Identification of the genes encoding candidate septate junction components expressed during early development of the sea urchin, Strongylocentrotus purpuratus, and evidence of a role for Mesh in the formation of the gut barrier.

Septate junctions (SJs) evolved as cell-cell junctions that regulate the paracellular barrier and integrity of epithelia in invertebrates. Multiple morphological variants of SJs exist specific to different epithelia and/or phyla but the biological significance of varied SJ morphology is unclear because the knowledge of the SJ associated proteins and their functions in non-insect invertebrates remains largely unknown. Here we report cell-specific expression of nine candidate SJ genes in the early life stages of the sea urchin Strongylocentrotus purpuratus. By use of in situ RNA hybridization and single cell RNA-seq we found that the expression of selected genes encoding putatively SJ associated transmembrane and cytoplasmic scaffold molecules was dynamically regulated during epithelial development in the embryos and larvae with different epithelia expressing different cohorts of SJ genes. We focused a functional analysis on SpMesh, a homolog of the Drosophila smooth SJ component Mesh, which was highly enriched in the endodermal epithelium of the mid- and hindgut. Functional perturbation of SpMesh by both CRISPR/Cas9 mutagenesis and vivo morpholino-mediated knockdown shows that loss of SpMesh does not disrupt the formation of the gut epithelium during gastrulation. However, loss of SpMesh resulted in a severely reduced gut-paracellular barrier as quantitated by increased permeability to 3-5 â€‹kDa FITC-dextran. Together, these studies provide a first look at the molecular SJ physiology during the development of a marine organism and suggest a shared role for Mesh-homologous proteins in forming an intestinal barrier in invertebrates. Results have implications for consideration of the traits underlying species-specific sensitivity of marine larvae to climate driven ocean change.

DNA at the whim of the water: environmental DNA as a course-based undergraduate research experience.

Course-based undergraduate research experiences (CUREs) increase student access to high impact research experiences. CUREs engage students in the scientific process by learning how to pose scientific questions, develop hypotheses, and generate data to test them. Environmental DNA (eDNA) is a growing field of research that is gaining accessibility through decreasing laboratory costs, which can make a foundation for multiple, engaging CUREs. This manuscript describes three case studies that used eDNA in an upper year undergraduate course. The first focusses on a systematic literature review of eDNA metadata reporting. The second describes the biomonitoring of brook trout in southern Ontario using eDNA. The third involves eDNA metabarcoding for freshwater fish detection in southern Ontario. Undergraduates were involved in the development and execution of experiments, scientific communication, the peer review process, and fundraising. Through this manuscript, we show the novel application of eDNA CUREs and provide a roadmap for other instructors interested in implementing similar projects. Interviews with seven students from these courses indicate the benefits experienced from taking these courses. We argue that the use of eDNA in CUREs should be expanded in undergraduate biology programs due to the benefit to students and the increasing accessibility of this technology.

Comparative transcriptomics analysis pipeline for the meta-analysis of phylogenetically divergent datasets (CoRMAP).

BACKGROUND: Transcriptional regulation is a fundamental mechanism underlying biological functions. In recent years, a broad array of RNA-Seq tools have been used to measure transcription levels in biological experiments, in whole organisms, tissues, and at the single cell level. Collectively, this is a vast comparative dataset on transcriptional processes across organisms. Yet, due to technical differences between the studies (sequencing, experimental design, and analysis) extracting usable comparative information and conducting meta-analyses remains challenging. RESULTS: We introduce Comparative RNA-Seq Metadata Analysis Pipeline (CoRMAP), a meta-analysis tool to retrieve comparative gene expression data from any RNA-Seq dataset using de novo assembly, standardized gene expression tools and the implementation of OrthoMCL, a gene orthology search algorithm. It employs the use of orthogroup assignments to ensure the accurate comparison of gene expression levels between experiments and species. Here we demonstrate the use of CoRMAP on two mouse brain transcriptomes with similar scope, that were collected several years from each other using different sequencing technologies and analysis methods. We also compare the performance of CoRMAP with a functional mapping tool, previously published. CONCLUSION: CoRMAP provides a framework for the meta-analysis of RNA-Seq data from divergent taxonomic groups. This method facilitates the retrieval and comparison of gene expression levels from published data sets using standardized assembly and analysis. CoRMAP does not rely on reference genomes and consequently facilitates direct comparison between diverse studies on a range of organisms.

