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BACKGROUND: The consequences of the earth's daily rotation have led to 24-h biological rhythms in most organisms. Even some parasites are known to have daily rhythms, which, when in synchrony with host rhythms, can optimise their fitness. Understanding these rhythms may enable the development of control strategies that take advantage of rhythmic vulnerabilities. Recent work on protozoan parasites has revealed 24-h rhythms in gene expression, drug sensitivity and the presence of an intrinsic circadian clock; however, similar studies on metazoan parasites are lacking. To address this, we investigated if a metazoan parasite has daily molecular oscillations, whether they reveal how these longer-lived organisms can survive host daily cycles over a lifespan of many years and if animal circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni that lives in the vasculature for decades and causes the tropical disease schistosomiasis. RESULTS: Using round-the-clock transcriptomics of male and female adult worms collected from experimentally infected mice, we discovered that ~ 2% of its genes followed a daily pattern of expression. Rhythmic processes included a stress response during the host's active phase and a 'peak in metabolic activity' during the host's resting phase. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include predicted drug targets and a vaccine candidate. These 24-h rhythms may be driven by host rhythms and/or generated by a circadian clock; however, orthologs of core clock genes are missing and secondary clock genes show no 24-h rhythmicity. CONCLUSIONS: There are daily rhythms in the transcriptomes of adult S. mansoni, but they appear less pronounced than in other organisms. The rhythms reveal temporally compartmentalised internal processes and host interactions relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other animals. We have shown which transcripts oscillate at this temporal scale and this will benefit the development and delivery of treatments against schistosomiasis.
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Relojes Circadianos , Parásitos , Animales , Relojes Circadianos/genética , Ritmo Circadiano/genética , Femenino , Humanos , Masculino , Ratones , Parásitos/genética , Schistosoma mansoni/genética , TranscriptomaRESUMEN
Chondrichthyans possess endoskeletal appendages called branchial rays that extend laterally from their hyoid and gill-bearing (branchial) arches. Branchial ray outgrowth, like tetrapod limb outgrowth, is maintained by Sonic hedgehog (Shh) signaling. In limbs, distal endoskeletal elements fail to form in the absence of normal Shh signaling, whereas shortened duration of Shh expression correlates with distal endoskeletal reduction in naturally variable populations. Chondrichthyans also exhibit natural variation with respect to branchial ray distribution--elasmobranchs (sharks and batoids) possess a series of ray-supported septa on their hyoid and gill arches, whereas holocephalans (chimaeras) possess a single hyoid arch ray-supported operculum. Here we show that the elongate hyoid rays of the holocephalan Callorhinchus milii grow in association with sustained Shh expression within an opercular epithelial fold, whereas Shh is only transiently expressed in the gill arches. Coincident with this transient Shh expression, branchial ray outgrowth is initiated in C. milii but is not maintained, yielding previously unrecognized vestigial gill arch branchial rays. This is in contrast to the condition seen in sharks, where sustained Shh expression corresponds to the presence of fully formed branchial rays on the hyoid and gill arches. Considered in light of current hypotheses of chondrichthyan phylogeny, our data suggest that the holocephalan operculum evolved in concert with gill arch appendage reduction by attenuation of Shh-mediated branchial ray outgrowth, and that chondrichthyan branchial rays and tetrapod limbs exhibit parallel developmental mechanisms of evolutionary reduction.
