The Complete Genome Sequences of seven species of sea slugs (Nudibranchia, Gastropoda, Mollusca).

We present the complete genome sequences of seven species of sea slugs. Illumina sequencing was performed on tissue from wild-collected museum specimens. The reads were assembled using a de novo method followed by a finishing step. The raw and assembled data are publicly available via Genbank.

Precocious Sperm Exchange in the Simultaneously Hermaphroditic Nudibranch, Berghia stephanieae.

Sexual systems vary greatly across molluscs. This diversity includes simultaneous hermaphroditism, with both sexes functional at the same time. Most nudibranch molluscs are thought to be simultaneous hermaphrodites, but detailed studies of reproductive development and timing remain rare as most species cannot be cultured in the lab. The aeolid nudibranch, Berghia stephanieae, is one such species that can be cultured through multiple generations on the benchtop. We studied B. stephanieae reproductive timing to establish when animals first exchange sperm and how long sperm can be stored. We isolated age- and size-matched individuals at sequential timepoints to learn how early individuals can exchange sperm. Individuals isolated at 10 weeks post initial feeding (wpf; ∼13 weeks postlaying [wpl]) can produce fertilized eggs. This is 6 weeks before animals first lay egg masses, indicating that sperm exchange occurs well before individuals are capable of laying eggs. Our results indicate that male gonads become functional for animals between 6 mm (∼6 wpf, ∼9 wpl) and 9 mm (∼12 wpf, ∼15 wpl) in length. That is much smaller (and sooner) than the size (and age) of individuals at first laying (12-19 mm; ∼16 wpf, ∼19 wpl), indicating that male and female functions do not develop simultaneously. We also tracked the number of fertilized eggs in each egg mass, which remained steady for the first 10-15 egg masses, followed by a decline to near-to-no fertilization. This dataset provides insights into the precise timing of the onset of functionality of the male and female reproductive systems in B. stephanieae. These data contribute to a broader understanding of reproductive development and the potential for understanding the evolution of diverse sexual systems in molluscs.

Traduit par Maryna Lesoway et Hereroa JohnstonLes stratégies de reproduction sont énormément variables chez les mollusques. Cette diversité inclut les hermaphrodites simultanés pouvant être mâle et femelle à la fois. La plupart des mollusques nudibranches sont considérés être des hermaphrodites simultanés, mais les études détaillées du développement reproductif restent rares, car la plupart des nudibranches ne peuvent pas être cultivés en laboratoire. Le nudibranche aeolid, Berghia stephanieae, est l'une des rares espèces pouvant être facilement cultivées sur plusieurs générations sur une paillasse de laboratoire. On a étudié le développement temporel reproductif de B. stephanieae dans le but d'établir à quel moment les individus font leurs premiers échanges de sperme et pour combien de temps ce dernier est gardé. Pour cela des individus ont été isolés en fonction de leur âge et de leurs tailles de manière séquentielle au cours de leurs développement afin de déterminer les premiers échanges de sperme.. Les individus isolés 10 semaines après avoir commencé de manger (∼13 semaines après avoir été pondu) sont capables de produire des œufs fertilisés. Cela se produisant 6 semaines avant que ces individus ne soient capables de déposer des masses d'œufs fécondés, indiquant que l'échange de sperme a eu lieu bien avant que ces individus aient la capacité de pondre des œufs. Nos résultats indiquent que la gonade mâle devient fonctionnelle quand les individus mesurent entre 6 mm et 9 mm de longueur. Par contraste, ces individus ne pondent pas d'œufs avant de mesurer 12 à 19 mm de longueur, indiquant que les fonctions mâles et femelles ne commencent pas en même temps. De plus, on a compté le nombre d'œufs fécondés par masse d'œufs, ce dernier restant inchangé pour les premières 10 à 15 masses d'œufs mais cela s'est suivi par un déclin rapide aboutissant à zéro œuf fécondé par masse d'œuf. Les résultats présentés ici fournissent des informations précises à propos du début du fonctionnement des systèmes reproductifs mâle et femelle chez B. stephanieae. Ces données contribuent à une compréhension approfondie du développement reproductif avec le potentiel d'une meilleure compréhension de l'évolution des diverses systèmes de reproductions.

