Hc-daf-2 encodes an insulin-like receptor kinase in the barber’s pole worm, Haemonchus contortus, and restores partial dauer regulation

Infective L3s (iL3s) of parasitic nematodes share common behavioural, morphological and developmental characteristics with the developmentally arrested (dauer) larvae of the free-living nematode Caenorhabditis elegans. It is proposed that similar molecular mechanisms regulate entry into or exit from the dauer stage in C. elegans, and the transition from free-living to parasitic forms of parasitic nematodes. In C. elegans, one of the key factors regulating the dauer transition is the insulin-like receptor (designated Ce-DAF-2) encoded by the gene Ce-daf-2. However, nothing is known about DAF-2 homologues in most parasitic nematodes. Here, using a PCR-based approach, we identified and characterised a gene (Hc-daf-2) and its inferred product (Hc-DAF-2) in Haemonchus contortus (a socioeconomically important parasitic nematode of ruminants). The sequence of Hc-DAF-2 displays significant sequence homology to insulin receptors (IR) in both vertebrates and invertebrates, and contains conserved structural domains. A sequence encoding an important proteolytic motif (RKRR) identified in the predicted peptide sequence of Hc-DAF-2 is consistent with that of the human IR, suggesting that it is involved in the formation of the IR complex. The Hc-daf-2 gene was transcribed in all life stages of H. contortus, with a significant up-regulation in the iL3 compared with other stages. To compare patterns of expression between Hc-daf-2 and Ce-daf-2, reporter constructs fusing the Ce-daf-2 or Hc-daf-2 promoter to sequence encoding GFP were microinjected into the N2 strain of C. elegans, and transgenic lines were established and examined. Both genes showed similar patterns of expression in amphidial (head) neurons, which relate to sensation and signal transduction. Further study by heterologous genetic complementation in a daf-2-deficient strain of C. elegans (CB1370) showed partial rescue of function by Hc-daf-2. Taken together, these findings provide a first insight into the roles of Hc-daf-2/Hc-DAF-2 in the biology and development of H. contortus, particularly in the transition to parasitism.     

Functional genomics of hsp-90 in parasitic and free-living nematodes

Heat shock protein 90 (Hsp-90) is a highly conserved essential protein in eukaryotes. Here we describe the molecular characterisation of hsp-90 from three nematodes, the free-living Caenorhabditis elegans (Ce) and the parasitic worms Brugia pahangi (Bp) and Haemonchus contortus (Hc). These molecules were functionally characterised by rescue of a Ce-daf-21 (hsp-90) null mutant. Our results show a gradient of rescue: the C. elegans endogenous gene provided full rescue of the daf-21 mutant, while Hc-hsp-90 provided partial rescue. In contrast, no rescue could be obtained using a variety of Bp-hsp-90 constructs, despite the fact that Bp-hsp-90 was transcribed and translated in the mutant worms. daf-21 RNA interference (RNAi) experiments were carried out to determine whether knock-down of the endogenous daf-21 mRNA in N2 worms could be complemented by expression of either parasite gene. However neither parasite gene could rescue the daf-21 (RNAi) phenotypes. These results indicate that factors other than the level of sequence identity are important for determining whether parasite genes can functionally complement in C. elegans.     

Atypical (RIO) protein kinases from Haemonchus contortus--promise as new targets for nematocidal drugs

Almost nothing is known about atypical kinases in multicellular organisms, including parasites. Supported by information and data available for the free-living nematode, Caenorhabditis elegans, and other eukaryotes, the present article describes three RIO kinase genes, riok-1, riok-2 and riok-3, from Haemonchus contortus, one of the most important parasitic nematodes of small ruminants. Analyses of these genes and their products predict that they each play critical roles in the developmental pathways of parasitic nematodes. The findings of this review indicate prospects for functional studies of these genes in C. elegans (as a surrogate) and opportunities for the design of a novel class of nematode-specific inhibitors of RIO kinases. The latter aspect is of paramount importance, given the serious problems linked to anthelmintic resistance in parasitic nematode populations of livestock.     