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Evolución Biológica , Elasmobranquios/embriología , Peces/embriología , Branquias/embriología , Animales , Australia , Tipificación del Cuerpo , Región Branquial/embriología , Región Branquial/metabolismo , Elasmobranquios/clasificación , Elasmobranquios/genética , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Proteínas de Peces/genética , Peces/clasificación , Peces/genética , Regulación del Desarrollo de la Expresión Génica , Geografía , Branquias/metabolismo , Proteínas Hedgehog/genética , Hibridación in Situ , Datos de Secuencia Molecular , Nueva Zelanda , Filogenia , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Schistosoma mansoni is a parasitic flatworm that causes the major neglected tropical disease schistosomiasis. The miracidium is the first larval stage of the life cycle. It swims and infects a freshwater snail, transforms into a mother sporocyst, where its stem cells generate daughter sporocysts that give rise to human-infective cercariae larvae. To understand the miracidium at cellular and molecular levels, we created a whole-body atlas of its ~365 cells. Single-cell RNA sequencing identified 19 transcriptionally distinct cell clusters. In situ hybridisation of tissue-specific genes revealed that 93% of the cells in the larva are somatic (57% neural, 19% muscle, 13% epidermal or tegument, 2% parenchyma, and 2% protonephridia) and 7% are stem. Whereas neurons represent the most diverse somatic cell types, trajectory analysis of the two main stem cell populations indicates that one of them is the origin of the tegument lineage and the other likely contains pluripotent cells. Furthermore, unlike the somatic cells, each of these stem populations shows sex-biased transcriptional signatures suggesting a cell-type-specific gene dosage compensation for sex chromosome-linked loci. The miracidium represents a simple developmental stage with which to gain a fundamental understanding of the molecular biology and spatial architecture of schistosome cells.
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Larva , Schistosoma mansoni , Análisis de la Célula Individual , Animales , Schistosoma mansoni/genética , Schistosoma mansoni/crecimiento & desarrollo , Larva/crecimiento & desarrollo , Larva/genética , Estadios del Ciclo de Vida/genéticaRESUMEN
Schistosomiasis is a major Neglected Tropical Disease, caused by the infection with blood flukes in the genus Schistosoma. To complete the life cycle, the parasite undergoes asexual and sexual reproduction within an intermediate snail host and a definitive mammalian host, respectively. The intra-molluscan phase provides a critical amplification step that ensures a successful transmission. However, the cellular and molecular mechanisms underlying the development of the intra-molluscan stages remain poorly understood. Here, single cell suspensions from S. mansoni mother sporocysts were produced and sequenced using the droplet-based 10X Genomics Chromium platform. Six cell clusters comprising two tegument, muscle, neuron, parenchyma and stem/germinal cell clusters were identified and validated by in situ hybridisation. Gene Ontology term analysis predicted key biological processes for each of the clusters, including three stem/germinal sub-clusters. Furthermore, putative transcription factors predicted for stem/germinal and tegument clusters may play key roles during parasite development and interaction with the intermediate host.
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Parásitos , Esquistosomiasis mansoni , Esquistosomiasis , Animales , Perfilación de la Expresión Génica , Mamíferos/genética , Moluscos/genética , Parásitos/genética , Schistosoma mansoni/genética , Esquistosomiasis/parasitología , Esquistosomiasis mansoni/parasitologíaRESUMEN
Platyhelminthes (flatworms) are a diverse invertebrate phylum useful for exploring life-history evolution. Within Platyhelminthes, only two clades develop through a larval stage: free-living polyclads and parasitic neodermatans. Neodermatan larvae are considered evolutionarily derived, whereas polyclad larvae are hypothesized to be ancestral due to ciliary band similarities among polyclad and other spiralian larvae. However, larval evolution has been challenging to investigate within polyclads due to low support for deeper phylogenetic relationships. To investigate polyclad life-history evolution, we generated transcriptomic data for 21 species of polyclads to build a well-supported phylogeny for the group. The resulting tree provides strong support for deeper nodes, and we recover a new monophyletic clade of early branching cotyleans. We then used ancestral state reconstructions to investigate ancestral modes of development within Polycladida and more broadly within flatworms. In polyclads, we were unable to reconstruct the ancestral state of deeper nodes with significant support because early branching clades show diverse modes of development. This suggests a complex history of larval evolution in polyclads that likely includes multiple losses and/or multiple gains. However, our ancestral state reconstruction across a previously published platyhelminth phylogeny supports a direct developing prorhynchid/polyclad ancestor, which suggests that a larval stage in the life cycle evolved along the polyclad stem lineage or within polyclads.
