Ann M. Burnell

Anhydrobiosis: Plant desiccation gene found in a nematode

When subjected to drought conditions, some organisms enter a state of suspended animation known as anhydrobiosis, surviving for indefinite periods until rehydration allows them to resume normal metabolism. We have identified a gene in the anhydrobiotic nematode Aphelenchus avenae that is upregulated in response to desiccation stress and whose encoded protein shares sequence similarity with a late-embryonic gene that is induced in many plants when they are deprived of water. This finding suggests that animals and plants that undergo anhydrobiosis may use common protective strategies against dehydration, and provides a unifying insight into the mechanism of anhydrobiosis.

Heterorhabditis, steinernema and their bacterial symbionts : lethal pathogens of insects

The entomopathogenic nematodes (EPN) Heterorhabditis and Steinernema together with their symbiont bacteria Photorhabdus and Xenorhabdus, respectively, are obligate and lethal parasites of insects. EPN can provide effective biological control of some important lepidopteran, dipteran and coleopteran pests of commercial crops and they are amenable to large-scale culture in liquid fermentors. They are unique among rhabditids in having a symbiotic relationship with an enteric bacterium species. The bacterial symbiont is required to kill the insect host and to digest the host tissues, thereby providing suitable nutrient conditions for nematode growth and development. This review describes the general biology of EPN and their symbionts and gives an overview of studies to date on EPN biodiversity, biogeography and phylogeny. The impetus for research in EPN and their symbionts has come about because of their biological control potential, with much of the focus in EPN research having been on applied aspects relating to pest control. However EPN and their symbionts are increasingly being viewed as exciting subjects for basic research in the areas of ecology, biodiversity, evolution, biochemistry, symbiosis and molecular genetics. Much progress has been made over the past 20 years in our understanding of the basic biology and genetics of EPN and their symbionts. We are now entering a new phase in which the tools of molecular genetics are being increasingly used to address a range of biological questions in EPN research. The knowledge gained from this endeavour should ensure that EPN will become even more effective biopesticides and should also ensure that EPN and their symbionts gain prominence as unique and intrinsically interesting biological systems. Les nematodes entomopathogenes (EPN) Heterorhabditis et Steinernema, avec leur bacteries symbiotes Photorhabdus et Xenorhabdus, respectivement, sont des parasites obliges et mortels des insectes. Les EPN peuvent servir a un controle biologique de quelques lepidopteres, dipteres et coleopteres importants pour les cultures commerciales et ils sont elevables a grande echelle dans des fermenteurs liquides. Ils sont uniques chez les rhabditides par leur relation symbiotique avec une espece de bacterie enterique. La bacterie symbiote est necessaire pour tuer l’insecte hote et pour digerer les tissus de l’hote, permettant ainsi des conditons de nutrition favorables a la croissance et au developpement du nematode. La presente revue decrit la biologie generale des EPN et de leur symbiotes et donne un etat des etudes actuelles sur la biodiversite, la biogeographie et la phylogenie des EPN. L’impulsion donnee aux recherches sur les EPN et leur symbiotes provient de leur potentialites pour le controle biologique, une grande partie des recherches sur les EPN ayant trait a des aspects appliques en relation avec ce controle des parasites. Cependant, les EPN et leur symbiotes bacteriens sont de plus en plus consideres comme des sujets interessants pour la recherche fondamentale dans les domaines de l’ecologie, de la biodiversite, de l’evolution, de la biochimie, des processus symbiotiques et de la genetique moleculaire. De nombreux progres ont ete realises ces 20 dernieres annees dans la comprehension de la biologie et de la genetique des EPN et de leur symbiotes. Nous entrons actuellement dans une nouvelle phase ou les moyens de la biologie moleculaire sont utilises de maniere croissante pour formuler une serie de questions biologiques pour la recherche sur les EPN. Les connaissances resultant de ces efforts doivent conduire a verifier que les EPN deviendront des biopesticides toujours plus efficaces et que les EPN et leur symbiotes prendront de l’importance en tant que systemes biologiques uniques et intrinsequement interessants.

Dehydration-Specific Induction of Hydrophilic Protein Genes in the Anhydrobiotic Nematode Aphelenchus avenae

ABSTRACT 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.

