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).

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.

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.

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.

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.

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.

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.

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.