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Whipworms are large metazoan parasites that inhabit multi-intracellular epithelial tunnels in the large intestine of their hosts, causing chronic disease in humans and other mammals. How first-stage larvae invade host epithelia and establish infection remains unclear. Here we investigate early infection events using both Trichuris muris infections of mice and murine caecaloids, the first in-vitro system for whipworm infection and organoid model for live helminths. We show that larvae degrade mucus layers to access epithelial cells. In early syncytial tunnels, larvae are completely intracellular, woven through multiple live dividing cells. Using single-cell RNA sequencing of infected mouse caecum, we reveal that progression of infection results in cell damage and an expansion of enterocytes expressing of Isg15, potentially instigating the host immune response to the whipworm and tissue repair. Our results unravel intestinal epithelium invasion by whipworms and reveal specific host-parasite interactions that allow the whipworm to establish its multi-intracellular niche.
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Helmintos , Tricuriasis , Animales , Mucosa Intestinal , Intestinos/parasitología , Mamíferos , Ratones , Trichuris/fisiologíaRESUMEN
Schistosomiasis, the most important helminthic disease of humanity, is caused by infection with parasitic flatworms of the genus Schistosoma. The disease is driven by parasite eggs becoming trapped in host tissues, followed by inflammation and granuloma formation. Despite abundant transcriptome data for most developmental stages of the three main human-infective schistosome species-Schistosoma mansoni, S. japonicum and S. haematobium-the transcriptomic profiles of developing eggs remain under unexplored. In this study, we performed RNAseq of S. mansoni eggs laid in vitro during early and late embryogenesis, days 1-3 and 3-6 post-oviposition, respectively. Analysis of the transcriptomes identified hundreds of up-regulated genes during the later stage, including venom allergen-like (VAL) proteins, well-established host immunomodulators, and genes involved in organogenesis of the miracidium larva. In addition, the transcriptomes of the in vitro laid eggs were compared with existing publicly available RNA-seq datasets from S. mansoni eggs collected from the livers of rodent hosts. Analysis of enriched GO terms and pathway annotations revealed cell division and protein synthesis processes associated with early embryogenesis, whereas cellular metabolic processes, microtubule-based movement, and microtubule cytoskeleton organization were enriched in the later developmental time point. This is the first transcriptomic analysis of S. mansoni embryonic development, and will facilitate our understanding of infection pathogenesis, miracidial development and life cycle progression of schistosomes.
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BACKGROUND: Planktonic life history stages of spiralians share some muscular, nervous and ciliary system characters in common. The distribution of these characters is patchy and can be interpreted either as the result of convergent evolution, or as the retention of primitive spiralian larval features. To understand the evolution of these characters adequate taxon sampling across the Spiralia is necessary. Polyclad flatworms are the only free-living Platyhelminthes that exhibit a continuum of developmental modes, with direct development at one extreme, and indirect development via a trochophore-like larval stage at the other. Here I present embryological and larval anatomical data from the indirect developing polyclad Maritrigrella crozieri, and consider these data within a comparative spiralian context. RESULTS: After 196 h hours of embryonic development, M. crozieri hatches as a swimming, planktotrophic larva. Larval myoanatomy consists of an orthogonal grid of circular and longitudinal body wall muscles plus parenchymal muscles. Diagonal body wall muscles develop over the planktonic period. Larval neuroanatomy consists of an apical plate, neuropile, paired nerve cords, a peri-oral nerve ring, a medial nerve, a ciliary band nerve net and putative ciliary photoreceptors. Apical neural elements develop first followed by posterior perikarya and later pharyngeal neural elements. The ciliated larva is encircled by a continuous, pre-oral band of longer cilia, which follows the distal margins of the lobes; it also possesses distinct apical and caudal cilia. CONCLUSIONS: Within polyclads heterochronic shifts in the development of diagonal bodywall and pharyngeal muscles are correlated with life history strategies and feeding requirements. In contrast to many spiralians, M. crozieri hatch with well developed nervous and muscular systems. Comparisons of the ciliary bands and apical organs amongst spiralian planktonic life-stages reveal differences; M. crozieri lack a distinct ciliary band muscle and flask-shaped epidermal serotonergic cells of the apical organ. Based on current phylogenies, the distribution of ciliary bands and apical organs between polyclads and other spiralians is not congruent with a hypothesis of homology. However, some similarities exist, and this study sets an anatomical framework from which to investigate cellular and molecular mechanisms that will help to distinguish between parallelism, convergence and homology of these features.
