Multiple lineage specific expansions within the guanylyl cyclase gene family

BACKGROUND: Guanylyl cyclases (GCs) are responsible for the production of the secondary messenger cyclic guanosine monophosphate, which plays important roles in a variety of physiological responses such as vision, olfaction, muscle contraction, homeostatic regulation, cardiovascular and nervous function. There are two types of GCs in animals, soluble (sGCs) which are found ubiquitously in cell cytoplasm, and receptor (rGC) forms which span cell membranes. The complete genomes of several vertebrate and invertebrate species are now available. These data provide a platform to investigate the evolution of GCs across a diverse range of animal phyla. RESULTS: In this analysis we located GC genes from a broad spectrum of vertebrate and invertebrate animals and reconstructed molecular phylogenies for both sGC and rGC proteins. The most notable features of the resulting phylogenies are the number of lineage specific rGC and sGC expansions that have occurred during metazoan evolution. Among these expansions is a large nematode specific rGC clade comprising 21 genes in C. elegans alone; a vertebrate specific expansion in the natriuretic receptors GC-A and GC-B; a vertebrate specific expansion in the guanylyl GC-C receptors, an echinoderm specific expansion in the sperm rGC genes and a nematode specific sGC clade. Our phylogenetic reconstruction also shows the existence of a basal group of nitric oxide (NO) insensitive insect and nematode sGCs which are regulated by O(2). This suggests that the primordial eukaryotes probably utilized sGC as an O(2 )sensor, with the ligand specificity of sGC later switching to NO which provides a very effective local cell-to-cell signalling system. Phylogenetic analysis of the sGC and bacterial heme nitric oxide/oxygen binding protein domain supports the hypothesis that this domain originated from a cyanobacterial source. CONCLUSION: The most salient feature of our phylogenies is the number of lineage specific expansions, which have occurred within the GC gene family during metazoan evolution. Our phylogenetic analyses reveal that the rGC and sGC multi-domain proteins evolved early in eumetazoan evolution. Subsequent gene duplications, tissue specific expression patterns and lineage specific expansions resulted in the evolution of new networks of interaction and new biological functions associated with the maintenance of organismal complexity and homeostasis

Characterization of Heterorhabditis isolates by PCR amplification of segments of mtDNA and rDNA genes

Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping

Characterization of \u3ci\u3eHeterorhabditis\u3c/i\u3e Isolates by PCR Amplification of Segments of mtDNA and rDNA Genes

Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping

Chemoreceptor genes: what can we learn from Caenorhabditis elegans and how can we apply this information to studies on other nematodes?

Soil dwelling nematodes encounter many types of volatile and water-soluble molecules in their environment. For free-living nematodes like Caenorhabdiris clegnns, successful foraging depends on the ability to detect a gradient in one odorant while ignoring extraneous odoun. The infectious stages of plant and animal parasitic nematodes also rely on chemoreception as their primary host finding cue. Using a combination of genetic, molecular and bioinformatic approaches chemoreceptor genes have been identified in C. clegans. These C. elegans chemoreceptor genes encode seven-transmembrane G-protein coupled receptors (GPCR) and comprise the largest gene family in this nematode. GPCR are also involved in olfactory signal transduction across a broad spectrum of animals including insects, crustaceans, fish and mammals, but the C. elegans (and Drosophila) chemoreceptor genes have no sequence homology to vertebrate GPCR odour receptor genes and they also differ from vertebrate odour receptor genes in their genomic structure. We review the genomic structure and diversity of odorant and chemoreceptor gene families in vertebrates and invertebrates and describe our attempts, using homology-based approaches, to isolate chemoreceptor genes in the entomopathogenic nematode Heterorhabditis bacteriophora

Chemoreceptor genes: what can we learn from Caenorhabditis elegans and how can we apply this information to studies on other nematodes?

