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.

PVC pellet leachates affect adult immune system and embryonic development but not reproductive capacity in the sea urchin Paracentrotus lividus.

Microplastic pollution is a major concern of our age, eliciting a range of effects on organisms including during embryonic development. Plastic preproduction pellets stunt the development of sea urchins through the leaching of teratogenic compounds. However, the effect of these leachates on adult sea urchins and their fertility is unknown. Here we investigate the effect of PVC leachates on the capacity to produce normal embryos, and demonstrate that adults kept in contaminated water still produce viable offspring. However, we observe a cumulative negative effect by continued exposure to highly polluted water: adult animals had lower counts and disturbed morphological profiles of immune cells, were under increased oxidative stress, and produced embryos less tolerant of contaminated environments. Our findings suggest that even in highly polluted areas, sea urchins are fertile, but that sublethal effects seen in the adults may lead to transgenerational effects that reduce developmental robustness of the embryos.

Plastic leachate-induced toxicity during sea urchin embryonic development: Insights into the molecular pathways affected by PVC.

Microplastics are now polluting all seas and, while studies have found numerous negative interactions between plastic pollution and marine animals, the effects on embryonic development are poorly understood. A potentially important source of developmental ecotoxicity comes from chemicals leached from plastic particles to the marine environment. Here we investigate the effects of leachates from new and beach-collected pellets on the embryonic and larval development of the sea urchin Strongylocentrotus purpuratus and demonstrate that exposure of developing embryos to these leachates elicits severe, consistent and treatment-specific developmental abnormalities including radialisation of the embryo and malformation of the skeleton, neural and immune cells. Using a multi-omics approach we define the developmental pathways disturbed upon exposure to PVC leachates and provide a mechanistic view that pinpoints cellular redox stress and energy production as drivers of phenotypic abnormalities following exposure to PVC leachates. Analysis of leachates identified high concentrations of zinc that are the likely cause of these observed defects. Our findings point to clear and specific detrimental effects of marine plastic pollution on the development of echinoderms, demonstrating that chemicals leached from plastic particles into sea water can produce strong developmental abnormalities via specific pathways, and therefore have the potential to impact on a wide range of organisms.

Genome-wide transcriptomic changes reveal the genetic pathways involved in insect migration.

Insects are capable of extraordinary feats of long-distance movement that have profound impacts on the function of terrestrial ecosystems. The ability to undertake these movements arose multiple times through the evolution of a suite of traits that make up the migratory syndrome, however the underlying genetic pathways involved remain poorly understood. Migratory hoverflies (Diptera: Syrphidae) are an emerging model group for studies of migration. They undertake seasonal movements in huge numbers across large parts of the globe and are important pollinators, biological control agents and decomposers. Here, we assembled a high-quality draft genome of the marmalade hoverfly (Episyrphus balteatus). We leveraged this genomic resource to undertake a genome-wide transcriptomic comparison of actively migrating Episyrphus, captured from a high mountain pass as they flew south to overwinter, with the transcriptomes of summer forms which were non-migratory. We identified 1543 genes with very strong evidence for differential expression. Interrogation of this gene set reveals a remarkable range of roles in metabolism, muscle structure and function, hormonal regulation, immunity, stress resistance, flight and feeding behaviour, longevity, reproductive diapause and sensory perception. These features of the migrant phenotype have arisen by the integration and modification of pathways such as insulin signalling for diapause and longevity, JAK/SAT for immunity, and those leading to octopamine production and fuelling to boost flight capabilities. Our results provide a powerful genomic resource for future research, and paint a comprehensive picture of global expression changes in an actively migrating insect, identifying key genomic components involved in this important life-history strategy.

Transgenerational effects on development following microplastic exposure in Drosophila melanogaster.

BACKGROUND: Plastic pollution affects all ecosystems, and detrimental effects to animals have been reported in a growing number of studies. However, there is a paucity of evidence for effects on terrestrial animals in comparison to those in the marine realm. METHODS: We used the fly Drosophila melanogaster to study the effects that exposure to plastics may have on life history traits and immune response. We reared flies in four conditions: In media containing 1% virgin polyethylene, with no chemical additives; in media supplemented with 1% or 4% polyvinyl chloride, known to have a high content of added chemicals; and control flies in non-supplemented media. Plastic particle size ranged from 23-500 µm. We studied fly survival to viral infection, the length of the larval and pupal stage, sex ratios, fertility and the size of the resultant adult flies. We then performed crossings of F1 flies in non-supplemented media and looked at the life history traits of the F2. RESULTS: Flies treated with plastics in the food media showed changes in fertility and sex ratio, but showed no differences in developmental times, adult size or the capacity to fight infections in comparison with controls. However, the offspring of treated flies reared in non-supplemented food had shorter life cycles, and those coming from both polyvinyl chloride treatments were smaller than those offspring of controls.

