Rapid acclimation of juvenile corals to CO2 -mediated acidification by upregulation of heat shock protein and Bcl-2 genes.

Corals play a key role in ocean ecosystems and carbonate balance, but their molecular response to ocean acidification remains unclear. The only previous whole-transcriptome study (Moya et al. Molecular Ecology, 2012; 21, 2440) documented extensive disruption of gene expression, particularly of genes encoding skeletal organic matrix proteins, in juvenile corals (Acropora millepora) after short-term (3 d) exposure to elevated pCO2 . In this study, whole-transcriptome analysis was used to compare the effects of such 'acute' (3 d) exposure to elevated pCO2 with a longer ('prolonged'; 9 d) period of exposure beginning immediately post-fertilization. Far fewer genes were differentially expressed under the 9-d treatment, and although the transcriptome data implied wholesale disruption of metabolism and calcification genes in the acute treatment experiment, expression of most genes was at control levels after prolonged treatment. There was little overlap between the genes responding to the acute and prolonged treatments, but heat shock proteins (HSPs) and heat shock factors (HSFs) were over-represented amongst the genes responding to both treatments. Amongst these was an HSP70 gene previously shown to be involved in acclimation to thermal stress in a field population of another acroporid coral. The most obvious feature of the molecular response in the 9-d treatment experiment was the upregulation of five distinct Bcl-2 family members, the majority predicted to be anti-apoptotic. This suggests that an important component of the longer term response to elevated CO2 is suppression of apoptosis. It therefore appears that juvenile A. millepora have the capacity to rapidly acclimate to elevated pCO2 , a process mediated by upregulation of specific HSPs and a suite of Bcl-2 family members.

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.

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 CO(2) 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 CO(2) 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 CO(2) -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 CO(2) -driven acidification, however, was on genes encoding candidate and known components of the skeletal organic matrix that controls CaCO(3) 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.

AmAMP1 from Acropora millepora and damicornin define a family of coral-specific antimicrobial peptides related to the Shk toxins of sea anemones.

A candidate antimicrobial peptide (AmAMP1) was identified by searching the whole genome sequence of Acropora millepora for short (<125AA) cysteine-rich predicted proteins with an N-terminal signal peptide but lacking clear homologs in the SwissProt database. It resembled but was not closely related to damicornin, the only other known AMP from a coral, and was shown to be active against both Gram-negative and Gram-positive bacteria. These proteins define a family of AMPs present in corals and their close relatives, the Corallimorpharia, and are synthesised as preproproteins in which the C-terminal mature peptide contains a conserved arrangement of six cysteine residues. Consistent with the idea of a common origin for AMPs and toxins, this Cys motif is shared between the coral AMPs and the Shk neurotoxins of sea anemones. AmAMP1 is expressed at late stages of coral development, in ectodermal cells that resemble the "ganglion neurons" of Hydra, in which it has recently been demonstrated that a distinct AMP known as NDA-1 is expressed.

Nucleotide sequence and expression of the sn-glycerol-3-phosphate dehydrogenase gene in Locusta migratoria.

The sn-glycerol-3-phosphate dehydrogenase gene (Gpdh) in the locust (Locusta migratoria) encodes three mature transcripts and a number of isozymes. Gpdh expression is tissue- and developmentally regulated such that two transcripts are unique to flight muscle. Identical proteins are encoded by two transcripts which are generated by alternative splicing downstream of the stop codon in the penultimate exon.

The promoters and spacers in the rDNAs of the melanogaster species subgroup of Drosophila.

The spacer sequences of rDNAs of members of the melanogaster species subgroup of Drosophila (melanogaster, simulans, mauritiana, teissieri, and yakuba) have been compared. The external transcribed spacers (ETSs; the region encoding the 5' end of the primary transcript, upstream from the 18S sequences) are highly conserved between in D. melanogaster, D. simulans and D. mauritiana, whereas the more distantly related D. yakuba and D. teissieri differ in having apparent deletions of 22 and 27 bp, respectively, in this region. The divergence of nucleotide sequence upstream from the transcription start points is consistent with the established phylogeny of the five species. The sequences between bp positions -47 and +24 from the primary transcription start point show extremely little variation between each species. This is also the case for sequences between the approximate bp positions -140 to -125 and -85 to -70. This could indicate a functional importance not only of the sequences next to the transcription start point, but also of these upstream regions. An array of 240-bp repeats can be found at a comparable distance upstream from the transcription start point in each species. Matrix homology comparisons indicate that for each species not only is the sequence at the primary transcription start point duplicated within the 240-bp repeats as previously reported for D. melanogaster, but that this is part of a longer interrupted duplication which includes a region of strong similarity with the sequence between the approximate positions -105 to -65. This region is contained within one of the regions upstream from the transcription start point that is strongly conserved between the species. This sequence may therefore have functional significance not only for the transcription of the rRNA precursor, but also for transcription of the so-called NTS sequences which is now known to occur. The 240-bp arrays are themselves highly conserved within a species indicating that homogenisation mechanisms are operative. The divergence of these arrays between species is consistent with the phylogenetic tree. The 3' sequences of the primary transcription unit, now known to be RNA-processing sites, are also highly similar between the species. Immediately downstream from these sites there is little homology between the rDNA of the different species, until 95-bp tandem arrays are reached in each case.