Traducción por Daniel Escobar-CamachoLos sistemas sexuales varían ampliamente entre los moluscos. Esta diversidad incluye el hermafroditismo simultáneo, ambos sexos funcionales al mismo tiempo. Se cree que la mayoría de los moluscos nudibranquios son hermafroditas simultáneos, pero los estudios detallados del desarrollo reproductivo y su sincronización temporal son raros ya que la mayoría de las especies no se pueden mantener en el laboratorio. El nudibranquio eólido, Berghia stephanieae, es una especie que se puede mantener en cautiverio durante varias generaciones en condiciones de laboratorio. En este estudio, se analizó la sincronización del estado reproductivo de B. stephanieae para establecer el momento en el cual los animales intercambian esperma por primera vez y la duración de cuánto tiempo se puede almacenar el esperma. Para aprender cómo los individuos juveniles intercambian esperma, aislamos individuos de la misma edad y tamaño, en diferentes puntos de tiempo en una secuencia temporal. Se pudo observar que los individuos aislados a las 10 semanas, después de la primera alimentación (wpf; ∼13 semanas después de la puesta, wpl), pueden producir huevos fertilizados. Esto es 6 semanas antes de que los animales desoven masas de óvulos por primera vez, lo cual sugiere que el intercambio de esperma ocurre mucho antes de que los individuos sean capaces de desovar óvulos. Nuestros resultados indican que las gónadas masculinas se vuelven funcionales para animales de entre 6 mm (∼6 wpf, ∼9 wpl) y 9 mm (∼12 wpf, ∼15 wpl) de longitud. Este tamaño es más pequeño (y más temprano) que el tamaño (y la edad) de los individuos en su primera puesta de huevos (12­19 mm; ∼16 wpf, ∼19 wpl), lo que indica que la funcionalidad de machos y hembras no se desarrollan simultáneamente. También analizamos la cantidad de huevos fertilizados en cada masa de huevos, que se mantuvo constante durante las primeras 10 a 15 masas de huevos, seguido de una disminución de fertilización hasta casi ser nula. Estos datos proporcionan información sobre el momento preciso del inicio de la funcionalidad de los sistemas reproductivos masculino y femenino en B. stephanieae, y contribuyen a una comprensión más amplia del desarrollo reproductivo y la evolución de los diversos sistemas sexuales en los moluscos.

Retinoids promote penis development in sequentially hermaphroditic snails.

Sexual systems are surprisingly diverse, considering the ubiquity of sexual reproduction. Sequential hermaphroditism, the ability of an individual to change sex, has emerged multiple times independently across the animal kingdom. In molluscs, repeated shifts between ancestrally separate sexes and hermaphroditism are generally found at the level of family and above, suggesting recruitment of deeply conserved mechanisms. Despite this, molecular mechanisms of sexual development are poorly known. In molluscs with separate sexes, endocrine disrupting toxins bind the retinoid X receptor (RXR), activating ectopic male development in females, suggesting the retinoid pathway as a candidate controlling sexual transitions in sequential hermaphrodites. We therefore tested the role of retinoic acid signaling in sequentially hermaphroditic Crepidula snails, which develop first into males, then change sex, maturing into females. We show that retinoid agonists induce precocious penis growth in juveniles and superimposition of male development in females. Combining RXR antagonists with retinoid agonists significantly reduces penis length in induced juveniles, while similar treatments using retinoic acid receptor (RAR) antagonists increase penis length. Transcripts of both receptors are expressed in the induced penis. Our findings therefore show that retinoid signaling can initiate molluscan male genital development, and regulate penis length. Further, we show that retinoids induce ectopic male development in multiple Crepidula species. Species-specific influence of conspecific induction of sexual transitions correlates with responsiveness to retinoids. We propose that retinoid signaling plays a conserved role in molluscan male development, and that shifts in the timing of retinoid signaling may have been important for the origins of sequential hermaphroditism within molluscs.

Ancestral form and function of larval feeding structures are retained during the development of non-planktotrophic gastropods.

Mode of development (MOD) is a key feature that influences the rate and direction of evolution of marine invertebrates. Although many groups include species with different MODs, the evolutionary loss of feeding larvae is thought to be irreversible, as the complex structures used for larval feeding and swimming are lost, reduced, or modified in many species lacking feeding larvae. This view is largely based on observations of echinoderms. Phylogenetic analysis suggests that feeding larvae have been re-gained in at least one species of calyptraeid gastropod. Further, its sister species has retained the velum, the structure used for larval feeding and swimming. Here, we document velar morphology and function in calyptraeids with 4 different MODs. Embryos of Crepidula navicella, Crepidula atrasolea, Bostrycapulus aculeatus, Bostrycapulus odites, Bostrycapulus urraca, Crepipatella dilatata, Crepipatella occulta, Crucibulum quiriquinae and Crepidula coquimbensis all hatch as crawling juveniles, yet only Crepidula coquimbensis does not make a well-formed velum during intracapsular development. The velar dimensions of 6 species with non-planktotrophic development were similar to those of planktotrophic species, while the body sizes were significantly larger. All of the species studied were able to capture and ingest particles from suspension, but several non-planktotrophic species may ingest captured particles only occasionally. Video footage suggests that some species with adelphophagic direct development capture but frequently fail to ingest particles compared to species with the other MODs. Together these lines of evidence show that, among calyptraeids at least, species that lack planktotrophic larvae often retain the structures and functions necessary to successfully capture and ingest particles, reducing the barriers to the re-evolution of planktotrophy.