Thyroid hormone-induced cell death in sea urchin metamorphic development.

Thyroid hormones (THs) are important regulators of development, metabolism and homeostasis in metazoans. Specifically, they have been shown to regulate the metamorphic transitions of vertebrates and invertebrates alike. Indirectly developing sea urchin larvae accelerate the formation of juvenile structures in response to thyroxine (T4) treatment, while reducing their larval arm length. The mechanisms underlying larval arm reduction are unknown and we hypothesized that programmed cell death (PCD) is linked to this process. To test this hypothesis, we measured larval arm retraction in response to different THs (T4, T3, rT3, Tetrac) and assessed cell death in larvae using three different methods (TUNEL, YO-PRO-1 and caspase-3 activity) in the sea urchin Strongylocentrotus purpuratus. We also compared the extent of PCD in response to TH treatment before and after the invagination of the larval ectoderm, which marks the initiation of juvenile development in larval sea urchin species. We found that T4 treatment results in the strongest reduction of larval arms but detected a significant increase of PCD in response to T4, T3 and Tetrac in post-ingression but not pre-ingression larvae. As post-ingression larvae have initiated metamorphic development and therefore allocate resources to both larval and the juvenile structures, these results provide evidence that THs regulate larval development differentially via PCD. PCD in combination with cell proliferation likely has a key function in sea urchin development.

Transcriptomic signature of extinction learning in the brain of the fire-bellied toad, Bombina orientalis.

Insight into the molecular and cellular mechanisms of learning and memory from a diverse array of taxa contributes to our understanding of the evolution of these processes. The fire-bellied toad, Bombina orientalis, is a basal anuran amphibian model species who could help us describe shared and divergent characteristics of learning and memory mechanisms between amphibians and other vertebrates, and hence answer questions about the evolution of learning. Utilizing next generation sequencing techniques, we profiled gene expression patterns associated with the extinction of prey-catching conditioning in the brain of the fire-bellied toad. For this purpose, gene expression was at first compared between toads sacrificed after acquisition and extinction of the conditioned response. A second comparison was done between toads submitted to extinction following either short or long acquisition training, which results in toads displaying response extinction or resistance to extinction, respectively. We analyzed brain tissue transcription profiles common to both acquisition and extinction learning, or unique to extinction learning and resistance to extinction, and found significant overlap in gene expression related to molecular pathways involving neuronal plasticity (e.g. structural modification, transcription). However, extinction learning induced a unique GABAergic transcriptomic signal, which may be responsible for suppression of the original response memory. Further, when comparing extinction learning in short- and long-trained groups, short training engaged many pathways related to neuronal plasticity, as expected, but long training engaged molecular pathways related to the suppression of learning through epigenetic mediated transcriptional suppression and inhibitory neurotransmission. Overall, gene expression patterns associated with extinction learning in the fire-bellied toad were similar to those found in mammals submitted to extinction, although some divergent profiles highlighted potential differences in the mechanisms of learning and memory among tetrapods.

Consequences of calcium decline on the embryogenesis and life history of Daphnia magna.