Effects of Lactobacillus salivarius, Lactobacillus reuteri, and Pediococcus acidilactici on the nematode Caenorhabditis elegans include possible antitumor activity

This study examined the effects of three lactic acid bacteria (LAB) strains on the nematode Caenorhabditis elegans. Lactobacillus salivarius, Lactobacillus reuteri, and Pediococcus acidilactici were found to inhibit the development and growth of the worm. Compared to Escherichia coli used as the control, L. reuteri and P. acidilactici reduced the lifespan of wild-type and short-lived daf-16 worms. On the contrary, L. salivarius extended the lifespan of daf-16 worms when used live, but reduced it as UV-killed bacteria. The three LAB induced the expression of genes involved in pathogen response and inhibited the growth of tumor-like germ cells, without affecting DAF16 localization or increasing corpse cells. Our results suggest the possible use of C. elegans as a model for studying the antitumor attributes of LAB. The negative effects of these LAB strains on the nematode also indicate their potential use against parasitic nematodes.     

Identification of an Ascaris G protein-coupled acetylcholine receptor with atypical muscarinic pharmacology

Acetylcholine (ACh) is a neurotransmitter/neuromodulator in the nematode nervous system and induces its effects through interaction with both ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs). The structure, pharmacology and physiological importance of LGICs have been appreciably elucidated in model nematodes, including parasitic species where they are targets for anthelmintic drugs. Significantly less, however, is understood about nematode ACh GPCRs, termed GARs (G protein-linked ACh receptors). What is known comes from the free-living Caenorhabditis elegans as no GARs have been characterized from parasitic species. Here we clone a putative GAR from the pig gastrointestinal nematode Ascaris suum with high structural homology to the C. elegans receptor GAR-1. Our GPCR, dubbed AsGAR-1, is alternatively spliced and expressed in the head and tail of adult worms but not in dorsal or ventral body wall muscle, or the ovijector. ACh activated AsGAR-1 in a concentration-dependent manner but the receptor was not activated by other small neurotransmitters. The classical muscarinic agonists carbachol, arecoline, oxotremorine M and bethanechol were also AsGAR-1 agonists but pilocarpine was ineffective. AsGAR-1 activation by ACh was partially antagonized by the muscarinic blocker atropine but pirenzepine and scopolamine were largely ineffective. Certain biogenic amine GPCR antagonists were also found to block AsGAR-1. Our conclusion is that Ascaris possesses G protein-coupled ACh receptors that are homologous in structure to those present in C. elegans, and that although they have some sequence homology to vertebrate muscarinic receptors, their pharmacology is atypically muscarinic.     

Haemonchus contortus and Trichostrongylus colubriformis did not adapt to long-term exposure to sheep that were genetically resistant or susceptible to nematode infections

We tested the hypothesis that Haemonchus contortus and Trichostrongylus colubriformis would adapt to long-term exposure to sheep that were either genetically resistant or susceptible to H. contortus. Sheep genotypes were from lines with 10 years prior selection for low (resistant, R) or high (susceptible, S) faecal worm egg count (WEC) following H. contortus infection. Long-term exposure of H. contortus and T.colubriformis to R or S genotypes was achieved using serial passage for up to 30 nematode generations. Thus, we generated four nematode strains; one strain of each species solely exposed to R sheep and one strain of each species solely exposed to S sheep. Considerable host genotype differences in mean WEC during serial passage confirmed adequate nematode selection pressure for both H. contortus (R 4900 eggs per gram (epg), S 19,900 epg) and T. colubriformis (R 5300 epg, S 13,500 epg). Adaptation of nematode strain to host genotype was tested using seven cross-classified tests for H. contortus, and two cross-classified and one outbred genotype test for T. colubriformis. In the cross-classified design, where each strain infects groups of R, S or randomly bred control sheep, parasite adaptation would be indicated by a significant host genotype by nematode strain interaction for traits indicating parasite reproductive success; specifically WEC and, for H. contortus strains, packed cell volume. We found no significant evidence of parasite adaptation to host genotype (P > 0.05) for either the H. contortus or T. colubriformis strains. Therefore, we argue that nematodes will not adapt quickly to sheep bred for nematode resistance, where selection is based on low WEC, although selecting sheep using a subset of immune functions may increase adaptation risk. Our results support the hypothesis that nematode resistance is determined by many genes each with relatively small effect. In conclusion, selection of sheep for nematode resistance using WEC should be sustainable in the medium to long-term.     

