Fred A. Lewis

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.

Comparative gene analysis of Biomphalaria glabrata hemocytes pre- and post-exposure to miracidia of Schistosoma mansoni☆

The internal defense mechanism of the snail Biomphalaria glabrata during a schistosome infection is activated and mediated via the immune effector cells known as hemocytes. Since resistance and susceptibility to schistosome infection is known to be genetically determined, our interest was to use the EST approach as a gene discovery tool to examine transcription profiles in hemocytes of resistant snails pre- and post-exposure to Schistosoma mansoni. Comparative analysis of the transcripts suggested that parasite exposure caused an active metabolic response in the hemocytes. The most abundant transcripts were those showing 23-74% similarity to known reverse transcriptases (RT). Further characterization by RT-PCR indicated the RT transcripts were expressed in normal snails, parasite exposed snails, and the embryonic cell line Bge. To determine whether the occurrence of RT transcripts correlates to the presence of functional enzyme activity in the snails, RT assays were performed from both resistant and susceptible snails, pre- and post-exposure to miracidia, using protein extracts from the head-foot and posterior region tissues. Results indicated that in the resistant snail, RT activity was greater in the posterior region than in the head-foot. After exposure, however, RT activity increased dramatically in the head-foot, with peak activity at 24 h post-exposure. The detection of RT activity in B. glabrata was unexpected and the role of this enzyme in the hemocyte-mediated killing of parasites is not yet known. However, identification of this and other transcripts from these cells by the EST approach provides a useful resource towards elucidating the molecular basis of resistance/susceptibility in this snail-host parasite relationship.

Ultrastructure of the Schistosoma mansoni cercaria.

The cercaria of the schistosome parasite is a short-lived, free-swimming larval stage that is infective for the mammalian, definitive host. This atlas describes the ultrastructure of the cells that comprise the cercaria of Schistosoma mansoni, a leading causative agent of human schistosomiasis. In addition to the cells which make up the various organ systems, such as the nervous, tegumental, osmoregulatory, muscular and primordial digestive systems, also we show the ultrastructure of those cells whose organization or location are not as well defined structurally but are essential nevertheless for the success of the parasite. These latter include the various support cells, and those cells that, upon differentiation into the adult worm, serve reproductive functions. A description is also given of the cells whose sole functions are realized only at the cercarial stage, chiefly involved in the vigorous act of skin penetration. Although we include a detailed review of the ultrastructure of S. mansoni cercariae, much of the information reported has not been previously published. In summary, this paper brings together an ultrastructural description of all the cell types presently known that make up the much studied larval stage of this medically important trematode.

Genetic diversity of Schistosoma mansoni: quantifying strain heterogeneity using a polymorphic DNA element

Intraspecific genetic variation among 14 geographic isolates of Schistosoma mansoni was quantified using a molecular marker to examine individual genotypes. Genetic crosses demonstrated maternal inheritance of S. mansoni DNA element pSM750. This element revealed diagnostic banding profiles, which allowed accurate strain identification. Most strains had similarity indices greater than 0.75 indicating that within-strain variation in these laboratory parasite populations was low. However, individual parasites from the NMRI strain were quite diverse (S = 0.40). Genetic heterogeneity among strains was quantified using a phenogram of mean genetic distance. Strain diversity between two geographic regions was quantified using a similarity index and was estimated to be substantial among isolates collected from a single local site.

Schistosoma mansoni infection of juvenile Biomphalaria glabrata induces a differential stress response between resistant and susceptible snails

Schistosomes develop successfully in susceptible snails but are encapsulated and killed in resistant ones. Mechanism(s) shaping these outcomes involves the parasites ability to evade the snails defenses. RNA analysis from resistant (BS-90), non-susceptible (LAC2) and susceptible (NMRI) juvenile Biomphalaria glabrata to Schistosoma mansoni revealed that stress-related genes, heat shock protein 70 (Hsp 70) and reverse transcriptase (RT), were dramatically co-induced early in susceptible snails, but not in resistant/non-susceptible ones. These transcripts were, however, down regulated upon exposure to irradiated parasites although penetration behavior of irradiated vs. normal parasites were the same, indicating that Hsp 70 and RT regulation was elicited by infection and not injury. Understanding molecular events involved in stress response transcriptional regulation of Hsp 70 in juvenile snails could pave a way towards the identification of genes involved in schistosome/snail interactions.

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.

DISTRIBUTION OF SCHISTOSOME INFECTIONS IN MOLLUSCAN HOSTS AT DIFFERENT LEVELS OF PARASITE PREVALENCE

Biomphalaria glabrata snails infected with Schistosoma mansoni were collected during consecutive seasons from a site in Brazil known to have a very high percentage of infected snails. Schistosoma mansoni cercariae from single snails were used to infect individual mice, and the recovered adult worms were genetically assessed using a mtVNTR marker. The number of unique parasite genotypes found per snail was compared to expected abundance values, based on the infection prevalence at the site, to determine the distribution of S. mansoni infections within the snail population. The observed distributions and those from previous studies were used to examine the relationship between schistosome prevalence and aggregation across a wide range of prevalence values. Our analysis showed that prevalence was inversely related to the degree of parasite overdispersion, and at high prevalence, S. mansoni infections were randomly distributed among snails.

