Eldon E. Ball

Whole Transcriptome Analysis of the Coral Acropora millepora Reveals Complex Responses to CO2‐driven Acidification during the Initiation of Calcification

The impact of ocean acidification (OA) on coral calcification, a subject of intense current interest, is poorly understood in part because of the presence of symbionts in adult corals. Early life history stages of Acropora spp. provide an opportunity to study the effects of elevated CO2 on coral calcification without the complication of symbiont metabolism. Therefore, we used the Illumina RNAseq approach to study the effects of acute exposure to elevated CO2 on gene expression in primary polyps of Acropora millepora, using as reference a novel comprehensive transcriptome assembly developed for this study. Gene ontology analysis of this whole transcriptome data set indicated that CO2‐driven acidification strongly suppressed metabolism but enhanced extracellular organic matrix synthesis, whereas targeted analyses revealed complex effects on genes implicated in calcification. Unexpectedly, expression of most ion transport proteins was unaffected, while many membrane‐associated or secreted carbonic anhydrases were expressed at lower levels. The most dramatic effect of CO2‐driven acidification, however, was on genes encoding candidate and known components of the skeletal organic matrix that controls CaCO3 deposition. The skeletal organic matrix effects included elevated expression of adult‐type galaxins and some secreted acidic proteins, but down‐regulation of other galaxins, secreted acidic proteins, SCRiPs and other coral‐specific genes, suggesting specialized roles for the members of these protein families and complex impacts of OA on mineral deposition. This study is the first exhaustive exploration of the transcriptomic response of a scleractinian coral to acidification and provides an unbiased perspective on its effects during the early stages of calcification.

Muscle development in the grasshopper embryo. I. Muscles, nerves, and apodemes in the metathoracic leg.

Much is known about the development of nerve pathways in the metathoracic limb bud of the grasshopper embryo. In this series of three papers, we report on the development of muscles in the same embryonic appendage. In a fourth paper (E. E. Ball, R. K. Ho, and C. S. Goodman, 1985, J. Neurosci, in press) we examine the development of specific neuromuscular connections for one of these muscles (coxal muscle 133a). In this first paper, we present an overview of the development of muscles, nerves, and apodemes (tendons). We previously reported on a class of large mesodermal cells, called muscle pioneers (MPs), that arises early in development and appears to act as a scaffold for developing muscles and guidance cue for motoneuron growth cones (R. K. Ho, E. E. Ball, and C. S. Goodman, 1983, Nature (London) 301, 66-69). We have used the I-5 monoclonal antibody (which specifically labels the MPs as well as the nerve pathways), HRP immunocytochemistry, and Normarski optics to visualize muscle, nerve, and apodeme development in the embryonic metathoracic limb bud from 27.5% (before the appearance of the MPs) to 55% (after the muscles have attained their basic adult pattern). Cell fusions, cell migration, and cell death all appear to play important roles in the development of MPs. The patterns of muscle development vary greatly, ranging from (i) single MPs for simple muscles (which in the adult have only one bundle of muscle fibers, e.g., coxal muscle 133a), to (ii) arrays of MPs for complex muscles [which in the adult have many bundles of muscle fibers each with separate sites of insertion, e.g., the extensor tibiae (ETi) and flexor tibiae (FlTi) muscles in the femur].

Localized expression of a dpp/BMP2/4 ortholog in a coral embryo

As the closest outgroup to the Bilateria, the Phylum Cnidaria is likely to be critical to understanding the origins and evolution of body axes. Proteins of the decapentaplegic (DPP)/bone morphogenetic protein (BMP) 2/4 subfamily are central to the specification of the dorsoventral (D/V) axis in bilateral animals, albeit with an axis inversion between arthropods and chordates. We show that a dpp/BMP2/4 ortholog (bmp2/4-Am) is present in the reef-building scleractinian coral, Acropora millepora (Class Anthozoa) and that it is capable of causing phenotypic effects in Drosophila that mimic those of the endogenous dpp gene. We also show that, during coral embryonic development, bmp2/4-Am expression is localized in an ectodermal region adjacent to the blastopore. Thus, a representative of the DPP/BMP2/4 subfamily of ligands was present in the common ancestor of diploblastic and triploblastic animals where it was probably expressed in a localized fashion during development. A localized source of DPP/BMP2/4 may have already been used in axis formation in this ancestor, or it may have provided a means by which an axis could evolve in triploblastic animals.