An automated aquatic rack system for rearing marine invertebrates.

BACKGROUND: One hundred years ago, marine organisms were the dominant systems for the study of developmental biology. The challenges in rearing these organisms outside of a marine setting ultimately contributed to a shift towards work on a smaller number of so-called model systems. Those animals are typically non-marine organisms with advantages afforded by short life cycles, high fecundity, and relative ease in laboratory culture. However, a full understanding of biodiversity, evolution, and anthropogenic effects on biological systems requires a broader survey of development in the animal kingdom. To this day, marine organisms remain relatively understudied, particularly the members of the Lophotrochozoa (Spiralia), which include well over one third of the metazoan phyla (such as the annelids, mollusks, flatworms) and exhibit a tremendous diversity of body plans and developmental modes. To facilitate studies of this group, we have previously described the development and culture of one lophotrochozoan representative, the slipper snail Crepidula atrasolea, which is easy to rear in recirculating marine aquaria. Lab-based culture and rearing of larger populations of animals remain a general challenge for many marine organisms, particularly for inland laboratories. RESULTS: Here, we describe the development of an automated marine aquatic rack system for the high-density culture of marine species, which is particularly well suited for rearing filter-feeding animals. Based on existing freshwater recirculating aquatic rack systems, our system is specific to the needs of marine organisms and incorporates robust filtration measures to eliminate wastes, reducing the need for regular water changes. In addition, this system incorporates sensors and associated equipment for automated assessment and adjustment of water quality. An automated feeding system permits precise delivery of liquid food (e.g., phytoplankton) throughout the day, mimicking real-life feeding conditions that contribute to increased growth rates and fecundity. CONCLUSION: This automated system makes laboratory culture of marine animals feasible for both large and small research groups, significantly reducing the time, labor, and overall costs needed to rear these organisms.

Sex Determination, Sexual Development, and Sex Change in Slipper Snails.

Sex determination and sexual development are highly diverse and controlled by mechanisms that are extremely labile. While dioecy (separate male and female functions) is the norm for most animals, hermaphroditism (both male and female functions within a single body) is phylogenetically widespread. Much of our current understanding of sexual development comes from a small number of model systems, limiting our ability to make broader conclusions about the evolution of sexual diversity. We present the calyptraeid gastropods as a model for the study of the evolution of sex determination in a sequentially hermaphroditic system. Calyptraeid gastropods, a group of sedentary, filter-feeding marine snails, are sequential hermaphrodites that change sex from male to female during their life span (protandry). This transition includes resorption of the penis and the elaboration of female genitalia, in addition to shifting from production of spermatocytes to oocytes. This transition is typically under environmental control and frequently mediated by social interactions. Males in contact with females delay sex change to transition at larger sizes, while isolated males transition more rapidly and at smaller sizes. This phenomenon has been known for over a century; however, the mechanisms that control the switch from male to female are poorly understood. We review here our current understanding of sexual development and sex determination in the calyptraeid gastropods and other molluscs, highlighting our current understanding of factors implicated in the timing of sex change and the potential mechanisms. We also consider the embryonic origins and earliest expression of the germ line and the effects of environmental contaminants on sexual development.

The genome of the water strider Gerris buenoi reveals expansions of gene repertoires associated with adaptations to life on the water.