Ambient calcium is declining in thousands of soft-water lake habitats in temperate regions as a consequence of unsustainable forestry practices, decreased atmospheric calcium deposition and acidic deposition. As their exoskeleton is heavily reinforced with calcium, freshwater crustaceans have a high specific calcium requirement relative to other aquatic organisms. Daphnia, in particular, is an ideal crustacean for investigating the consequences of calcium decline because it is an abundant and important member of freshwater zooplankton communities. Although it has been established that adult and juvenile Daphnia have different tolerances to low ambient calcium as a result of their different life stage-specific calcium requirements, the consequences of declining calcium on embryonic development have never been investigated. Here, we describe the distribution of calcium in embryonic stages of D. magna and introduce a novel and easy to use staging scheme. We tested whether calcium can be traced from mothers to their offspring. Finally, we assessed the fitness consequences of maternal provisioning in limiting calcium environments. We found that while embryos require calcium for their development and moulting, they do not equilibrate with environmental calcium levels. Instead, we were able to trace calcium from mothers to their offspring. Furthermore, our data strongly suggest that females are faced with an allocation trade-off between providing calcium to their offspring and using it for growth and moulting. Together, these data provide novel insights into the consequences of calcium decline for freshwater zooplankton.

A detailed staging scheme for late larval development in Strongylocentrotus purpuratus focused on readily-visible juvenile structures within the rudiment.

BACKGROUND: The purple sea urchin, Strongylocentrotus purpuratus, has long been the focus of developmental and ecological studies, and its recently-sequenced genome has spawned a diversity of functional genomics approaches. S. purpuratus has an indirect developmental mode with a pluteus larva that transforms after 1-3 months in the plankton into a juvenile urchin. Compared to insects and frogs, mechanisms underlying the correspondingly dramatic metamorphosis in sea urchins remain poorly understood. In order to take advantage of modern techniques to further our understanding of juvenile morphogenesis, organ formation, metamorphosis and the evolution of the pentameral sea urchin body plan, it is critical to assess developmental progression and rate during the late larval phase. This requires a staging scheme that describes developmental landmarks that can quickly and consistently be used to identify the stage of individual living larvae, and can be tracked during the final two weeks of larval development, as the juvenile is forming. RESULTS: Notable structures that are easily observable in developing urchin larvae are the developing spines, test and tube feet within the juvenile rudiment that constitute much of the oral portion of the adult body plan. Here we present a detailed staging scheme of rudiment development in the purple urchin using soft structures of the rudiment and the primordia of these juvenile skeletal elements. We provide evidence that this scheme is robust and applicable across a range of temperature and feeding regimes. CONCLUSIONS: Our proposed staging scheme provides both a useful method to study late larval development in the purple urchin, and a framework for developing similar staging schemes across echinoderms. Such efforts will have a high impact on evolutionary developmental studies and larval ecology, and facilitate research on this important deuterostome group.

Spontaneous unraveling of hagfish slime thread skeins is mediated by a seawater-soluble protein adhesive.

Hagfishes are known for their ability to rapidly produce vast quantities of slime when provoked. The slime is formed via the interaction between seawater and two components released by the slime glands: mucin vesicles from gland mucous cells, which swell and rupture in seawater to form a network of mucus strands, and intermediate filament-rich threads, which are produced within gland thread cells as tightly coiled bundles called skeins. A previous study showed that the unraveling of skeins from Atlantic hagfish (Myxine glutinosa) requires both the presence of mucins and hydrodynamic mixing. In contrast, skeins from Pacific hagfish (Eptatretus stoutii) unravel in the absence of both mucins and mixing. We tested the hypothesis that spontaneous unraveling of E. stoutii skeins is triggered by the dissolution of a seawater-soluble protein adhesive and the release of stored strain energy within the coiled thread. Here we show that, as predicted by this hypothesis, unraveling can be initiated by a protease under conditions in which unraveling does not normally occur. We also demonstrate, using high resolution scanning electron microscopy, that the treatment of skeins with solutions that cause unraveling also leads to the disappearance of surface and inter-thread features that remain when skeins are washed with stabilizing solutions. Our study provides a mechanism for the deployment of thread skeins in Pacific hagfish slime, and raises the possibility of producing novel biomimetic protein adhesives that are salt, temperature and kosmotrope sensitive.

