Hybridisation has shaped a recent radiation of grass-feeding aphids.

BACKGROUND: Aphids are common crop pests. These insects reproduce by facultative parthenogenesis involving several rounds of clonal reproduction interspersed with an occasional sexual cycle. Furthermore, clonal aphids give birth to live young that are already pregnant. These qualities enable rapid population growth and have facilitated the colonisation of crops globally. In several cases, so-called "super clones" have come to dominate agricultural systems. However, the extent to which the sexual stage of the aphid life cycle has shaped global pest populations has remained unclear, as have the origins of successful lineages. Here, we used chromosome-scale genome assemblies to disentangle the evolution of two global pests of cereals-the English (Sitobion avenae) and Indian (Sitobion miscanthi) grain aphids. RESULTS: Genome-wide divergence between S. avenae and S. miscanthi is low. Moreover, comparison of haplotype-resolved assemblies revealed that the S. miscanthi isolate used for genome sequencing is likely a hybrid, with one of its diploid genome copies closely related to S. avenae (~ 0.5% divergence) and the other substantially more divergent (> 1%). Population genomics analyses of UK and China grain aphids showed that S. avenae and S. miscanthi are part of a cryptic species complex with many highly differentiated lineages that predate the origins of agriculture. The complex consists of hybrid lineages that display a tangled history of hybridisation and genetic introgression. CONCLUSIONS: Our analyses reveal that hybridisation has substantially contributed to grain aphid diversity, and hence, to the evolutionary potential of this important pest species. Furthermore, we propose that aphids are particularly well placed to exploit hybridisation events via the rapid propagation of live-born "frozen hybrids" via asexual reproduction, increasing the likelihood of hybrid lineage formation.

First chromosome scale genomes of ithomiine butterflies (Nymphalidae: Ithomiini): Comparative models for mimicry genetic studies.

The ithomiine butterflies (Nymphalidae: Danainae) represent the largest known radiation of Müllerian mimetic butterflies. They dominate by number the mimetic butterfly communities, which include species such as the iconic neotropical Heliconius genus. Recent studies on the ecology and genetics of speciation in Ithomiini have suggested that sexual pheromones, colour pattern and perhaps hostplant could drive reproductive isolation. However, no reference genome was available for Ithomiini, which has hindered further exploration on the genetic architecture of these candidate traits, and more generally on the genomic patterns of divergence. Here, we generated high-quality, chromosome-scale genome assemblies for two Melinaea species, M. marsaeus and M. menophilus, and a draft genome of the species Ithomia salapia. We obtained genomes with a size ranging from 396 to 503 Mb across the three species and scaffold N50 of 40.5 and 23.2 Mb for the two chromosome-scale assemblies. Using collinearity analyses we identified massive rearrangements between the two closely related Melinaea species. An annotation of transposable elements and gene content was performed, as well as a specialist annotation to target chemosensory genes, which is crucial for host plant detection and mate recognition in mimetic species. A comparative genomic approach revealed independent gene expansions in ithomiines and particularly in gustatory receptor genes. These first three genomes of ithomiine mimetic butterflies constitute a valuable addition and a welcome comparison to existing biological models such as Heliconius, and will enable further understanding of the mechanisms of adaptation in butterflies.

Role of Acrostyle Cuticular Proteins in the Retention of an Aphid Salivary Effector.

To avoid the activation of plant defenses and ensure sustained feeding, aphids are assumed to use their mouthparts to deliver effectors into plant cells. A recent study has shown that effectors detected near feeding sites are differentially distributed in plant tissues. However, the precise process of effector delivery into specific plant compartments is unknown. The acrostyle, a cuticular organ located at the tip of maxillary stylets that transiently binds plant viruses via its stylin proteins, may participate in this specific delivery process. Here, we demonstrate that Mp10, a saliva effector released into the plant cytoplasm during aphid probing, binds to the acrostyles of Acyrthosiphon pisum and Myzus persicae. The effector probably interacts with Stylin-03 as a lowered Mp10-binding to the acrostyle was observed upon RNAi-mediated reduction in Stylin-03 production. In addition, Stylin-03 and Stylin-01 RNAi aphids exhibited changes in their feeding behavior as evidenced by electrical penetration graph experiments showing longer aphid probing behaviors associated with watery saliva release into the cytoplasm of plant cells. Taken together, these data demonstrate that the acrostyle also has effector binding capacity and supports its role in the delivery of aphid effectors into plant cells.

Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network.

In plants, nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NLR required for cell death (NRCs), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesised that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 165 bacterial, oomycete, nematode, and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among 5 of the identified suppressors, 1 cyst nematode protein and 1 oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network.

Chromosome-Scale Genome Assemblies of Aphids Reveal Extensively Rearranged Autosomes and Long-Term Conservation of the X Chromosome.

Chromosome rearrangements are arguably the most dramatic type of mutations, often leading to rapid evolution and speciation. However, chromosome dynamics have only been studied at the sequence level in a small number of model systems. In insects, Diptera and Lepidoptera have conserved genome structure at the scale of whole chromosomes or chromosome arms. Whether this reflects the diversity of insect genome evolution is questionable given that many species exhibit rapid karyotype evolution. Here, we investigate chromosome evolution in aphids-an important group of hemipteran plant pests-using newly generated chromosome-scale genome assemblies of the green peach aphid (Myzus persicae) and the pea aphid (Acyrthosiphon pisum), and a previously published assembly of the corn-leaf aphid (Rhopalosiphum maidis). We find that aphid autosomes have undergone dramatic reorganization over the last 30 My, to the extent that chromosome homology cannot be determined between aphids from the tribes Macrosiphini (Myzus persicae and Acyrthosiphon pisum) and Aphidini (Rhopalosiphum maidis). In contrast, gene content of the aphid sex (X) chromosome remained unchanged despite rapid sequence evolution, low gene expression, and high transposable element load. To test whether rapid evolution of genome structure is a hallmark of Hemiptera, we compared our aphid assemblies with chromosome-scale assemblies of two blood-feeding Hemiptera (Rhodnius prolixus and Triatoma rubrofasciata). Despite being more diverged, the blood-feeding hemipterans have conserved synteny. The exceptional rate of structural evolution of aphid autosomes renders them an important emerging model system for studying the role of large-scale genome rearrangements in evolution.

A chromosome-level genome assembly of the woolly apple aphid, Eriosoma lanigerum Hausmann (Hemiptera: Aphididae).

Woolly apple aphid (WAA, Eriosoma lanigerum Hausmann) (Hemiptera: Aphididae) is a major pest of apple trees (Malus domestica, order Rosales) and is critical to the economics of the apple industry in most parts of the world. Here, we generated a chromosome-level genome assembly of WAA-representing the first genome sequence from the aphid subfamily Eriosomatinae-using a combination of 10X Genomics linked-reads and in vivo Hi-C data. The final genome assembly is 327 Mb, with 91% of the assembled sequences anchored into six chromosomes. The contig and scaffold N50 values are 158 kb and 71 Mb, respectively, and we predicted a total of 28,186 protein-coding genes. The assembly is highly complete, including 97% of conserved arthropod single-copy orthologues based on Benchmarking Universal Single-Copy Orthologs (busco) analysis. Phylogenomic analysis of WAA and nine previously published aphid genomes, spanning four aphid tribes and three subfamilies, reveals that the tribe Eriosomatini (represented by WAA) is recovered as a sister group to Aphidini + Macrosiphini (subfamily Aphidinae). We identified syntenic blocks of genes between our WAA assembly and the genomes of other aphid species and find that two WAA chromosomes (El5 and El6) map to the conserved Macrosiphini and Aphidini X chromosome. Our high-quality WAA genome assembly and annotation provides a valuable resource for research in a broad range of areas such as comparative and population genomics, insect-plant interactions and pest resistance management.

Genome Sequence of the Banana Aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae) and Its Symbionts.

