Nathan J. Kenny

Genomic Sequence and Experimental Tractability of a New Decapod Shrimp Model, Neocaridina denticulata

The speciose Crustacea is the largest subphylum of arthropods on the planet after the Insecta. To date, however, the only publically available sequenced crustacean genome is that of the water flea, Daphnia pulex, a member of the Branchiopoda. While Daphnia is a well-established ecotoxicological model, previous study showed that one-third of genes contained in its genome are lineage-specific and could not be identified in any other metazoan genomes. To better understand the genomic evolution of crustaceans and arthropods, we have sequenced the genome of a novel shrimp model, Neocaridina denticulata, and tested its experimental malleability. A library of 170-bp nominal fragment size was constructed from DNA of a starved single adult and sequenced using the Illumina HiSeq2000 platform. Core eukaryotic genes, the mitochondrial genome, developmental patterning genes (such as Hox) and microRNA processing pathway genes are all present in this animal, suggesting it has not undergone massive genomic loss. Comparison with the published genome of Daphnia pulex has allowed us to reveal 3750 genes that are indeed specific to the lineage containing malacostracans and branchiopods, rather than Daphnia-specific (E-value: 10−6). We also show the experimental tractability of N. denticulata, which, together with the genomic resources presented here, make it an ideal model for a wide range of further aquacultural, developmental, ecotoxicological, food safety, genetic, hormonal, physiological and reproductive research, allowing better understanding of the evolution of crustaceans and other arthropods.

Right across the tree of life: The evolution of left–right asymmetry in the Bilateria

Directional left/right (LR) asymmetries, in which there are consistent, heritable differences in morphology between the left and right sides of bilaterally symmetrical organisms, are found in animals across the Bilateria. For many years, we have lacked evidence for shared mechanisms underlying their development. This led to the supposition that the mechanisms driving establishment of LR asymmetries, and consequently the asymmetries themselves, had evolved separately in the three major Superphyla that constitute the Bilateria. The recent discovery that the transforming growth factor‐beta (TGF‐B) ligand Nodal plays a role in the regulation of LR asymmetry in both Deuterostomia and Lophotrochozoa has reignited debate in this field, as it suggests that at least this aspect of the development of the LR axis is conserved. In this review, we discuss evidence for shared mechanisms of LR asymmetry establishment across the bilaterian tree of life and consider how these mechanisms might have diverged across the Metazoa over the last 500 million years or so of evolution. As well as the likelihood that Nodal is an ancestral mechanism for regulating LR asymmetry, we reemphasize cytoskeletal architecture as a potential shared mechanism underlying symmetry breaking. However, convergent evolution remains a distinct possibility and study of a wider diversity of species will be needed to distinguish between conserved and lineage‐specific mechanisms. genesis 52:458–470, 2014.

Ancestral whole-genome duplication in the marine chelicerate horseshoe crabs

Whole-genome duplication (WGD) results in new genomic resources that can be exploited by evolution for rewiring genetic regulatory networks in organisms. In metazoans, WGD occurred before the last common ancestor of vertebrates, and has been postulated as a major evolutionary force that contributed to their speciation and diversification of morphological structures. Here, we have sequenced genomes from three of the four extant species of horseshoe crabs—Carcinoscorpius rotundicauda, Limulus polyphemus and Tachypleus tridentatus. Phylogenetic and sequence analyses of their Hox and other homeobox genes, which encode crucial transcription factors and have been used as indicators of WGD in animals, strongly suggests that WGD happened before the last common ancestor of these marine chelicerates >135 million years ago. Signatures of subfunctionalisation of paralogues of Hox genes are revealed in the appendages of two species of horseshoe crabs. Further, residual homeobox pseudogenes are observed in the three lineages. The existence of WGD in the horseshoe crabs, noted for relative morphological stasis over geological time, suggests that genomic diversity need not always be reflected phenotypically, in contrast to the suggested situation in vertebrates. This study provides evidence of ancient WGD in the ecdysozoan lineage, and reveals new opportunities for studying genomic and regulatory evolution after WGD in the Metazoa.

Identification of putative ecdysteroid and juvenile hormone pathway genes in the shrimp Neocaridina denticulata.

