Identification of genes involved in interactions between Biomphalaria glabrata and Schistosoma mansoni by suppression subtractive hybridization.

Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, a medically important schistosome. In order to identify transcripts involved in snail-schistosome interactions, subtractive cDNA libraries were prepared, using suppression subtractive hybridization (SSH) between a parasite-exposed schistosome-resistant and a susceptible strain of B. glabrata, and also between schistosome-exposed and unexposed snails from the resistant snail line. Separate libraries were made from both haemocytes and the haemopoietic organ. Subtraction was performed in both directions enriching for cDNAs differentially expressed between parasite-exposed resistant and susceptible samples and up or down-regulated in the resistant line after challenge. The resulting eight libraries were screened and eight genes, differentially expressed between the haemocytes of resistant and susceptible snail strains, were identified and confirmed with reverse transcriptase PCR, including two transcripts expected to be involved in the stress response mechanism for regulating the damaging oxidative burst pathways involved in cytotoxic killing of the parasite: the iron-storage and immunoregulatory molecule, ferritin, and HtrA2, a serine protease involved in the cellular stress response. Transcripts with elevated levels in the resistant strain, had the same expression patterns in the subtracted libraries and unsubtracted controls; higher levels in exposed resistant snails compared to susceptible ones and down-regulated in exposed compared with unexposed resistant snails. Differential expression of two of the transcripts with no known function from the susceptible strain, was independently confirmed in a repeat exposure experiment.

Biomphalaria glabrata transcriptome: identification of cell-signalling, transcriptional control and immune-related genes from open reading frame expressed sequence tags (ORESTES).

Biomphalaria glabrata is the major intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. Much remains to be discovered concerning specific molecules mediating the defence events in these intermediate hosts, triggered by invading schistosomes. An expressed sequence tag (EST) gene discovery strategy known as ORESTES has been employed to identify transcripts that might be involved in snail-schistosome interactions in order to examine gene expression patterns in infected B. glabrata. Over 3930 ESTs were sequenced from cDNA libraries made from both schistosome-exposed and unexposed snails using different tissue types, producing a database of 1843 non-redundant clones. The non-redundant set has been assessed for gene ontology and KEGG pathway assignments. This approach has revealed a number of signalling, antioxidant and immune-related gene homologues that, based on current understanding of molluscan and other comparative systems, might play an important role in the molluscan defence response towards infection.

Extraordinary host switching in siphonostomatoid copepods and the demise of the Monstrilloida: integrating molecular data, ontogeny and antennulary morphology.

Copepods exhibit an astounding variety of lifestyles, host associations and morphology, to the extent that their crustacean affinities may be obscured. Relationships among the ten copepod orders based on morphological characters remain equivocal. Here we test the ordinal status of the enigmatic Monstrilloida using SSU rDNA gene sequences, comparative morphological data (antennulary sensory interface) and ontogenetic data (caudal ramus setation patterns). Bayesian analysis unexpectedly revealed the Monstrilloida are nested within a fish-parasitic clade of the Siphonostomatoida and share a common ancestor with the stem species of the caligiform families (sea-lice). This unforeseen relationship is congruent with both antennulary and caudal ramus morphology. The divergence of the monstrilloids from an ectoparasitic, vertebrate-associated ancestor involved radical changes in host utilization, body plan and life cycle strategy, a combination rarely observed and probably unique in metazoan parasites. Adult monstrilloids secondarily returned to a free-living, predator-exposed mode of life and we postulate the pressure on maintaining a functional approaching-predator detection system has progenetically delayed the suppression (as in post-copepodid caligiform instars) of the 5-point antennulary sensory array. The homoplastic evolution of the frontal filament in Siphonostomatoida is discussed.