Pearls of wisdom: synergising leadership and expertise in molluscan genomics

The great pondsnail is a model organism in various research domains. Its relatively simple central nervous system makes it a model of choice to decipher molecular mechanisms underlying neurological determinants of physiological behaviors such as feeding, locomotion, learning and memory. In evolutionary biology, L. stagnalis is used to compare hermaphroditism as a reproductive strategy with separate sexes, thereby contributing to the understanding of the evolution of hermaphroditism in general. As both a selfer and outcrosser, it also offers the possibility to test major hypotheses about mating system evolution in animals, a topic traditionally addressed in flowering plants. Regarding development, L. stagnalis is a model for the study of left-right asymmetry, and has been at the centre of the description of the genetic underpinnings of chirality in animals for nearly a century. Likewise, evo-devo approaches also use L. stagnalis with the aim of improving the understanding of the evolution of biomineralization, as a key innovation in Metazoa. Finally, in the domain of environmental sciences, L. stagnalis is widely used in ecotoxicology and is a new model for the regulation of chemicals. A high-quality reference genome will be presented, as generated by the multidisciplinary consortium STAGING (lymnaea STAGnalis INternational Genome initiative).

Marie-Agnès Coutellec, INRA, France

Marie-Agnès Coutellec, INRA, France

Marie-Agnès Coutellec is a researcher at INRA (French National Institute for Agricultural Research). She works at the Joint Research Unit Ecology and Ecosystem Health, in the team EPIX, which focuses on the Evolutionary Ecology of Systems Perturbed by Biological Invasions and Xenobiotics (https://www6.rennes.inra.fr/ese_eng/ABOUT-US/Research-Groups). After completing a PhD in population genetics at Rennes 1 University, France, and a postdoctoral position in molecular phylogeography at the University of Copenhagen, Denmark, she has worked for seven years as lecturer in ecology and animal biology at the University of Rennes 1 before joining INRA. She is interested in evolutionary effects of chemical contaminants on freshwater invertebrates, including underlying molecular processes. Using the hermaphroditic snail Lymnaea stagnalis as main model species, she also studies how environmental stress may interact with inbreeding and drive the evolution of self-fertilization.

Potamopyrgus antipodarum, a New Zealand freshwater snail, is a powerful system for the study of host-parasite coevolution and invasion biology to the maintenance of sexual reproduction. This talk will describe an ongoing effort to generate de novo genome assemblies for P. antipodarum and a close outgroup species, P. estuarinus, using a mixture of short- and long-read sequencing technologies and library types. The initial genome assembly of a reference sexual P. antipodarum lineage indicated high fractions (~35%) of scaffolds containing extended and nearly identical duplicated regions. Flow cytometry data also showed that the diploid genome size of P. estuarinus is ~0.6X of the size of diploid P. antipodarum. Together, this data inspired the hypothesis that P. antipodarum had experienced a recent whole-genome duplication (WGD) event prior to the diversification of its many sexual and asexual lineages. Subsequent analyses supported this hypothesis and mean that P. antipodarum can be used to evaluate the short-term evolutionary consequences of genome duplication. A recent duplication would also explain the general difficulty with assembling the genome, despite generating >100X genome coverage using multiple methodologies. Beyond WGD, this talk describes the role of both selective and non-selective forces and sexual vs asexual reproduction in shaping the genomes of the focal species. Though the process of attaining a high-quality genome of a non-model species has streamlined considerably in recent years, this talk also underlines the challenges that await future attempts to characterize the genomes of a wider range of mollusks.

Dr Maurine Neiman, University of Iowa, USA

Dr Maurine Neiman, University of Iowa, USA

Maurine Neiman (bioweb.biology.uiowa.edu/neiman/index.php) is an Associate Professor in the Department of Biology at the University of Iowa. Her main research interest is a central unanswered question in evolutionary biology: why most organisms reproduce sexually even though asexual reproduction is much simpler and less costly. Much of her research program is focused on using genomic approaches to address the sex question in her snail system, the New Zealand freshwater snail Potamopyrgus antipodarum. Dr Neiman has led the effort to produce the first-ever genome assembly for these snails, and she is now using these genomic data to perform powerful tests of hypotheses for sex.