Developmental toxicity of plastic leachates on the sea urchin Paracentrotus lividus.

Microplastic pollution has become ubiquitous, affecting a wide variety of biota. Although microplastics are known to alter the development of a range of marine invertebrates, no studies provide a detailed morphological characterisation of the developmental defects. Likewise, the developmental toxicity of chemicals leached from plastic particles is understudied. The consequences of these developmental effects are likely underestimated, and the effects on ecosystems are unknown. Using the sea urchin Paracentrotus lividus as a model, we studied the effects of leachates of three forms of plastic pellet: new industrial pre-production plastic nurdles, beached pre-production nurdles, and floating filters, known as biobeads, also retrieved from the environment. Our chemical analyses show that leachates from beached pellets (biobead and nurdle pellets) and highly plasticised industrial pellets (PVC) contain polycyclic aromatic hydrocarbons and polychlorinated biphenyls, which are known to be detrimental to development and other life stages of animals. We also demonstrate that these microplastic leachates elicit severe, consistent and treatment-specific developmental abnormalities in P. lividus at embryonic and larval stages. Those embryos exposed to virgin polyethylene leachates with no additives nor environmental contaminants developed normally, suggesting that the abnormalities observed are the result of exposure to either environmentally adsorbed contaminants or pre-existing industrial additives within the polymer matrix. In the light of the chemical contents of the leachates and other characteristics of the plastic particles used, we discuss the phenotypes observed during our study, which include abnormal gastrulation, impaired skeletogenesis, abnormal neurogenesis, redistribution of pigmented cells and embryo radialisation.

tarsal-less is expressed as a gap gene but has no gap gene phenotype in the moth midge Clogmia albipunctata.

Gap genes are involved in segment determination during early development of the vinegar fly Drosophila melanogaster and other dipteran insects (flies, midges and mosquitoes). They are expressed in overlapping domains along the antero-posterior (A-P) axis of the blastoderm embryo. While gap domains cover the entire length of the A-P axis in Drosophila, there is a region in the blastoderm of the moth midge Clogmia albipunctata, which lacks canonical gap gene expression. Is a non-canonical gap gene functioning in this area? Here, we characterize tarsal-less (tal) in C. albipunctata. The homologue of tal in the flour beetle Tribolium castaneum (called milles-pattes, mlpt) is a bona fide gap gene. We find that Ca-tal is expressed in the region previously reported as lacking gap gene expression. Using RNA interference, we study the interaction of Ca-tal with gap genes. We show that Ca-tal is regulated by gap genes, but only has a very subtle effect on tailless (Ca-tll), while not affecting other gap genes at all. Moreover, cuticle phenotypes of Ca-tal depleted embryos do not show any gap phenotype. We conclude that Ca-tal is expressed and regulated like a gap gene, but does not function as a gap gene in C. albipunctata.

A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila.

Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects.

Non-canonical dorsoventral patterning in the moth midge Clogmia albipunctata.

BACKGROUND: Bone morphogenetic proteins (BMPs) are of central importance for dorsal-ventral (DV) axis specification. They are core components of a signalling cascade that includes the BMP ligand decapentaplegic (DPP) and its antagonist short gastrulation (SOG) in Drosophila melanogaster. These components are very ancient, with orthologs involved in DV patterning in both protostomes and deuterostomes. Despite such strong conservation, recent comparative work in insects has revealed interesting differences in the way the patterning function of the DV system is achieved in different species. RESULTS: In this paper, we characterise the expression patterns of the principal components of the BMP DV patterning system, as well as its signalling outputs and downstream targets, in the non-cyclorrhaphan moth midge Clogmia albipunctata (Diptera: Psychodidae). We previously reported ventral expression patterns of dpp in the pole regions of C. albipunctata blastoderm embryos. Strikingly, we also find ventral sog and posteriorly restricted tkv expression, as well as expanded polar activity of pMad. We use our results from gene knock-down by embryonic RNA interference to propose a mechanism of polar morphogen shuttling in C. albipunctata. We compare these results to available data from other species and discuss scenarios for the evolution of DV signalling in the holometabolan insects. CONCLUSIONS: A comparison of gene expression patterns across hemipteran and holometabolan insects reveals that expression of upstream signalling factors in the DV system is very variable, while signalling output is highly conserved. This has two major implications: first, as long as ligand shuttling and other upstream regulatory mechanisms lead to an appropriately localised activation of BMP signalling at the dorsal midline, it is of less importance exactly where the upstream components of the DV system are expressed. This, in turn, explains why the early-acting components of the DV patterning system in insects exhibit extensive amounts of developmental systems drift constrained by highly conserved downstream signalling output.

