Amphipod Crustacean Parhyale Hawaiensis Research Articles

Identification and classification of cis-regulatory elements in the amphipod crustacean Parhyale hawaiensis.

Emerging research organisms enable the study of biology that cannot be addressed using classical 'model' organisms. New data resources can accelerate research in such animals. Here, we present new functional genomic resources for the amphipod crustacean Parhyale hawaiensis, facilitating the exploration of gene regulatory evolution using this emerging research organism. We use Omni-ATAC-seq to identify accessible chromatin genome-wide across a broad time course of Parhyale embryonic development. This time course encompasses many major morphological events, including segmentation, body regionalization, gut morphogenesis and limb development. In addition, we use short- and long-read RNA-seq to generate an improved Parhyale genome annotation, enabling deeper classification of identified regulatory elements. We discover differential accessibility, predict nucleosome positioning, infer transcription factor binding, cluster peaks based on accessibility dynamics, classify biological functions and correlate gene expression with accessibility. Using a Minos transposase reporter system, we demonstrate the potential to identify novel regulatory elements using this approach. This work provides a platform for the identification of novel developmental regulatory elements in Parhyale, and offers a framework for performing such experiments in other emerging research organisms.

The \u201camphi\u201d-brains of amphipods: new insights from the neuroanatomy of Parhyale hawaiensis (Dana, 1853)

BackgroundOver the last years, the amphipod crustacean Parhyale hawaiensis has developed into an attractive marine animal model for evolutionary developmental studies that offers several advantages over existing experimental organisms. It is easy to rear in laboratory conditions with embryos available year-round and amenable to numerous kinds of embryological and functional genetic manipulations. However, beyond these developmental and genetic analyses, research on the architecture of its nervous system is fragmentary. In order to provide a first neuroanatomical atlas of the brain, we investigated P. hawaiensis using immunohistochemical labelings combined with laser-scanning microscopy, X-ray microcomputed tomography, histological sectioning and 3D reconstructions.ResultsAs in most amphipod crustaceans, the brain is dorsally bent out of the body axis with downward oriented lateral hemispheres of the protocerebrum. It comprises almost all prominent neuropils that are part of the suggested ground pattern of malacostracan crustaceans (except the lobula plate and projection neuron tract neuropil). Beyond a general uniformity of these neuropils, the brain of P. hawaiensis is characterized by an elaborated central complex and a modified lamina (first order visual neuropil), which displays a chambered appearance. In the light of a recent analysis on photoreceptor projections in P. hawaiensis, the observed architecture of the lamina corresponds to specialized photoreceptor terminals. Furthermore, in contrast to previous descriptions of amphipod brains, we suggest the presence of a poorly differentiated hemiellipsoid body and an inner chiasm and critically discuss these aspects.ConclusionsDespite a general uniformity of amphipod brains, there is also a certain degree of variability in architecture and size of different neuropils, reflecting various ecologies and life styles of different species. In contrast to other amphipods, the brain of P. hawaiensis does not display any striking modifications or bias towards processing one particular sensory modality. Thus, we conclude that this brain represents a common type of an amphipod brain. Considering various established protocols for analyzing and manipulating P. hawaiensis, this organism is a suitable model to gain deeper understanding of brain anatomy e.g. by using connectome approaches, and this study can serve as first solid basis for following studies.

The amphipod crustacean Parhyale hawaiensis: An emerging comparative model of arthropod development, evolution, and regeneration.

Recent advances in genetic manipulation and genome sequencing have paved the way for a new generation of research organisms. The amphipod crustacean Parhyale hawaiensis is one such system. Parhyale are easy to rear and offer large broods of embryos amenable to injection, dissection, and live imaging. Foundational work has described Parhyale embryonic development, while advancements in genetic manipulation using CRISPR‐Cas9 and other techniques, combined with genome and transcriptome sequencing, have enabled its use in studies of arthropod development, evolution, and regeneration. This study introduces Parhyale development and life history, a catalog of techniques and resources for Parhyale research, and two case studies illustrating its power as a comparative research system.This article is categorized under:Comparative Development and Evolution > Evolutionary NoveltiesAdult Stem Cells, Tissue Renewal, and Regeneration > RegenerationComparative Development and Evolution > Model SystemsComparative Development and Evolution > Body Plan Evolution

The genome of the crustacean Parhyale hawaiensis, a model for animal development, regeneration, immunity and lignocellulose digestion.