The banana aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae), is a major pest of cultivated bananas (Musa spp., order Zingiberales), primarily due to its role as a vector of Banana bunchy top virus (BBTV), the most severe viral disease of banana worldwide. Here, we generated a highly complete genome assembly of P. nigronervosa using a single PCR-free Illumina sequencing library. Using the same sequence data, we also generated complete genome assemblies of the P. nigronervosa symbiotic bacteria Buchnera aphidicola and Wolbachia To improve our initial assembly of P. nigronervosa we developed a k-mer based deduplication pipeline to remove genomic scaffolds derived from the assembly of haplotigs (allelic variants assembled as separate scaffolds). To demonstrate the usefulness of this pipeline, we applied it to the recently generated assembly of the aphid Myzus cerasi, reducing the duplication of conserved BUSCO genes by 25%. Phylogenomic analysis of P. nigronervosa, our improved M. cerasi assembly, and seven previously published aphid genomes, spanning three aphid tribes and two subfamilies, reveals that P. nigronervosa falls within the tribe Macrosiphini, but is an outgroup to other Macrosiphini sequenced so far. As such, the genomic resources reported here will be useful for understanding both the evolution of Macrosphini and for the study of P. nigronervosa. Furthermore, our approach using low cost, high-quality, Illumina short-reads to generate complete genome assemblies of understudied aphid species will help to fill in genomic black spots in the diverse aphid tree of life.

Complete Genome Sequence of "Candidatus Phytoplasma asteris" RP166, a Plant Pathogen Associated with Rapeseed Phyllody Disease in Poland.

The complete genome sequence of "Candidatus Phytoplasma asteris" RP166, which consists of one 829,546-bp circular chromosome, is presented in this work. This bacterium is associated with rapeseed phyllody disease in Poland and belongs to the 16SrI-B (i.e., aster yellows) group.

An aphid RNA transcript migrates systemically within plants and is a virulence factor.

Aphids are sap-feeding insects that colonize a broad range of plant species and often cause feeding damage and transmit plant pathogens, including bacteria, viruses, and viroids. These insects feed from the plant vascular tissue, predominantly the phloem. However, it remains largely unknown how aphids, and other sap-feeding insects, establish intimate long-term interactions with plants. To identify aphid virulence factors, we took advantage of the ability of the green peach aphid Myzus persicae to colonize divergent plant species. We found that a M. persicae clone of near-identical females established stable colonies on nine plant species of five representative plant eudicot and monocot families that span the angiosperm phylogeny. Members of the novel aphid gene family Ya are differentially expressed in aphids on the nine plant species and are coregulated and organized as tandem repeats in aphid genomes. Aphids translocate Ya transcripts into plants, and some transcripts migrate to distal leaves within several plant species. RNAi-mediated knockdown of Ya genes reduces M. persicae fecundity, and M. persicae produces more progeny on transgenic plants that heterologously produce one of the systemically migrating Ya transcripts as a long noncoding (lnc) RNA. Taken together, our findings show that beyond a range of pathogens, M. persicae aphids translocate their own transcripts into plants, including a Ya lncRNA that migrates to distal locations within plants, promotes aphid fecundity, and is a member of a previously undescribed host-responsive aphid gene family that operate as virulence factors.

Sex-specific changes in the aphid DNA methylation landscape.

Aphids present an ideal system to study epigenetics as they can produce diverse, but genetically identical, morphs in response to environmental stimuli. Here, using whole genome bisulphite sequencing and transcriptome sequencing of the green peach aphid (Myzus persicae), we present the first detailed analysis of cytosine methylation in an aphid and investigate differences in the methylation and transcriptional landscapes of male and asexual female morphs. We found that methylation primarily occurs in a CG dinucleotide (CpG) context and that exons are highly enriched for methylated CpGs, particularly at the 3' end of genes. Methylation is positively associated with gene expression, and methylated genes are more stably expressed than unmethylated genes. Male and asexual female morphs have distinct methylation profiles. Strikingly, these profiles are divergent between the sex chromosome and the autosomes; autosomal genes are hypomethylated in males compared to asexual females, whereas genes belonging to the sex chromosome, which is haploid in males, are hypermethylated. Overall, we found correlated changes in methylation and gene expression between males and asexual females, and this correlation was particularly strong for genes located on the sex chromosome. Our results suggest that differential methylation of sex-biased genes plays a role in aphid sexual differentiation.

Towards take-all control: a C-21ß oxidase required for acylation of triterpene defence compounds in oat.

