A major weakness of the Baermann funnel technique for extracting nematode larvae from feces is the funnel. As many as 67% of Parelaphostrongylus tenuis first-stage larvae lodged on the sloping surface of glass Baermann funnels. The number of larvae collected after 24 hr was not significantly correlated with total numbers in the samples, whether feces were supported over tissue paper or over window screening. Instead, we collected about 8 times as many larvae and achieved a significant relationship between larvae collected and the total numbers present when pelleted fecal material was submerged over screening in vertical-sided beakers. The methodology of this more efficient and more accurate way of estimating numbers of protostrongylid larvae is described. Most larvae were located on and in the mucous layer covering fecal pellets and readily left fresh pellets emersed in water; 72% of these larvae left after 6 min and only 11% remained after 1 hr. Larvae in water at room temperature sank as fast as 6 cm/min, but those close to a vertical glass surface sank more slowly (97% sank 18.5 cm in 105 min).

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Helminth parasites rely on fast-synaptic transmission in their neuromusculature to experience the outside world and respond to it. Acetylcholine plays a pivotal role in this and its receptors are targeted by a wide variety of both natural and synthetic compounds used in human health and for the control of parasitic disease. The model, Caenorhabditis elegans is characterized by a large number of acetylcholine receptor subunit genes, a feature shared across the nematodes. This dynamic family is characterized by both gene duplication and loss between species. The pentameric levamisole-sensitive acetylcholine receptor has been characterized from C. elegans, comprised of five different subunits. More recently, cognate receptors have been reconstituted from multiple parasitic nematodes that are found to vary in subunit composition. In order to understand the implications of receptor composition change and the origins of potentially novel drug targets, we investigated a specific example of subunit duplication based on analysis of genome data for 25 species from the 50 helminth genome initiative. We found multiple independent duplications of the unc-29, acetylcholine receptor subunit, where codon substitution rate analysis identified positive, directional selection acting on amino acid positions associated with subunit assembly. Characterization of four gene copies from a model parasitic nematode, Haemonchus contortus, demonstrated that each copy has acquired unique functional characteristics based on phenotype rescue of transgenic C. elegans and electrophysiology of receptors reconstituted in Xenopus oocytes. We found evidence that a specific incompatibility has evolved for two subunits co-expressed in muscle. We demonstrated that functional divergence of acetylcholine receptors, driven by directional selection, can occur more rapidly than previously thought and may be mediated by alteration of receptor assembly. This phenomenon is common among the clade V parasitic nematodes and this work provides a foundation for understanding the broader context of changing anthelmintic drug targets across the parasitic nematodes.

J. Neurochem. (2010) 113, 1113–1122. Abstract We have identified two genes from the parasitic nematode Haemonchus contortus, Hco‐unc‐49B and Hco‐unc‐49C that encode two GABA‐gated chloride channel subunits. Electrophysiological analysis revealed that this channel has properties similar to those of the UNC‐49 channel from the free‐living nematode Caenorhabditis elegans. For example, the Hco‐UNC‐49B subunit forms a functional homomeric channel that responds to GABA and is highly sensitive to picrotoxin. Hco‐UNC‐49C alone does not respond to GABA but can assemble with Hco‐UNC‐49B to form a heteromeric channel with a lower sensitivity to picrotoxin. However, we did find that the Hco‐UNC‐49B/C heteromeric channel is significantly more responsive to agonists compared to the Hco‐UNC‐49B homomeric channel, which is the opposite trend to what has been found previously for the C. elegans channel. To investigate the subunit requirements for high agonist sensitivity, we generated cross‐assembled channels by co‐expressing the H. contortus subunits with UNC‐49 subunits from C. elegans (Cel‐UNC‐49). Co‐expressing Cel‐UNC‐49B with Hco‐UNC‐49C produced a heteromeric channel with a reduced sensitivity to GABA compared to that of the Cel‐UNC‐49B homomeric channel. In contrast, co‐expressing Hco‐UNC‐49B with Cel‐UNC‐49C produced a heteromeric channel that, like the Hco‐UNC‐49B/C heteromeric channel, exhibits an increased sensitivity to GABA. These results suggest that the Hco‐UNC‐49B subunit is the key determinant for the high agonist sensitivity of heteromeric channels.