BACKGROUND: Having conquered water surfaces worldwide, the semi-aquatic bugs occupy ponds, streams, lakes, mangroves, and even open oceans. The diversity of this group has inspired a range of scientific studies from ecology and evolution to developmental genetics and hydrodynamics of fluid locomotion. However, the lack of a representative water strider genome hinders our ability to more thoroughly investigate the molecular mechanisms underlying the processes of adaptation and diversification within this group. RESULTS: Here we report the sequencing and manual annotation of the Gerris buenoi (G. buenoi) genome; the first water strider genome to be sequenced thus far. The size of the G. buenoi genome is approximately 1,000 Mb, and this sequencing effort has recovered 20,949 predicted protein-coding genes. Manual annotation uncovered a number of local (tandem and proximal) gene duplications and expansions of gene families known for their importance in a variety of processes associated with morphological and physiological adaptations to a water surface lifestyle. These expansions may affect key processes associated with growth, vision, desiccation resistance, detoxification, olfaction and epigenetic regulation. Strikingly, the G. buenoi genome contains three insulin receptors, suggesting key changes in the rewiring and function of the insulin pathway. Other genomic changes affecting with opsin genes may be associated with wavelength sensitivity shifts in opsins, which is likely to be key in facilitating specific adaptations in vision for diverse water habitats. CONCLUSIONS: Our findings suggest that local gene duplications might have played an important role during the evolution of water striders. Along with these findings, the sequencing of the G. buenoi genome now provides us the opportunity to pursue exciting research opportunities to further understand the genomic underpinnings of traits associated with the extreme body plan and life history of water striders.

Beyond the sea: Crepidula atrasolea as a spiralian model system.

This paper introduces the black-footed slipper snail, Crepidula atrasolea, as a new model for biological studies in the Spiralia. C. atrasolea is a calyptraeid gastropod, and congener of the Atlantic slipper snail, C. fornicata. Like C. fornicata, C. atrasolea shares a sedentary, filter-feeding, protandrous lifestyle, but is preferable as a developmental model because of its short generation time, year-round reproduction, and direct development. In our lab, individuals go from egg to reproductive females in under six months, as compared to an estimated 1-2 years for C. fornicata. Here we provide details for collecting and transporting animals, setting up inland aquaria, and maintaining laboratory colonies of C. atrasolea. We also describe early development, which is similar to that in other calyptraeids. Females brood encapsulated embryos for three weeks, which hatch as "crawl-away" juveniles. We also present a developmental transcriptome for C. atrasolea, covering early cleavage through late organogenesis stages, as a useful tool for future studies of gene expression and function. We provide this information to the broader developmental community to facilitate widespread use of this system.

Early Activation of MAPK and Apoptosis in Nutritive Embryos of Calyptraeid Gastropods.

Investigation of alternative phenotypes, different morphologies produced by a single genome, has contributed novel insights into development and evolution. Yet, the mechanisms underlying developmental switch points between alternative phenotypes remain poorly understood. The calyptraeid snails Crepidula navicella and Calyptraea lichen produce two phenotypes: viable and nutritive embryos, where nutritive embryos arrest their development after gastrulation and are ingested by their viable siblings as a form of intracapsular nutrition. Here, we investigate the activity of mitogen-activated protein kinase (MAPK, ERK1/2) and apoptosis during early cleavage. MAPK and apoptosis, found in a previous transcriptomic study, are known to be involved in organization of other spiralian embryos and nutritive embryo development, respectively. In the model Crepidula fornicata, MAPK activation begins at the 16-cell stage. In contrast, we discovered in C. navicella and C. lichen that many embryos begin MAPK activation at the one-cell stage. A subset of embryos shows a similar pattern of MAPK activation to C. fornicata at later stages. In all stages where MAPK is detected, the activation pattern is highly variable, frequently occurring in all quadrants or in multiple tiers of cells. We also detected apoptosis in cleaving embryos, while C. fornicata and Crepidula lessoni, which do not produce nutritive embryos, show no signs of apoptosis during cleavage. Our results show that MAPK and apoptosis are expressed during early development in species with nutritive embryos, and raises the possibility that these processes may play a role and even interact with one another in producing the nutritive embryo phenotype.

The future of Evo-Devo: the inaugural meeting of the Pan American Society for evolutionary developmental biology.

What is the future of evolutionary developmental biology? This question and more were discussed at the inaugural meeting for the Pan American Society for Evolutionary Developmental Biology, held August 5-9, 2015, in Berkeley, California, USA. More than 300 participants attended the first meeting of the new society, representing the current diversity of Evo-Devo. Speakers came from throughout the Americas, presenting work using an impressive range of study systems, techniques, and approaches. Current research draws from themes including the role of gene regulatory networks, plasticity and the role of the environment, novelty, population genetics, and regeneration, using new and emerging techniques as well as traditional tools. Multiple workshops and a discussion session covered subjects both practical and theoretical, providing an opportunity for members to discuss the current challenges and future directions for Evo-Devo. The excitement and discussion generated over the course of the meeting demonstrates the current dynamism of the field, suggesting that the future of Evo-Devo is bright indeed.