Gap Gene Regulatory Dynamics Evolve along a Genotype Network.

Developmental gene networks implement the dynamic regulatory mechanisms that pattern and shape the organism. Over evolutionary time, the wiring of these networks changes, yet the patterning outcome is often preserved, a phenomenon known as "system drift." System drift is illustrated by the gap gene network-involved in segmental patterning-in dipteran insects. In the classic model organism Drosophila melanogaster and the nonmodel scuttle fly Megaselia abdita, early activation and placement of gap gene expression domains show significant quantitative differences, yet the final patterning output of the system is essentially identical in both species. In this detailed modeling analysis of system drift, we use gene circuits which are fit to quantitative gap gene expression data in M. abdita and compare them with an equivalent set of models from D. melanogaster. The results of this comparative analysis show precisely how compensatory regulatory mechanisms achieve equivalent final patterns in both species. We discuss the larger implications of the work in terms of "genotype networks" and the ways in which the structure of regulatory networks can influence patterns of evolutionary change (evolvability).

Rearing the scuttle fly Megaselia scalaris (Diptera: Phoridae) on industrial compounds: implications on size and lifespan.

Megaselia scalaris (Loew, 1866) (Diptera, phoridae) is a cosmopolitan fly species used in forensic science, and has been developed as a laboratory model species. They feed on decaying corpses as well as a wide variety of organic matter, and previous studies have even found them feeding on liquid paint or shoe polish, suggesting the possibility that they could breakdown industrial compounds. To test this possibility, we fed M. scalaris on a variety of industrially obtained materials and found that it was unable to complete its life cycle, dying at the larval stage, with the majority of compounds tested. However, when fed on modeling clay, a substrate that contains starch and inedible compounds, it was able to complete its life cycle. On this diet we observed increased larval development time, decreased pupal development time and a shortened adult life span. Additionally, pupae and adult flies were smaller than control flies. Contrary to previous reports, we find no evidence that M. scalaris is able to survive on modern formulations of liquid paint.

High-resolution gene expression data from blastoderm embryos of the scuttle fly Megaselia abdita.

Gap genes are involved in segment determination during early development in dipteran insects (flies, midges, and mosquitoes). We carried out a systematic quantitative comparative analysis of the gap gene network across different dipteran species. Our work provides mechanistic insights into the evolution of this pattern-forming network. As a central component of our project, we created a high-resolution quantitative spatio-temporal data set of gap and maternal co-ordinate gene expression in the blastoderm embryo of the non-drosophilid scuttle fly, Megaselia abdita. Our data include expression patterns in both wild-type and RNAi-treated embryos. The data-covering 10 genes, 10 time points, and over 1,000 individual embryos-consist of original embryo images, quantified expression profiles, extracted positions of expression boundaries, and integrated expression patterns, plus metadata and intermediate processing steps. These data provide a valuable resource for researchers interested in the comparative study of gene regulatory networks and pattern formation, an essential step towards a more quantitative and mechanistic understanding of developmental evolution.

Maternal co-ordinate gene regulation and axis polarity in the scuttle fly Megaselia abdita.

Axis specification and segment determination in dipteran insects are an excellent model system for comparative analyses of gene network evolution. Antero-posterior polarity of the embryo is established through systems of maternal morphogen gradients. In Drosophila melanogaster, the anterior system acts through opposing gradients of Bicoid (Bcd) and Caudal (Cad), while the posterior system involves Nanos (Nos) and Hunchback (Hb) protein. These systems act redundantly. Both Bcd and Hb need to be eliminated to cause a complete loss of polarity resulting in mirror-duplicated abdomens, so-called bicaudal phenotypes. In contrast, knock-down of bcd alone is sufficient to induce double abdomens in non-drosophilid cyclorrhaphan dipterans such as the hoverfly Episyrphus balteatus or the scuttle fly Megaselia abdita. We investigate conserved and divergent aspects of axis specification in the cyclorrhaphan lineage through a detailed study of the establishment and regulatory effect of maternal gradients in M. abdita. Our results show that the function of the anterior maternal system is highly conserved in this species, despite the loss of maternal cad expression. In contrast, hb does not activate gap genes in this species. The absence of this activatory role provides a precise genetic explanation for the loss of polarity upon bcd knock-down in M. abdita, and suggests a general scenario in which the posterior maternal system is increasingly replaced by the anterior one during the evolution of the cyclorrhaphan dipteran lineage.

