RNA-mediated gene suppression and in vitro culture in Hymenolepis microstoma.

Hymenolepis microstoma, the mouse bile-duct tapeworm, is a classical rodent-hosted model that provides easy laboratory access to all stages of the life cycle. Recent characterisation of its genome has greatly advanced its utility for molecular research, albeit contemporary techniques such as those for assaying gene function have yet to be developed in the system. Here we present research on the development of RNA-mediated gene suppression via RNA interference (RNAi), and on in vitro culture of the enteric, adult phase of the life cycle to support this work. We demonstrate up to 80% quantitative suppression of a Hox transcript via soaking activated juvenile worms with double-stranded RNAs. However, we were unable to achieve segmentation of the worms in culture despite extensive manipulations of the culture media and supplements, preventing functional interpretation. An alternative, in vivo approach to RNAi was also tested by exposing cysticercoids prior to inoculation in mice, but fluorescent labelling showed that the RNAs did not sufficiently penetrate the cyst body and no difference in expression was found between exposed and control groups grown in vivo. Genomic and transcriptomic data revealed that H. microstoma has two orthologs each of Dicer, Drosha and Ago-1-like genes and that expression of one of the Ago-1 genes appears exclusive to germline development, suggesting that two or more independent RNA-mediated pathways are in operation. These studies demonstrate the viability of RNAi in H. microstoma and extend the utility of the model for research in the genomic era.

Spatial and temporal consistency of putative reference genes for real-time PCR in a model tapeworm.

Relative quantification of gene expression by real-time PCR relies on the use of reference genes whose expressed levels remain consistent across experimental conditions. Here we compare expression levels of commonly employed endogenous housekeeping genes against a developmental regulatory gene in the model tapeworm Hymenolepis microstoma, examining variation both spatially across regions of the adult worm and temporally through the course of larval metamorphosis. ß-Tubulin, RNA polymerase II and 60S ribosomal subunit L28 showed the most variance among candidate reference genes when comparing changes in expression along the anteroposterior gradient of development represented by the adult body, whereas expression of 18S rDNA and cyclic AMP were highly consistent and could be used reliably for relative quantification. The transcription factor Hox4, referenced to either 18S or cAMP, showed 3-fold higher expression levels in the neck region than in more mature regions of the strobila. In contrast, variance among samples representing progressive stages of larval metamorphosis were greater for candidate reference genes than for Hox4, indicating that none of the candidates can be used reliably for quantifying relative changes in gene expression during metamorphosis.

Functional divergence of former alleles in an ancient asexual invertebrate.

Theory suggests it should be difficult for asexual organisms to adapt to a changing environment because genetic diversity can only arise from mutations accumulating within direct antecedents and not through sexual exchange. In an asexual microinvertebrate, the bdelloid rotifer, we have observed a mechanism by which such organisms could acquire the diversity needed for adaptation. Gene copies most likely representing former alleles have diverged in function so that the proteins they encode play complementary roles in survival of dry conditions. One protein prevents desiccation-sensitive enzymes from aggregating during drying, whereas its counterpart does not have this activity, but is able to associate with phospholipid bilayers and is potentially involved in maintenance of membrane integrity. The functional divergence of former alleles observed here suggests that adoption of asexual reproduction could itself be an evolutionary mechanism for the generation of diversity.

Spliced leader RNA-mediated trans-splicing in phylum Rotifera.

In kinetoplastids, Euglena, and four metazoan phyla, trans-splicing has been described as a mechanism for the generation of mature messenger RNAs (mRNAs): 5'-ends of precursor mRNAs are replaced by a short spliced leader (SL) exon from a small SL RNA. Although the full phylogenetic range is unknown, trans-splicing has not been found in vertebrates, insects, plants, or yeast. In animal groups where it does occur, i.e., nematodes, cnidarians, platyhelminths, and primitive chordates, SL RNAs do not show sequence relatedness across phyla. The apparently sporadic phylogenetic distribution and the lack of SL RNA homology have led to opposing hypotheses on its evolution, involving either an ancient origin followed by loss in multiple lineages or independent acquisition in several taxa. Here we present evidence for the occurrence of trans-splicing in bdelloid rotifers (Bdelloidea, Rotifera). A common 23-nt sequence, representing the SL exon-diagnostic of SL RNA-mediated trans-splicing-was found at the 5'-end of at least 50%-65% of mRNAs from Adineta ricciae and Philodina sp. The trans-splicing pattern in bdelloid rotifers can be unusually complex, as observed in transcripts from a heat shock protein gene, hsp82-1, where the SL exon was spliced to three alternative positions. Bdelloid rotifer SL RNAs were found to be 105 or 106 nt long and comprised the SL sequence, a conserved splice donor site and an intron containing a putative spliceosome-binding motif. Intriguingly, some similarity of rotifer SL RNA sequence and predicted secondary structure was seen to that of the predominant SL1 RNA of nematodes, although it is unlikely that this demonstrates homology. In addition, sequence corresponding to the rotifer SL exon was found at the 5'-end of a number of full-length complementary DNA (cDNA) clones in a rice (Oryza sativa) database. None of these cDNAs gave a close match with homologous plant genes, suggesting that a small but significant portion of the rice expressed sequence tag database represents sequences derived from rotifers. In summary, the description of SL-mediated trans-splicing in Rotifera extends its representation to at least five metazoan phyla, making it increasingly probable that this is a phylogenetically widespread and therefore ancient phenomenon.

Molecular studies of a novel dragline silk from a nursery web spider, Euprosthenops sp. (Pisauridae).

Various spider species produce dragline silks with different mechanical properties. The primary structure of silk proteins is thought to contribute to the elasticity and strength of the fibres. Previously published work has demonstrated that the dragline silk of Euprosthenops sp. is stiffer then comparable silk of Nephila edulis, Araneus diadematus and Latrodectus mactans. Our studies of Euprosthenops dragline silk at the molecular level have revealed that nursery web spider fibroin has the highest polyalanine content among previously characterised silks and this is likely to contribute to the superior qualities of pisaurid dragline.