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Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic flatworms known as schistosomes. Humans become infected by free-swimming, water-borne larvae, which penetrate the skin. The earliest intra-mammalian stage, called the schistosomulum, undergoes a series of developmental transitions. These changes are critical for the parasite to adapt to its new environment as it navigates through host tissues to reach its niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive intra-mammalian development requires knowledge of the spatial organisation and transcriptional dynamics of different cell types that comprise the schistomulum body. To fill these important knowledge gaps, we perform single-cell RNA sequencing on two-day old schistosomula of Schistosoma mansoni. We identify likely gene expression profiles for muscle, nervous system, tegument, oesophageal gland, parenchymal/primordial gut cells, and stem cells. In addition, we validate cell markers for all these clusters by in situ hybridisation in schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-type atlas for the early intra-mammalian stage of this devastating metazoan parasite.
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Mamíferos/parasitología , Parásitos/citología , Parásitos/crecimiento & desarrollo , Schistosoma mansoni/citología , Schistosoma mansoni/crecimiento & desarrollo , Análisis de la Célula Individual , Animales , Esófago/metabolismo , Exones/genética , Regulación de la Expresión Génica , Humanos , Células Musculares/metabolismo , Sistema Nervioso/citología , Neuronas/citología , Parásitos/genética , Schistosoma mansoni/genética , Células Madre/citología , Células Madre/metabolismo , Transcripción GenéticaRESUMEN
Animals detect light using opsin photopigments. Xenopsin, a recently classified subtype of opsin, challenges our views on opsin and photoreceptor evolution. Originally thought to belong to the Gαi-coupled ciliary opsins, xenopsins are now understood to have diverged from ciliary opsins in pre-bilaterian times, but little is known about the cells that deploy these proteins, or if they form a photopigment and drive phototransduction. We characterized xenopsin in a flatworm, Maritigrella crozieri, and found it expressed in ciliary cells of eyes in the larva, and in extraocular cells around the brain in the adult. These extraocular cells house hundreds of cilia in an intra-cellular vacuole (phaosome). Functional assays in human cells show Maritigrella xenopsin drives phototransduction primarily by coupling to Gαi. These findings highlight similarities between xenopsin and c-opsin and reveal a novel type of opsin-expressing cell that, like jawed vertebrate rods, encloses the ciliary membrane within their own plasma membrane.
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Péptidos/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Platelmintos/fisiología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Encéfalo , Membrana Celular/metabolismo , Evolución Molecular , Ojo/citología , Ojo/metabolismo , Subunidades alfa de la Proteína de Unión al GTP , Humanos , Larva , Fototransducción/fisiología , Opsinas/clasificación , Opsinas/genética , Opsinas/metabolismo , Células Fotorreceptoras/citología , Células Fotorreceptoras/fisiología , Células Fotorreceptoras de Vertebrados/fisiología , Filogenia , Células Fotorreceptoras Retinianas Bastones/citología , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
Schistosomiasis is an important parasitic disease, touching roughly 200 million people worldwide. The causative agents are different Schistosoma species. Schistosomes have a complex life cycle, with a freshwater snail as intermediate host. After infection, sporocysts develop inside the snail host and give rise to human dwelling larvae. We present here a detailed step-by-step video instruction in English, French, Spanish and Portuguese that shows how these sporocysts can be manipulated and transferred from one snail to another. This procedure provides a technical basis for different types of ex vivo modifications, such as those used in functional genomics studies.
