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Overview

Theo Murphy international scientific meeting organised by Dr Angus Davison and Dr Maurine Neiman.

Although molluscs are ecologically, economically, and medically important, progress in understanding their biology has been limited by problems associated with genome sequencing and assembly. As technology breakthroughs overcome these challenges, this meeting brought together experts in molluscan biology and genomics to share best practice in how to use these developments in elucidating the biology of this diverse and ill-understood phylum.

Speaker biographies and abstracts can be found below. Recorded audio of the presentations are also available below. 

Enquiries: contact the Scientific Programmes team.

Organisers

Schedule


Chair

09:05-09:35
Unravelling the supergene for colour polymorphism in the grove snail Cepaea nemoralis

Abstract

The land snail Cepaea nemoralis has been one of the most important models in studying colour polymorphism in an ecological and evolutionary context. The shell colour is partially controlled by a supergene, ie a cluster of genes inherited as a single locus due to tight physical linkage. Functional dissection of this locus can provide important insights into supergene evolution and the role of genome structure in adaptation, but has so far been prevented by lack of genomic resources. Obtaining high-quality genome assembly, however, is challenging because of the size and repetitive nature of snail genomes. Here, the results of C. nemoralis whole genome sequencing using PacBio long-read technology and the (dis)advantages of several genome assemblers will be discussed. Furthermore, the preliminary data on the identification of the shell colour supergene will be presented as well.

Speakers


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09:35-09:45
Discussion
09:45-10:15
Evolution of light-sensing proteins informs the multiple origins of eyes in bivalves

Abstract

Understanding the evolutionary origin of eyes is of great interest to biologists because eyes provide insight into how complex phenotypes can arise through redeployment and elaboration of genetic pathways. Yet, this goal cannot be realised without studying closely related lineages that include eyeless and eyed taxa with independently-derived eye types. To understand how spatiotemporal changes to gene expression have played a role in the evolution of eyes, a combination of tissue-specific transcriptomes and genome assembly will be used to identify genes involved in phototransduction from species spanning the taxonomic and ocular diversity of pteriomorphian bivalves. Data will be analysed in a phylogenetic framework to address the following questions: 1) Do lineages with eyes also have expansions of phototransduction gene families? 2) How similar are the phototransduction pathways between the different photoreceptive structures within and among species? and 3) Do eyes acquire phototransduction pathways using the same evolutionary process or does each eye type have a unique evolutionary trajectory to photoreception? The project will identify molecular mechanisms underlying mantle, eyespot, and ocular photoreception across morphologically diverse structures while determining the roles of adaptation and exaptation in eye evolution.

Speakers


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10:15-10:25
Discussion
10:25-10:55
Coffee
10:55-11:25
MolluscDB: open access to genomes and transcriptomes of many species

Abstract

The mollusc research community has generated large scale genomic and transcriptomic datasets for many species. These data, generated for diverse purposes, from deep phylogeny to supporting shellfish farming, are largely dispersed and inaccessible in an integrated way. We have developed GenomeHubs, a system for the display, sharing and interrogation of genomic data based on the stable Ensembl database model, GenomeHubs make the integration of data from diverse sources possible through public application programming interfaces. We have built MolluscDB, a GenomeHubs database for Mollusca. Importantly, for MolluscDB, we have adapted the underlying database to be able to include species that only have transcriptome data. Currently, MolluscDB presents genomes and reassembled transcriptomes for 23 species, including 15 for which only transcriptome data are available. I will illustrate the exploitation of these data in phylogenetics and in identifying clade-specific gene family expansions. As the Earth BioGenome Project and the UK Darwin Tree of Life projects get underway, with the prospect of many hundreds of new mollusc genomes, resources such as MolluscDB will become invaluable for the wider community. MolluscDB v1 was built by Carlos Caurcel and Richard Challis with assistance from Dominik Laetsch and Kevin Cocot. Development was funded by an EU ITN award.

Speakers


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11:25-11:35
Discussion
11:35-12:05
Cephalopod genomics and the evolution of novelty

Abstract

Coleoid cephalopods (octopuses, squid, and cuttlefish) have a suite of evolutionary innovations, including complex centralised nervous systems and camera-like eyes, which are classic examples of convergent evolution with vertebrates. Coleoids also present a number of true novelties that have no obvious correlations in other animals, such as their adaptive coloration system and sucker-lined arm crown. To study the genetic bases underlying these morphological innovations, the Albertin group sequenced the genome of the longfin inshore squid Doryteuthis pealeii. The group found that the D. pealeii genome is substantially larger than that of Octopus bimaculoides. The expansions of protocadherins, a family of cell adhesion molecules important for wiring vertebrate brains, and of C2H2 zinc-finger transcription factors described in octopus appears to be even larger in squid. Most importantly, our chromosome-scale assembly also identifies many local expansions of genes expressed in novel cephalopod structures. Some of these gene clusters are cephalopod-, or even squid-specific, while others appear to be local expansions of genes found in distantly related animals. This data highlights a major role for the acquisition of novel genes and the selective expansion of known gene families in the evolution of cephalopod morphological innovations.

