strains of B. glabrata. Results of this study clearly demonstrate that circulating B. glabrata hemocytes, consisting of a single, predominant population of adherent cells, is composed of several distinct antigenic subpopulations based on the specific binding of anti-hemocyte mAB probes. Our successful application of hybridoma techniques to the study of molluscan hemocyte surface antigens underscores further the great potential usefulness of this method in analysing the molecular basis of hemocyte reactivity.

Schistosoma mansoni: Relationship between cercarial production levels and snail host susceptibility

Two populations of Biomphalaria glabrata snails differing slightly in their susceptibility to Schistosoma mansoni infection showed dramatic differences in cercarial output per snail. Exposed to five or more miracidia, snails from a group with a 90-100% susceptibility rate (Group A) produced nearly twice the number of cercariae as those from a group with a 70-80% susceptibility rate (Group B). Exposure of individual snails to known numbers of miracidia resulted in higher numbers of primary (mother) sporocysts in Group A snails than in Group B snails. However, monomiracidial exposure of snails from both groups resulted in equivalent numbers of cercariae produced per positive snail, indicating that, once established, all primary sporocysts possess a similar reproductive potential. Morphometric analysis of serially sectioned 9-day-old primary sporocysts supported this conclusion; the size of the primary sporocysts and the size and numbers of secondary (daughter) sporocysts within each primary sporocyst were comparable in snails from both groups. The data indicate cercarial production in this system is regulated prior to, and/or during, early development of the primary sporocyst.

Schistosoma mansoni: Origin and expression of a tegumental surface antigen on the miracidium and primary sporocyst

A monoclonal antibody, recognizing a carbohydrate epitope associated with several tegumental surface components on Schistosoma mansoni primary sporocysts, was used to follow tegumental formation during transformation of the miracidium to sporocyst and its subsequent development in vitro and in vivo. Indirect fluorescent antibody and direct immunogold labeling methods confirm a structural connection between the intercellular ridges and a submuscular, multinucleate syncytium in the miracidium. Immunoreactive vesicles within this latter system directly contribute to elaboration of the tegumental surface membrane, through the process of membrane fusion. Lateral expansion of intercellular ridges by vesicular fusion ultimately result in fully transformed sporocysts exhibiting vesicular membrane epitopes as prominent tegumental surface components. Light microscopical and ultrastructural observations, together with Western immunoblot analyses, suggest a gradual depletion of intracellular and surface immunoreactive material of vesicular origin in primary sporocysts grown in culture for up to 12 days. In contrast, similar immunoreactive vesicles appear to be continuously synthesized throughout in vivo primary sporocyst development. Monoclonal antibody reactive epitopes appear to be uniquely expressed in the miracidium/primary sporocyst since similar molecules are absent from daughter sporocysts, cercariae, adults, and snail tissues.

Soluble mediators of cytolytic activity in hemocytes of the asian clam, Corbicula fluminea

Serum-free hemocytes from the clam, Corbicula fluminea, are cytolytic to mammalian erythrocytes (RBCs) as demonstrated in a hemoglobin release assay. The reaction is hemocyte dose- and temperature-dependent, and is mediated by the release of soluble hemolytic factors from clam cells. Initiation of hemocyte secretory activity does not appear to require contact with target RBCs. The chemical nature of the secreted lysin(s) has not been determined; however, PAGE analysis of hemocyte extracts (lysates) reveals the presence of at least five peaks of lytic activity. This activity is sensitive to heat and proteolytic enzyme treatment, and can be removed by absorption with fixed, homologous RBCs. Thus it is likely that the secreted hemocyte lysin is a heat-labile protein which exerts its hemolytic activity by binding directly to target RBC membrane constituents.

Lectins and antibodies as molecular probes of molluscan hemocyte surface membranes

Abstract A variety of carbohydrate-specific lectins and antigen-specific antibodies have been employed to assess the occurrence and topographic distribution of surface membrane determinants on hemocytes of two stocks of the snail, Biomphalaria glabrata , which differ in their susceptibilities to larval blood fluke ( Schistosoma mansoni ) infections. Using fluorescence labeling methods, several “classes” of hemocyte membrane components have been identified. These include (1) macromolecules, primarily glycoproteins, possessing reactive sites for concanavalin A and Ricinus communis agglutinin, (2) snail hemolymph or hemolymph-like determinants, (3) fibronectin-like membrane components, and (4) surface determinants with structural similarities to murine Thy-1 antigen. No qualitative differences in surface molecular composition were observed between hemocytes of the two B . glabrata stocks, although the broad reaction specificity of most of the probe reagents employed preclude the detection of small or minor differences in hemocyte membrane structure. Results of these investigations indicate that snail hemocytes possess a complex array of surface membrane components some of which appear to be shared with higher vertebrate species.

Role of divalent cations in plasma opsonin-dependent and-independent erythrophagocytosis by hemocytes of the Asian clam, Corbicula fluminea

Abstract The in vitro phagocytosis-promoting properties of hemolymph from the freshwater clam, Corbicula fluminea , are described. Hemocytes were capable of phagocytosing aldehyde-fixed erythrocytes (RBCs) of seven vertebrate species with equal facility, but only in the presence of homologous clam plasma. The plasma factors mediating erythrophagocytosis were heat sensitive. Pretreatment (opsonizing) of target RBCs with plasma also resulted in enhancement of hemocyte particle uptake in the absence of plasma. Opsonin-dependent phagocytosis required the presence of divalent cations, especially calcium, although not in free ionic form. Evidence suggests that the plasma opsonin may normally exist as a divalent cation-macromolecular complex since opsonizing activity was retained after dialysis against Tris-buffered saline (TBS), but was lost following TBS/EDTA or TBS/EGTA dialysis. We also have identified an opsonin-independent phagocytosis mechanism in which Corbicula hemocytes are able to ingest nonopsonized RBCs in the absence of homologous plasma. Extracellular calcium or magnesium in the incubation medium is needed for particle uptake, although the direct binding of free ions to the target RBC surface does not appear to be mediating enhanced phagocytosis. From the present data, it is concluded that hemocyte recognition of aldehyde-fixed RBCs can be accomplished by either of two mechanisms: (1) by the coating of cells with plasma factors capable of triggering the phagocytic process (opsonization) or (2) by a plasma opsonin-independent mechanism in which extracellular divalent cations (e.g., Ca 2+ or Mg 2+ ) in the incubation buffer stimulate uptake of nonopsonized RBCs. The factors regulating in vitro erythrophagocytosis by clam hemocytes are considered to be analogous to those involved in nonimmune opsonin-dependent and -independent phagocytosis in mammalian macrophages.