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The interrelationships of the flatworms (phylum Platyhelminthes) are poorly resolved despite decades of morphological and molecular phylogenetic studies. The earliest-branching clades (Catenulida, Macrostomorpha, and Polycladida) share spiral cleavage and entolecithal eggs with other lophotrochozoans. Lecithoepitheliata have primitive spiral cleavage but derived ectolecithal eggs. Other orders (Rhabdocoela, Proseriata, Tricladida and relatives, and Bothrioplanida) all have derived ectolecithal eggs but have uncertain affinities to one another. The orders of parasitic Neodermata emerge from an uncertain position from within these ectolecithal classes. To tackle these problems, we have sequenced transcriptomes from 18 flatworms and 5 other metazoan groups. The addition of published data produces an alignment of >107,000 amino acids with less than 28% missing data from 27 flatworm taxa in 11 orders covering all major clades. Our phylogenetic analyses show that Platyhelminthes consist of the two clades Catenulida and Rhabditophora. Within Rhabditophora, we show the earliest-emerging branch is Macrostomorpha, not Polycladida. We show Lecithoepitheliata are not members of Neoophora but are sister group of Polycladida, implying independent origins of the ectolecithal eggs found in Lecithoepitheliata and Neoophora. We resolve Rhabdocoela as the most basally branching euneoophoran taxon. Tricladida, Bothrioplanida, and Neodermata constitute a group that appears to have lost both spiral cleavage and centrosomes. We identify Bothrioplanida as the long-sought closest free-living sister group of the parasitic Neodermata. Among parasitic orders, we show that Cestoda are closer to Trematoda than to Monogenea, rejecting the concept of the Cercomeromorpha. Our results have important implications for understanding the evolution of this major phylum.
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Filogenia , Platelmintos/genética , Animales , Evolución Biológica , Centrómero/genética , Femenino , Perfilación de la Expresión Génica , Masculino , Óvulo/fisiología , Planarias/genética , Platelmintos/fisiología , Espermatozoides/fisiología , Terminología como AsuntoRESUMEN
Polyclad flatworms offer an excellent system with which to explore the evolution of larval structures and the ecological and developmental mechanisms driving flatworm and marine invertebrate life history evolution. Although the most common mode of development in polyclads might be direct development (where the embryo develops directly into a form resembling the young adult), there are many species that develop indirectly, through a planktonic phase with transient larval features, before settling to the sea floor. In this review, I introduce polyclad life history strategies, larval diversity and larval anatomical features (presenting previously unpublished micrographs of a diversity of polyclad larvae). I summarize what is known about polyclad larval development during the planktonic phase and the transition to the benthic juvenile. Finally, I discuss evolutionary and developmental scenarios on the origin of polyclad larval characters.The most prominent characters that are found exclusively in the larval stages are lobes that protrude from the body and a ciliary band, or ciliary tufts, at the peripheral margins of the lobes. Larvae with 4-8 and 10 lobes have been described, with most indirect developing species hatching with 8 lobes. A ventral sucker develops in late stage larvae, and I put forward the hypothesis that this is an organ for larval settlement for species belonging to the Cotylea. Historically, the biphasic life cycle of polyclads was thought to be a shared primitive feature of marine invertebrates, with similarities in larval features among phyla resulting from evolutionary conservation. However, our current understanding of animal phylogeny suggests that indirect development in polyclads has evolved independently of similar life cycles found in parasitic flatworms and some other spiralian taxa, and that morphological similarities between the larvae of polyclads and other spiralians are likely a result of convergent evolution.