The amphipod crustacean Parhyale hawaiensis is a blossoming model system for studies of developmental mechanisms and more recently regeneration. We have sequenced the genome allowing annotation of all key signaling pathways, transcription factors, and non-coding RNAs that will enhance ongoing functional studies. Parhyale is a member of the Malacostraca clade, which includes crustacean food crop species. We analysed the immunity related genes of Parhyale as an important comparative system for these species, where immunity related aquaculture problems have increased as farming has intensified. We also find that Parhyale and other species within Multicrustacea contain the enzyme sets necessary to perform lignocellulose digestion ('wood eating'), suggesting this ability may predate the diversification of this lineage. Our data provide an essential resource for further development of Parhyale as an experimental model. The first malacostracan genome will underpin ongoing comparative work in food crop species and research investigating lignocellulose as an energy source.

Comprehensive analysis of Hox gene expression in the amphipod crustacean Parhyale hawaiensis

Hox genes play crucial roles in establishing regional identity along the anterior–posterior axis in bilaterian animals, and have been implicated in generating morphological diversity throughout evolution. Here we report the identification, expression, and initial genomic characterization of the complete set of Hox genes from the amphipod crustacean Parhyale hawaiensis. Parhyale is an emerging model system that is amenable to experimental manipulations and evolutionary comparisons among the arthropods. Our analyses indicate that the Parhyale genome contains a single copy of each canonical Hox gene with the exception of fushi tarazu, and preliminary mapping suggests that at least some of these genes are clustered together in the genome. With few exceptions, Parhyale Hox genes exhibit both temporal and spatial colinearity, and expression boundaries correlate with morphological differences between segments and their associated appendages. This work represents the most comprehensive analysis of Hox gene expression in a crustacean to date, and provides a foundation for functional studies aimed at elucidating the role of Hox genes in arthropod development and evolution.

Codon and Amino Acid Usage Are Shaped by Selection Across Divergent Model Organisms of the Pancrustacea.

In protein-coding genes, synonymous codon usage and amino acid composition correlate to expression in some eukaryotes, and may result from translational selection. Here, we studied large-scale RNA-seq data from three divergent arthropod models, including cricket (Gryllus bimaculatus), milkweed bug (Oncopeltus fasciatus), and the amphipod crustacean Parhyale hawaiensis, and tested for optimization of codon and amino acid usage relative to expression level. We report strong signals of AT3 optimal codons (those favored in highly expressed genes) in G. bimaculatus and O. fasciatus, whereas weaker signs of GC3 optimal codons were found in P. hawaiensis, suggesting selection on codon usage in all three organisms. Further, in G. bimaculatus and O. fasciatus, high expression was associated with lowered frequency of amino acids with large size/complexity (S/C) scores in favor of those with intermediate S/C values; thus, selection may favor smaller amino acids while retaining those of moderate size for protein stability or conformation. In P. hawaiensis, highly transcribed genes had elevated frequency of amino acids with large and small S/C scores, suggesting a complex dynamic in this crustacean. In all species, the highly transcribed genes appeared to favor short proteins, high optimal codon usage, specific amino acids, and were preferentially involved in cell-cycling and protein synthesis. Together, based on examination of 1,680,067, 1,667,783, and 1,326,896 codon sites in G. bimaculatus, O. fasciatus, and P. hawaiensis, respectively, we conclude that translational selection shapes codon and amino acid usage in these three Pancrustacean arthropods.

Transgenesis in non-model organisms: the case of Parhyale.

One of the most striking manifestations of Hox gene activity is the morphological and functional diversity of arthropod body plans, segments, and associated appendages. Among arthropod models, the amphipod crustacean Parhyale hawaiensis satisfies a number of appealing biological and technical requirements to study the Hox control of tissue and organ morphogenesis. Parhyale embryos undergo direct development from fertilized eggs into miniature adults within 10 days and are amenable to all sorts of embryological and functional genetic manipulations. Furthermore, each embryo develops a series of specialized appendages along the anterior-posterior body axis, offering exceptional material to probe the genetic basis of appendage patterning, growth, and differentiation. Here, we describe the methodologies and techniques required for transgenesis-based gain-of-function studies of Hox genes in Parhyale embryos. First, we introduce a protocol for efficient microinjection of early-stage Parhyale embryos. Second, we describe the application of fast and reliable assays to test the activity of the Minos DNA transposon in embryos. Third, we present the use of Minos-based transgenesis vectors to generate stable and transient transgenic Parhyale. Finally, we describe the development and application of a conditional heat-inducible misexpression system to study the role of the Hox gene Ultrabithorax in Parhyale appendage specialization. Beyond providing a useful resource for Parhyalists, this chapter also aims to provide a road map for researchers working on other emerging model organisms. Acknowledging the time and effort that need to be invested in developing transgenic approaches in new species, it is all worth it considering the wide scope of experimentation that opens up once transgenesis is established.

