Trees, fungi and bacteria: tripartite metatranscriptomics of a root microbiome responding to soil contamination.

BACKGROUND: One method for rejuvenating land polluted with anthropogenic contaminants is through phytoremediation, the reclamation of land through the cultivation of specific crops. The capacity for phytoremediation crops, such as Salix spp., to tolerate and even flourish in contaminated soils relies on a highly complex and predominantly cryptic interacting community of microbial life. METHODS: Here, Illumina HiSeq 2500 sequencing and de novo transcriptome assembly were used to observe gene expression in washed Salix purpurea cv. 'Fish Creek' roots from trees pot grown in petroleum hydrocarbon-contaminated or non-contaminated soil. All 189,849 assembled contigs were annotated without a priori assumption as to sequence origin and differential expression was assessed. RESULTS: The 839 contigs differentially expressed (DE) and annotated from S. purpurea revealed substantial increases in transcripts encoding abiotic stress response equipment, such as glutathione S-transferases, in roots of contaminated trees as well as the hallmarks of fungal interaction, such as SWEET2 (Sugars Will Eventually Be Exported Transporter). A total of 8252 DE transcripts were fungal in origin, with contamination conditions resulting in a community shift from Ascomycota to Basidiomycota genera. In response to contamination, 1745 Basidiomycota transcripts increased in abundance (the majority uniquely expressed in contaminated soil) including major monosaccharide transporter MST1, primary cell wall and lamella CAZy enzymes, and an ectomycorrhiza-upregulated exo-ß-1,3-glucanase (GH5). Additionally, 639 DE polycistronic transcripts from an uncharacterised Enterobacteriaceae species were uniformly in higher abundance in contamination conditions and comprised a wide spectrum of genes cryptic under laboratory conditions but considered putatively involved in eukaryotic interaction, biofilm formation and dioxygenase hydrocarbon degradation. CONCLUSIONS: Fungal gene expression, representing the majority of contigs assembled, suggests out-competition of white rot Ascomycota genera (dominated by Pyronema), a sometimes ectomycorrhizal (ECM) Ascomycota (Tuber) and ECM Basidiomycota (Hebeloma) by a poorly characterised putative ECM Basidiomycota due to contamination. Root and fungal expression involved transcripts encoding carbohydrate/amino acid (C/N) dialogue whereas bacterial gene expression included the apparatus necessary for biofilm interaction and direct reduction of contamination stress, a potential bacterial currency for a role in tripartite mutualism. Unmistakable within the metatranscriptome is the degree to which the landscape of rhizospheric biology, particularly the important but predominantly uncharacterised fungal genetics, is yet to be discovered.

Prolyl 4-hydroxylase is an essential procollagen-modifying enzyme required for exoskeleton formation and the maintenance of body shape in the nematode Caenorhabditis elegans.

The multienzyme complex prolyl 4-hydroxylase catalyzes the hydroxylation of proline residues and acts as a chaperone during collagen synthesis in multicellular organisms. The beta subunit of this complex is identical to protein disulfide isomerase (PDI). The free-living nematode Caenorhabditis elegans is encased in a collagenous exoskeleton and represents an excellent model for the study of collagen biosynthesis and extracellular matrix formation. In this study, we examined prolyl 4-hydroxylase alpha-subunit (PHY; EC 1.14.11.2)- and beta-subunit (PDI; EC 5.3.4.1)-encoding genes with respect to their role in collagen modification and formation of the C. elegans exoskeleton. We identified genes encoding two PHYs and a single associated PDI and showed that all three are expressed in collagen-synthesizing ectodermal cells at times of maximal collagen synthesis. Disruption of the pdi gene via RNA interference resulted in embryonic lethality. Similarly, the combined phy genes are required for embryonic development. Interference with phy-1 resulted in a morphologically dumpy phenotype, which we determined to be identical to the uncharacterized dpy-18 locus. Two dpy-18 mutant strains were shown to have null alleles for phy-1 and to have a reduced hydroxyproline content in their exoskeleton collagens. This study demonstrates in vivo that this enzyme complex plays a central role in extracellular matrix formation and is essential for normal metazoan development.

