Developmental toxicity of pre-production plastic pellets affects a large swathe of invertebrate taxa.

Microplastics pose risks to marine organisms through ingestion, entanglement, and as carriers of toxic additives and environmental pollutants. Plastic pre-production pellet leachates have been shown to affect the development of sea urchins and, to some extent, mussels. The extent of those developmental effects on other animal phyla remains unknown. Here, we test the toxicity of environmental mixed nurdle samples and new PVC pellets for the embryonic development or asexual reproduction by regeneration of animals from all the major animal superphyla (Lophotrochozoa, Ecdysozoa, Deuterostomia and Cnidaria). Our results show diverse, concentration-dependent impacts in all the species sampled for new pellets, and for molluscs and deuterostomes for environmental samples. Embryo axial formation, cell specification and, specially, morphogenesis seem to be the main processes affected by plastic leachate exposure. Our study serves as a proof of principle for the potentially catastrophic effects that increasing plastic concentrations in the oceans and other ecosystems can have across animal populations from all major animal superphyla.

Early embryogenesis and organogenesis in the annelid Owenia fusiformis.

BACKGROUND: Annelids are a diverse group of segmented worms within Spiralia, whose embryos exhibit spiral cleavage and a variety of larval forms. While most modern embryological studies focus on species with unequal spiral cleavage nested in Pleistoannelida (Sedentaria + Errantia), a few recent studies looked into Owenia fusiformis, a member of the sister group to all remaining annelids and thus a key lineage to understand annelid and spiralian evolution and development. However, the timing of early cleavage and detailed morphogenetic events leading to the formation of the idiosyncratic mitraria larva of O. fusiformis remain largely unexplored. RESULTS: Owenia fusiformis undergoes equal spiral cleavage where the first quartet of animal micromeres are slightly larger than the vegetal macromeres. Cleavage results in a coeloblastula approximately 5 h post-fertilization (hpf) at 19 °C. Gastrulation occurs via invagination and completes 4 h later, with putative mesodermal precursors and the chaetoblasts appearing 10 hpf at the dorso-posterior side. Soon after, at 11 hpf, the apical tuft emerges, followed by the first neurons (as revealed by the expression of elav1 and synaptotagmin-1) in the apical organ and the prototroch by 13 hpf. Muscles connecting the chaetal sac to various larval tissues develop around 18 hpf and by the time the mitraria is fully formed at 22 hpf, there are FMRFamide+ neurons in the apical organ and prototroch, the latter forming a prototrochal ring. As the mitraria feeds, it grows in size and the prototroch expands through active proliferation. The larva becomes competent after ~ 3 weeks post-fertilization at 15 °C, when a conspicuous juvenile rudiment has formed ventrally. CONCLUSIONS: Owenia fusiformis embryogenesis is similar to that of other equal spiral cleaving annelids, supporting that equal cleavage is associated with the formation of a coeloblastula, gastrulation via invagination, and a feeding trochophore-like larva in Annelida. The nervous system of the mitraria larva forms earlier and is more elaborated than previously recognized and develops from anterior to posterior, which is likely an ancestral condition to Annelida. Altogether, our study identifies the major developmental events during O. fusiformis ontogeny, defining a conceptual framework for future investigations.

General Principles of Planarian Embryogenesis and Its Analysis by In Situ Hybridization and Immunohistochemistry Methods.

Thanks to their ability to regrow any missing body part after injury, planarians have become a well-established invertebrate model system in regenerative studies. However, planarians are also unique in their embryonic development, displaying ectolecithality, or the accumulation of embryonic nutrients into accessory cells accompanying the zygotes. Gaining a better understanding of their peculiar embryogenesis can offer answers to some fundamental questions regarding the appearance and evolution of planarian regenerative capacities, and in a broader context, the diversification of embryonic and postembryonic development in animals. In this chapter, I give an overview of the present knowledge of planarian embryogenesis and the methodologies applied to its study. I describe and comment on protocols to fix and dissect planarian egg capsules, and perform whole-mount in situ hybridization and whole-mount immunohistochemistry on planarian embryos.