Iodine accumulation in sea urchin larvae is dependent on peroxide.

Iodine has many important biological functions and its concentrations vary with the environment. Recent research has provided novel insights into iodine uptake mechanisms in marine bacteria and kelp through hydrogen peroxide-dependent diffusion (PDD). This mechanism is distinct from sodium-dependent mechanisms known from vertebrates. In vertebrates, iodine accumulates in the thyroid gland by the action of the apical iodide transporter (AIT) and the sodium/iodide symporter (NIS). Neither of these proteins has, thus far, been identified outside of the chordates, and PDD (as an iodine uptake mechanism) has never been studied in animals. Using (125)I as a marker for total iodine influx, we tested iodine uptake via sodium-dependent transport versus PDD in embryos and larvae of the sea urchin Strongylocentrotus purpuratus. We found that iodine uptake in S. purpuratus is largely independent of NIS/AIT. Instead, we found that uptake is dependent on the presence and production of hydrogen peroxide, indicating that sea urchin larvae use PDD as a mechanism for iodine acquisition. Our data, for the first time, provide conclusive evidence for this mechanism in an animal. Furthermore, our data provide preliminary evidence that sodium-dependent iodine uptake via active transporter proteins is a synapomorphy of vertebrates.

Thyroid hormone membrane receptor binding and transcriptional regulation in the sea urchin Strongylocentrotus purpuratus.

Thyroid hormones (THs) are small amino acid derived signaling molecules with broad physiological and developmental functions in animals. Specifically, their function in metamorphic development, ion regulation, angiogenesis and many others have been studied in detail in mammals and some other vertebrates. Despite extensive reports showing pharmacological responses of invertebrate species to THs, little is known about TH signaling mechanisms outside of vertebrates. Previous work in sea urchins suggests that non-genomic mechanisms are activated by TH ligands. Here we show that several THs bind to sea urchin (Strongylocentrotus purpuratus) cell membrane extracts and are displaced by ligands of RGD-binding integrins. A transcriptional analysis across sea urchin developmental stages shows activation of genomic and non-genomic pathways in response to TH exposure, suggesting that both pathways are activated by THs in sea urchin embryos and larvae. We also provide evidence associating TH regulation of gene expression with TH response elements in the genome. In ontogeny, we found more differentially expressed genes in older larvae compared to gastrula stages. In contrast to gastrula stages, the acceleration of skeletogenesis by thyroxine in older larvae is not fully inhibited by competitive ligands or inhibitors of the integrin membrane receptor pathway, suggesting that THs likely activate multiple pathways. Our data confirms a signaling function of THs in sea urchin development and suggests that both genomic and non-genomic mechanisms play a role, with genomic signaling being more prominent during later stages of larval development.

Histamine is a modulator of metamorphic competence in Strongylocentrotus purpuratus (Echinodermata: Echinoidea).

BACKGROUND: A metamorphic life-history is present in the majority of animal phyla. This developmental mode is particularly prominent among marine invertebrates with a bentho-planktonic life cycle, where a pelagic larval form transforms into a benthic adult. Metamorphic competence (the stage at which a larva is capable to undergo the metamorphic transformation and settlement) is an important adaptation both ecologically and physiologically. The competence period maintains the larval state until suitable settlement sites are encountered, at which point the larvae settle in response to settlement cues. The mechanistic basis for metamorphosis (the morphogenetic transition from a larva to a juvenile including settlement), i.e. the molecular and cellular processes underlying metamorphosis in marine invertebrate species, is poorly understood. Histamine (HA), a neurotransmitter used for various physiological and developmental functions among animals, has a critical role in sea urchin fertilization and in the induction of metamorphosis. Here we test the premise that HA functions as a developmental modulator of metamorphic competence in the sea urchin Strongylocentrotus purpuratus. RESULTS: Our results provide strong evidence that HA leads to the acquisition of metamorphic competence in S. purpuratus larvae. Pharmacological analysis of several HA receptor antagonists and an inhibitor of HA synthesis indicates a function of HA in metamorphic competence as well as programmed cell death (PCD) during arm retraction. Furthermore we identified an extensive network of histaminergic neurons in pre-metamorphic and metamorphically competent larvae. Analysis of this network throughout larval development indicates that the maturation of specific neuronal clusters correlates with the acquisition of metamorphic competence. Moreover, histamine receptor antagonist treatment leads to the induction of caspase mediated apoptosis in competent larvae. CONCLUSIONS: We conclude that HA is a modulator of metamorphic competence in S. purpuratus development and hypothesize that HA may have played an important role in the evolution of settlement strategies in echinoids. Our findings provide novel insights into the evolution of HA signalling and its function in one of the most important and widespread life history transitions in the animal kingdom--metamorphosis.