Identification of a putative germ plasm in the amphipod Parhyale hawaiensis

BackgroundSpecification of the germ line is an essential event during the embryonic development of sexually reproducing animals, as germ line cells are uniquely capable of giving rise to the next generation. Animal germ cells arise through either inheritance of a specialized, maternally supplied cytoplasm called 'germ plasm’ or though inductive signaling by somatic cells. Our understanding of germ cell determination is based largely on a small number of model organisms. To better understand the evolution of germ cell specification, we are investigating this process in the amphipod crustacean Parhyale hawaiensis. Experimental evidence from previous studies demonstrated that Parhyale germ cells are specified through inheritance of a maternally supplied cytoplasmic determinant; however, this determinant has not been identified.ResultsHere we show that the one-cell stage Parhyale embryo has a distinct cytoplasmic region that can be identified by morphology as well as the localization of germ line-associated RNAs. Removal of this cytoplasmic region results in a loss of embryonic germ cells, supporting the hypothesis that it is required for specification of the germ line. Surprisingly, we found that removal of this distinct cytoplasm also results in aberrant somatic cell behaviors, as embryos fail to gastrulate.ConclusionsParhyale hawaiensis embryos have a specialized cytoplasm that is required for specification of the germ line. Our data provide the first functional evidence of a putative germ plasm in a crustacean and provide the basis for comparative functional analysis of germ plasm formation within non-insect arthropods.

ASGARD: an open-access database of annotated transcriptomes for emerging model arthropod species

The increased throughput and decreased cost of next-generation sequencing (NGS) have shifted the bottleneck genomic research from sequencing to annotation, analysis and accessibility. This is particularly challenging for research communities working on organisms that lack the basic infrastructure of a sequenced genome, or an efficient way to utilize whatever sequence data may be available. Here we present a new database, the Assembled Searchable Giant Arthropod Read Database (ASGARD). This database is a repository and search engine for transcriptomic data from arthropods that are of high interest to multiple research communities but currently lack sequenced genomes. We demonstrate the functionality and utility of ASGARD using de novo assembled transcriptomes from the milkweed bug Oncopeltus fasciatus, the cricket Gryllus bimaculatus and the amphipod crustacean Parhyale hawaiensis. We have annotated these transcriptomes to assign putative orthology, coding region determination, protein domain identification and Gene Ontology (GO) term annotation to all possible assembly products. ASGARD allows users to search all assemblies by orthology annotation, GO term annotation or Basic Local Alignment Search Tool. User-friendly features of ASGARD include search term auto-completion suggestions based on database content, the ability to download assembly product sequences in FASTA format, direct links to NCBI data for predicted orthologs and graphical representation of the location of protein domains and matches to similar sequences from the NCBI non-redundant database. ASGARD will be a useful repository for transcriptome data from future NGS studies on these and other emerging model arthropods, regardless of sequencing platform, assembly or annotation status. This database thus provides easy, one-stop access to multi-species annotated transcriptome information. We anticipate that this database will be useful for members of multiple research communities, including developmental biology, physiology, evolutionary biology, ecology, comparative genomics and phylogenomics.Database URL: asgard.rc.fas.harvard.edu

Independent migration of cell populations in the early gastrulation of the amphipod crustacean Parhyale hawaiensis

Cells are the principal component of tissues and can drive morphogenesis through dynamic changes in structure and interaction. During gastrulation, the primary morphogenetic event of early development, cells change shape, exchange neighbors, and migrate long distances to establish cell layers that will form the tissues of the adult animal. Outside of Drosophila, little is known about how changes in cell behavior might drive gastrulation among arthropods. Here, we focus on three cell populations that form two aggregations during early gastrulation in the crustacean Parhyale hawaiensis. Using cytoskeletal markers and lineage tracing we observe bottle cells in anterior and visceral mesoderm precursors as gastrulation commences, and find that both Cytochalasin D, an inhibitor of actin polymerization, and ROCKOUT, an inhibitor of Rho-kinase activity, prevent gastrulation. Furthermore, by ablating specific cells, we show that each of the three populations acts independently during gastrulation, confirming previous hypotheses that cell behavior during Parhyale gastrulation relies on intrinsic signals instead of an inductive mechanism.