Although the sesquiterpenoid juvenile hormone (JH) and the steroidal ecdysteroids are of vital importance to the development and reproduction of insects, our understanding of the evolution of these crucial hormonal regulators in other arthropods is limited. To better understand arthropod hormone evolution and regulation, here we describe the hormonal pathway genes (e.g. those involved in hormone biosynthesis, degradation, regulation and signal transduction) of a new decapod model, the shrimp Neocaridina denticulata. The majority of known insect sesquiterpenoid and ecdysteroid pathway genes and their regulators are contained in the N. denticulata genome. In the sesquiterpenoid pathway, these include biosynthetic pathway components: juvenile hormone acid methyltransferase (JHAMT); hormone binding protein: juvenile hormone binding protein (JHBP); and degradation pathway components: juvenile hormone esterase (JHE), juvenile hormone esterase binding protein (JHEBP) and juvenile hormone epoxide hydrolase (JHEH), with the JHBP, JHEBP and JHEH genes being discovered in a crustacean for the first time here. Ecdysteroid biosynthetic pathway genes identified include spook, phantom, disembodied, shadow and CYP18. Potential hormonal regulators and signal transducers such as allatostatins (ASTs), Methoprene-tolerant (Met), Retinoid X receptor (RXR), Ecdysone receptor (EcR), calponin-like protein Chd64, FK509-binding protein (FKBP39), Broad-complex (Br-c), and crustacean hyperglycemic hormone/molt-inhibiting hormone/gonad-inhibiting hormone (CHH/MIH/GIH) genes are all present in the shrimp N. denticulata. To our knowledge, this is the first report of these hormonal pathways and their regulatory genes together in a single decapod, providing a vital resource for further research into development, reproduction, endocrinology and evolution of crustaceans, and arthropods in general.

How Did Arthropod Sesquiterpenoids and Ecdysteroids Arise? Comparison of Hormonal Pathway Genes in Noninsect Arthropod Genomes

The phylum Arthropoda contains the largest number of described living animal species, with insects and crustaceans dominating the terrestrial and aquatic environments, respectively. Their successful radiations have long been linked to their rigid exoskeleton in conjunction with their specialized endocrine systems. In order to understand how hormones can contribute to the evolution of these animals, here, we have categorized the sesquiterpenoid and ecdysteroid pathway genes in the noninsect arthropod genomes, which are known to play important roles in the regulation of molting and metamorphosis in insects. In our analyses, the majority of gene homologs involved in the biosynthetic, degradative, and signaling pathways of sesquiterpenoids and ecdysteroids can be identified, implying these two hormonal systems were present in the last common ancestor of arthropods. Moreover, we found that the “Broad-Complex” was specifically gained in the Pancrustacea, and the innovation of juvenile hormone (JH) in the insect linage correlates with the gain of the JH epoxidase (CYP15A1/C1) and the key residue changes in the binding domain of JH receptor (“Methoprene-tolerant”). Furthermore, the gain of “Phantom” differentiates chelicerates from the other arthropods in using ponasterone A rather than 20-hydroxyecdysone as molting hormone. This study establishes a comprehensive framework for interpreting the evolution of these vital hormonal pathways in these most successful animals, the arthropods, for the first time.

Components of the dorsal-ventral pathway also contribute to anterior-posterior patterning in honeybee embryos (Apis mellifera)

BackgroundA key early step in embryogenesis is the establishment of the major body axes; the dorsal-ventral (DV) and anterior-posterior (AP) axes. Determination of these axes in some insects requires the function of different sets of signalling pathways for each axis. Patterning across the DV axis requires interaction between the Toll and Dpp/TGF-β pathways, whereas patterning across the AP axis requires gradients of bicoid/orthodenticle proteins and the actions of a hierarchy of gene transcription factors. We examined the expression and function of Toll and Dpp signalling during honeybee embryogenesis to assess to the role of these genes in DV patterning.ResultsPathway components that are required for dorsal specification in Drosophila are expressed in an AP-restricted pattern in the honeybee embryo, including Dpp and its receptor Tkv. Components of the Toll pathway are expressed in a more conserved pattern along the ventral axis of the embryo. Late-stage embryos from RNA interference (RNAi) knockdown of Toll and Dpp pathways had both DV and AP patterning defects, confirmed by staining with Am-sna, Am-zen, Am-eve, and Am-twi at earlier stages. We also identified two orthologues of dorsal in the honeybee genome, with one being expressed during embryogenesis and having a minor role in axis patterning, as determined by RNAi and the other expressed during oogenesis.ConclusionsWe found that early acting pathways (Toll and Dpp) are involved not only in DV patterning but also AP patterning in honeybee embryogenesis. Changes to the expression patterns and function of these genes may reflect evolutionary changes in the placement of the extra-embryonic membranes during embryogenesis with respect to the AP and DV axes.

