BMC Biology

BackgroundBiological methylation requires S-adenosylmethionine (SAM) and participates in a range of processes from modulation of gene expression via histone modifications to neurotransmitter synthesis. An important factor in all methylation reactions is the concentration ratio of SAM to methylation byproduct S-adenosylhomocysteine (SAH). SAH hydrolase, also known as adenosylhomocysteinase, depletes SAH and thereby facilitates metabolite recycling and maintains the methylation permissive SAM/SAH ratio. While the importance of SAH hydrolase in sustaining methylation is obvious on the cellular level, the function of this metabolic process on the organismal scale is not clear. ResultsWe used planarian Dugesia japonica to investigate the role SAH hydrolase in physiological homeostasis on the body-wide scale. Remarkably, pharmacological inhibition of the SAH hydrolase results in regression of anterior tissues and is accompanied by extensive apoptosis throughout the planarian body. Moreover, exposure to the SAHH inhibitor AdOx leads to changes in brain morphology and spatial shift in the expression of Wnt-modulator Notum. Strikingly, planarians are able to overcome these destructive patterning defects through regeneration of the anterior tissues and adaptation to the used inhibitor. Transcriptome analysis indicates that resistance to the SAHH inhibitor is at least partly mediated by changes in folate cycle and lipid metabolism. ConclusionsSAH hydrolase plays a critical role in planarian homeostasis and anterior patterning potentially through modulation of Wnt signaling. Moreover, planarian adaptation to the SAHH inhibitor via metabolic reprogramming suggests potential targets for addressing methylation-related human conditions.

Jellyfish have complex life-cycles, but there has been limited exploration of how this is achieved at the cellular level. We used single-cell transcriptomics to assemble a cell atlas for the planula larva of Clytia hemisphaerica, and compared it to an updated cell atlas for the medusa (jellyfish) stage. The cells of the planula fell into the same broad categories as for the medusa: ectoderm, gastroderm, interstitial cells (i-cells), nematocytes (stinging cells), neurons and secretory cells. Although the planula cells generally showed less diversity than medusae within each category, cells with specialized features unique to their stage could be distinguished by their transcriptional profiles as well as by ultrastructure. Some planula-specific types were identified: aboral secretory cells involved in settlement, and a cell type attributed a role in immunity or post-metamorphic theca production. Distinct transcriptome profiles within different regions of the ciliated planula ectoderm reflected different post-metamorphosis fates of domains along the oral-aboral axis. Inspection of the cell clusters showing significant similarity of marker genes between planula and medusa, and inference of similarity using a statistical model of marker gene presence/absence, revealed correspondences between families of cells from planula and medusa rather than precise cell identities.

BackgroundThe polychaete Capitella teleta is a commonly used annelid for studies in evolutionary developmental biology, comparative genomics, conservation, and ecotoxicology. Over a decade ago, it was the first polychaete to have its genome sequenced and assembled, contributing to pioneering studies that transformed our understanding of animal genomes and their evolution. However, this early resource is now outdated compared to current genome sequencing standards, limiting the use of modern functional genomic tools that could further our understanding of numerous biological processes. ResultsWe combine long-read and short-read sequencing with Hi-C chromatin conformation capture data to assemble the chromosome-level nuclear and mitochondrial genomes of the laboratory strain of C. teleta. This reference assembly more accurately reflects the expected genome size for this polychaete ([~]243.6 Mb) and contains a highly complete, evolutionarily conserved gene repertoire. Notably, the nuclear and mitochondrial genomes are heavily rearranged, indicating a decoupling between gene family repertoire and chromosomal evolution. The analyses of multi-omic datasets available for C. teleta, including developmental time courses of bulk and single-cell RNA-seq, ATAC-seq, and EM-seq, using the new reference assembly, resulted in a significant quality improvement, allowing us to identify new cell-type-specific gene markers and gain additional insights of biological relevance. Finally, we generated a publicly available genome browser that ensures all these resources are easily findable, accessible, interoperable, and reusable. ConclusionsOur study provides state-of-the-art genomic resources for the polychaete model C. teleta, addressing a pressing community need that will open new research opportunities in animal and genome evolution.