Soil dwelling nematodes encounter many types of volatile and water-soluble molecules in their environment. For free-living nematodes like Caenorhabdiris clegnns, successful foraging depends on the ability to detect a gradient in one odorant while ignoring extraneous odoun. The infectious stages of plant and animal parasitic nematodes also rely on chemoreception as their primary host finding cue. Using a combination of genetic, molecular and bioinformatic approaches chemoreceptor genes have been identified in C. clegans. These C. elegans chemoreceptor genes encode seven-transmembrane G-protein coupled receptors (GPCR) and comprise the largest gene family in this nematode. GPCR are also involved in olfactory signal transduction across a broad spectrum of animals including insects, crustaceans, fish and mammals, but the C. elegans (and Drosophila) chemoreceptor genes have no sequence homology to vertebrate GPCR odour receptor genes and they also differ from vertebrate odour receptor genes in their genomic structure. We review the genomic structure and diversity of odorant and chemoreceptor gene families in vertebrates and invertebrates and describe our attempts, using homology-based approaches, to isolate chemoreceptor genes in the entomopathogenic nematode Heterorhabditis bacteriophora

Systemic RNAi mediated gene silencing in the anhydrobiotic nematode Panagrolaimus superbus

<p>Abstract</p> <p>Background</p> <p>Gene silencing by RNA interference (RNAi) is a powerful tool for functional genomics. Although RNAi was first described in <it>Caenorhabditis elegans</it>, several nematode species are unable to mount an RNAi response when exposed to exogenous double stranded RNA (dsRNA). These include the satellite model organisms <it>Pristionchus pacificus </it>and <it>Oscheius tipulae</it>. Available data also suggest that the RNAi pathway targeting exogenous dsRNA may not be fully functional in some animal parasitic nematodes. The genus <it>Panagrolaimus </it>contains bacterial feeding nematodes which occupy a diversity of niches ranging from polar, temperate and semi-arid soils to terrestrial mosses. Thus many <it>Panagrolaimus </it>species are adapted to tolerate freezing and desiccation and are excellent systems to study the molecular basis of environmental stress tolerance. We investigated whether <it>Panagrolaimus </it>is susceptible to RNAi to determine whether this nematode could be used in large scale RNAi studies in functional genomics.</p> <p>Results</p> <p>We studied two species: <it>Panagrolaimus </it>sp. PS1159 and <it>Panagrolaimus superbus</it>. Both nematode species displayed embryonic lethal RNAi phenotypes following ingestion of <it>Escherichia coli </it>expressing dsRNA for the <it>C. elegans </it>embryonic lethal genes <it>Ce-lmn-1 </it>and <it>Ce-ran-4</it>. Embryonic lethal RNAi phenotypes were also obtained in both species upon ingestion of dsRNA for the <it>Panagrolaimus </it>genes <it>ef1b </it>and <it>rps-2</it>. Single nematode RT-PCR showed that a significant reduction in mRNA transcript levels occurred for the target <it>ef1b </it>and <it>rps-2 </it>genes in RNAi treated <it>Panagrolaimus </it>sp. 1159 nematodes. Visible RNAi phenotypes were also observed when <it>P. superbus </it>was exposed to dsRNA for structural genes encoding contractile proteins. All RNAi phenotypes were highly penetrant, particularly in <it>P. superbus</it>.</p> <p>Conclusion</p> <p>This demonstration that <it>Panagrolaimus </it>is amenable to RNAi by feeding will allow the development of high throughput methods of RNAi screening for <it>P. superbus</it>. This greatly enhances the utility of this nematode as a model system for the study of the molecular biology of anhydrobiosis and cryobiosis and as a possible satellite model nematode for comparative and functional genomics. Our data also identify another nematode infraorder which is amenable to RNAi and provide additional information on the diversity of RNAi phenotypes in nematodes.</p

The effect of day of emergence from the insect cadaver on the behaviour and environmental tolerances of infective juveniles of the entomopathogenic nematode Heterorhabditis Megidis (Strain UK211)