Evolution, divergence and loss of the Nodal signalling pathway: new data and a synthesis across the Bilateria

Since the discovery that the TGF-β signalling molecule Nodal and its downstream effector Pitx have a parallel role in establishing asymmetry between molluscs and deuterostomes the debate over the degree to which this signalling pathway is conserved across the Bilateria as a whole has been ongoing. Further taxon sampling is critical to understand the evolution and divergence of this signalling pathway in animals. Using genome and transcriptome mining we confirmed the presence of nodal and Pitx in a range of additional animal taxa for which their presence has not yet been described. In situ hybridization was used to show the embryonic expression of these genes in brachiopods and planarians. We show that both nodal and Pitx genes are broadly conserved across the Spiralia, and nodal likely appeared in the Bilaterian stem lineage after the divergence of the Acoelomorpha. Furthermore, both nodal and Pitx mRNA appears to be expressed in an asymmetric fashion in the brachiopod Terebratalia transversa. No evidence for the presence of a Lefty ortholog could be found in the non-deuterostome genomic resources examined. Nodal expression is asymmetric in a number of spiralian lineages, indicating a possible ancestral role of the Nodal/Pitx cascade in the establishment of asymmetries across the Bilateria.

How are comparative genomics and the study of microRNAs changing our views on arthropod endocrinology and adaptations to the environment

As the last few decades of work has shown, precise regulation of biosynthesis and release of arthropod hormones is essential to cope with environmental stresses and challenges. In crustaceans and insects, the sesquiterpenoids methyl farnesoate (MF), farnesoic acid (FA) and juvenile hormone (JH) regulate many developmental, physiological, and reproductive processes. In this review, we discuss how comparative genomics has and will impact our views on arthropod endocrinology. We will also highlight the current knowledge of regulation of genes involved in arthropod hormone biosynthesis by microRNAs, and describe the potential insights into arthropod endocrinology, evolution, and adaptation that are likely to come from the study of microRNAs.

Genome of the Rusty Millipede, Trigoniulus corallinus, Illuminates Diplopod, Myriapod, and Arthropod Evolution

The increasing availability of genomic information from the Arthropoda continues to revolutionize our understanding of the biology of this most diverse animal phylum. However, our sampling of arthropod diversity remains uneven, and key clade such as the Myriapoda are severely underrepresented. Here we present the genome of the cosmopolitanly distributed Rusty Millipede Trigoniulus corallinus, which represents the first diplopod genome to be published, and the second example from the Myriapoda as a whole. This genomic resource contains the majority of core eukaryotic genes (94.3%), and key transcription factor classes that were thought to be lost in the Ecdysozoa. Mitochondrial genome and gene family (transcription factor, Dscam, circadian clock-driving protein, odorant receptor cassette, bioactive compound, and cuticular protein) analyses were also carried out to shed light on their states in the Diplopoda and Myriapoda. The ready availability of T. corallinus recommends it as a new model for evolutionary developmental biology, and the data set described here will be of widespread utility in investigating myriapod and arthropod genomics and evolution.

Draft genome assemblies and predicted microRNA complements of the intertidal lophotrochozoans Patella vulgata (Mollusca, Patellogastropoda) and Spirobranchus (Pomatoceros) lamarcki (Annelida, Serpulida)

MicroRNAs (miRNA) are small non-coding RNAs that act post-transcriptionally to regulate gene expression levels. Some studies have indicated that microRNAs may have low homoplasy, and as a consequence the phylogenetic distribution of microRNA families has been used to study animal evolutionary relationships. Limited levels of lineage sampling, however, may distort such analyses. Lophotrochozoa is an under-sampled taxon that includes molluscs, annelids and nemerteans, among other phyla. Here, we present two novel draft genomes, those of the limpet Patella vulgata and polychaete Spirobranchus (Pomatoceros) lamarcki. Surveying these genomes for known microRNAs identifies numerous potential orthologues, including a number that have been considered to be confined to other lineages. RT-PCR demonstrates that some of these (miR-1285, miR-1287, miR-1957, miR-1983 and miR-3533), previously thought to be found only in vertebrates, are expressed. This study provides genomic resources for two lophotrochozoans and reveals patterns of microRNA evolution that could be hidden by more restricted sampling.