Deuterostome phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida.

Deuterostomes comprise vertebrates, the related invertebrate chordates (tunicates and cephalochordates) and three other invertebrate taxa: hemichordates, echinoderms and Xenoturbella. The relationships between invertebrate and vertebrate deuterostomes are clearly important for understanding our own distant origins. Recent phylogenetic studies of chordate classes and a sea urchin have indicated that urochordates might be the closest invertebrate sister group of vertebrates, rather than cephalochordates, as traditionally believed. More remarkable is the suggestion that cephalochordates are closer to echinoderms than to vertebrates and urochordates, meaning that chordates are paraphyletic. To study the relationships among all deuterostome groups, we have assembled an alignment of more than 35,000 homologous amino acids, including new data from a hemichordate, starfish and Xenoturbella. We have also sequenced the mitochondrial genome of Xenoturbella. We support the clades Olfactores (urochordates and vertebrates) and Ambulacraria (hemichordates and echinoderms). Analyses using our new data, however, do not support a cephalochordate and echinoderm grouping and we conclude that chordates are monophyletic. Finally, nuclear and mitochondrial data place Xenoturbella as the sister group of the two ambulacrarian phyla. As such, Xenoturbella is shown to be an independent phylum, Xenoturbellida, bringing the number of living deuterostome phyla to four.

Evolution of non-genomic nuclear receptor function.

Nuclear receptors (NRs) are responsible for the regulation of diverse developmental and physiological systems in metazoans. NR actions can be the result of genomic and non-genomic mechanisms depending on whether they act inside or outside of the nucleus respectively. While the actions of both mechanisms have been shown to be crucial to NR functions, non-genomic actions are considered less frequently than genomic actions. Furthermore, hypotheses on the origin and evolution of non-genomic NR signaling pathways are rarely discussed in the literature. Here we summarize non-genomic NR signaling mechanisms in the context of NR protein family evolution and animal phyla. We find that NRs across groups and phyla act via calcium flux as well as protein phosphorylation cascades (MAPK/PI3K/PKC). We hypothesize and discuss a possible synapomorphy of NRs in the NR1 and NR3 families, including the thyroid hormone receptor, vitamin D receptor, ecdysone receptor, retinoic acid receptor, steroid receptors, and others. In conclusion, we propose that the advent of non-genomic NR signaling may have been a driving force behind the expansion of NR diversity in Cnidarians, Placozoans, and Bilaterians.

Latent effect of larval rearing environment on post-metamorphic brain growth in an anuran amphibian.