Infective juveniles (Us) of entomopathogenic nematodes (EPNs) are obligate parasites of insect larvae. Inside the host they develop into sexually mature adult stages and complete their life cycle. Two or 3 adult nematode generations can occur in the insect host. The increase in nematode population density in the insect cadaver, together with limiting nutrient conditions, result in the formation of IJs. These IJs emerge into the soil to search for a new host. It typically takes 7-8 days for all Us to emerge from a parasitized insect. We have investigated the effect of the day of emergence of Us from insect cadavers on the environmental tolerance and behavior of the EPN Heterorhabditis megidis strain UK211. The Us that emerge early display good initial host-finding ability and increased temperature tolerance but disperse poorly and have poor tolerance to desiccation. Conversely, the IJs that emerge later display poor initial host-finding ability and poor temperature tolerance but they disperse well and possess increased desiccation tolerance. These phenotypic differences are likely to facilitate early-emerging Us in locating and infecting hosts in the vicinity of the cadaver, whereas IJs that emerge late are adapted to disperse away from their natal cadaver. We hypothesize that adaptive phenotypic plasticity rather than allelic variability may provide the genetic basis for the different physiological and behavioral phenotypes of the early- and late-emerging IJs

Expression profiling and cross-species RNA interference (RNAi) of desiccation-induced transcripts in the anhydrobiotic nematode Aphelenchus avenae.

BACKGROUND: Some organisms can survive extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living mycophagous nematode Aphelenchus avenae can be induced to enter anhydrobiosis by pre-exposure to moderate reductions in relative humidity (RH) prior to extreme desiccation. This preconditioning phase is thought to allow modification of the transcriptome by activation of genes required for desiccation tolerance. RESULTS: To identify such genes, a panel of expressed sequence tags (ESTs) enriched for sequences upregulated in A. avenae during preconditioning was created. A subset of 30 genes with significant matches in databases, together with a number of apparently novel sequences, were chosen for further study. Several of the recognisable genes are associated with water stress, encoding, for example, two new hydrophilic proteins related to the late embryogenesis abundant (LEA) protein family. Expression studies confirmed EST panel members to be upregulated by evaporative water loss, and the majority of genes was also induced by osmotic stress and cold, but rather fewer by heat. We attempted to use RNA interference (RNAi) to demonstrate the importance of this gene set for anhydrobiosis, but found A. avenae to be recalcitrant with the techniques used. Instead, therefore, we developed a cross-species RNAi procedure using A. avenae sequences in another anhydrobiotic nematode, Panagrolaimus superbus, which is amenable to gene silencing. Of 20 A. avenae ESTs screened, a significant reduction in survival of desiccation in treated P. superbus populations was observed with two sequences, one of which was novel, while the other encoded a glutathione peroxidase. To confirm a role for glutathione peroxidases in anhydrobiosis, RNAi with cognate sequences from P. superbus was performed and was also shown to reduce desiccation tolerance in this species. CONCLUSIONS: This study has identified and characterised the expression profiles of members of the anhydrobiotic gene set in A. avenae. It also demonstrates the potential of RNAi for the analysis of anhydrobiosis and provides the first genetic data to underline the importance of effective antioxidant systems in metazoan desiccation tolerance.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Anhydrobiosis and Freezing-Tolerance:Adaptations That Facilitate the Establishment of Panagrolaimus Nematodes in Polar Habitats

