Intraspecific variations in the hemolymph of Biomphalaria glabrata, a snail host of Schistosoma mansoni.

An attempt was made to characterize the hemolymph of Biomphalaria glabrata with reference to "normal" intra-specific variation, i.e., both inter- and intra-strain differences. Total protein concentration, per cent hemoglobin, pH, and osmolarity were studied. Seven geographic strains of B, glabrata were examined. In addition, observations were made on the hemolymph of Biomphalaria straminea, several strains of Helisoma caribaeum, and on B. glabrata subjected to infection with Schistosoma mansoni or to periods of starvation. Intra-strain differences in total protein concentration and total hemoglobin concentration in B. glabrata appeared to be more closely related with snail size than with absolute age. Inter-strain variation in B. glabrata was also noted, but the differences were of the same magnitude as those from intra-strain samples. Significant differences in total protein concentration were observed, however, between the means of similar size B. glabrata, B. straminea and H. caribaeum. The osmolatity of the hemolymph from different size B. glabrata was similar as were the osmolalities of the hemolymph from similar size snails of different strains. However, all B. glabrata strains exhibited hemolymph osmolalities lower than observed in strains of H. caribaeum. Infection with S. mansoni reduced the protein concentration of B. glabrata hemolymph. Differences were noted as early as 1.5-24 hr post-infection, with significant alterations occurring at about 11 days post-infection. To a lesser extent, starvation also depleted the protein content of the hemolymph.     

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.     

A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (http://biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 x coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.     

Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations

This review discusses the large-scale laboratory maintenance of Schistosoma mansoni. Emphasized are features which increase efficiency in such facilities, and problems most frequently encountered. Profiles are given of the long-term, high-level production of 3 strains of S. mansoni. Two of the strains, NMRI and PR-1, were of Puerto Rican origin and the other, LE, was from Brazil. Three to 8 million cercariae of each strain were usually obtained per week. The most obvious differences between the 3 strains were cercarial output per snail and snail mortality rates. Maintenance problems encountered were usually related to water quality, temperature, genetics of the parasite or snail host, predators or contaminants, feeding, or crowding of snails. Examination of the production data from these 3 life cycles led to identification of features that could be of benefit for increasing the productivity and efficiency of other S. mansoni life cycles used in research activities.     

Factors affecting adoptive transfer of resistance to Schistosoma mansoni in the snail intermediate host, Biomphalaria glabrata

We examined potential variables affecting adoptive transfer of resistance to Schistosoma mansoni in Biomphalaria glabrata implanted with amebocyte-producing organs (APOs) from resistant snails. Transplants of 7 tissues other than the APO (heart, kidney, mantle, albumin gland, brain, digestive gland, and gonad) did not transfer resistance, suggesting a unique property of this structure. Only APOs from donors previously exposed to miracidia transferred resistance, although whether this is evidence for a priming effect or merely the elimination of susceptible donors is not known. Variability in the donor and in the implant itself apparently was unimportant, inasmuch as implants from small or large snails or from 2 separate donors all conferred similar levels of resistance. Recipients of APOs from 2 additional resistant strains of B. glabrata, 10-R2 and Salvador, also displayed resistance. However, no resistance was transferred by APOs from schistosome-refractory B. obstructa. Histological examination of implants removed from recipients that either did or did not show transferred resistance revealed no differences in mitotic activity. Furthermore, implanted APOs from B. obstructa displayed no mitotic activity. Finally, reexposure of snails with transferred resistance to a large dose of miracidia caused infection in 70%, suggesting that either transferred resistance is transitory or it can be overwhelmed.     

Ki-67 is expressed in multiplying forms of Schistosoma mansoni, but not in snail host tissues

Ki-67 is a protein expressed in the nucleus of several species during cell-division, being absent during the GO resting phase of the cellular cycle. During attempts to disclose mitosis in the so-called " amebocyte-producing organ " in Biomphalaria glabrata infected with Schistosoma mansoni, the parasite multiplying forms appeared strongly marked for Ki-67, while the snail tissues were completely negative. These data are worth registering to complement general data on Ki-67, and to help future studies on the relationship of the parasite and of its intermediate host.     