Patterns of cell lineage, movement, and migration from germ layer specification to gastrulation in the amphipod crustacean Parhyale hawaiensis

The acquisition of specific cell fates throughout embryonic development is one of the core problems in developmental and evolutionary biology. In the amphipod Parhyale hawaiensis all three germ layers and the germ line are determined by the eight-cell stage. Despite this early fate determination, multiple cell types can be replaced following ablation of their founder cells, showing that this embryo also has significant regulative properties. Here we present a cellular-level resolution lineage analysis for P. hawaiensis embryos between fertilization and gastrulation, including analysis of cleavage patterns, division times, and clonal behaviors. We compare these cellular behaviors in wild type embryos with those in embryos where specific founder cells have been ablated, or where zygotic transcription has been inhibited. We observe that when germ line, endoderm or mesoderm founder cells are ablated, the remaining cells do not alter their cleavage or migration behaviors before the onset of gastrulation. In the absence of zygotic transcription, ingression movements proceed normally, but epibolic movements are disrupted. This indicates that the embryo's regulative response to germ layer founder loss, in the form of altered cell behavior, is realized in the ~32h between gastrulation and early germ band elongation, and is likely to require zygotic reprogramming rather than alternative deployment of maternally supplied determinants. Combining these data with the observations of previous studies, we propose a framework to elucidate the molecular mechanisms that regulate the determinative and regulative properties of the P. hawaiensis embryo.

A prominent requirement forsingle-mindedand the ventral midline in patterning the dorsoventral axis of the crustaceanParhyale hawaiensis

In bilaterians, establishing the correct spatial positioning of structures along the dorsoventral (DV) axis is essential for proper embryonic development. Insects such as Drosophila rely on the Dorsal activity gradient and Bone morphogenetic protein (BMP) signaling to establish cell fates along the DV axis, leading to the distinction between tissues such as mesoderm, neurogenic ectoderm and dorsal ectoderm in the developing embryo. Subsequently, the ventral midline plays a more restricted role in DV patterning by establishing differential cell fates in adjacent regions of the neurogenic ectoderm. In this study, we examine the function of the ventral midline and the midline-associated gene single-minded (Ph-sim) in the amphipod crustacean Parhyale hawaiensis. Remarkably, we found that Ph-sim and the ventral midline play a central role in establishing proper fates along the entire DV axis in this animal; laser ablation of midline cells causes a failure to form neurogenic ectoderm and Ph-sim RNAi results in severely dorsalized embryos lacking both neurogenic ectoderm and the appendage-bearing lateral ectoderm. Furthermore, we hypothesize that this role of midline cells was present in the last common ancestor of crustaceans and insects. We predict that the transition to a Dorsal-dependent DV patterning system in the phylogenetically derived insect lineage leading to Drosophila has led to a more restricted role of the ventral midline in patterning the DV axis of these insects.

The Crustacean Parhyale hawaiensis: A New Model for Arthropod Development

The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. In addition to its phylogenetically strategic position, P. hawaiensis has proven to be highly amenable to experimental manipulation, is straightforward to rear in the laboratory, and has large numbers of embryos that are available year-round. A detailed staging system has been developed to characterize P. hawaiensis embryogenesis. Robust protocols exist for the collection and fixation of all embryonic stages, in situ hybridization to study mRNA localization, and immunohistochemistry to study protein localization. Microinjection of blastomeres enables detailed cell-lineage analyses, transient and transgenic introduction of recombinant genetic material, and targeted knockdowns of gene function using either RNA interference (RNAi) or morpholino methods. Directed genome sequencing will generate important data for comparative studies aimed at understanding cis-regulatory evolution. Bacterial artificial chromosome (BAC) clones containing genes of interest to the developmental and evolutionary biology communities are being targeted for sequencing. An expressed sequence tag (EST) database will facilitate discovery of additional developmental genes and should broaden our understanding of the genetic controls of body patterning. A reference genome from the related amphipod crustacean Jassa slatteryi will shortly be available.

In Situ Hybridization of Labeled RNA Probes to Fixed Parhyale hawaiensis Embryos

The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol describes in situ hybridization of fluorescein- or digoxigenin (DIG)-labeled RNA probes to fixed P. hawaiensis embryos. Standard techniques of molecular biology should be used to produce an appropriate template for generation of antisense RNA probes. RNA-labeling mixes designed to produce fluorescein- or DIG-labeled RNA probes using T3, T7, or SP6 RNA polymerases are commercially available. Probes should be purified using QIAGEN RNeasy columns or similar means. Considerations for double-labeling experiments using both fluorescein- and DIG-labeled RNA probes are included.

