Unravelling the genetic basis of renal diseases; from single gene to multifactorial disorders

Chronic kidney disease is common with up to 5% of the adult population reported to have an estimated glomerular filtration rate of

Phylogeographic analysis of North American populations of the parasitic herbaceous plant Monotropa hypopitys reveals a complex history of range expansion from multiple late glacial refugia

Aim We carried out a phylogeographic study across the range of the herbaceous plant species Monotropa hypopitys L. in North America to determine whether its current disjunct distribution is due to recolonization from separate eastern and western refugia after the Last Glacial Maximum (LGM). Location North America: Pacific Northwest and north-eastern USA/south-eastern Canada. Methods Palaeodistribution modelling was carried out to determine suitable climatic regions for M. hypopitys at the LGM. We analysed between 155 and 176 individuals from 39 locations spanning the species' entire range in North America. Sequence data were obtained for the chloroplast rps2 gene (n=168) and for the nuclear ITS region (n=158). Individuals were also genotyped for eight microsatellite loci (n=176). Interpolation of diversity values was used to visualize the range-wide distribution of genetic diversity for each of the three marker classes. Minimum spanning networks were constructed showing the relationships between the rps2 and ITS haplotypes, and the geographical distributions of these haplotypes were plotted. The numbers of genetic clusters based on the microsatellite data were estimated using Bayesian clustering approaches. Results The palaeodistribution modelling indicated suitable climate envelopes for M. hypopitys at the LGM in both the Pacific Northwest and south-eastern USA. High levels of genetic diversity and endemic haplotypes were found in Oregon, the Alexander Archipelago, Wisconsin, and in the south-eastern part of the species' distribution range. Main conclusions Our results suggest a complex recolonization history for M. hypopitys in North America, involving persistence in separate eastern and western refugia. A generally high degree of congruence between the different marker classes analysed indicated the presence of multiple refugia, with at least two refugia in each area. In the west, putative refugia were identified in Oregon and the Alexander Archipelago, whereas eastern refugia may have been located in the southern part of the species' current distribution, as well as in the 'Driftless Area'. These findings are in contrast to a previous study on the related species Orthilia secunda, which has a similar disjunct distribution to M. hypopitys, but which appears to have recolonized solely from western refugia. © 2011 Blackwell Publishing Ltd.

RNAi Effector Diversity in Nematodes

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.

Novel bioassays to examine the host-finding ability of plant-parasitic nematodes

We present two novel bioassays to be used in the examination of plant-parasitic nematode host-finding ability. The host-finding 'pipette-bulb assay' was constructed from modelled Pasteur pipette bulbs and connecting barrels using parafilm fastenings. This assay examines the direction of second-stage juvenile (J2) migration in response to a host seedling, through a moistened sand substrate, which underlies terminal upward-facing 'seedling bulbs', one containing a host seedling in potting compost, the other with only potting compost. An equal watering regime through both upward-facing seedling bulbs creates a directional concentration gradient of host diffusate chemotactic factors. Positive chemotactic stimuli cause the J2 to orientate and migrate towards the host plant. We present validation data collected from assays of the root-knot nematode, Meloidogyne incognita, and the potato cyst nematode, Globodera pallida, which indicate a highly significant positive attraction of J2 of both species to respective host plants. This represents a simple, quick and inexpensive method of assessing host-finding behaviour in the laboratory. We consider that the pipette-bulb assay improves on previous host-finding/chemo-attraction assays through creating a more biologically relevant environment for experimental J2; analysis is quick and easy, allowing the straightforward interpretation of results. In addition, we have developed an 'agar trough' sensory assay variant which we believe can be used rapidly to ratify nematode responses to chemical gustatory or olfactory cues. This was constructed from a water agar substrate such that two counting wells were connected by a raised central trough, all flooded with water. Two small water agar plugs were dehydrated briefly in an oven and then hydrated in either an attractant, repellent or water control; these plugs were then placed in the terminal counting wells and subsequently leached the attractant or repellent to form a concentration gradient along the central trough, which contained the initial J2 innoculum. Our data show that both M. incognita and G. pallida J2 are positively attracted to host diffusates. In addition, they displayed a strong repulsion in response to 1 M NaCl2. J2 of M. incognita displayed a mild aversion to a non-host oak root diffusate, whereas G. pallida J2 displayed a strong aversion to the same non-host diffusate; neither species responded to a compost leachate. We believe that the agar trough assay improves on previous methods by facilitating rapid diffusion of attractant or repellents. Both of the aforementioned assays were designed as tools to assess the impact of RNAi-based reverse genetics screens for gene targets involved in chemosensory orientation.

THE ACTION OF SEROTONIN AND THE NEMATODE NEUROPEPTIDE KSAYMRFAMIDE ON THE PHARYNGEAL MUSCLE OF THE PARASITIC NEMATODE, ASCARIS-SUUM

The pharyngeal component of the enteric nervous system of the parasitic nematode, Ascaris suum exhibits immunoreactivity for serotonin (5-hydroxytryptamine or 5-HT) and for FMRFamide-like peptides. This paper describes the application of an in vitro pharmacological approach to investigate the functional role of 5-HT and FMRFamide-like peptides. The pharyngeal pumping behaviour of Ascaris suum was monitored using a modified pressure transducer system which measures pharyngeal pressure changes and therefore pumping. The pharynx did not contract spontaneously; however, 5-HT (10-1000 mu M) stimulated pumping at a frequency of 0 . 5 Hz. FMRFamide had no apparent effect on pharyngeal pumping. The native nematode FMRFamide-related peptide (FaRP), KSAYMRFamide inhibited the pumping elicited by 5-HT. The duration of inhibition was dose-dependent (0 . 1-1000 nM) with a threshold of 0 . 1 nM. In 4 preparations, the inhibition of the pharyngeal muscle was preceded by an initial excitation and increase in the amplitude of pharyngeal pressure changes. The pharynx is involved in various nematode processes, including feeding, regulation of hydrostatic pressure and excretion. The role of 5-HT and KSAYMRFamide in the pharyngeal function of nematodes is discussed.