Signatures of the Evolution of Parthenogenesis and Cryptobiosis in the Genomes of Panagrolaimid Nematodes.

Most animal species reproduce sexually and fully parthenogenetic lineages are usually short lived in evolution. Still, parthenogenesis may be advantageous as it avoids the cost of sex and permits colonization by single individuals. Panagrolaimid nematodes have colonized environments ranging from arid deserts to Arctic and Antarctic biomes. Many are obligatory meiotic parthenogens, and most have cryptobiotic abilities, being able to survive repeated cycles of complete desiccation and freezing. To identify systems that may contribute to these striking abilities, we sequenced and compared the genomes and transcriptomes of parthenogenetic and outcrossing panagrolaimid species, including cryptobionts and non-cryptobionts. The parthenogens are triploids, most likely originating through hybridization. Adaptation to cryptobiosis shaped the genomes of panagrolaimid nematodes and is associated with the expansion of gene families and signatures of selection on genes involved in cryptobiosis. All panagrolaimids have acquired genes through horizontal gene transfer, some of which are likely to contribute to cryptobiosis.

Anhydrobiosis and freezing-tolerance: adaptations that facilitate the establishment of Panagrolaimus nematodes in polar habitats.

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. Panagrolaimus davidi, 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 Panagrolaimus strains from tropical, temperate, continental and polar habitats and we analysed their phylogenetic relationships. We found that several other Panagrolaimus 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 P. davidi belongs to a clade of anhydrobiotic and freezing-tolerant panagrolaimids containing strains from temperate and continental regions and that P. superbus, 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 Panagrolaimus. The early-diverging Panagrolaimus lineages are strongly anhydrobiotic but weakly freezing-tolerant, suggesting that freezing tolerance is most likely a derived trait. The common ancestors of the davidi and the superbus 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 P. davidi do not show evidence of an evolved response to polar conditions. Thus we suggest that the colonization of Antarctica by P. davidi and of Surtsey by P. superbus may be examples of recent "ecological fitting" of freezing-tolerant anhydrobiotic propagules to the respective abiotic conditions in Ross Island and Surtsey.

A role for the Parkinson's disease protein DJ-1 as a chaperone and antioxidant in the anhydrobiotic nematode Panagrolaimus superbus.

Mutations in the human DJ-1/PARK7 gene are associated with familial Parkinson's disease. DJ-1 belongs to a large, functionally diverse family with homologues in all biological kingdoms. Several activities have been demonstrated for DJ-1: an antioxidant protein, a redox-regulated molecular chaperone and a modulator of multiple cellular signalling pathways. The majority of functional studies have focussed on human DJ-1 (hDJ-1), but studies on DJ-1 homologues in Drosophila melanogaster, Caenorhabditis elegans, Dugesia japonica and Escherichia coli also provide evidence of a role for DJ-1 as an antioxidant. Here, we show that dehydration is a potent inducer of a dj-1 gene in the anhydrobiotic nematode Panagrolaimus superbus. Our secondary structure and homology modelling analyses shows that recombinant DJ-1 protein from P. superbus (PsuDJ-1.1) is a well-folded protein, which is similar in structure to the hDJ-1. PsuDJ-1.1 is a heat stable protein; with T1/2 unfolding transition values of 76 and 70 °C obtained from both circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) measurements respectively. We found that PsuDJ-1.1 is an efficient antioxidant that also functions as a 'holdase' molecular chaperone that can maintain its chaperone function in a reducing environment. In addition to its chaperone activity, PsuDJ-1.1 may also be an important non-enzymatic antioxidant, capable of providing protection to P. superbus from oxidative damage when the nematodes are in a desiccated, anhydrobiotic state.