Dr Peter Fields, University of Basel, Switzerland

Dr Peter Fields, University of Basel, Switzerland

Peter Fields is originally from Kentucky, United States of America. He received his undergraduate degree from Alice Lloyd College and completed his PhD at the University of Virginia in the lab of Douglas Taylor. After completion of his PhD, Peter moved to Basel, Switzerland (present residence) as a post-doctoral researcher with Dieter Ebert. His research focuses primarily on the population genetics/genomics of the freshwater crustacean Dapnnia magna. His previous research projects have included the implementation of de novo assembly strategies in order to answer questions focused on the evolutionary biology of bacteria, microsporidians, crustaceans, and mollusks. This work included the application of high-throughput Illumina sequencing, PacBio long-read sequencing, and the construction of genetic maps (both traditional and optical mapping).

The Biomphalaria glabrata (Hygrophila, Panpulmonata) genome characterisation was initiated in the mid-2000s as a traditional, multi-institute project, and generated an amalgam of several types of short read sequence data (<800 bases): BES, Sanger, 454 and PE (short,3k,8k) Illumina. The resulting genome assembly (Newbler-SOAP) was fragmented, also due to genome complexity (~1Gbp, 63% AT content, ~50% repetitive content). While effective for annotation of genes functioning in immunity and other aspects of snail biology, the assembly includes gaps and inversions within scaffolds, split-, incomplete- and missing genes. Long reads (Nanopore MinION, ≤79kb) from select BAC clones (~136kb average, restriction digested) help greatly to resolve such problems. For comparative immunogenomics, genomes of additional panpulmonata were captured (Illumina Nextseq, 150b PE reads, modest coverage), including several Stylommatophora and hygrophilids from the families Planorbidae, Lymnaeidae and Physidae. With public NGS data this enables the tracking of phylogenetic distribution of snail immune genes. Examplified by revealing intraspecifically diverse mitogenomes in Physella acuta; identifying phylogenetic branch points for evolution of gastropod immunity (eg the Laevapex fuscus (Ancylidae) genome for tracking FREP gene expansion in Planorbidae); and connecting molecular immunology to field study by ecogenomics, “omics” data inspire and compel broad and in-depth, collaborative consideration of molluscan biology.

Professor Coen Adema, University of New Mexico, USA

Professor Coen Adema, University of New Mexico, USA

Professor Coen M Adema has studied gastropods since 1988, obtaining his PhD (Vrije Universiteit Amsterdam, 1992) for characterisation of haemocyte (cell)-mediated cytotoxicity in the pond snail Lymnaea stagnalis as determinant of snail-trematode parasite immunocompatibility. At the University of New Mexico (USA) from 1993, he contributed to the first molecular-level characterisation of anti-parasite snail immune factors from Biomphalaria glabrata (ramshorn snail), FREP lectins that co-determine parasite-host compatibility. The discovery that somatic mutations of FREP genes provide B. glabrata snails with individual immunological identities underscores the realization of unanticipated sophistication of invertebrate innate immunity. Adema developed transcriptomic and genomic resources to further unlock the biology of B. glabrata, ultimately as founding member/chair of the international B. glabrata genome initiative, completed in 2017. Integrating NGS capabilities and bioinfomatics, he collaboratively studies a widening taxonomic range of snails to pursue the evolution of immune function and basic biology in gastropods.

Parasites threaten all free-living species. The immune system is the most important barrier against infections and understanding the form and strength of natural selection on immune activity is crucial for predicting the evolution of parasite resistance. Ecoimmunological studies that examine selection on immune defence typically measure the end products of one or maximum few immunological cascades in invertebrates. This approach was initially motivated by the assumed simplicity of invertebrate immune systems. Over recent decades, however, comparative immunology with aid from genomics has shown that invertebrate immune systems are highly complex and diverse. This provides new challenges for evolutionary ecological research on immune function. Here,  Otto Seppälä presents ongoing work on natural selection on immune activity in Lymnaea stagnalis snails. First, Professor Seppälä shows how natural selection operates on two phenotypic immune traits. Then, Professor Seppälä will show how this work is expanded to cover a broader range of immunological mechanisms at the gene expression level. To identify relevant components of snail immune system their transcriptome responses to bacterial and trematode pathogens were characterised by applying Illumina based RNAseq. In current experiments, the expression levels of selected target genes that cover different branches of snail immune system are used to estimate selection on immune activity.