Biochemical and structural characterization of a divergent loop cyclophilin from Caenorhabditis elegans.

Cyclophilin 3 (CYP-3) is one of the most abundantly expressed cyclophilin isoforms in the free living nematode Caenorhabditis elegans. The detailed post-embryonic expression pattern of the cyp-3 transcript is unusual, peaking during early larval development. The spatial expression pattern was examined via reporter gene analysis demonstrating that the cyp-3 transcript is exclusively expressed in the single anterior excretory cell. Recombinant cyclophilin 3 has been purified, crystallized and solved to a resolution of 1.8 A. The peptidyl-prolyl isomerase activity of CYP-3 has been characterized against the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and gives a k(cat)/K(m) value of 2.4 x 10(6) M(-1) s(-1). The immunosuppressive drug cyclosporin A binds and inhibits CYP-3 with an IC(50) value of 16 nM, comparable with the range of values found for human cyclophilin A. The x-ray structure shows that the overall fold and active site geometry is similar to other cyclophilin structures. There are however a number of distinctive features, and we use this structure and amino acid sequence alignment analysis to identify a subgroup of "divergent-loop cyclophilins". This subgroup has a number of uniquely conserved features: an additional loop between residues 48 and 54 (KSGKPLH); two cysteine residues (Cys(40) and Cys(168)) that are in close proximity but remain in the unoxidized form, and two other conserved residues, His(54) and Glu(83). We suggest that these features are functionally important for the role played by this class of cyclophilins during cellular responses to stress caused by changes in the redox environment or by up-regulation of cellular activity. This study represents a detailed biological, biochemical, and structural characterization of a single cyclophilin isoform in the model organism Caenorhabditis elegans.

A highly conserved nematode protein folding operon in Caenorhabditis elegans and Caenorhabditis briggsae.

In the free-living model nematode, Caenorhabditis elegans, a protein-folding co-transcribed gene pair has previously been described. The degree and form of trans-splicing, orientation and spacing of the genes, and the co-ordinate co-expression of protein folding catalysts in the nematode's hypodermis indicated this to be a functionally important operon. This gene pair has now been cloned and compared in the related organism Caenorhabditis briggsae to identify evolutionarily conserved, functionally important features. The corresponding C. briggsae gene pair was found to share the operon-specific features, including sequence homology blocks in the upstream 5' flanking regions. The intergenic regions were not conserved. The homology block closest to the translational initiation codon of the upstream gene was found to contain a known Ceanorhabbitis promoter element site, and may therefore be an important cis-regulatory region directing the hypodermis-specific expression of this operon gene of C. elegans. This study also provides further confirmation of the high degree of chromosomal synteny between these nematode species.

A divergent multi-domain cyclophilin is highly conserved between parasitic and free-living nematode species and is important in larval muscle development.

A divergent multi-domain cyclophilin from the filarial nematodes Brugia malayi, Onchocerca volvulus and Dirofilaria immitis has a highly conserved orthologue in the free-living nematodes Caenorhabditis elegans and C. briggsae. Cyclophilins are the receptors for the immunosuppressive and anti-parasitic agent cyclosporin A and additionally these ubiquitously expressed proteins have protein folding capabilities, and exhibit proline isomerase activity. These divergent nematode cyclophilins (CYP-4 isoforms) are three domain proteins, which share 63-88% identity and have highly conserved differences present in their functionally important cyclosporin A binding and proline isomerase domains. This unusual class of nematode cyclophilins has been studied in the model nematode C. elegans, revealing a unique temporal and spatial expression pattern. The cyp-4 transcript is most abundantly expressed in the early larval stages and is expressed exclusively in the body-wall striated muscle cells. An important functional role was established for this divergent enzyme, as specific double-stranded RNA interference experiments resulted in progeny with a phenotypically lumpy appearance. This morphological defect was predominantly expressed in the early larval stages and is consistent with an effect on body-wall muscle cell development. This study has established that this highly conserved family of nematode cyclophilins has a tissue-specific, functional role in early larval development and supports the use of C. elegans as a model for the study of orthologues in the experimentally less amenable parasitic nematodes.