Speakers

12:05-12:15
Discussion

Chair

13:15-13:45
The Scaly-foot Snail genome and the ancient origins of biomineralised armour

Abstract

The appearance of biomineralised skeletons in the Cambrian precipitated an evolutionary arms race and the original explosive diversification of modern animal forms. Understanding the genomic toolkit that enabled innovations in skeletons and armour is critical to reconstructing the early radiation of major animal clades. The Scaly-foot Snail, Chrysomallon squamiferum, presents a combination of biomineralised features, reminiscent of enigmatic early fossil taxa with complex shells and sclerites such as sachtids, but in a recently-diverged living species which even has iron-infused hard parts. Whether such cases represent recurring ancestral phenomes or adaptive novelties, however, remains unclear. Using a high-quality whole-genome assembly and tissue-specific transcriptomics, we show that scale and shell formation in the Scaly-foot Snail employ independent subsets of 25 highly-expressed transcription factors, and illuminate potential genomic mechanisms for the nano-scale biosynthesis of iron. Comparisons with other lophotrochozoan genomes indicate that the biomineralisation toolkit is ancient but with different expression patterns across major lineages. The ability of lophotrochozoan lineages to generate a wide range of hard parts, exemplified by the remarkable morphological disparity in Mollusca, draws on a capacity to dynamically modify the expression and positioning of elements of biomineralisation toolkit across the genome.

Speakers


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13:45-13:55
Discussion
13:55-14:25
Long reads and large genomes

Speakers


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14:25-14:35
Discussion
14:35-15:05
Short reads, long repeats and high heterozygosity: assembly and annotation of the invasive quagga mussel genome

Abstract

Like many molluscs, the genome of the quagga mussel Dreissena rostriformis is long, repetitive and highly heterozygous. These three factors combine to make accurate and contiguous genome assembly a difficult and resource intensive task, especially when utilising short-read sequencing technology. This talk will outline the technical challenges involved in the quagga mussel genome project and will discuss how improvements to modern long-read and scaffolding technologies have diminished the impact of some but not all of the difficulties involved in large eukaryotic genome assembly projects. Finally, a defence will be made of moderately contiguous genome assemblies for practical use in genomic biology.

Speakers


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15:05-15:15
Discussion
15:15-15:45
Tea

Chair

15:45-16:15
Littorina saxatilis: challenges to produce a genome draft to study genomic landscape of divergence

Abstract

Genome sequencing project of the intertidal gastropod Littorina saxatilis was initiated in 2010. The main objective for the genome project was the outstanding ability of this snail to evolve locally adapted ecotypes, and the genome draft was intended as a platform to study genomic landscapes of divergence. The project proved to be much more difficult than anticipated. Challenges included obtaining high quality DNA and assembling sequence data from multiple Illumina libraries in combination with PacBio. The Littorina genome draft v.1 has a size of 1.6 Gb (the estimated genome size is 1.35 Gb). Despite the large amount of data, the assembly is still quite fragmented (NG50 = 55Kb) due to many repetitive elements in the genome. The real progress in understanding the genomics of divergence in Littorina came with a genetic map. Further, 47% of predicted Littorina genes could be annotated. Among them, 79% of the reference metazoan genes were found as complete and single-copy. However, several target genes are present as multiple functional and/or non-functional gene copies that could be true segmental duplications or assembly artefacts. Future plans include Littorina genome draft v.2 based on high coverage long-read sequencing and improving the genetic map.

Speakers


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16:15-16:25
Discussion
16:25-16:55
The pearl oyster genome: present, problem and future

Abstract

Pinctada fucata has the longest history of any cultured pearl oyster and stands as the iconic species in the pearl industry not only in Japan but also in the world. The complete genome sequence is essential for future studies to develop healthy pearl oysters and high-quality pearls. In 2012, the version-1 sequences of 1.4 Gb P. fucata genome was assembled (Takeuchi et al DNA Res. 19: 177), in which contigs N50 was 1.6 kb (total contig assembly reached to 1024 Mb) and scaffolds N50 14.5 kb, respectively. The pearl oyster genome is AT-rich, with a GC content of 34%, and contains 23,257 complete gene models, 70% of which are supported by the corresponding expressed sequence tags. DNA transposons, retrotransposons, and tandem repeat elements occupied 0.4, 1.5, and 7.9% of the genome, respectively (a total of 9.8%). By additional sequencing, the version-2 achieved contig and scaffold N50 sizes of 21.3 kb and 167 kb, respectively (Takeuchi et al. Zool Lett 2:3, 2016). This version estimates 29,353 gene models, 23,516 (80.1 %) of which represent putative, full-length genes with both start and stop codons. At present, a chromosome-level assembly of the P. fucata genome is challenged.

Speakers


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16:55-17:05
Discussion
17:05-18:00
Poster session

Chair

09:30-09:40
Discussion
09:00-09:30
Reference genome of the freshwater snail Lymnaea stagnalis, a multidisciplinary invertebrate model: overall rationale of the STAGING project

Abstract

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).

Speakers


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09:40-10:10
Genomes gone wild: a tale of (a)sex and duplicity

Abstract

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.

Speakers


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10:10-10:20
Discussion
10:20-10:50
Coffee

Chair

10:50-11:20
Biomphalaria and beyond, gastropod immunogenomics

Abstract

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.

Speakers


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11:20-11:30
Discussion
11:30-12:00
Natural selection on immune function

Abstract

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.

Speakers

12:00-12:10
Discussion
12:10-12:40
The genome of the invasive zebra mussel, Dreissena polymorpha

Abstract

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.

Speakers


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12:40-12:50
Discussion

Chair

13:50-14:20
Genomic analysis of the quagga mussel, Dreissena rostriformis bugensis: searching for vulnerabilities

Abstract

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.

Speakers


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14:20-14:30
Discussion
14:30-15:00
Gene editing in the slipper snail, Crepidula

Abstract

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.

Speakers


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15:00-15:10
Discussion
15:10-15:40
Tea
15:40-16:10
Gene duplication in bivalve genomes and implications for adaptation

Abstract

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.

Speakers


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16:10-16:20
Discussion
16:20-17:00
Summary of discussions and closing remarks

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