Otto Seppälä, University of Innsbruck, Austria

Otto Seppälä, University of Innsbruck, Austria

Otto Seppälä is a University Professor of Aquatic Evolutionary Ecology at the University of Innsbruck, Austria. He received his PhD at the University of Jyväskylä, Finland. Before his current position, he worked as a postdoc and a group leader at ETH Zürich and Eawag, Switzerland. His work addresses fundamental questions of adaptation in natural populations with the main focus on selective agents with high applied relevance, namely disease and human-induced environmental change. He is currently running complementary research projects examining (1) natural selection on immune function and parasite resistance, (2) selection imposed by climate change and chemical pollution, and (3) potential of natural populations to respond to the above mentioned selective pressures in a quantitative genetic framework. His work uses divergent populations of a freshwater snail Lymnaea stagnalis that differ in genetic variation and selection they experience in the wild.

The Eurasian zebra mussel (Dreissena polymorpha) continues to spread across Europe and North America, causing billions of dollars in damage and dramatically altering aquatic ecosystems. Yet its genome has not been sequenced, and zebra mussels diverged from their closest sequenced relative more than 450 million years ago. Here we use long-read sequencing and Hi-C scaffolding, and generate the most contiguous chromosome-level mollusk assembly to date. Using comparative analysis and transcriptomics, we shed light on processes that influence invasive spread, including shell formation, byssal thread synthesis, and high temperature tolerance. The McCartney group identify several Steamer-Like Elements, retrotransposons linked to transmissible cancer in marine clams. Finally, the talk describes the unique D. polymorpha mitochondrial genome— the longest ever reported in Eumetazoa, with its abundant tandem repeats. Together these findings create a rich resource for research and control of this destructive invasive, and for genomic studies of several underexplored branches of the bivalve tree of life.

Dr Michael McCartney, University of Minnesota, USA

Dr Michael McCartney, University of Minnesota, USA

Since 1990, Dr McCartney has studied molecular ecology and evolution of marine and freshwater invertebrates and fishes, with a focus on mollusks.  Over the past five years, Dr McCartney led the zebra mussel research programme in the Minnesota Aquatic Invasive Species (AIS) Research Center at the University of Minnesota (UMN). Using genetic and genomic approaches, his lab described the pathways of zebra mussel spread in North America. The goal of this work was to help AIS managers in Minnesota direct their efforts towards pathways and vectors that carry the highest risks of invasion. He also conducts research on managing zebra mussels. Shorter-term goals are to reduce populations with chemical treatments, and long-term goals are to develop genetic biocontrols that target weaknesses in zebra mussel biology, revealed through the genome sequencing project that is the subject of his presentation.

The quagga mussel (Dreissena rostriformis bugeneis) is a freshwater heterodont indigenous to the Dnieper River drainage in the Ponto-Caspian region. Over the last 30 years human activity has inadvertently spread the species throughout Europe and North America, where it has become established in numerous river systems and waterbodies. Once established it often reaches high populations densities, altering community structures and potentially impacting human activities and water usage. Sequencing of the quagga genome has been undertaken using both Illumina HiSeq and PacBio Sequel technologies. Additional sequencing is underway using Phase Genomics H-C technology, with the aim of developing a highly complete assembly of the quagga genome. The primary aims of this project are to gain a better understanding of the quagga mussels invasion biology and to identify potential vulnerabilities that can be targeted for the development of population control, including through the development of genetic biocontrols.

Dr Yale Passamaneck, US Bureau of Reclamation, USA

Dr Yale Passamaneck, US Bureau of Reclamation, USA

Yale Passamaneck has been studying the evolution, development, and ecology of invertebrates for 20 years. He is currently a research biologist at the US Bureau of Reclamation, working on mitigating the impacts of dreissenid mussels and other invasive species in the western United States. He completed his doctoral degree in the MIT/Woods Hole Oceanographic Institution Joint Program, studying the molecular phylogenetics of molluscs and bryozoans. He conducted postdoctoral research at Weill Cornell Medical College and the University of Hawaii, studying the developmental evolution of tunicates, brachiopods, and anthozoans.