Schistosoma mansoni: Use of a cloned ribosomal RNA gene probe to detect restriction fragment length polymorphisms in the intermediate host Biomphalaria glabrata

Adult susceptibility of Biomphalaria glabrata to Schistosoma mansoni infection is controlled by simple Mendelian genetics. In this study a molecular approach was used to determine the degree of genetic variation between well-defined lines of B. glabrata which are either resistant (10-R2) or susceptible (M-line) to S. mansoni infection. A cloned probe pSM389, which contains part of the S. mansoni small ribosomal RNA gene and a portion of the nontranscribed spacer was found to cross-hybridize with B. glabrata DNA and was used in Southern hybridizations to detect restriction fragment length polymorphisms (RFLPs) between the above snail stocks. Polymorphisms were noted with a variety of restriction enzymes, namely Bg/II, BamHI, AccI, AvaII, ClaI, EcoRI, EcoRV, KpnI, PvuII, and NcoI. Although most RFLPs were relatively minor, a significant difference was observed with EcoRV. Further analysis of the EcoRV RFLPs among other isolates of the resistant stock demonstrated that a high frequency of genetic variation exists even among isolates of the same origin, but maintained in separate laboratories. Interestingly, RFLPs in the EcoRV site were detected in DNA isolated from a single generation of selfed progeny of a single 10-R2 parent. RFLPs associated with this site were found to occur between B. glabrata and B. tenagophila, B. straminea, and B. schrammi, indicating that Southern blot analysis using ribosomal gene probes may be useful for the molecular differentiation of B. glabrata from other intermediate hosts and from morphologically similar species that are refractory to infection.

Use of RAPD-PCR to differentiate genetically defined lines of an intermediate host of Schistosoma mansoni, Biomphalaria glabrata.

The genetic differentiation among several laboratory-maintained pedigree snail lines of Biomphalaria glabrata (with different susceptibility phenotypes to Schistosoma mansoni infection) was assessed with the random amplified polymorphic DNA method. Out of the 20 primers tested, 2 (OPA-01 and OPA-06) gave reproducible markers with either individual or bulked DNA samples from resistant (BS-90, 10-R2, LAC-line) and susceptible (M-line) snails. Arbitrary primer, OPA-01, amplification of BS-90 DNA identified a 180-bp strain-specific fragment and a 400-bp marker in the susceptible M-line stock. In the 10-R2 and LAC snail lines, OPA-01 specific markers of 200 bp and 550 bp were identified. Amplification with primer OPA-06 identified several major strain-specific markers in the BS-90 (150 bp, 400 bp, 800 bp) and M-line (1,100 bp) snails. The heritability of the RAPD markers was evaluated in progeny snails derived from a cross between the BS-90 and M-line stocks. Results showed that markers were inherited in a dominant or codominant fashion. The 1,100-bp M-line marker was inherited in all susceptible progeny snails analyzed.

Genetic manipulation of Schistosoma haematobium, the neglected schistosome.

Background Minimal information on the genome and proteome of Schistosoma haematobium is available, in marked contrast to the situation with the other major species of human schistosomes for which draft genome sequences have been reported. Accordingly, little is known about functional genomics in S. haematobium, including the utility or not of RNA interference techniques that, if available, promise to guide development of new interventions for schistosomiasis haematobia. Methods/Findings Here we isolated and cultured developmental stages of S. haematobium, derived from experimentally infected hamsters. Targeting different developmental stages, we investigated the utility of soaking and/or square wave electroporation in order to transfect S. haematobium with nucleic acid reporters including Cy3-labeled small RNAs, messenger RNA encoding firefly luciferase, and short interfering RNAs (siRNAs). Three hours after incubation of S. haematobium eggs in 50 ng/µl Cy3-labeled siRNA, fluorescent foci were evident indicating that labeled siRNA had penetrated into miracidia developing within the egg shell. Firefly luciferase activity was detected three hours after square wave electroporation of the schistosome eggs and adult worms in 150 ng/µl of mRNA. RNA interference knockdown (silencing) of reporter luciferase activity was seen following the introduction of dsRNA specific for luciferase mRNA in eggs, schistosomules and mixed sex adults. Moreover, introduction of an endogenous gene-specific siRNA into adult schistosomes silenced transcription of tetraspanin 2 (Sh-tsp-2), the apparent orthologue of the Schistosoma mansoni gene Sm-tsp-2 which encodes the surface localized structural and signaling protein Sm-TSP-2. Together, knockdown of reporter luciferase and Sh-tsp-2 indicated the presence of an intact RNAi pathway in S. haematobium. Also, we employed laser scanning confocal microscopy to view the adult stages of S. haematobium. Conclusions These findings and approaches should facilitate analysis of gene function in S. haematobium, which in turn could facilitate the characterization of prospective intervention targets for this neglected tropical disease pathogen.