Microarray analysis identifies candidate genes for key roles in coral development

BackgroundAnthozoan cnidarians are amongst the simplest animals at the tissue level of organization, but are surprisingly complex and vertebrate-like in terms of gene repertoire. As major components of tropical reef ecosystems, the stony corals are anthozoans of particular ecological significance. To better understand the molecular bases of both cnidarian development in general and coral-specific processes such as skeletogenesis and symbiont acquisition, microarray analysis was carried out through the period of early development – when skeletogenesis is initiated, and symbionts are first acquired.ResultsOf 5081 unique peptide coding genes, 1084 were differentially expressed (P ≤ 0.05) in comparisons between four different stages of coral development, spanning key developmental transitions. Genes of likely relevance to the processes of settlement, metamorphosis, calcification and interaction with symbionts were characterised further and their spatial expression patterns investigated using whole-mount in situ hybridization.ConclusionThis study is the first large-scale investigation of developmental gene expression for any cnidarian, and has provided candidate genes for key roles in many aspects of coral biology, including calcification, metamorphosis and symbiont uptake. One surprising finding is that some of these genes have clear counterparts in higher animals but are not present in the closely-related sea anemone Nematostella. Secondly, coral-specific processes (i.e. traits which distinguish corals from their close relatives) may be analogous to similar processes in distantly related organisms. This first large-scale application of microarray analysis demonstrates the potential of this approach for investigating many aspects of coral biology, including the effects of stress and disease.

The sequence of Locusta RXR, homologous to Drosophila Ultraspiracle, and its evolutionary implications

Abstract The cellular response to steroid hormones is mediated by nuclear receptors which act by regulating transcription. In Drosophila melanogaster, the receptor for the insect molting hormone, 20-hydroxyecdysone, is a heterodimer composed of the Ecdysone Receptor and Ultraspiracle (USP) proteins. The DNA binding domains of arthropod USPs and their vertebrate homologs, the retinoid X receptor (RXR) family, are highly conserved. The ligand binding domain sequences, however, divide into two distinct groups. One group consists of sequences from members of the holometabolous higher insect orders Diptera and Lepidoptera, the other of sequences from vertebrates, a crab and a tick. We here report the sequence of an RXR/USP from the hemimetabolous orthopteran, Locusta migratoria. The locust RXR/USP ligand binding domain clearly falls in the vertebrate-crab-tick rather than the dipteran-lepidopteran group. The reason for the evolutionarily abrupt divergence of the dipteran and lepidopteran sequences is unknown, but it could be a change in the type of ligand bound or the loss of ligand altogether.

Gene structure and larval expression of cnox-2Am from the coral Acropora millepora

Abstract. We have cloned a Hox-like gene, cnox-2Am, from a staghorn coral, Acropora millepora, an anthozoan cnidarian, and characterised its embryonic and larval expression. cnox-2Am and its orthologs in other cnidarians and Trichoplax most closely resemble the Gsx and, to a lesser extent, Hox 3/4 proteins. Developmental northern blots and in situ hybridisation are consistent in showing that cnox-2Am message appears in the planula larva shortly after the oral/aboral axis is formed following gastrulation. Expression is localised in scattered ectodermal cells with a restricted distribution along the oral/aboral body axis. They are most abundant along the sides of the cylindrical larva, rare in the oral region and absent from the aboral region. These cells, which on morphological grounds we believe to be neurons, are of two types; one tri-or multipolar near the basement membrane and a second extending projections in both directions from a mid-ectodermal nucleus. Anti-RFamide staining reveals neurons with a similar morphology to the cnox-2Am-expressing cells. However, RFamide-expressing neurons are more abundant, especially at the aboral end of the planula, where there is no cnox-2Am expression. The pattern of expression of cnox-2Am resembles that of Gsx orthologs in Drosophila and vertebrates in being expressed in a spatially restricted portion of the nervous system.

Structure and development of the auditory system in the prothoracic leg of the cricket Teleogryllus commodus (walker)

SummaryThe microanatomy of the adult auditory system of the cricket, Teleogryllus commodus, is described with special attention to the number and arrangement of the groups of scolopidia forming the tympanal organ.In the proximal part of the prothoracic tibia, there is a large posterior tympanum and a smaller anterior tympanum, which are associated internally with two expanded chambers of the tracheal system. The tympanal organ, which is identical in both sexes, is situated on the dorsal surface of the anterior tympanal trachea. It contains about 70 scolopidia, divided into two main groups: the proximal group, whose attachment cells insert on large accessory cells dorsally within the tympanal organ, and the distal group, whose attachment cells insert on modified dorsal hypodermal cells. These groups can be further divided into five main types, each containing a relatively constant number of scolopidia: 1. proximal group A; 2. proximal main group with proximal neurons; 3. proximal main group with distal neurons; 4. proximal part of distal group; 5. distal part of distal group. These types differ consistently in the following features: location of sensory neuron, orientation of dendrite and scolopale cell, structure of scolopale cell, shape and orientation of attachment cell and its connection to the secondary attachment cell.