Fixation and Dissection of Parhyale hawaiensis Embryos

The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol describes the dissection and fixation of P. hawaiensis embryos. Embryonic tissue fixed in the following manner is suitable for in situ hybridization experiments to study mRNA expression or for immunocytochemistry to study protein localization.

Injection of Parhyale hawaiensis Blastomeres with Fluorescently Labeled Tracers

The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol describes the injection of P. hawaiensis blastomeres with fluorescently labeled tracers for the purpose of cell-lineage analysis. The total (holoblastic) cleavages that characterize early embryogenesis in P. hawaiensis generate an eight-cell embryo with a stereotypical arrangement of blastomeres, each of which already possesses an invariant cell fate. Fluorochrome-conjugated dextran solutions, mRNAs encoding fluorescent proteins, and biotin-dextran have all proven to be useful lineage markers. The relative merits of various tracers are considered.

Antibody Staining of Parhyale hawaiensis Embryos

The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol provides a simplified protocol for antibody staining of P. hawaiensis embryos. The method also works well for other arthropods and phyla. Fixed embryos are rehydrated, washed, blocked with normal goat serum, and incubated overnight with primary antibody. Embryos are then washed and incubated with a peroxidase-conjugated secondary antibody that binds to the primary antibody. A subsequent histochemical reaction produces a black stain in those cells where antibodies have localized.

Cuticle differentiation in the embryo of the amphipod crustacean Parhyale hawaiensis

The arthropod cuticle is a multilayered extracellular matrix produced by the epidermis during embryogenesis and moulting. Molecularly and histologically, cuticle differentiation has been extensively investigated in the embryo of the insect Drosophila melanogaster. To learn about the evolution of cuticle differentiation, we have studied the histology of cuticle differentiation during embryogenesis of the amphipod crustacean Parhyale hawaiensis, which had a common ancestor with Drosophila about 510 million years ago. The establishment of the layers of the Parhyale juvenile cuticle is largely governed by mechanisms observed in Drosophila, e.g. as in Drosophila, the synthesis and arrangement of chitin in the inner procuticle are separate processes. A major difference between the cuticle of Parhyale and Drosophila concerns the restructuring of the Parhyale dorsal epicuticle after deposition. In contrast to the uniform cuticle of the Drosophila larva, the Parhyale cuticle is subdivided into two regions, the ventral and the dorsal cuticles. Remarkably, the boundary between the ventral and dorsal cuticles is sharp suggesting active extracellular regionalisation. The present analysis of Parhyale cuticle differentiation should allow the characterisation of the cuticle-producing and -organising factors of Parhyale (by comparison with the branchiopod crustacean Daphnia pulex) in order to contribute to the elucidation of fundamental questions relevant to extracellular matrix organisation and differentiation.

The fate of isolated blastomeres with respect to germ cell formation in the amphipod crustacean Parhyale hawaiensis

Germ cells may be specified through the localization of germ line determinants to specific cells in early embryogenesis, or by inductive signals from neighboring cells to germ cell precursors in later embryogenesis. Such determinants can be produced and localized during or after oogenesis, either autonomously by oocytes or by associated nutritive cells. In Drosophila, each oocyte is connected to nurse cells by cytoplasmic bridges, and determinants synthesized in nurse cells are transported through these bridges to the oocyte. However, the Drosophila model may not be applicable to all arthropods, since in many species of all four extant arthropod classes, gametogenesis functions without nurse cells. In this paper, I use immunodetection of Vasa protein to study germ cell development in the amphipod crustacean Parhyale hawaiensis, a species whose ovaries lack nurse cells and whose eggs lack obvious polarity. Previous cell lineage analyses have shown that all three germ layers and the germ line are exclusively specified by third cleavage. In the present study, I use a molecular marker to follow germ cell development during P. hawaiensis embryogenesis. I determine the capacity of individual blastomeres to form germ cells by isolating blastomeres at early cleavage stages and provide experimental evidence for localized germ cell determinants at the two-cell stage in P. hawaiensis. These experiments indicate that many aspects of early amphipod development, including timing and symmetry of cell division, the transition from holoblastic to superficial cleavage, and possibly some gastrulation movements, are cell autonomous following first cleavage.