Molecular Anhydrobiology: Identifying Molecules Implicated in Invertebrate Anhydrobiosis

Abstract 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.

An investigation of chemotaxis in the insect parasitic nematode Heterorhabditis bacteriophora

We tested the chemotactic responses of dauer juvenile stages (DJs) of the insect parasitic nematode Heterorhabditis bacteriophora to a variety of compounds that are known to be highly attractive or highly repellent to Caenorhabditis elegans. While H. bacteriophora DJs respond to alcohols and some aromatic compounds as well as to host metabolites such as uric acid and CO2, the most notable difference in the responses of these two nematodes is that H. bacteriophora DJs are unresponsive to a large number of compounds which C. elegans finds highly attractive. The latter compounds are typical by-products of bacterial metabolism and include aldehydes, esters, ketones and short-chain alcohols. While C. elegans finds long-chain alcohols (e.g. 1-heptanol and 1-octanol) repellent and short-chain alcohols highly attractive, H. bacteriophora DJs are strongly attracted to 1-heptanol, 1-octanol and 1-nonanol and find short-chain alcohols to be only slightly attractive. Parasitic-stage H. bacteriophora nematodes show a very weak chemotactic response to volatile molecules that DJs find highly attractive. Our results suggest that, associated with the adoption of a parasitic mode of life by Heterorhabditis, there was an adaptive change in chemotactic behaviour of the infective stages, resulting in a decreased sensitivity to volatile by-products of bacterial metabolism and an increased sensitivity towards long-chain alcohols and other insect-specific volatiles and possibly also to herbivore-induced plant volatiles.

Multiple lineage specific expansions within the guanylyl cyclase gene family

BackgroundGuanylyl 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.ResultsIn 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.ConclusionThe 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.

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

BackgroundSome 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.ResultsTo 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.ConclusionsThis 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.

Detection of changes occurring during recovery from the dauer stage in Heterorhabditis bacteriophora.

Nematodes of the genus Heterorhabditis are insect parasites that are widely used as biological control agents. When conditions are unfavourable for reproduction in H. bacteriophora, a long-lived, non-feeding, survival and dispersal stage, the dauer juvenile (DJ), is formed. This DJ stage is also adapted for host finding and infection. When it infects a suitable host, the DJ recovers and resumes growth and development. We describe a series of methods for improved detection of recovery in H. bacteriophora. We also describe some of the physiological changes that occur immediately after the onset of recovery in these nematodes as revealed using fluorescent nucleic acid binding SYTO dyes. Although recovery could be monitored using morphological changes, we found that observation of the uptake of fluorescent latex microspheres by recovering nematodes was a far more sensitive and efficient means of detecting recovery. SYTO dyes were also found to be useful indicators of recovery, binding to the pharyngeal glands and genital primordia as little as 3 h after the onset of recovery. The use of SYTO dyes also indicated that the pharyngeal glands produce large quantities of RNA following the onset of recovery, implying that these structures may produce proteins important in the infection and/or feeding process of H. bacteriophora.

Systemic RNAi mediated gene silencing in the anhydrobiotic nematode Panagrolaimus superbus

BackgroundGene 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.ResultsWe 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.ConclusionThis 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.

Molecular characterisation of Heterorhabditis indica isolates from India, Kenya, Indonesia and Cuba

Summary - Isolates of Heterorhabditis were identiA½ ed as H. indica using the following molecular diagnostic features: hybridisation to a H. indica speciA½ c satellite DNA probe; AluI and MboI restriction proA½ les of the rDNA ITS PCR product and the AluI proA½ le of the rDNA IGS PCR product. The Kenyan isolates represent a distinct subgroup of H. indica. These isolates lacked one of the two Hinf I restriction sites which are present in the rDNA ITS product of all the other isolates tested and they also differed from other H. indica isolates in their rDNA IGS HaeIII restriction proA½ le. The Indian isolates are interfertile. The Kenyan isolates are interfertile but only one Kenyan isolate, Ki3, produced viable progeny when crossed with H. indica LN2. The four Indonesian isolates are interfertile, but only one Indonesian isolate (INA H1) produced viable hybrids when crossed with H. indica LN2. INA H1 was also interfertile with the Kenyan isolate Ki3.