Planarians are organisms with a unique capacity to regenerate any part of their body. New tissues are generated in a process that requires many swift cell divisions. How costly is this process to an animal in terms of mutational load remains unknown. Using whole genome sequencing, we defined the mutational profile of the process of regeneration in the planarian species Schmidtea polychroa. We assembled de novo the genome of S. polychroa and analyzed mutations in animals that have undergone regeneration. We observed a threefold increase in the number of mutations and an altered mutational spectrum. High allele frequencies of subclonal mutations in regenerated animals suggested that relatively few stem cells with high expansion potential regenerated the animal. We provide, for the first time, the draft genome assembly of S. polychroa, an estimation of the germline mutation rate for a planarian species and the mutational spectrum of the regeneration process of a living organism.

The apical pole of eumetazoan ciliated larvae acts as a neurosensory structure and is principally composed of sensory-secretory cells. Cnidarians like the sea anemone Nematostella vectensis are the only non-bilaterian group to evolve ciliated larvae with a neural integrated sensory organ that is likely homologous to bilaterians. Here, we uncovered the molecular signature of the larval sensory organ in Nematostella by generating a transcriptome of the apical tissue. We characterised the cellular identity of the apical domain by integrating larval single-cell data with the apical transcriptome and further validated this through in-situ hybridisation. We discovered that the apical domain comprises a minimum of 6 distinct cell types, including apical cells, neurons, peripheral flask-shaped gland/secretory cells, and undifferentiated cells. By profiling the spatial expression of neuronal genes, we showed that the apical region has a unique neuronal signature distinct from the rest of the body. By combining the planula cilia proteome with the apical transcriptome data, we revealed the sheer complexity of the non-motile apical tuft. Overall, we present comprehensive spatial/molecular data on the Nematostella larval sensory organ and open new directions for elucidating the functional role of the apical organ and larval nervous system.

Neurons function by interacting with each other and with other cell types, often exerting organism-wide regulation. Serotonergic neurons play a systemic role in processes such as appetite, sleep and motor control. Functional studies in the planarian Schmidtea mediterranea have shown that impairment of serotonergic neurons results in systemic effects. Studying neurons and the tissues they interact with is challenging using either bulk or single-cell analysis techniques. While bulk methods merge the information from all cell types, single-cell methods show promise in overcoming this limitation. However, current single-cell approaches encounter other challenges including stress of cell dissociation, high cost, multiplexing capacity, batch effects, replication and statistical analysis. Here we used ACME and SPLiT-seq to generate a multiplex single-cell analysis of serotonergic neuron function in planarians by inhibiting pitx and lhx1/5-1, two transcription factors expressed in them. We recovered single-cell transcriptomic profiles of 47,292 cells from knockdown and control animals, including biological and technical replicates. Our results show that epidermal, muscular and the recently described parenchymal cell types are affected the most by serotonergic neuron impairment. By computationally dissecting each cell type, we elucidated gene expression changes in each, including changes in epidermis cilia genes and myofiber genes in muscle. Interestingly, parenchymal cells downregulate genes involved in neurotransmitter recycling, suggesting a glial-like function of these recently described enigmatic cell types. Our results will allow disentangling the complexity of serotonergic neuron inhibition by studying the downstream effectors and the affected tissues, and offer new data on the function of parenchymal cells in planarians. Ultimately, our results pave the way for dissecting complex phenotypes through multiplex single-cell transcriptomics.