Oats produce avenacins, antifungal triterpenes that are synthesized in the roots and provide protection against take-all and other soilborne diseases. Avenacins are acylated at the carbon-21 position of the triterpene scaffold, a modification critical for antifungal activity. We have previously characterized several steps in the avenacin pathway, including those required for acylation. However, transfer of the acyl group to the scaffold requires the C-21ß position to be oxidized first, by an as yet uncharacterized enzyme. We mined oat transcriptome data to identify candidate cytochrome P450 enzymes that may catalyse C-21ß oxidation. Candidates were screened for activity by transient expression in Nicotiana benthamiana. We identified a cytochrome P450 enzyme AsCYP72A475 as a triterpene C-21ß hydroxylase, and showed that expression of this enzyme together with early pathway steps yields C-21ß oxidized avenacin intermediates. We further demonstrate that AsCYP72A475 is synonymous with Sad6, a previously uncharacterized locus required for avenacin biosynthesis. sad6 mutants are compromised in avenacin acylation and have enhanced disease susceptibility. The discovery of AsCYP72A475 represents an important advance in the understanding of triterpene biosynthesis and paves the way for engineering the avenacin pathway into wheat and other cereals for control of take-all and other diseases.

Multiple circadian clock outputs regulate diel turnover of carbon and nitrogen reserves.

Plants accumulate reserves in the daytime to support growth at night. Circadian regulation of diel reserve turnover was investigated by profiling starch, sugars, glucose 6-phosphate, organic acids, and amino acids during a light-dark cycle and after transfer to continuous light in Arabidopsis wild types and in mutants lacking dawn (lhy cca1), morning (prr7 prr9), dusk (toc1, gi), or evening (elf3) clock components. The metabolite time series were integrated with published time series for circadian clock transcripts to identify circadian outputs that regulate central metabolism. (a) Starch accumulation was slower in elf3 and prr7 prr9. It is proposed that ELF3 positively regulates starch accumulation. (b) Reducing sugars were high early in the T-cycle in elf3, revealing that ELF3 negatively regulates sucrose recycling. (c) The pattern of starch mobilization was modified in all five mutants. A model is proposed in which dawn and dusk/evening components interact to pace degradation to anticipated dawn. (d) An endogenous oscillation of glucose 6-phosphate revealed that the clock buffers metabolism against the large influx of carbon from photosynthesis. (e) Low levels of organic and amino acids in lhy cca1 and high levels in prr7 prr9 provide evidence that the dawn components positively regulate the accumulation of amino acid reserves.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor.

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding.

A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction.

Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species.

BACKGROUND: The prevailing paradigm of host-parasite evolution is that arms races lead to increasing specialisation via genetic adaptation. Insect herbivores are no exception and the majority have evolved to colonise a small number of closely related host species. Remarkably, the green peach aphid, Myzus persicae, colonises plant species across 40 families and single M. persicae clonal lineages can colonise distantly related plants. This remarkable ability makes M. persicae a highly destructive pest of many important crop species. RESULTS: To investigate the exceptional phenotypic plasticity of M. persicae, we sequenced the M. persicae genome and assessed how one clonal lineage responds to host plant species of different families. We show that genetically identical individuals are able to colonise distantly related host species through the differential regulation of genes belonging to aphid-expanded gene families. Multigene clusters collectively upregulate in single aphids within two days upon host switch. Furthermore, we demonstrate the functional significance of this rapid transcriptional change using RNA interference (RNAi)-mediated knock-down of genes belonging to the cathepsin B gene family. Knock-down of cathepsin B genes reduced aphid fitness, but only on the host that induced upregulation of these genes. CONCLUSIONS: Previous research has focused on the role of genetic adaptation of parasites to their hosts. Here we show that the generalist aphid pest M. persicae is able to colonise diverse host plant species in the absence of genetic specialisation. This is achieved through rapid transcriptional plasticity of genes that have duplicated during aphid evolution.

An Immuno-Suppressive Aphid Saliva Protein Is Delivered into the Cytosol of Plant Mesophyll Cells During Feeding.