Intermediate host specificity in Schistosoma mansoni

Miracidia of Schistosoma mansoni penetrate into many kinds of snails, but development of normal sporocysts takes place only in certain species of Biomphalaria. Different populations of this snail vary greatly in laboratory infection rates with S. mansoni originating from diverse geographic localities. Cross-exposure experiments show that compatibility factors exist in both snails and parasites. Susceptibility of stocks of Biomphalaria to particular strains of S. mansoni is genetically determined and may be modified by selection in the laboratory. In a compatible snail, the sporocyst develops without host tissue reaction; in incompatible snails the early larvae are rapidly surrounded by amebocytes and fibroblasts, and destroyed. This reaction resembles the generalized host cellular response elicited by any foreign body. An individual snail exposed to many miracidia may have both developing and encapsulated sporocysts side by side within its tissues. The weight of current evidence suggests that elicitation or absence of this cellular response resides in the recognition or nonrecognition of the sporocyst as a foreign body. The sporocyst tegument surface, which forms within a few hours after miracidial penetration, may have a molecular conformation identical with that of the snail, or may be able to bind specific host molecules, so that detection and subsequent encapsulation by host cells are averted. Presuming genetic determination of the sporocyst surface structure and of the host cell detection capability, differing infection rates would result from the particular frequencies of relevant genes in the populations concerned.     

Fine-scale population structure and dispersal in Biomphalaria glabrata, the intermediate snail host of Schistosoma mansoni, in Venezuela

Biomphalaria glabrata is the main intermediate host of Schistosoma mansoni in America and one of the most intensely studied species of freshwater snails, yet very little is known about its population biology. Here, we used seven highly polymorphic microsatellite loci to analyse genetic diversity in the Valencia lake basin, which represents the core of the endemic area for schistosomiasis in Venezuela. Populations were sampled at short spatial scale (a few kilometres), both inside the lake and in ponds or rivers near the lake. Our results indicate that B. glabrata essentially cross-fertilizes, with little variation in selfing rates among populations. Our markers detected considerable genetic variation, with an average heterozygosity of 0.60. More diversity per population was found within than outside the lake, suggesting an influence of connectivity among populations on the levels of genetic diversity. A marked population structure was detected and lake populations were less structured than other populations. Most individuals were assigned to their population of origin using an assignment test. No strong demographic signal (e.g. bottleneck) was detected, though lake populations are likely to experience bottlenecks more frequently than the other populations analysed. Differences in gene flow therefore seem to play an important role in population differentiation and in the restoring of genetic diversity in demographically unstable populations.     

Schistosoma mansoni and Biomphalaria glabrata share epitopes: antibodies to sporocysts bind host snail hemocytes

Using an independent protocol, we have confirmed that sporocysts of the human blood fluke, Schistosoma mansoni, synthesize antigens which stimulate rabbit antibody activity to epitopes on infermediate snail host hemocytes. This molecular mimicry may aid S. mansoni to escape the innate immune system of this host, Biomphalaria glabrata.     

Uptake of [14C]oxamniquine by Schistosoma mansoni

The uptake and retention of drug-related material by Schistosoma mansoni was studied in the mouse host following a single oral or intramuscular dose (50 mg/kg) of [14C]oxamniquine. Male worms took up more labelled material than did female worms but the amount in each particular sex of worm was found to be similar after both routes of administration. Exposure of worms was therefore independent of the route of administration. Six days after drug administration, at the time of an hepatic shift, significantly more drug-related material was present in male worms than in female worms. Examination of worms recovered from mice 4 h after treatment showed that metabolites of oxamniquine constituted 70--90% of the drug-related material present in the worms. Both sexes of worms were able to take up metabolites of oxamniquine in vitro.     

Involvement of the cytokine MIF in the snail host immune response to the parasite Schistosoma mansoni

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions.     

Effect of miracidial dose on adoptively transferred resistance to Schistosoma mansoni in the snail intermediate host, Biomphalaria glabrata

Adoptively transferred resistance to Schistosoma mansoni in the snail intermediate host Biomphalaria glabrata was measured as a function of miracidial challenge dose. Schistosome-susceptible snails implanted with the amebocyte-producing organ (APO) from resistant donors showed 29 and 39% prevalences of infection after challenge with 5 and 10 miracidia, respectively, but 68-83% prevalences when exposed to 25-200 miracidia. Prevalences in control (untampered) susceptible snails ranged from 97 to 100% at the different miracidial doses. Higher infection prevalences at elevated doses suggest that a range of transferred resistance occurs and possibly that low levels of APO-derived plasma factors or hemocytes in some recipients can be overwhelmed by larger numbers of parasites.