BackgroundToll-like receptors (TLRs) play a crucial role in immunity and development. They contain leucine-rich repeat domains, one transmembrane domain, and one Toll/IL-1 receptor domain. TLRs have been classified into V-type/scc and P-type/mcc TLRs, based on differences in the leucine-rich repeat domain region. Although TLRs are widespread in animals, detailed phylogenetic studies of this gene family are lacking. Here we aim to uncover TLR evolution by conducting a survey and a phylogenetic analysis in species across Bilateria. To discriminate between their role in development and immunity we furthermore analyzed stage-specific transcriptomes of the ecdysozoans Priapulus caudatus and Hypsibius exemplaris, and the spiralians Crassostrea gigas and Terebratalia transversa. ResultsWe detected a low number of TLRs in ecdysozoan species, and multiple independent radiations within the Spiralia. V-type/scc and P-type/mcc type-receptors are present in cnidarians, protostomes and deuterostomes, and therefore they emerged early in TLR evolution, followed by a loss in xenacoelomorphs. Our phylogenetic analysis shows that TLRs cluster into three major clades: clade is present in cnidarians, ecdysozoans, and spiralians; clade {beta} in deuterostomes, ecdysozoans, and spiralians; and clade {gamma} is only found in spiralians. Our stage-specific transcriptome and in situ hybridization analyses show that TLRs are expressed during development in all species analyzed, which indicates a broad role of TLRs during animal development. ConclusionsOur findings suggest that the bilaterian TLRs likely emerged by duplication from a single TLR encoding gene (proto-TLR) present in the last common cnidarian-bilaterian ancestor. This proto-TLR gene duplicated before the split of protostomes and deuterostomes; a second duplication occurred in the lineage to the Trochozoa. While all three clades further radiated in several spiralian lineages, specific TLRs clades have been presumably lost in others. Furthermore, the expression of the majority of these genes during protostome ontogeny suggests their involvement in immunity and development.

Sex chromosomes impact chromatin organization and histone modification dynamics differently between males and females, particularly those involved in dosage compensation (DC). The stability of DC mechanisms may shape sex-specific phenotypes and traits such as lifespan. However, the tissue- and age-dependent variations as well as the evolutionary diversity of DC mechanisms are incompletely understood. Here, we investigate the occurrence of histone H4 lysine 16 acetylation (H4K16ac), previously known for its role in sex chromosome DC in the male-heterogametic fruit fly Drosophila melanogaster and the green anole lizard Anolis carolinensis. By sampling multiple arthropods, we find the convergent evolution of H4K16ac for DC in a female-heterogametic (ZW) species, the crustacean Artemia franciscana. CUT&Tag analysis demonstrates that H4K16ac is confined to the non-recombining stratum of the Z chromosome in females. H4K16ac-mediated DC is established during embryogenesis, sustained until adulthood, but then becomes reconfigured and variable in aged females. Interfering with acetylation alleviates male-female differences in lifespan. Our findings shed light on the evolutionary diversity of DC across species. They highlight histone acetylation as a sex-specific vulnerability linked to the chromatin landscape of sex chromosomes and its potential role in driving male-female differences.

Long-read RNA sequencing has great potential to improve genomic characterization of non-model organisms due to its ability to yield full-length genes. Coupled with absolute gene expression quantification, dynamics of development orchestrated at transcript level can be elucidated with high precision. The resolution of this precision can be further improved by studying organisms as close as possible to their basic entities, single cells for example or single embryos. Here, we collected developing embryos of the Mediterranean fruit fly (medfly, Ceratitis capitata) at hourly time-points for the first 15 hours of development. The medfly is an organism of huge economic importance in agriculture due to its wide host range including apples, pear, citrus, olives, etc. We simultaneously isolated total RNA and genomic DNA from single embryos and sexed the embryos using Y-specific PCR assays. The RNA, spiked with external ERCC standards to aid in absolute quantification, was used to perform Nanopore long-read RNA-seq. We developed a genome-guided transcriptome assembly based on full-length transcripts and identified a total of 22,875 transcripts comprising 3879 novel genes, missed in the current NCBI predicted gene models. We show that, indeed, the absolute quantification of gene expression performs superiorly to relative quantification in highly dynamic systems such as developing embryos. Further, we used unsupervised clustering and lineage tracing algorithms to group and accurately place embryos along a pseudo-temporal development trajectory. We show that medfly embryos undergo successive waves of zygotic genome activation. We discover a dramatic reorganization of maternally deposited mRNA occurring within the first 3 hours of egg laying followed by maternal-to-zygotic transition. We finally identify modules of temporal synexpression and elucidate the biological role of these modules. Together, these results provide the first detailed look at early embryo development in the medfly and should aid in future control efforts of this pest.