Early development is highly susceptible to environmental influence. We evaluated the role of larval visual environment on brain morphology plasticity in late larval and juvenile stages of Bombina orientalis, an anuran amphibian changing from an aquatic to a terrestrial habitat after metamorphosis. Manipulation of the visual environment was achieved by rearing larvae in normal and darkened water. The juveniles were exposed to normal lighting conditions after metamorphosis, allowing to assess if plastic effects persisted or emerged after metamorphosis. The darkness treatment accelerated development before slowing it down substantially, allowing controls to metamorphose earlier. Although larvae reared in darkened water had the same relative brain size as controls by the end of the larval period, juveniles that had been reared in darkened water as larvae had brains that were 14.4% smaller than juveniles that had been reared under control conditions. Conversely, relative telencephalon size was 6.7% larger in juveniles previously reared in darkened water compared with controls, again with no effect of darkened water seen by the end of the larval period. Unlike the latent effects seen on whole brain and telencephalon size, relative size of the optic tectum was significantly smaller in both larvae and juveniles exposed to the darkened water treatment. Therefore, the effects of visual restriction on juvenile brain form were a combination of latent (whole brain and telencephalon) and carry-over (optic tectum) developmental effects.

Developmental transcriptome of Aplysia californica.

Genome-wide transcriptional changes in development provide important insight into mechanisms underlying growth, differentiation, and patterning. However, such large-scale developmental studies have been limited to a few representatives of Ecdysozoans and Chordates. Here, we characterize transcriptomes of embryonic, larval, and metamorphic development in the marine mollusc Aplysia californica and reveal novel molecular components associated with life history transitions. Specifically, we identify more than 20 signal peptides, putative hormones, and transcription factors in association with early development and metamorphic stages-many of which seem to be evolutionarily conserved elements of signal transduction pathways. We also characterize genes related to biomineralization-a critical process of molluscan development. In summary, our experiment provides the first large-scale survey of gene expression in mollusc development, and complements previous studies on the regulatory mechanisms underlying body plan patterning and the formation of larval and juvenile structures. This study serves as a resource for further functional annotation of transcripts and genes in Aplysia, specifically and molluscs in general. A comparison of the Aplysia developmental transcriptome with similar studies in the zebra fish Danio rerio, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and other studies on molluscs suggests an overall highly divergent pattern of gene regulatory mechanisms that are likely a consequence of the different developmental modes of these organisms.

Functional Studies of Trichoplax adhaerens Voltage-Gated Calcium Channel Activity.

Trichoplax adhaerens is a member of the phylum Placozoa, an enigmatic group of benthic animals with remarkably simple morphology. While initial work on these organisms has primarily focused on their morphology and the development of genomic resources, Trichoplax has received increased attention as a model for studying the evolution of nervous and sensory systems. This work is motivated by the fact that Trichoplax features distinct behaviours and responses to environmental stimuli. Therefore, much progress has been made in recent years on the molecular, cellular, and behavioral understanding of this organism. Methods outlined here provide hands-on approaches to cutting edge molecular and cellular techniques to record cellular activities in Trichoplax.

Mass transfer and flow characterization of novel algae-based nutrient removal system.

BACKGROUND: Recirculating aquaculture systems (RAS) are an essential component of sustainable inland seafood production. Still, nutrient removal from these systems can result in substantial environmental problems, or present a major cost factor with few added value options. In this study, an innovative and energy-efficient algae based nutrient removal system (NRS) was developed that has the potential to generate revenue through algal commercialization. We optimized mass transfer in our NRS design using novel aeration and mixing technology, using air lift pumps and developed an original membrane cartridge for the continuous operation of nutrient removal and algae production. Specifically, we designed, manufactured and tested a 60-L NRS prototype. Based on specific airlift mixing conditions as well as concentration gradients, we assessed NRS nutrient removal capacity. We then examined the effects of different internal bioreactor geometries and radial orientations on the mixing efficiency. RESULTS: Using the start-up dynamic method, the overall mass transfer coefficient was found to be in the range of 0.00164-0.0074 [Formula: see text], depending on flow parameters and we confirmed a scaling relationship of mass transfer across concentration gradients. We found the optimal Reynolds number to be 500 for optimal mass transfer, as higher Reynolds numbers resulted in a relatively reduced increase of mass transfer. This relationship between mass transfer and Reynolds number is critical to assess scalability of our system. Our results demonstrate an even distribution of dissolved oxygen levels across the reactor core, demonstrating adequate mixing by the airlift pump, a critical consideration for optimal algal growth. Distribution of dissolved gases in the reactor was further assessed using flow visualization in order to relate the bubble distribution to the mass transfer capabilities of the reactor. We run a successful proof of principle trial using the green alga Dunaliella tertiolecta to assess mass transfer of nutrients across the membrane and biomass production. CONCLUSIONS: Manipulation of the concentration gradient across the membrane demonstrates a more prominent role of airlift mixing at higher concentration gradients. Specifically, the mass transfer rate increased threefold when the concentration gradient was increased 2.5-fold. We found that we can grow algae in the reactor chamber at rates comparable to those of other production systems and that the membrane scaffolds effectively remove nutrients form the wastewater. Our findings provide support for scalability of the design and support the use of this novel NRS for nutrient removal in aquaculture and potentially other applications.