The X-ray structure of a divergent cyclophilin from the nematode parasite Brugia malayi.

A structure of residues 1-177 of the cyclophilin domain of a large divergent cyclophilin from the filarial nematode parasite Brugia malayi has been crystallised and solved in two different crystal forms. The active site has a similar structure to that of human cyclophilin A. Two of the 13 residues important in forming the human cyclophilin A/cyclosporin A complex are altered in the B. malayi cyclophilin and explain the relatively poor inhibition of peptidyl prolyl isomerase activity by cyclosporin A.

Cyclophilin and protein disulfide isomerase genes are co-transcribed in a functionally related manner in Caenorhabditis elegans.

The ubiquitous enzymes peptidyl prolyl cis-trans isomerase (PPI, EC 5.2.1.8) and protein disulfide isomerase (PDI, EC 5.3.4.1) are important rate-limiting catalysts of protein-folding events in the cell. In the free-living nematode Caenorhabditis elegans, two genes encoding these enzymes (cyp-9 and pdi-1, respectively) are clustered together on chromosome III. In work described elsewhere, the encoded enzymes have been expressed as recombinant proteins and have been determined to possess in vitro PPI and PDI activity. Taken together, this organization of the two genes and the related functions of their transcripts indicate that they may be cotranscribed as a polycistronic unit, similar to bacterial operons. This study details the very close linkage of pdi-1 and cyp-9, which are in the same orientation. pdi-1 is the upstream gene, and the putative polyadenylation cleavage signal of this gene is separated from the trans-splice acceptor site of cyp-9 by only 103 bp. pdi-1 is trans-spliced by the ubiquitous nematode trans-spliced leader SL1, whereas cyp-9 was found to be predominantly trans-spliced by the "operon-specific" trans-spliced leader SL2. Similar trends in relative transcript abundance were demonstrated with synchronously produced mRNA for both genes during larval development, supporting the contention that the genes are co-expressed. Finally, reporter gene analysis provides strong evidence that both genes are controlled by a single upstream regulatory element, which directs expression of both enzymes in the hypodermal cells that synthesize the cuticle.

Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis elegans and characterization of prolyl 4-hydroxylases containing one of these polypeptides as their beta subunit.

Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional polypeptide that is identical to the beta subunit of prolyl 4-hydroxylases. We report here on the cloning and expression of the Caenorhabditis elegans PDI/beta polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. elegans PDI/beta polypeptides are 61% and 85% respectively, and those between the C. elegans PDI/beta polypeptide and the PDI isoform 46% and 73%. The isoform differs from the PDI/beta and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/beta polypeptide forms an active prolyl 4-hydroxylase alpha 2 beta 2 tetramer with the human alpha subunit and an alpha beta dimer with the C. elegans alpha subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either alpha subunit. Removal of the 32-residue C-terminal extension from the C. elegans alpha subunit totally eliminated alpha beta dimer formation. The C. elegans PDI/beta polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans alpha subunits than did the human PDI/beta polypeptide, being particularly ineffective with the C. elegans alpha subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/beta polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/beta polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase alpha beta dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the K(m) for the hydroxylation of long polypeptide substrates.

Cloning and biochemical characterization of the cyclophilin homologues from the free-living nematode Caenorhabditis elegans.

Cyclosporin A (CsA) is the most widely used immunosuppressive agent, whose properties are exerted via an interaction with cyclophilin, resulting in down-regulation of signal-transduction events in the T-cell. Cyclophilin is identical with peptidylprolyl cis-trans isomerase (PPI; EC 5.2.1.8), an enzyme which catalyses the isomerization between the two proline conformations in proteins, thereby acting as a catalyst in protein-folding events. Several reports indicate that CsA has potent anti-parasitic activity, effective against both protozoan and helminth species. In order to understand the various biological roles that cyclophilins play we have initiated a study of these proteins in the genetically tractable nematode Caenorhabditis elegans. Here we describe the cloning and characterization of 11 cyclophilin genes (cyp-1 to -11) derived from this nematode; this is currently the greatest number of isoforms described in a single species. Southern blotting and physical mapping indicated that these genes are dispersed throughout the nematode genome. A high degree of conservation exists between several isoforms, which also share characteristics with the ubiquitous isoforms previously described. The remaining isoforms are divergent, having altered CsA-binding domains and additional non-cyclophilin domains, which may impart compartmental specificity. Ten of these isoforms have been expressed in Escherichia coli, and the resultant fusion proteins have been examined biochemically for PPI activity, which they all possess. Isomerase activity is highest in the conserved and lowest in divergent isoforms, perhaps indicating a more specific substrate for the latter. Analysis of the C. elegans cyp genes will provide answers as to the roles played by cyclophilins in protein folding and signal transduction.