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

BACKGROUND: Some organisms can survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. Panagrolaimus superbus is a free-living anhydrobiotic nematode that can survive rapid environmental desiccation. The mechanisms that P. superbus 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. RESULTS: To identify constitutively expressed candidate anhydrobiotic genes we obtained 9,216 ESTs from an unstressed mixed stage population of P. superbus. We derived 4,009 unigenes from these ESTs. These unigene annotations and sequences can be accessed at http://www.nematodes.org/nembase4/species_info.php?species=PSC. We manually annotated a set of 187 constitutively expressed candidate anhydrobiotic genes from P. superbus. Notable among those is a putative lineage expansion of the lea (late embryogenesis abundant) gene family. The most abundantly expressed sequence was a member of the nematode specific sxp/ral-2 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-ß-1,3-glucanase (GHF5 family), most similar to a sequence from Phytophthora infestans. 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 P. superbus 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 glutathione peroxidase, dj-1 and 1-Cys peroxiredoxin, an shsp sequence and an lea gene. CONCLUSIONS: P. superbus appears to utilise a strategy of combined constitutive and inducible gene expression in preparation for entry into anhydrobiosis. The apparent lineage expansion of lea genes, together with their constitutive and inducible expression, suggests that LEA3 proteins are important components of the anhydrobiotic protection repertoire of P. superbus.

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.

Systemic RNAi mediated gene silencing in the anhydrobiotic nematode Panagrolaimus superbus.

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

Gene induction by desiccation stress in the entomopathogenic nematode Steinernema carpocapsae reveals parallels with drought tolerance mechanisms in plants.

The dauer juvenile (DJ) stage of the insect parasitic nematode Steinernema carpocapsae is the only stage in the life cycle which is capable of surviving outside its host and it is adapted for tolerating environmental stresses and for host finding. We have isolated 45 unique expressed sequence tags (ESTs) that are up-regulated in response to desiccation in S. carpocapsae DJs. The majority of these ESTs were co-expressed in response to desiccation and osmotic stress and were generally not induced in response to heat and cold stress. Thirty-two ESTs showed similarity to known sequences. Among these were sequences which encode putative signalling molecules or transcription factors, sequences which detoxify reactive oxygen species, two C-type lectin sequences, ESTs which encode membrane-associated proteins and seven distinct late embryogenic abundant (LEA) sequences. We also isolated 13 novel ESTs. These data show that the molecular response to desiccation stress in entomopathogenic nematode DJs is complex and parallels many of the adaptive changes which occur in drought tolerant plants during exposure to desiccation and osmotic stress. A notable feature of the desiccation response of plants is the number and diversity of hydrophilic LEA proteins synthesised in response to desiccation. All of the LEA sequences detected in animals to date, including those reported in this study, belong to LEA3 group. We show that S. carpocapsae expresses several novel sequences which encode putative hydrophilic and natively unfolded proteins. It is likely that these novel and putative proteins play an important role in desiccation tolerance, possibly by carrying out analogous roles in nematodes to those carried out by the other LEA protein classes in plants.

The molecular phylogeny of a nematode-specific clade of heterotrimeric G-protein alpha-subunit genes.

In animal olfactory systems, odorant molecules are detected by olfactory receptors (ORs). ORs are part of the G-protein-coupled receptor (GPCR) superfamily. Heterotrimeric guanine nucleotide binding G-proteins (G-proteins) relay signals from GPCRs to intracellular effectors. G-proteins are comprised of three peptides. The G-protein alpha subunit confers functional specificity to G-proteins. Vertebrate and insect Galpha-subunit genes are divided into four subfamilies based on functional and sequence attributes. The nematode Caenorhabditis elegans contains 21 Galpha genes, 14 of which are exclusively expressed in sensory neurons. Most individual mammalian cells express multiple distinct GPCR gene products, however, individual mammalian and insect olfactory neurons express only one functional odorant OR. By contrast C. elegans expresses multiple ORs and multiple Galpha subunits within each olfactory neuron. Here we show that, in addition to having at least one member of each of the four mammalian Galpha gene classes, C. elegans and other nematodes also possess two lineage-specific Galpha gene expansions, homologues of which are not found in any other organisms examined. We hypothesize that these novel nematode-specific Galpha genes increase the functional complexity of individual chemosensory neurons, enabling them to integrate odor signals from the multiple distinct ORs expressed on their membranes. This neuronal gene expansion most likely occurred in nematodes to enable them to compensate for the small number of chemosensory cells and the limited emphasis on cephalization during nematode evolution.