Quantitative system drift compensates for altered maternal inputs to the gap gene network of the scuttle fly Megaselia abdita.

The segmentation gene network in insects can produce equivalent phenotypic outputs despite differences in upstream regulatory inputs between species. We investigate the mechanistic basis of this phenomenon through a systems-level analysis of the gap gene network in the scuttle fly Megaselia abdita (Phoridae). It combines quantification of gene expression at high spatio-temporal resolution with systematic knock-downs by RNA interference (RNAi). Initiation and dynamics of gap gene expression differ markedly between M. abdita and Drosophila melanogaster, while the output of the system converges to equivalent patterns at the end of the blastoderm stage. Although the qualitative structure of the gap gene network is conserved, there are differences in the strength of regulatory interactions between species. We term such network rewiring 'quantitative system drift'. It provides a mechanistic explanation for the developmental hourglass model in the dipteran lineage. Quantitative system drift is likely to be a widespread mechanism for developmental evolution.

SuperFly: a comparative database for quantified spatio-temporal gene expression patterns in early dipteran embryos.

We present SuperFly (http://superfly.crg.eu), a relational database for quantified spatio-temporal expression data of segmentation genes during early development in different species of dipteran insects (flies, midges and mosquitoes). SuperFly has a special focus on emerging non-drosophilid model systems. The database currently includes data of high spatio-temporal resolution for three species: the vinegar fly Drosophila melanogaster, the scuttle fly Megaselia abdita and the moth midge Clogmia albipunctata. At this point, SuperFly covers up to 9 genes and 16 time points per species, with a total of 1823 individual embryos. It provides an intuitive web interface, enabling the user to query and access original embryo images, quantified expression profiles, extracted positions of expression boundaries and integrated datasets, plus metadata and intermediate processing steps. SuperFly is a valuable new resource for the quantitative comparative study of gene expression patterns across dipteran species. Moreover, it provides an interesting test set for systems biologists interested in fitting mathematical gene network models to data. Both of these aspects are essential ingredients for progress toward a more quantitative and mechanistic understanding of developmental evolution.

A quantitative atlas of Even-skipped and Hunchback expression in Clogmia albipunctata (Diptera: Psychodidae) blastoderm embryos.

BACKGROUND: Comparative studies of developmental processes are one of the main approaches to evolutionary developmental biology (evo-devo). Over recent years, there has been a shift of focus from the comparative study of particular regulatory genes to the level of whole gene networks. Reverse-engineering methods can be used to computationally reconstitute and analyze the function and dynamics of such networks. These methods require quantitative spatio-temporal expression data for model fitting. Obtaining such data in non-model organisms remains a major technical challenge, impeding the wider application of data-driven mathematical modeling to evo-devo. RESULTS: We have raised antibodies against four segmentation gene products in the moth midge Clogmia albipunctata, a non-drosophilid dipteran species. We have used these antibodies to create a quantitative atlas of protein expression patterns for the gap gene hunchback (hb), and the pair-rule gene even-skipped (eve). Our data reveal differences in the dynamics of Hb boundary positioning and Eve stripe formation between C. albipunctata and Drosophila melanogaster. Despite these differences, the overall relative spatial arrangement of Hb and Eve domains is remarkably conserved between these two distantly related dipteran species. CONCLUSIONS: We provide a proof of principle that it is possible to acquire quantitative gene expression data at high accuracy and spatio-temporal resolution in non-model organisms. Our quantitative data extend earlier qualitative studies of segmentation gene expression in C. albipunctata, and provide a starting point for comparative reverse-engineering studies of the evolutionary and developmental dynamics of the segmentation gene system.

A staging scheme for the development of the scuttle fly Megaselia abdita.