Parasite cyclophilins and antiparasite activity of cyclosporin A.

Cyclosporin A (CsA) was initially developed as an immunosuppressive drug. In the past several years, it has been shown to possess antiparasite activity independent of the immune system. It is not known how the drug exerts these antiparasite effects, or why it is stage and/or species specific. The answers may lie in the enzymatic function of cyclophilins. The cyclophilins are a growing family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPiase) activity and bid CsA to varying degrees. PPiases have been shown to play a role in the folding of many essential proteins. Antony Page, Sanjay Kumar and Clotilde Carlow here review parasite cyclophilins and their association with CsA. The possible biological function of parasite cyclophilins and their potential role in future drug discovery are also discussed.

Molecular characterization of a cyclosporin A-insensitive cyclophilin from the parasitic nematode Brugia malayi.

The cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) activity and bind the immunosuppressive agent cyclosporin A (CsA) to varying degrees. We have isolated a cDNA clone encoding a novel cyclophilin from the human filarial parasite Brugia malayi. This gene possesses an N-terminal domain homologous to cyclophilins from diverse phyla (49-60% amino acid sequence identity) and a hydrophilic C-terminal domain. The cyclophilin domain was overexpressed in Escherichia coli and found to possess peptidyl-prolyl cis-trans isomerase (PPIase) activity, with a kcat/Km value of 7.9 x 10(6) M-1 s-1. A histidine residue in lieu of tryptophan in the highly conserved CsA-binding site suggests that B. malayi cyclophilin is more closely related to the cyclophilin-like proteins described recently from natural killer (NK) cells, plants, and the 40 kDa cyclophilins from mammals. In accordance with the histidine-containing CsA-binding domain, the B. malayi enzyme was relatively insensitive to inhibition by CsA, since an IC50 value of 860 nM (compared to 19 nM for human cyclophilin A) was determined.

An abundant, trans-spliced mRNA from Toxocara canis infective larvae encodes a 26-kDa protein with homology to phosphatidylethanolamine-binding proteins.

A full-length mRNA encoding a secreted 26-kDa antigen of infective larvae of the ascarid nematode parasite Toxocara canis has been identified. This was characterized as a 1,082-base pair clone highly abundant (0.8-1.9%) in cDNA prepared from infective stage larvae but absent from cDNA from adult male worms. Sequence analysis revealed an open reading frame corresponding to a hydrophilic 263-amino acid residue polypeptide with a 20-residue N-terminal signal peptide, indicating that it is secreted. The 5' end of the cDNA was isolated by polymerase chain reaction using a primer containing the nematode-spliced leader sequence, SL1, showing that the mRNA is trans-spliced. The molecular mass of the putative protein with the signal peptide removed is 26.01 kDa, and antibody to the recombinant protein expressed in bacterial vectors reacts with a similarly sized protein in T. canis excretory/secretory (TES) products. An identical sequence was obtained from a genomic clone isolated by expression screening with mouse antibody to TES. The 72 amino acid residues adjacent to the signal peptide form two homologous 36-residue motifs containing 6 cysteine residues; this motif is found also in the T. canis-secreted glycoprotein TES-120 and in genes of Caenorhabditis elegans. Sequence data base searches revealed significant similarity to 7 other sequences in a newly recognized gene family of phosphatidylethanolamine-binding proteins that includes yeast, Drosophila, rat, bovine, simian, and human genes and a representative from the filarial nematode Onchocerca volvulus. Assays with the T. canis recombinant 26-kDa protein expressed as a fusion with maltose-binding protein have confirmed phosphatidylethanolamine-binding specificity for this novel product.