Developing grasshopper neurons show variable levels of guanylyl cyclase activity on arrival at their targets.

The ability of certain grasshopper neurons to respond to exogenously applied donors of nitric oxide (NO) by producing cyclic GMP (cGMP) depends on their developmental state. ODQ, a selective blocker of NO‐sensitive guanylyl cyclase, blocks cGMP production at 10−5 M, thus confirming the nature of the response. Experiments in which the distal axon is separated from its proximal stump before application of an NO donor show that guanylyl cyclase is distributed uniformly throughout the neuron. In the locust abdomen, where segments are formed sequentially, the pattern of guanylyl cyclase up‐regulation is predictable and sequential from anterior to posterior. There are two patterns of innervation by cGMP‐expressing motor neurons. In the first, typified by muscle 187, an innervating neuron begins to be NO responsive on arrival at its muscle and continues to be so over most of the remainder of embryonic development, including the formation of motor end plates. In the second, typified by a neuron innervating muscle 191, the neuron extends well along the muscle, apparently laying down a number of sites of contact with it, before it becomes NO responsive. In both patterns, however, NO responsiveness marks the neurons transition from growth cone elongation to the production of lateral branches. Individual muscles receive innervation from multiple motor neurons, some of which express transient NO sensitivity during development and others which do not. With the exception of the leg motor neuron SETi, the first motor neuron to reach any muscle is usually not NO responsive. We suggest that cGMP plays a role in, or reflects, the early stages of communication between a target and specific innervating neurons. J. Comp. Neurol. 394:1–13, 1998.

The biology of coral metamorphosis: Molecular responses of larvae to inducers of settlement and metamorphosis

Like many other cnidarians, corals undergo metamorphosis from a motile planula larva to a sedentary polyp. In some sea anemones such as Nematostella this process is a smooth transition requiring no extrinsic stimuli, but in many corals it is more complex and is cue-driven. To better understand the molecular events underlying coral metamorphosis, competent larvae were treated with either a natural inducer of settlement (crustose coralline algae chips/extract) or LWamide, which bypasses the settlement phase and drives larvae directly into metamorphosis. Microarrays featuring >8000 Acropora unigenes were used to follow gene expression changes during the 12h period after these treatments, and the expression patterns of specific genes, selected on the basis of the array experiments, were investigated by in situ hybridization. Three patterns of expression were common-an aboral pattern restricted to the searching/settlement phase, a second phase of aboral expression corresponding to the beginning of the development of the calicoblastic ectoderm and continuing after metamorphosis, and a later orally-restricted pattern.

Patterns of Gene Expression in a Scleractinian Coral Undergoing Natural Bleaching

Coral bleaching is a major threat to coral reefs worldwide and is predicted to intensify with increasing global temperature. This study represents the first investigation of gene expression in an Indo-Pacific coral species undergoing natural bleaching which involved the loss of algal symbionts. Quantitative real-time polymerase chain reaction experiments were conducted to select and evaluate coral internal control genes (ICGs), and to investigate selected coral genes of interest (GOIs) for changes in gene expression in nine colonies of the scleractinian coral Acropora millepora undergoing bleaching at Magnetic Island, Great Barrier Reef, Australia. Among the six ICGs tested, glyceraldehyde 3-phosphate dehydrogenase and the ribosomal protein genes S7 and L9 exhibited the most constant expression levels between samples from healthy-looking colonies and samples from the same colonies when severely bleached a year later. These ICGs were therefore utilised for normalisation of expression data for seven selected GOIs. Of the seven GOIs, homologues of catalase, C-type lectin and chromoprotein genes were significantly up-regulated as a result of bleaching by factors of 1.81, 1.46 and 1.61 (linear mixed models analysis of variance, P < 0.05), respectively. We present these genes as potential coral bleaching response genes. In contrast, three genes, including one putative ICG, showed highly variable levels of expression between coral colonies. Potential variation in microhabitat, gene function unrelated to the stress response and individualised stress responses may influence such differences between colonies and need to be better understood when designing and interpreting future studies of gene expression in natural coral populations.