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Polyclad flatworms are an early branching clade within the rhabditophoran Platyhelminthes. They provide an interesting system with which to explore the evolution of development within Platyhelminthes and amongst Spiralia (Lophotrochozoa). Unlike most other flatworms, polyclads undergo spiral cleavage (similar to that seen in some other spiralian taxa), they are the only free-living flatworms where development via a larval stage occurs, and they are the only flatworms in which embryos can be reared outside of their protective egg case, enabling embryonic manipulations. Past work has focused on comparing early cleavage patterns and larval anatomy between polyclads and other spiralians. We have selected Maritigrella crozieri, the tiger flatworm, as a suitable polyclad species for developmental studies, because it is abundant and large in size compared to other species. These characteristics have facilitated the generation of a transcriptome from embryonic and larval material and are enabling us to develop methods for gene expression analysis and immunofluorescence techniques. Here we give an overview of M. crozieri and its development, we highlight the advantages and current limitations of this animal as a potential evo-devo model and discuss current lines of research.
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The role of corallivory is becoming increasingly recognised as an important factor in coral health at a time when coral reefs around the world face a number of other stressors. The polyclad flatworm, Amakusaplana acroporae, is a voracious predator of Indo-Pacific acroporid corals in captivity, and its inadvertent introduction into aquaria has lead to the death of entire coral colonies. While this flatworm has been a pest to the coral aquaculture community for over a decade, it has only been found in aquaria and has never been described from the wild. Understanding its biology and ecology in its natural environment is crucial for identifying viable biological controls for more successful rearing of Acropora colonies in aquaria, and for our understanding of what biotic interactions are important to coral growth and fitness on reefs. Using morphological, histological and molecular techniques we determine that a polyclad found on Acropora valida from Lizard Island, Australia is A. acroporae. The presence of extracellular Symbiodinium in the gut and parenchyma and spirocysts in the gut indicates that it is a corallivore in the wild. The examination of a size-range of individuals shows maturation of the sexual apparatus and increases in the number of eyes with increased body length. Conservative estimates of abundance show that A. acroporae occurred on 7 of the 10 coral colonies collected, with an average of 2.6±0.65 (mean ±SE) animals per colony. This represents the first report of A. acroporae in the wild, and sets the stage for future studies of A. acroporae ecology and life history in its natural habitat.
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Arrecifes de Coral , Turbelarios/fisiología , Animales , Antozoos/fisiología , AustraliaRESUMEN
1. Diel vertical migration (DVM) is a widespread phenomenon among marine and freshwater organisms and many studies with various taxa have sought to understand its adaptive significance. Among crustacean zooplankton and juveniles of some fish species DVM is accepted widely as an antipredator behaviour, but little is known about its adaptive value for relatively large-bodied, adult predatory fish such as sharks. Moreover, the majority of studies have focused on pelagic forms, which raises the question of whether DVM occurs in bottom-living predators. 2. To investigate DVM in benthic predatory fish in the marine environment and to determine why it might occur we tracked movements of adult male dogfish (Scyliorhinus canicula) by short- and long-term acoustic and archival telemetry. Movement studies were complemented with measurements of prey abundance and availability and thermal habitat within home ranges. A thermal choice experiment and energy budget modelling was used to investigate trade-offs between foraging and thermal habitat selection. 3. Male dogfish undertook normal DVM (nocturnal ascent) within relatively small home ranges (-100 x 100 m) comprising along-bottom movements up submarine slopes from deeper, colder waters occupied during the day into warmer, shallow prey-rich areas above the thermocline at night. Few daytime vertical movements occurred. Levels of activity were higher during the night above the thermocline compared to below it during the day indicating they foraged in warm water and rested in colder depths. 4. A thermal choice experiment using environmentally realistic temperatures supported the field observation that dogfish positively avoided warmer water even when it was associated with greater food availability. Males in laboratory aquaria moved into warm water from a cooler refuge only to obtain food, and after food consumption they preferred to rest and digest in cooler water. 5. Modelling of energy budgets under different realistic thermal-choice scenarios indicated dogfish adopting a 'hunt warm - rest cool' strategy could lower daily energy costs by just over 4%. Our results provide the first clear evidence that are consistent with the hypothesis that a benthic marine-fish predator utilizes DVM as an energy conservation strategy that increases bioenergetic efficiency.