Biosynthesis and glycosylation of serine/threonine-rich secreted proteins from Toxocara canis larvae.

Toxocara canis infective stage larvae continually produce excretory-secretory (TES) glycoproteins in long-term in vitro culture. The kinetics of synthesis and secretion were studied by metabolic labelling with radioactive [35S]methionine, [14C]serine and [14C]threonine. Maximal incorporation rates required overnight pre-incubation of parasites in medium depleted of the appropriate amino acid. Larvae rapidly incorporated isotope into their somatic tissues, but there was a minimum delay of 10 h before secretion of labelled antigens. Labelling with [14C]serine and [14C]threonine demonstrated a relative abundance of these amino acids in the major surface/secreted glycoproteins of this nematode (TES-32 and 120). Pulse-chase experiments suggested that TES-120 may be derived from a 58 kDa precursor, reflecting extensive posttranslational glycosylation. Inhibition of N-glycosylation with tunicamycin and digestion with N-glycanase provided evidence of N-glycosylation in the lower molecular weight ES components (TES-32, 55 and 70). These agents had no effect on the higher molecular weight components (TES-120 and 400) implying that for these molecules glycosylation is predominantly O-linked. The largest ES component (TES-400) was unusual, in incorporating serine and threonine but not methionine, and by exhibiting increased apparent molecular weight following pronase digestion; it is suggested that this molecule is a proteoglycan.

Lectin binding to secretory structures, the cuticle and the surface coat of Toxocara canis infective larvae.

Toxocara canis infective larvae are known to produce abundant glycosylated molecules which may be found associated with the surface or secreted into their environment. Using a range of fluorescein-conjugated and gold-conjugated lectins, the localization of particular carbohydrates was defined on the surface of live parasites, and internally at the ultrastructural level. Surface exposure of N-acetyl galactosamine and N-acetyl glucosamine was deduced by binding of FITC-conjugated Helix pomatia (HPA) and wheat-germ agglutinins (WGA). These sugars appear to be associated with a densely staining surface coat as conventional immuno-electron microscopy procedures dissipate this coat and reveal no surface binding site for these lectins. However, by using cryo-immuno-electron microscopical (C-IEM) techniques, the surface coat is retained and can be shown to bind WGA. The fluorescent lectins also revealed strong WGA binding to the secretory and amphidial pores, while the buccal opening and the cuticular alae bound HPA. Corresponding results were obtained at the ultra-structural level. Thus, HPA bound to the electron-dense area of the cuticle, areas of local cuticular thickening such as the alae and buccal labia, as well as to the oesophageal lumen. WGA also bound to the thickened cuticle of the alae and the buccal opening, but showed no reaction to either the electron-dense layer of the cuticle or the oesophageal lumen. Unlike HPA, WGA did bind specifically to the secretory column contents and the electron-dense regions of the lips associated with the chemosensory amphids. The compartmentalization of the sugars N-acetyl galactosamine and N-acetyl glucosamine, their sources and routes of surface expression and the possible association with the TES glycoprotein antigens are discussed.

Toxocara canis: a labile antigenic surface coat overlying the epicuticle of infective larvae.

An electron-dense coat covering the surface of Toxocara canis infective-stage larvae is described. This coat readily binds to cationized ferritin and ruthenium red, indicating a net negative charge and mucopolysaccharide content, and can be visualized by immuno-electron microscopy only if cryosectioning is employed. Monoclonal antibodies reactive to the surface of live larvae bind the surface coat but not the underlying cuticle in ultrathin cryosections. The surface coat is dissipated on exposure to ethanol, explaining the lack of surface reactivity of conventionally prepared immunoelectron microscopy sections of T. canis. Differential ethanol extraction of surface-iodinated larvae demonstrates that the major component associated with the coat is TES-120, a 120-kDa glycoprotein previously identified by surface iodination, which is also a dominant secreted product. The surface-labeled TES-70 glycoprotein is linked with a more hydrophobic stratum at the surface, while a prominent 32-kDa glycoprotein, TES-32, is more strongly represented within the cuticle itself. Antibody binding to the coat under physiological conditions results in the loss of the surface coat, but this process is arrested at 4 degrees C. This result gives a physical basis to earlier observations on the shedding of surface-bound antibodies by this parasite. An extracuticular surface coat has been demonstrated on Toxocara larvae prior to hatching from the egg and during all stages of in vitro culture, suggesting that it may play a role both in protecting the parasite on hatching in the gastrointestinal tract and on subsequent tissue invasion in evading host immune responses directed at surface antigens.