The larva of the ascidian Ciona intestinalis controls a small repertoire of behaviours with a simple nervous system in which each cell is identifiable. As such it offers the prospect of building a cohesive cell-level picture of how a nervous system integrates sensory inputs to produce specific behavioural outcomes. Here, we report the development of a microfluidic chip in which larvae can be immobilised and exposed to chemical stimuli. We generate transgenic larvae in which the calcium ion reporter GCaMP6m is expressed in a defined population of cells, allowing us to record real-time neural activity following stimulation. We then use this to establish that some cell populations can sense dissolved carbon dioxide. We also leverage genome and transcriptome data coupled with molecular evolutionary analysis to identify putative chemoreceptors of the MS4A family in Ciona. Our study demonstrates that Ciona larvae can respond to dissolved carbon dioxide, identifies the cells that are likely responsible for chemosensation, and establishes a chip based imaging platform coupled with transgenic technology that could be adapted to establish where other stimuli are sensed and how such incoming signals are processed in the brain to yield behavioural output.

The choanoflagellate Salpingoeca rosetta can differentiate into at least five morphologically and behaviorally distinct cell types during its lifetime, going from individual motile cells to linear and rosette-shaped colonies. Due to the capability to form colonies and its close relationship with Metazoa, this organism is considered a model for studying multicellular evolution. The gene regulatory programs underlying these transformations are, however, unknown. Here we analyze transcriptomic data obtained from Salpingoeca rosetta in different states to identify a core of genes associated with the formation of multicellular colonies. We then compare the results with other organisms which display simple forms of multicellularity, highlighting commonalities and differences.

The evolutionary arms race between transposable elements (TEs) and their hosts contributes to genomic complexity. As TEs mobilization is deleterious for individual cells and organisms, their activity is restricted. During stress, TEs can be reactivated; however, the exact mechanisms vary. We discovered that in the flatworm Macrostomum lignano, LTR retrotransposons hijack the heat shock response pathway to boost their transcription at elevated temperatures. While it has been well-described in cruciferous plants, this is the first report of this mechanism in animal LTR retrotransposons. Our results suggest a convergent evolution of the heat stress response in LTR retrotransposons from animals and plants.

Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs) or danger signals released by damaged tissues. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically related to themselves, such as parasitoid wasps infecting insects. Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we find that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we find that the activation of melanization response that kills parasitoids also requires exposure to a parasitoid wasp molecule that acts as a PAMP. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body that includes Tep1, which is essential for efficient melanization. We conclude that a combination of danger signals and PAMPs are required activate Drosophilas immune response against parasitic insects.

Invertebrates, animals (metazoans) without backbones, encompass [~]97% of all animal yet remains understudied. They have provided insights into molecular mechanisms underlying fundamentally identical mechanisms in phylogenetically diverse animals, including vertebrates. Marine invertebrates have long fascinated researchers due to their abundance, diversity, adaptations, and impact on ecosystems and human economies. Here, we report a compendium and appraisal of 190 marine invertebrate genomes spanning 21 phyla, 43 classes, 92 orders, and 134 families. We identify a high proportion and long unit size of tandem repeats, likely contributing to reported difficulties in invertebrate genome assembly. A well-supported phylogenetic tree of marine invertebrates from 974 single-copy orthologous genes resolved topological controversies. We show that Ctenophora is at the basal phylum and Porifera is the sister group of Parahoxozoa; that Xenacoelomorpha is within Bilateria and is the sister group to Protostomia, rejecting three out of four hypotheses in the field; and that Bryozoa is at the basal position of Lophotrochozoa, not grouped into Lophophorata. We also present insights into the genetic underpinnings of metazoans from Hox genes, innate immune gene families, and nervous system gene families. Our marine invertebrate genome compendium provides a unified foundation for studies on their evolution and effects on ecological systems and human life.