Herbivore selection of plant hosts and plant responses to insect colonization have been subjects of intense investigations. A growing body of evidence suggests that, for successful colonization to occur, (effector/virulence) proteins in insect saliva must modulate plant defense responses to the benefit of the insect. A range of insect saliva proteins that modulate plant defense responses have been identified, but there is no direct evidence that these proteins are delivered into specific plant tissues and enter plant cells. Aphids and other sap-sucking insects of the order Hemiptera use their specialized mouthparts (stylets) to probe plant mesophyll cells until they reach the phloem cells for long-term feeding. Here, we show, by immunogold-labeling of ultrathin sections of aphid feeding sites, that an immuno-suppressive aphid effector localizes in the cytoplasm of mesophyll cells near aphid stylets but not in cells further away from aphid feeding sites. In contrast, another aphid effector protein localizes in the sheaths composed of gelling saliva that surround the aphid stylets. Thus, insects deliver effectors directly into plant tissue. Moreover, different aphid effectors locate extracellularly in the sheath saliva or are introduced into the cytoplasm of plant cells. [Formula: see text] Copyright © 2016 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .

Complete Genome Sequence of "Candidatus Sulcia muelleri" ML, an Obligate Nutritional Symbiont of Maize Leafhopper (Dalbulus maidis).

"Candidatus Sulcia muelleri" is a symbiont of sap-feeding insects in the suborder Auchenorrhyncha. The strain "Ca. Sulcia muelleri" ML is associated with the maize leafhopper (Dalbulus maidis), collected in Brazil, which is a disease vector that affects corn production. Here, we report the complete genome sequence of this bacterium.

Investigation of terpene diversification across multiple sequenced plant genomes.

Plants produce an array of specialized metabolites, including chemicals that are important as medicines, flavors, fragrances, pigments and insecticides. The vast majority of this metabolic diversity is untapped. Here we take a systematic approach toward dissecting genetic components of plant specialized metabolism. Focusing on the terpenes, the largest class of plant natural products, we investigate the basis of terpene diversity through analysis of multiple sequenced plant genomes. The primary drivers of terpene diversification are terpenoid synthase (TS) "signature" enzymes (which generate scaffold diversity), and cytochromes P450 (CYPs), which modify and further diversify these scaffolds, so paving the way for further downstream modifications. Our systematic search of sequenced plant genomes for all TS and CYP genes reveals that distinct TS/CYP gene pairs are found together far more commonly than would be expected by chance, and that certain TS/CYP pairings predominate, providing signals for key events that are likely to have shaped terpene diversity. We recover TS/CYP gene pairs for previously characterized terpene metabolic gene clusters and demonstrate new functional pairing of TSs and CYPs within previously uncharacterized clusters. Unexpectedly, we find evidence for different mechanisms of pathway assembly in eudicots and monocots; in the former, microsyntenic blocks of TS/CYP gene pairs duplicate and provide templates for the evolution of new pathways, whereas in the latter, new pathways arise by mixing and matching of individual TS and CYP genes through dynamic genome rearrangements. This is, to our knowledge, the first documented observation of the unique pattern of TS and CYP assembly in eudicots and monocots.

Regulatory properties of ADP glucose pyrophosphorylase are required for adjustment of leaf starch synthesis in different photoperiods.

Arabidopsis (Arabidopsis thaliana) leaves synthesize starch faster in short days than in long days, but the mechanism that adjusts the rate of starch synthesis to daylength is unknown. To understand this mechanism, we first investigated whether adjustment occurs in mutants lacking components of the circadian clock or clock output pathways. Most mutants adjusted starch synthesis to daylength, but adjustment was compromised in plants lacking the GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F BOX1 components of the photoperiod-signaling pathway involved in flowering. We then examined whether the properties of the starch synthesis enzyme adenosine 5'-diphosphate-glucose pyrophosphorylase (AGPase) are important for adjustment of starch synthesis to daylength. Modulation of AGPase activity is known to bring about short-term adjustments of photosynthate partitioning between starch and sucrose (Suc) synthesis. We found that adjustment of starch synthesis to daylength was compromised in plants expressing a deregulated bacterial AGPase in place of the endogenous AGPase and in plants containing mutant forms of the endogenous AGPase with altered allosteric regulatory properties. We suggest that the rate of starch synthesis is in part determined by growth rate at the end of the preceding night. If growth at night is low, as in short days, there is a delay before growth recovers during the next day, leading to accumulation of Suc and stimulation of starch synthesis via activation of AGPase. If growth at night is fast, photosynthate is used for growth at the start of the day, Suc does not accumulate, and starch synthesis is not up-regulated.