Toxocara canis: monoclonal antibodies to carbohydrate epitopes of secreted (TES) antigens localize to different secretion-related structures in infective larvae.

The major secreted glycoproteins of Toxocara canis larvae appear to be derived from two specialized organs within the nematode organism. Using immunogold electron microscopy, we have analyzed the binding patterns of a panel of monoclonal antibodies (Tcn-1 to Tcn-8) reactive with Toxocara excretory-secretory (TES) antigens. We find, first, that the esophageal gland and lumen are strongly reactive with monoclonals Tcn-4, Tcn-5, and Tcn-8, and because the posterior portion of the gut is closed, we hypothesize that products of this gland are released through the oral aperture. Second, a distinct anti-TES antibody (Tcn-2) localizes solely to the midbody secretory column, which opens onto the cuticle at a secretory pore. Thus, the secretory apparatus is probably functional in this stage of parasite as an important source of TES products. Only one monoclonal, Tcn-7, can bind to both esophageal and secretory structures. In addition, another antibody, Tcn-3, binds both to the epicuticle and to a TES antigen, but our data do not directly determine whether antigens located in the cuticle are subsequently released. Thus there are at least two, and possibly three, independent sources of TES antigens within Toxocara larvae.

Nematode surface coats: actively evading immunity.

The classical view of nematode parasites depicts their surface as the epicuticle, the outermost layer of a thick extracellular cuticle. However, many stages and species of nematode have been found to bear an electron-dense cuter envelope distinct from and distal to the epicuticle itself. In this review, Mark Blaxter and colleagues summarize some wide-ranging studies in both free-living and parasitic nematodes, and suggest that, in many cases, it is the surface coat rather than the cuticle that displays dynamic properties thought to be involved in immune evasion by parasites.

Comparison of isolates and species of Toxocara and Toxascaris by biosynthetic labelling of somatic and ES proteins from infective larvae.

Infective-stage larvae of three different isolates of Toxocara canis were intrinsically ([35S]methionine) labelled in culture, to determine the presence of similarities or differences in the somatic and ES antigens expressed between larvae derived from different hosts and different geographical regions. Two other closely related ascaridids, Toxascaris leonina which infects cats and dogs, and Toxocara vitulorum (Neoascaris vitulorum) which infects cattle, were also compared to T. canis larvae by this method. Overall comparisons were made by 1- and 2-dimensional electrophoresis, while immunological cross-reactivities between the different species were analysed by radio-immunoprecipitation. Our results show that extensive physicochemical characteristics are shared between T. canis isolates, both from different hosts and different geographical locations. A substantial overlap was revealed when T. canis and T. vitulorum antigens were compared, whereas Toxascaris was found to produce a distinct antigen profile: this result was independent of whether methionine- or Iodogen-labelled products were being considered. Antigen recognition by polyclonal antibodies raised to all three species and to the cat ascaridid Toxocara cati, revealed considerable cross-reactivities. The cross-reactions were especially prominent between the Toxocara species, a fact further substantiated when reactivity of T. canis ES-specific monoclonal antibodies were tested against T. leonina and T. vitulorum antigens. The ES antigens of T. leonina were not recognized by the T. canis monoclonals, whereas the majority of these antibodies precipitated antigens of T. vitulorum. One which did not react with T. vitulorum was monoclonal antibody Tcn 2, indicating its species-specific reactivity and therefore its potential for the specific diagnosis of human toxocariasis.