Host-driven selection can be considered a strong driver of pathogen evolution. To successfully infect, colonize and complete their life cycle, plant pathogens are under constant selective pressures imposed by hosts, leading to genetic adaptation and possibly lineage radiation or speciation. Population and comparative genomics approaches are powerful tools to identify signatures of selection associated with host specialization in pathogen genomes and further allow recapturing population histories. Implementing such approaches, we identified evolutionary signatures of divergent host specialisation in distinct lineages of the fungal pathogen Zymoseptoria tritici, a major disease causing-agent of wheat. Unique collections of Z. tritici were isolated from wild (Aegilops spp.) and domesticated (Triticum aestivum) host grasses in the Middle East and whole-genome sequencing was performed in a selected subset of isolates from each collection. We observed distinct population structure between the two host-diverging pathogens and identified particular genomic features in the Aegilops-infecting isolates that may have shaped their evolutionary history. Phylogenomic analyses revealed that A. cylindrica and A. tauchii -infecting populations of Z. tritici form separate clusters, possibly reflecting incipient speciation driven by divergent host specialization. Using infection experiments, we confirm that Z. tritici isolates collected from Aegilops spp. only infect their respective host species and not T. aestivum. Population genomics analyses and demographic inference furthermore allowed us to detect signatures of recent selection and show that divergence of the wheat-infecting lineage likely coincided with wheat domestication. At last, we confirm a virulence-related role for one candidate effector located in a selective sweep region of the A. cylindrica-infecting pathogen. Taken together, our findings highlight the interplay between agricultural and wild hosts on the evolution of fungal plant pathogens and illustrate host specialization as a possible route of rapid pathogen emergence.

Long non-coding RNAs (lncRNAs) are regulatory molecules interacting in a wide array of biological processes. LncRNAs in fungal pathogens can be responsive to stress and play roles in regulating growth and nutrient acquisition. Recent evidence suggests that lncRNAs may also play roles in virulence, such as regulating pathogenicity-associated enzymes and on-host reproductive cycles. Despite the importance of lncRNAs, only few model fungi have well-documented inventories of lncRNA. In this study, we apply a machine-learning based pipeline to predict high-confidence lncRNA candidates in Zymoseptoria tritici, an important global pathogen of wheat impacting global food production. We analyzed genomic features of lncRNAs and the most likely associated processes through analyses of expression over a host infection cycle. We find that lncRNAs are frequently expressed during early infection, before the switch to necrotrophic growth. They are mostly located in facultative heterochromatic regions, which are known to contain many genes associated with pathogenicity. Furthermore, we find that lncRNAs are frequently co-expressed with genes that may be involved in responding to host signals, such as those responses to oxidative stress. Finally, we assess pangenome features of lncRNAs using four additional reference-quality genomes. We find evidence that the repertoire of expressed lncRNAs varies substantially between individuals, even though lncRNA loci tend to be shared at the genomic level. Overall, this study provides a repertoire and putative functions of lncRNAs in Z. tritici enabling molecular genetics and functional analyses in an important pathogen. Impact statementLong non-coding RNAs (lncRNAs) serve distinct roles from messenger RNA. Despite not encoding proteins, lncRNAs can control important cellular processes such as growth and response to stress. In fungal pathogens, lncRNAs are particularly interesting because they can influence how pathogens infect and harm their hosts. Yet, only very few fungal pathogens have high-quality repertoires of lncRNA established. Here, we used machine learning to identify lncRNA in the major wheat pathogen Zymoseptoria tritici. We found that lncRNAs are highly active during the early stages of infection, before the pathogen switches to necrotrophic growth. These lncRNAs are mainly located in regions of the genome associated with pathogenicity. The repertoire of expressed lncRNAs varies substantially among individuals highlighting the potential for pathogen adaptation based on variation in lncRNAs. By expanding our knowledge of lncRNAs in important pathogen models, we enable research to comprehensively investigating their roles across fungi.

The types of genomic change needed for environmental adaptation are of great interest. Annelid worms are a large phylum found in a rich diversity of habitats, giving opportunities to explore this issue. We report the chromosome level genome sequence of the Pompeii worm, the annelid Alvinella pompejana, an inhabitant of an extreme deep-sea hydrothermal vent environment. We find strong but heterogeneously distributed genetic divergence between populations taken from either side of the equator. Using transcript data, we produced a set of gene models and analysed the predicted protein set in the light of past hypotheses about the thermotolerance of Alvinella, and compared it to other recently sequenced annelid vent worms. We do not find evidence of a more extreme genome wide amino acid composition than other species, neither do we find evidence for rapid genome evolution in the form of disrupted synteny. We discount the hypothesis of loss of amino acid biosynthesis genes associated with obligate symbioses reported in siboglinid annelids. We do find evidence of a parallel increase in the number of globin encoding genes and loss of light sensitive opsins and cryptochromes. Alvinella encodes several respiratory enzymes unusual for bilaterian animals, suggesting an ability to better tolerate hypoxic environments.

Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout a copy of the ciliate germline genome are deleted during development of the streamlined somatic genome. Blepharisma represents one of the two earliest diverging ciliate classes, and, unusually, has dual pathways of somatic nuclear development, making it ideal for investigating the functioning and evolution of these processes. Here, we report the somatic genome assembly of Blepharisma stoltei strain ATCC 30299 (41 Mb), arranged as numerous alternative telomere-capped minichromosomes. This genome encodes eight PiggyBac transposase homologs liberated from transposons. All are subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We propose PiggyBac homologs were ancestral excisases that enabled evolution of extensive, natural genome editing.

The role of mitochondria in both adaptive and innate immune responses is increasingly recognized, but the role of mitochondrial DNA (mtDNA) variation as an immunomodulatory factor has received less attention. One reason for this is the difficulty of separating the effect of mtDNA from that of the nuclear genome. By utilizing the fruit fly Drosophila melanogaster, a powerful model system, we created cytoplasmic hybrids, aka. cybrid lines, where unique mtDNAs (mitotypes) were introgressed into a controlled isogenic nuclear background. We harnessed a panel of cybrid lines to study the effect of mtDNA variation on humoral and cell-mediated innate immune responses. Mitotypes exhibited heterogeneity in infection outcomes upon bacterial, viral and parasitoid infections. One mitotype of note (mtKSA2) was more immunocompetent when compared to other mitotypes. We performed transcriptomic profiling of uninfected and infected flies to find the mechanistic basis of the immunocompetence of the mtKSA2 line. We found that in uninfected flies mtKSA2 caused an upregulation of oxidative phosphorylation (OXPHOS) and tricarboxylic acid cycle (TCA) related genes and a downregulation of a set of antimicrobial peptides (AMPs). Upon infection, mtKSA2 flies produced transcriptomic changes that were infection type and duration specific. When we examined immune cells (hemocytes) in mtKSA2 larvae, we noted an increase in hemocyte numbers. These hemocytes were activated in the absence of infection, increased their production of ROS, and showed evidence of increased encapsulation efficiency upon parasitoid wasp infection. Overall, our results show that mtDNA variation acts as an immunomodulatory factor in both humoral and cell-mediated innate immunity and that specific mitotypes can provide enhanced protection against various infections.

Placozoa are small disc-shaped animals, representing the simplest known, possibly ancestral, organization of free-living animals. With only six morphological distinct cell types, without any recognized neurons or muscle, placozoans exhibit fast effector reactions and complex behaviors. However, little is known about electrogenic mechanisms in these animals. Here, we showed the presence of rapid action potentials in four species of placozoans (Trichoplax adhaerens [H1 haplotype], Trichoplax sp.[H2], Hoilungia hongkongensis [H13], and Hoilungia sp. [H4]). These action potentials are sodium-dependent and can be inducible. The molecular analysis suggests the presence of 5-7 different types of voltage-gated sodium channels, which showed substantial evolutionary radiation compared to many other metazoans. Such unexpected diversity of sodium channels in early-branched animal lineages reflect both duplication events and parallel evolution of unique behavioral integration in these nerveless animals. HighlightsO_LIPlacozoans are the simplest known animals without recognized neurons and muscles C_LIO_LIWith only six morphological cell types, placozoans showed complex & rapid behaviors C_LIO_LISodium-dependent action potentials have been discovered in intact animals C_LIO_LIVoltage-gated sodium channels (Nav) in Placozoa support a rapid behavioral integration C_LIO_LIPlacozoans have more Nav channels that any studied invertebrate animal so far C_LIO_LIDiversification of Nav-channels highlight the unique evolution of these nerveless animals C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/243113v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@4d9c5forg.highwire.dtl.DTLVardef@15539bforg.highwire.dtl.DTLVardef@4246faorg.highwire.dtl.DTLVardef@141e5fe_HPS_FORMAT_FIGEXP M_FIG C_FIG