Eco-evo-devo: the time has come.

The major goal of ecological evolutionary developmental biology, also known as "eco-evo-devo," is to uncover the rules that underlie the interactions between an organism's environment, genes, and development and to incorporate these rules into evolutionary theory. In this chapter, we discuss some key and emerging concepts within eco-evo-devo. These concepts show that the environment is a source and inducer of genotypic and phenotypic variation at multiple levels of biological organization, while development acts as a regulator that can mask, release, or create new combinations of variation. Natural selection can subsequently fix this variation, giving rise to novel phenotypes. Combining the approaches of eco-evo-devo and ecological genomics will mutually enrich these fields in a way that will not only enhance our understanding of evolution, but also of the genetic mechanisms underlying the responses of organisms to their natural environments.

The development of viable and nutritive embryos in the direct developing gastropod Crepidula navicella.

Adelphophagy occurs when encapsulated embryos complete development by feeding on their developing siblings, which are known as nutritive embryos. Nutritive embryos are found in a variety of animal groups, and are especially common in some groups of marine invertebrates. Although they have evolved numerous times independently in the calyptraeid gastropods, adelphophagic development with nutritive embryos has not been described in detail. Using light microscopy and time-lapse imaging of laboratory-reared embryos, we describe the development of Crepidula navicella, a direct developer with nutritive embryos that cleave and gastrulate. Early stages of nutritive and viable embryos do not show any obvious morphological differences, but do show asynchrony in early cleavage among embryos from the same capsule. We discovered that two classes of nutritive embryos are produced; gastrula-like nutritive embryos, which arrest after gastrulation, and post-gastrula-like nutritive embryos that are more variable in morphology, and show evidence of minor differentiation. This study provides a framework for future research on the developmental and molecular mechanisms of nutritive embryo development of C. navicella, which will allow us to address the role of nutritive embryos in the origins of developmental polyphenisms. Careful description of the developmental sequence is necessary before adaptive hypotheses can be addressed, and comparisons with other taxa can be made. Understanding the different ways that embryos and their development are disrupted to produce nutritive embryos will provide important insights into the normal process of development.

Cleavage pattern and fate map of the mesentoblast, 4d, in the gastropod Crepidula: a hallmark of spiralian development.

BACKGROUND: Animals with a spiral cleavage program, such as mollusks and annelids, make up the majority of the superphylum Lophotrochozoa. The great diversity of larval and adult body plans in this group emerges from this highly conserved developmental program. The 4d micromere is one of the most conserved aspects of spiralian development. Unlike the preceding pattern of spiral divisions, cleavages within the 4d teloblastic sublineages are bilateral, representing a critical transition towards constructing the bilaterian body plan. These cells give rise to the visceral mesoderm in virtually all spiralians examined and in many species they also contribute to the endodermal intestine. Hence, the 4d lineage is an ideal one for studying the evolution and diversification of the bipotential endomesodermal germ layer in protostomes at the level of individual cells. Little is known of how division patterns are controlled or how mesodermal and endodermal sublineages diverge in spiralians. Detailed modern fate maps for 4d exist in only a few species of clitellate annelids, specifically in glossiphoniid leeches and the sludge worm Tubifex. We investigated the 4d lineage in the gastropod Crepidula fornicata, an established model system for spiralian biology, and in a closely related direct-developing species, C. convexa. RESULTS: High-resolution cell lineage tracing techniques were used to study the 4d lineage of C. fornicata and C. convexa. We present a new nomenclature to name the progeny of 4d, and report the fate map for the sublineages up through the birth of the first five pairs of teloblast daughter cells (when 28 cells are present in the 4d sublineage), and describe each clone's behavior during gastrulation and later stages as these undergo differentiation. We identify the precise origin of the intestine, two cells of the larval kidney complex, the larval retractor muscles and the presumptive germ cells, among others. Other tissues that arise later in the 4d lineage include the adult heart, internal foot tissues, and additional muscle and mesenchymal cells derived from later-born progeny of the left and right teloblasts. To test whether other cells can compensate for the loss of these tissues (that is, undergo regulation), specific cells were ablated in C. fornicata. CONCLUSIONS: Our results present the first fate map of the 4d micromere sublineages in a mollusk. The fate map reveals that endodermal and mesodermal fates segregate much later than previously thought. We observed little evidence of regulation between sublineages, consistent with a lineage-driven cell specification process. Our results provide a framework for comparisons with other spiralians and lay the groundwork for investigation of the molecular mechanisms of endomesoderm formation, germ line segregation and bilateral differentiation in Crepidula.