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 O2. This suggests that the primordial eukaryotes probably utilized sGC as an O2 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.

Alternate metabolism during the dauer stage of the nematode Caenorhabditis elegans.

When environmental conditions are unsuitable to support nematode reproduction, Caenorhabditis elegans arrests development before the onset of sexual maturity and specialised 'dauer' larvae, adapted for dispersal, and extended diapause are formed. Dauer larvae do not feed and their metabolism is dependent on internal food reserves. Adult worms which express defects in the insulin/insulin-like growth factor receptor DAF-2 also display enhanced longevity. Whole genome mRNA expression profiling has demonstrated that C. elegans dauer larvae and daf-2 adults have similar transcription profiles for a cohort of longevity genes. Important components of this enhanced longevity system are the alpha-crystallin family of small heat shock proteins, anti-ROS defence systems, increased activity of cellular detoxification processes and possibly also increased chromatin stability and decreased protein turnover. Anaerobic fermentation pathways are upregulated in dauer larvae, while long-lived daf-2 adults appear to have normal oxidative metabolism. Anabolic pathways are down regulated in dauer larvae (and possibly in daf-2 adults as well), and energy consumption appears to be diverted to enhanced cellular maintenance and detoxification processes in both systems.

The anhydrobiotic potential and molecular phylogenetics of species and strains of Panagrolaimus (Nematoda, Panagrolaimidae).

Members of the genus Panagrolaimus are bacterial-feeding nematodes that occupy a diversity of niches ranging from Antarctic and temperate soils to terrestrial mosses. Some members of this genus are able to survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. We have assembled a collection of Panagrolaimus species and strains and have investigated their anhydrobiotic phenotypes. Our data show that within the genus Panagrolaimus there is a continuum of strains ranging from those unable to survive exposure to low relative humidity (RH) without prior preconditioning at high RH (slow desiccation strategists), through strains that have limited ability to survive rapid desiccation but whose anhydrobiotic ability improves upon preconditioning, to strains such as P. superbus that can readily survive immediate exposure to severe desiccation (fast desiccation strategists). Using this panel of nematodes we investigated the effect of preincubation at high RH on the accumulation of trehalose and on the nematodes' anhydrobiotic potential. We found that there is a strong correlation between trehalose induction and anhydrobiotic survival in Panagrolaimus. Furthermore, the high trehalose levels observed in fully hydrated P. superbus (10% dry mass) suggest that constitutive expression of trehalose pre-adapts this fast dehydration strategist to combat desiccation. All the strains observed, regardless of survival rates, undertook both coiling and clumping, which has the effect of reducing surface area and slowing the rate of water loss during desiccation. Phylogenetic analyses were carried out to investigate whether the observed anhydrobiotic phenotypes were the result of convergent evolution or represented a single phylogenetic lineage. These analyses, derived from alignments of the rDNA ITS and D3 sequences, indicate that the strongly anhydrobiotic strains of Panagrolaimus form a single phylogenetic lineage, which is separate from the weakly anhydrobiotic strains. The weakly anhydrobiotic strains are also phylogenetically divergent from each other. Our data indicate that Panagrolaimus has the potential to be an excellent model system for the investigation of molecular aspects of nematode anhydrobiosis.

Molecular anhydrobiology: identifying molecules implicated in invertebrate anhydrobiosis.