Evolution, divergence and loss of the Nodal signalling pathway: new data and a synthesis across the Bilateria.

Since the discovery that the TGF-ß signalling molecule Nodal and its downstream effector Pitx have a parallel role in establishing asymmetry between molluscs and deuterostomes the debate over the degree to which this signalling pathway is conserved across the Bilateria as a whole has been ongoing. Further taxon sampling is critical to understand the evolution and divergence of this signalling pathway in animals. Using genome and transcriptome mining we confirmed the presence of nodal and Pitx in a range of additional animal taxa for which their presence has not yet been described. In situ hybridization was used to show the embryonic expression of these genes in brachiopods and planarians. We show that both nodal and Pitx genes are broadly conserved across the Spiralia, and nodal likely appeared in the Bilaterian stem lineage after the divergence of the Acoelomorpha. Furthermore, both nodal and Pitx mRNA appears to be expressed in an asymmetric fashion in the brachiopod Terebratalia transversa. No evidence for the presence of a Lefty ortholog could be found in the non-deuterostome genomic resources examined. Nodal expression is asymmetric in a number of spiralian lineages, indicating a possible ancestral role of the Nodal/Pitx cascade in the establishment of asymmetries across the Bilateria.

Developmental diversity in free-living flatworms.

Flatworm embryology has attracted attention since the early beginnings of comparative evolutionary biology. Considered for a long time the most basal bilaterians, the Platyhelminthes (excluding Acoelomorpha) are now robustly placed within the Spiralia. Despite having lost their relevance to explain the transition from radially to bilaterally symmetrical animals, the study of flatworm embryology is still of great importance to understand the diversification of bilaterians and of developmental mechanisms. Flatworms are acoelomate organisms generally with a simple centralized nervous system, a blind gut, and lacking a circulatory organ, a skeleton and a respiratory system other than the epidermis. Regeneration and asexual reproduction, based on a totipotent neoblast stem cell system, are broadly present among different groups of flatworms. While some more basally branching groups - such as polyclad flatworms - retain the ancestral quartet spiral cleavage pattern, most flatworms have significantly diverged from this pattern and exhibit unique strategies to specify the common adult body plan. Most free-living flatworms (i.e. Platyhelminthes excluding the parasitic Neodermata) are directly developing, whereas in polyclads, also indirect developers with an intermediate free-living larval stage and subsequent metamorphosis are found. A comparative study of developmental diversity may help understanding major questions in evolutionary biology, such as the evolution of cleavage patterns, gastrulation and axial specification, the evolution of larval types, and the diversification and specialization of organ systems. In this review, we present a thorough overview of the embryonic development of the different groups of free-living (turbellarian) platyhelminths, including the Catenulida, Macrostomorpha, Polycladida, Lecithoepitheliata, Proseriata, Bothrioplanida, Rhabdocoela, Fecampiida, Prolecithophora and Tricladida, and discuss their main features under a consensus phylogeny of the phylum.

Morphological and molecular development of the eyes during embryogenesis of the freshwater planarian Schmidtea polychroa.

Photoreception is one of the most primitive sensory functions in metazoans. Despite the diversity of forms and components of metazoan eyes, many studies have demonstrated the existence of a common cellular and molecular basis for their development. Genes like pax6, sine oculis, eyes absent, dachshund, otx, Rx and atonal are known to be associated with the specification and development of the eyes. In planarians, sine oculis, eyes absent and otxA play an essential role during the formation of the eye after decapitation, whereas pax6, considered by many authors as a master control gene for eye formation, does not seem to be involved in adult eye regeneration. Whether this is a peculiarity of adult planarians or, on the contrary, is also found in embryogenesis remains unknown. Herein, we characterize embryonic eye development in the planarian species Schmidtea polychroa using histological sections and molecular markers. Additionally, we analyse the expression pattern of the pax6-sine oculis-eyes absent-dachshund network, and the genes Rx, otxA, otxB and atonal. We demonstrate that eye formation in planarian embryos shows great similarities to adult eye regeneration, both at the cellular and molecular level. We thus conclude that planarian eyes exhibit divergent molecular patterning mechanisms compared to the prototypic ancestral metazoan eye.

Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa.

The formation of a through-gut was a key innovation in the evolution of metazoans. There is still controversy regarding the origin of the anus and how it may have been either gained or lost during evolution in different bilaterian taxa. Thus, the study of groups with a blind gut is of great importance for understanding the evolution of this organ system. Here, we describe the morphogenesis and molecular patterning of the blind gut in the sexual triclad Schmidtea polychroa. We identify and analyze the expression of goosecoid, commonly associated with the foregut, and the GATA, ParaHox and T-box genes, members of which commonly are associated with gut regionalization. We show that GATA456a is expressed in the blind gut of triclads, while GATA456b is localized in dorsal parenchymal cells. Goosecoid is expressed in the central nervous system, and the unique ParaHox gene identified, Xlox, is detected in association with the nervous system. We have not isolated any brachyury gene in the T-box complement of S. polychroa, which consists of one tbx1/10, three tbx2/3 and one tbx20. Furthermore, the absence of genes like brachyury and caudal is also present in other groups of Platyhelminthes. This study suggests that GATA456, in combination with foxA, is a gut-specific patterning mechanism conserved in the triclad S. polychroa, while the conserved gut-associated expression of foregut, midgut and hindgut markers is absent. Based on these data and the deviations in spiral cleavage found in more basal flatworms, we propose that the lack of an anus is an innovation of Platyhelminthes. This may be associated with loss of gut gene expression or even gene loss.

Germ layer specification and axial patterning in the embryonic development of the freshwater planarian Schmidtea polychroa.

Although patterning during regeneration in adult planarians has been studied extensively, very little is known about how the initial planarian body plan arises during embryogenesis. Herein, we analyze the process of embryo patterning in the species Schmidtea polychroa by comparing the expression of genes involved in the establishment of the metazoan body plan. Planarians present a derived ectolecithic spiralian development characterized by dispersed cleavage within a yolk syncytium and an early transient embryo capable of feeding on the maternally supplied yolk cells. During this stage of development, we only found evidence of canonical Wnt pathway, mostly associated with the development of its transient pharynx. At these stages, genes involved in gastrulation (snail) and germ layer determination (foxA and twist) are specifically expressed in migrating blastomeres and those giving rise to the temporary gut and pharyngeal muscle. After yolk ingestion, the embryo expresses core components of the canonical Wnt pathway and the BMP pathway, suggesting that the definitive axial identities are established late. These data support the division of planarian development into two separate morphogenetic stages: a highly divergent gastrulation stage, which segregates the three germ layers and establishes the primary organization of the feeding embryo; and subsequent metamorphosis, based on totipotent blastomeres, which establishes the definitive adult body plan using mechanisms that are similar to those used during regeneration and homeostasis in the adult.

New method to deliver exogenous material into developing planarian embryos.

The ability to report or modify the embryological processes in living embryos is pivotal for developmental biology research. Planarian embryology has experienced renewed interest as the genetic pathways that drive adult regeneration were found to be involved in the development of embryos. The major drawback to the study of planarian embryology is the absence of methods that give access to the embryos and enable their manipulation. Herein, we report on a novel method for delivering external material into developing embryos using nanosecond laser pulses. When focused on the eggshell surface under optimal parameters, laser pulses ablate the protective case and open a pathway throughout which foreign material can be delivered. In this study, we used egg capsules from Schmidtea polychroa (Schmidt, 1861) to microinject 1 microm fluorescein isothiocyanate fluorescent beads into the live embryos. We obtained viability values ranging from 15% in early egg capsules to 100% in late developmental stages. Moreover, we measured the delivery effectiveness as the number of hatchlings containing fluorescent beads per microinjected egg capsule, reaching 100% in early stages and almost 40% in late stages. This is the first time that planarian embryos have been modified without compromising normal development. We consider that this technique will be of extreme value to future work on planarian developmental biology and regeneration, enabling the application of modern functional tools to the study of this Lophotrochozoan.