Crepidula atrasolea or black-footed slipper snail is a useful, direct-developing gastropod mollusc for research programs. The year-round availability of embryos, rapid sexual maturity, and amenability to embryonic manipulations make it well-suited for experimental investigations. Genomic tools include a number of developmental transcriptomes, and efforts are underway to sequence the genome (3.8 gigabases). Initial short reads produced a low-quality assembly due to high levels of heterozygosity and repetitive content (estimated at 4.5% and 70%, respectively). Therefore, long-read sequencing is now being planned to produce a contiguous assembly. Despite these challenges, we have identified genomic features from our preliminary assemblies to develop powerful genomic tools. We have employed successful gene-editing techniques, including microinjection and electroporation of plasmid DNA containing fluorescently tagged proteins driven by various promoters, CRISPR/Cas9 for knockdowns and knock-ins, as well as expression of artificial mRNA for both gain- and loss-of-function studies. Efforts are underway to identify and clone C. atrasolea promoters, in order to modify endogenous gene expression and raise stable transgenic lines. The development of these genomic tools, as well as a fully automated marine aquarium and feeding system allow us to produce large numbers of animals to answer questions about the development of this and other spiralians.

Kimberly Perry, University of Illinois, Urbana-Champaign, USA

Kimberly Perry, University of Illinois, Urbana-Champaign, USA

Specialist in the Department of Cell & Developmental Biology at the University of Illinois. For the last 16 years, she has worked with her colleagues in the lab of Jonathan Henry. Her early studies involved screening and examining genes that are expressed during cornea-lens regeneration, and to evaluate gene function in the context of embryonic lens development. This work led to more recent studies exploring the role of stem cells and sensory innervation in the context of cornea tissue homeostasis and lens regeneration. Additionally, her research focuses on the marine mollusc, Crepidula, and the molecular controls that direct cell determination, germ layer formation and axis specification during development. Ongoing studies aim to analyze transcriptional gene regulatory networks and cell signaling pathways that are active during development, and these networks are currently being examined through expression analyses, morpholino gene knock-downs and CRISPR-Cas9 genome editing.

Bivalve molluscs are well adapted to marine benthic life with many living in intertidal zones and tolerant wide fluctuations in temperature, salinity and air exposure. Bivalves have no adaptive immunity but thrive in microbe-rich environments as filter-feeders. Molecular bases of bivalve adaptations are not well understood, but recent sequencing of several bivalve genomes has provided some insights. All bivalve genomes are highly polymorphic and contain a surprisingly large numbers of genes due to duplication. Gene duplications are mostly lineage-specific and derived from retroposition and tandem-repetition. The duplicated genes, which are often associated with shell formation, stress and immune responses, show diverse expression profiles, suggesting neofunctionalization or specialisation for different environmental conditions. High gene diversity due to both paralogous duplication and allelic polymorphism is important to bivalve’s adaptation to heterogenous environments. With planktonic larvae capable of wide dispersal over diverse environments and stationary adults incapable of avoidance, bivalves must rely on genetic diversity and phenotypic plasticity to coup with unpredictable and wildly fluctuating environmental conditions. The unique life history of bivalves and environmental heterogeneity they face create strong balancing selection that favor the retention and diversification of certain duplicated genes, which is critical to the adaptation of bivalve molluscs.

Professor Ximing Guo, Rutgers University, USA

Professor Ximing Guo, Rutgers University, USA

Ximing Guo received his PhD from University of Washington, Seattle, USA. He is currently a Professor of Marine and Coastal Sciences and the Director of the Shellfish Genetics and Breeding Program at Rutgers University (New Jersey, USA). Professor Guo has been studying molluscs for over 30 years. He has published over 160 peer-reviewed articles on biology, genetics and genomics of marine molluscs. Professor Guo is the primary inventor of tetraploid oysters that have revolutionised triploid production and transformed oyster aquaculture. Professor Guo is co-director of the International Oyster Genome Project and participated in the sequencing of several bivalve genomes. Professor Guo’s interest in molluscan genomics is centered on genetics of bivalve adaptation and the involvement of gene duplication particularly in immune and stress response pathways. He is also interested in genetic variations underlying production traits that can be used for genetic improvement of aquaculture species.