Microscopy Studies of Placozoans.

Trichoplax adhaerens is an enigmatic animal with an extraordinarily simple morphology and a cellular organization, which are the focus of current research. Protocols outlined here provide detailed descriptions of advanced techniques for light and electron microscopic studies of Trichoplax. Studies using these techniques have enhanced our understanding of cell type diversity and function in placozoans and have provided insight into the evolution, development, and physiology of this little understood group.

Endocrine interactions between plants and animals: Implications of exogenous hormone sources for the evolution of hormone signaling.

Hormones are central to animal physiology, metabolism and development. Details on signal transduction systems and regulation of hormone synthesis, activation and release have only been studied for a small number of animal groups, notably arthropods and chordates. However, a significant body of literature suggests that hormonal signaling systems are not restricted to these phyla. For example, work on several echinoderm species shows that exogenous thyroid hormones (THs) affect larval development and metamorphosis and our new data provide strong evidence for endogenous synthesis of THs in sea urchin larvae. In addition to these endogenous sources, these larvae obtain THs when they consume phytoplankton. Another example of an exogenously acquired hormone or their precursors is in insect and arthropod signaling. Sterols from plants are essential for the synthesis of ecdysteroids, a crucial group of insect morphogenic steroids. The availability of a hormone or hormone precursor from food has implications for understanding hormone function and the evolution of hormonal signaling in animals. For hormone function, it creates an important link between the environment and the regulation of internal homeostatic systems. For the evolution of hormonal signaling it helps us to better understand how complex endocrine mechanisms may have evolved.

Transgenerational reproductive effects of two serotonin reuptake inhibitors after acute exposure in Daphnia magna embryos.

The release of pharmaceuticals and personal care products (PPCPs) into aquatic environments has been a major concern for the health of ecosystems. Transgenerational plasticity is a potential mechanism for organisms to respond to changing environmental conditions, including climate change and environmental contaminants. The purpose of the present study was to determine the long-term transgenerational effects of an abundant freshwater zooplankton, Daphnia magna, to acute embryonic exposures of serotonin re-uptake inhibitors (SSRI - fluoxetine and sertraline). Both SSRIs have been used extensively to treat depression and anxiety disorders for decades and persist in freshwater ecosystems at physiologically relevant concentrations. Our results revealed that even short (72 h) embryonic exposures of D. magna embryos had long lasting consequences on life history and expression of 5HT related genes in the unexposed generation (F3). Moreover, we identified direct effects of SSRIs on heart rate and swimming behavior in the first generation that carried over from embryonic exposure. We also found that SSRI exposure resulted in a transient increase of ephippia formation in the F1 and F2 . Our results suggest that SSRI exposure has transgenerational consequences to the unexposed generation and potentially beyond, even at low concentration (10-100× lower than what can be found in natural ecosystems) and as a result of embryonic exposure. Because of the short reproductive period of D. magna and their integral role in aquatic food webs, these findings have population-level implications and deserve further investigation.