Model organisms, such as Drosophila melanogaster, provide powerful experimental tools for the study of development. However, approaches using model systems need to be complemented by comparative studies for us to gain a deeper understanding of the functional properties and evolution of developmental processes. New model organisms need to be established to enable such comparative work. The establishment of new model system requires a detailed description of its life cycle and development. The resulting staging scheme is essential for providing morphological context for molecular studies, and allows us to homologise developmental processes between species. In this paper, we provide a staging scheme and morphological characterisation of the life cycle for an emerging non-drosophilid dipteran model system: the scuttle fly Megaselia abdita. We pay particular attention to early embryogenesis (cleavage and blastoderm stages up to gastrulation), the formation and retraction of extraembryonic tissues, and the determination and formation of germ (pole) cells. Despite the large evolutionary distance between the two species (approximately 150 million years), we find that M. abdita development is remarkably similar to D. melanogaster in terms of developmental landmarks and their relative timing.

A staging scheme for the development of the moth midge Clogmia albipunctata.

Model organisms, such as Drosophila melanogaster, allow us to address a wide range of biological questions with experimental rigour. However, studies in model species need to be complemented by comparative studies if we are to fully understand the functional properties and evolutionary history of developmental processes. The establishment of new model organisms is crucial for this purpose. One of the first essential steps to establish a species as an experimental model is to carefully describe its life cycle and development. The resulting staging scheme serves as a framework for molecular studies, and allows us to homologise developmental processes between species. In this paper, we have characterised the life cycle and development of an emerging non-drosophilid dipteran model system: the moth midge Clogmia albipunctata. In particular, we focus on early embryogenesis (cleavage and blastoderm cycles before gastrulation), on formation and retraction of extraembryonic tissues, and on formation of the germ line. Considering the large evolutionary distance between the two species (approximately 250 million years), we find that the development of C. albipunctata is remarkably conserved compared to D. melanogaster. On the other hand, we detect significant differences in morphology and timing affecting the development of extraembryonic tissues and the germ line. Moreover, C. albipunctata shows several heterochronic shifts, and lacks head involution and associated processes during late stages of development.

Evolution and expression of BMP genes in flies.

Bone morphogenetic proteins (BMPs) play key roles in development. In Drosophila melanogaster, there are three BMP-encoding genes: decapentaplegic (dpp), glass bottom boat (gbb) and screw (scw). dpp and gbb are found in all groups of insects. In contrast, the origin of scw via duplication of an ancestral gbb homologue is more recent, with new evidence placing it within the Diptera. Recent studies show that scw appeared basal to the Schizophora, since scw orthologues exist in aschizan cyclorrhaphan flies. In order to further localise the origin of scw, we have utilised new genomic resources for the nematoceran moth midge Clogmia albipunctata (Psychodidae). We identified the BMP subclass members dpp and gbb from an early embryonic transcriptome and show that their expression patterns in the blastoderm differ considerably from those seen in cyclorrhaphan flies. Further searches of the genome of C. albipunctata were unable to identify a scw-like gbb duplicate, but confirm the presence of dpp and gbb. Our phylogenetic analysis shows these to be clear orthologues of dpp and gbb from other non-cyclorrhaphan insects, with C. albipunctata gbb branching ancestrally to the cyclorrhaphan gbb/scw split. Furthermore, our analysis suggests that scw is absent from all Nematocera, including the Bibionomorpha. We conclude that the gbb/scw duplication occurred between the separation of the lineage leading to Brachycera and the origin of cyclorrhaphan flies 200-150 Ma ago.

Comparative transcriptomics of early dipteran development.

BACKGROUND: Modern sequencing technologies have massively increased the amount of data available for comparative genomics. Whole-transcriptome shotgun sequencing (RNA-seq) provides a powerful basis for comparative studies. In particular, this approach holds great promise for emerging model species in fields such as evolutionary developmental biology (evo-devo). RESULTS: We have sequenced early embryonic transcriptomes of two non-drosophilid dipteran species: the moth midge Clogmia albipunctata, and the scuttle fly Megaselia abdita. Our analysis includes a third, published, transcriptome for the hoverfly Episyrphus balteatus. These emerging models for comparative developmental studies close an important phylogenetic gap between Drosophila melanogaster and other insect model systems. In this paper, we provide a comparative analysis of early embryonic transcriptomes across species, and use our data for a phylogenomic re-evaluation of dipteran phylogenetic relationships. CONCLUSIONS: We show how comparative transcriptomics can be used to create useful resources for evo-devo, and to investigate phylogenetic relationships. Our results demonstrate that de novo assembly of short (Illumina) reads yields high-quality, high-coverage transcriptomic data sets. We use these data to investigate deep dipteran phylogenetic relationships. Our results, based on a concatenation of 160 orthologous genes, provide support for the traditional view of Clogmia being the sister group of Brachycera (Megaselia, Episyrphus, Drosophila), rather than that of Culicomorpha (which includes mosquitoes and blackflies).