Plant-parasitic Nematode Acetylcholinesterase Inhibition by Carbamate and Organophosphate Nematicides

The sensitivity of acetylcholinesterases (ACHE) isolated from the plant-parasitic nematodes Meloidogyne arenaria, M. incognita, and Heterodera glycines and the free-living nematode Caenorhabditis elegans to carbamate and organophosphate nematicides was examined. The AChE from plant-parasitic nematode species were more sensitive to carbamate inhibitors than was AChE from C. elegans, but response to the organophosphates was approximately equivalent. The sulfur-containing phosphate nematicides were poor inhibitors of nematode acetylcholinesterase, but treatment with an oxidizing agent greatly improved inhibition. Behavioral bioassays with living nematodes revealed a poor relationship between enzyme inhibition and expression of symptoms in live nematodes.     

Functional characterisation of a cyst nematode acetylcholinesterase gene using Caenorhabditis elegans as a heterologous system

Migration of plant-parasitic nematode infective larval stages through soil and invasion of roots requires perception and integration of sensory cues culminating in particular responses that lead to root penetration and parasite establishment. Components of the chemoreceptive neuronal circuitry involved in these responses are targets for control measures aimed at preventing infection. Here we report, to our knowledge, the first isolation of cyst nematode ace-2 genes encoding acetylcholinesterase (AChE). The ace-2 genes from Globodera pallida (Gp-ace-2) and Heterodera glycines (Hg-ace-2) show homology to ace-2 of Caenorhabditis elegans (Ce-ace-2). Gp-ace-2 is expressed most highly in the infective J2 stage with lowest expression in the early parasitic stages. Expression and functional analysis of the Globodera gene were carried out using the free-living nematode C. elegans in order to overcome the refractory nature of the obligate parasite G. pallida to many biological studies. Caenorhabditis elegans transformed with a GFP reporter construct under the control of the Gp-ace-2 promoter exhibited specific and restricted GFP expression in neuronal cells in the head ganglia. Gp-ACE-2 protein can functionally complement its C. elegans homologue. A chimeric construct containing the Ce-ace-2 promoter region and the Gp-ace-2 coding region and 3′ untranslated region was able to restore a normal phenotype to the uncoordinated C. elegans double mutant ace-1;ace-2. This study demonstrates conservation of AChE function and expression between free-living and plant-parasitic nematode species, and highlights the utility of C. elegans as a heterologous system to study neuronal aspects of plant-parasitic nematode biology.     

Adjustments,extinction, and remains of selenocysteine incorporation machinery in the nematode lineage

Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Sec-incorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.     

Ultraviolet light increases mortality of nematode larvae and can explain patterns of larval availability at pasture

Despite its documented effects on the viability of living organisms, the impact of ultraviolet (UV) light on the survival of parasitic nematode larvae has received surprisingly little attention. Infective L3s of the trichostrongyloid nematodes Haemonchus contortus, Teladorsagia circumcinta and Nematodirus battus, suspended in water, were exposed to direct UV irradiation in two experiments. In the first, during 6 days of constant illumination with UVA lamps at intensities simulating sunlight at ground level, the mortality rate was increased up to 100-fold compared with controls sheltered by UV-impermeable perspex. Significant differences in mortality rates were detected between the three species, with H. contortus the least sensitive. In the second experiment, larvae were exposed to natural sunlight during the temperate spring and summer, for 24-h periods on seven separate days representing a range of weather and UV doses. Mortality was again increased by UV exposure in all species, but was less in H. contortus than in T. circumcincta or N. battus. At higher daily UV doses, the mortality rate was on average 2.27 times higher in exposed larvae than in sheltered controls. Increased mortality caused by UV irradiation could help to explain patterns of abundance of infective stages at pasture, especially pronounced population declines in spring when solar radiation rises rapidly and temperature is still low. Implications for the epidemiology of gastrointestinal nematode infection in ruminants, and for trade-offs in parasite life history, are discussed.