<div><p>Anhydrobiotic animals can survive the loss of both free and bound water from their cells. While in this state they are also resistant to freezing. This physiology adapts anhydrobiotes to harsh environments and it aids their dispersal. <i>Panagrolaimus davidi</i>, a bacterial feeding anhydrobiotic nematode isolated from Ross Island Antarctica, can survive intracellular ice formation when fully hydrated. A capacity to survive freezing while fully hydrated has also been observed in some other Antarctic nematodes. We experimentally determined the anhydrobiotic and freezing-tolerance phenotypes of 24 <i>Panagrolaimus</i> strains from tropical, temperate, continental and polar habitats and we analysed their phylogenetic relationships. We found that several other <i>Panagrolaimus</i> isolates can also survive freezing when fully hydrated and that tissue extracts from these freezing-tolerant nematodes can inhibit the growth of ice crystals. We show that <i>P. davidi</i> belongs to a clade of anhydrobiotic and freezing-tolerant panagrolaimids containing strains from temperate and continental regions and that <i>P. superbus</i>, an early colonizer at Surtsey island, Iceland after its volcanic formation, is closely related to a species from Pennsylvania, USA. Ancestral state reconstructions show that anhydrobiosis evolved deep in the phylogeny of <i>Panagrolaimus</i>. The early-diverging <i>Panagrolaimus</i> lineages are strongly anhydrobiotic but weakly freezing-tolerant, suggesting that freezing tolerance is most likely a derived trait. The common ancestors of the <i>davidi</i> and the <i>superbus</i> clades were anhydrobiotic and also possessed robust freezing tolerance, along with a capacity to inhibit the growth and recrystallization of ice crystals. Unlike other endemic Antarctic nematodes, the life history traits of <i>P. davidi</i> do not show evidence of an evolved response to polar conditions. Thus we suggest that the colonization of Antarctica by <i>P. davidi</i> and of Surtsey by <i>P. superbus</i> may be examples of recent “ecological fitting” of freezing-tolerant anhydrobiotic propagules to the respective abiotic conditions in Ross Island and Surtsey.</p></div

A molecular analysis of desiccation tolerance mechanisms in the anhydrobiotic nematode Panagrolaimus superbus using expressed sequenced tags

<p>Abstract</p> <p>Background</p> <p>Some organisms can survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. <it>Panagrolaimus superbus </it>is a free-living anhydrobiotic nematode that can survive rapid environmental desiccation. The mechanisms that <it>P. superbus </it>uses to combat the potentially lethal effects of cellular dehydration may include the constitutive and inducible expression of protective molecules, along with behavioural and/or morphological adaptations that slow the rate of cellular water loss. In addition, inducible repair and revival programmes may also be required for successful rehydration and recovery from anhydrobiosis.</p> <p>Results</p> <p>To identify constitutively expressed candidate anhydrobiotic genes we obtained 9,216 ESTs from an unstressed mixed stage population of <it>P. superbus</it>. We derived 4,009 unigenes from these ESTs. These unigene annotations and sequences can be accessed at <url>http://www.nematodes.org/nembase4/species_info.php?species=PSC</url>. We manually annotated a set of 187 constitutively expressed candidate anhydrobiotic genes from <it>P. superbus</it>. Notable among those is a putative lineage expansion of the <it>lea </it>(late embryogenesis abundant) gene family. The most abundantly expressed sequence was a member of the nematode specific <it>sxp/ral-2 </it>family that is highly expressed in parasitic nematodes and secreted onto the surface of the nematodes' cuticles. There were 2,059 novel unigenes (51.7% of the total), 149 of which are predicted to encode intrinsically disordered proteins lacking a fixed tertiary structure. One unigene may encode an exo-β-1,3-glucanase (GHF5 family), most similar to a sequence from <it>Phytophthora infestans</it>. GHF5 enzymes have been reported from several species of plant parasitic nematodes, with horizontal gene transfer (HGT) from bacteria proposed to explain their evolutionary origin. This <it>P. superbus </it>sequence represents another possible HGT event within the Nematoda. The expression of five of the 19 putative stress response genes tested was upregulated in response to desiccation. These were the antioxidants <it>glutathione peroxidase, dj-1 </it>and <it>1-Cys peroxiredoxin</it>, an <it>shsp </it>sequence and an <it>lea </it>gene.</p> <p>Conclusions</p> <p><it>P. superbus </it>appears to utilise a strategy of combined constitutive and inducible gene expression in preparation for entry into anhydrobiosis. The apparent lineage expansion of <it>lea </it>genes, together with their constitutive and inducible expression, suggests that LEA3 proteins are important components of the anhydrobiotic protection repertoire of <it>P. superbus</it>.</p