Studies in anhydrobiotic plants have defined many genes which are upregulated during desiccation, but comparable studies in invertebrates are at an early stage. To develop a better understanding of invertebrate anhydrobiosis, we have begun to characterise dehydration-inducible genes and their proteins in anhydrobiotic nematodes and bdelloid rotifers; this review emphasises recent findings with a hydrophilic nematode protein. Initial work with the fungivorous nematode Aphelenchus avenae led to the identification of two genes, both of which were markedly induced on slow drying (90-98% relative humidity, 24 hr) and also by osmotic stress, but not by heat or cold or oxidative stresses. The first of these genes encodes a novel protein we have named anhydrin; it is a small, basic polypeptide, with no counterparts in sequence databases, which is predicted to be natively unstructured and highly hydrophilic. The second is a member of the Group 3 LEA protein family; this and other families of LEA proteins are widely described in plants, where they are most commonly associated with the acquisition of desiccation tolerance in maturing seeds. Like anhydrin, the nematode LEA protein, Aav-LEA-1, is highly hydrophilic and a recombinant form has been shown to be unstructured in solution. In vitro functional studies suggest that Aav-LEA-1 is able to stabilise other proteins against desiccation-induced aggregation, which is in keeping with a role of LEA proteins in anhydrobiosis. In vivo, however, Aav-LEA-1 is apparently processed into smaller forms during desiccation. A processing activity was found in protein extracts of dehydrated, but not hydrated, nematodes; these shorter polypeptides are also active anti-aggregants and we hypothesise that processing LEA protein serves to increase the number of active molecules available to the dehydrating animal. Other LEA-like proteins are being identified in nematodes and it seems likely therefore that they will play a major role in the molecular anhydrobiology of invertebrates, as they are thought to do in plants.

Dehydration-specific induction of hydrophilic protein genes in the anhydrobiotic nematode Aphelenchus avenae.

Some organisms can survive exposure to extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living nematode Aphelenchus avenae can be induced to enter the anhydrobiotic state by exposure to a moderate reduction in relative humidity. During this preconditioning period, the nematode accumulates large amounts of the disaccharide trehalose, which is thought to be necessary, but not sufficient, for successful anhydrobiosis. To identify other adaptations that are required for anhydrobiosis, we developed a novel SL1-based mRNA differential display technique to clone genes that are upregulated by dehydration in A. avenae. Three such genes, Aav-lea-1, Aav-ahn-1, and Aav-glx-1, encode, respectively, a late embryogenesis abundant (LEA) group 3 protein, a novel protein that we named anhydrin, and the antioxidant enzyme glutaredoxin. Strikingly, the predicted LEA and anhydrin proteins are highly hydrophilic and lack significant secondary structure in the hydrated state. The dehydration-induced upregulation of Aav-lea-1 and Aav-ahn-1 was confirmed by Northern hybridization and quantitative PCR experiments. Both genes were also upregulated by an osmotic upshift, but not by cold, heat, or oxidative stress. Experiments to investigate the relationship between mRNA levels and protein expression for these genes are in progress. LEA proteins occur commonly in plants, accumulating during seed maturation and desiccation stress; the presence of a gene encoding an LEA protein in an anhydrobiotic nematode suggests that some mechanisms of coping with water loss are conserved between plants and animals.

Morphological characterisation of three isolates of Heterorhabditis Poinar, 1976 from the 'Irish group' (Nematoda: Rhabditida: Heterorhabditidae) and additional evidence supporting their recognition as a distinct species, H.downesi n.sp.

The morphological variation of three representative isolates of the 'Irish group' of Heterorhabditis was examined. First generation hermaphrodites were characterised by having a blunt and mucronate tail. Females (second generation) and third-stage infective juveniles were also distinguished by the morphology of the tail and the presence of a refractile projection in the tail tip. Males were characterised by the position of the excretory pore and by the value of ratio SW. These morphological features do not fit the description of currently recognised Heterorhabditis species, and provide additional evidence in support for the consideration of the Irish group as a new species. A description of this species, as H. downesi n. sp., is provided.