Molecular cloning and expression analysis of a cDNA encoding a glutamate transporter in the honeybee brain.

We have cloned and characterized a cDNA encoding a putative glutamate transporter, Am-EAAT, from the brain of the honeybee, Apis mellifera. The 543-amino-acid AmEAAT gene product shares the highest sequence identity (54%) with the human EAAT2 subtype. Am-EAAT is expressed predominantly in the brain, and its transcripts are abundant in the optic lobes and inner compact Kenyon cells of the mushroom bodies (MBs), with most other regions of the brain showing lower levels of Am-EAAT expression. High levels of Am-EAAT message are found in pupal stages, possibly indicating a role for glutamate in the developing brain.

Segments of chromosomal DNA from Rhynchosciara americana that undergo additional rounds of DNA replication in the salivary gland DNA puffs have only weak ARS activity in yeast.

We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of 'scrambled' moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.

Light-responsive cryptochromes from a simple multicellular animal, the coral Acropora millepora.

Hundreds of species of reef-building corals spawn synchronously over a few nights each year, and moonlight regulates this spawning event. However, the molecular elements underpinning the detection of moonlight remain unknown. Here we report the presence of an ancient family of blue-light-sensing photoreceptors, cryptochromes, in the reef-building coral Acropora millepora. In addition to being cryptochrome genes from one of the earliest-diverging eumetazoan phyla, cry1 and cry2 were expressed preferentially in light. Consistent with potential roles in the synchronization of fundamentally important behaviors such as mass spawning, cry2 expression increased on full moon nights versus new moon nights. Our results demonstrate phylogenetically broad roles of these ancient circadian clock-related molecules in the animal kingdom.

The structure of the USP/RXR of Xenos pecki indicates that Strepsiptera are not closely related to Diptera.

The receptor for the insect molting hormone, ecdysone, is a heterodimer consisting of the Ecdysone Receptor and Ultraspiracle (USP) proteins. The ligand binding domain sequences of arthropod USPs divide into two distinct groups. One group consists of sequences from members of the holometabolous Lepidoptera and Diptera, while the other arthropod sequences group with vertebrate retinoid-X-receptors (RXRs). We therefore wondered whether USP/RXR structure could be used to clarify the contentious phylogenetic position of the order Strepsiptera, which has proposed affinities with either Diptera or Coleoptera. We have cloned and sequenced the USP/RXR from the strepsipteran Xenos pecki. Phylogenetic analyses are not consistent with a close affinity between Strepsiptera and Diptera.

Analysis of the Drosophila rDNA promoter by transient expression.

We have examined the expression of the bacterial gene chloramphenicol acetyl transferase (CAT) under the control of the Drosophila rDNA promoter following transfection into Drosophila tissue culture cells. Constructs having an entire NTS, corresponding to approximately 3640 base pairs of upstream rDNA sequence, or constructs with 306 base pairs of upstream sequence respectively, are transcribed at 5 fold or 2 fold higher levels than a construct with 43 base pairs of upstream DNA. In co-transfection experiments, the construct with the entire NTS competes for transcription 20 fold more effectively than the construct with 306 base pairs of upstream sequence. Constructs having either 72 base pairs or 60 base pairs of upstream rDNA sequences, on the other hand, are transcribed very much less efficiently than constructs with either 306 bp or with only 43 bp of upstream DNA. These sequences, which reduce levels of rDNA transcription in the absence of additional upstream DNA, lie in a region in which the rDNA promoter differs from its duplications within the NTS.

Transcription of the 'non-transcribed' spacer of Drosophila melanogaster rDNA.

We have detected a set of transcripts in Drosophila melanogaster cells which are homologous to repeating elements within the 'non-transcribed' spacer region of rDNA. The RNA molecules range from 240 to 1680 nucleotides, differing in length by an integral value of 240 nucleotides. We have sequenced several AluI fragments which characterise the main 240 nucleotide repeating element. We find that each of these fragments contain a segment of approximately 50 nucleotides, which is homologous to the transcription initiation site for pre-rRNA.

Molecular evolution of integrins: genes encoding integrin beta subunits from a coral and a sponge.

The integrin family of cell surface receptors is strongly conserved in higher animals, but the evolutionary history of integrins is obscure. We have identified and sequenced cDNAs encoding integrin beta subunits from a coral (phylum Cnidaria) and a sponge (Porifera), indicating that these proteins existed in the earliest stages of metazoan evolution. The coral betaCn1 and, especially, the sponge betaPo1 sequences are the most divergent of the "beta1-class" integrins and share a number of features not found in any other vertebrate or invertebrate integrins. Perhaps the greatest difference from other beta subunits is found in the third and fourth repeats of the cysteine-rich stalk, where the generally conserved spacings between cysteines are highly variable, but not similar, in betaCn1 and betaPo1. Alternatively spliced cDNAs, containing a stop codon about midway through the full-length translated sequence, were isolated from the sponge library. These cDNAs appear to define a boundary between functional domains, as they would encode a protein that includes the globular ligand-binding head but would be missing the stalk, transmembrane, and cytoplasmic domains. These and other sequence comparisons with vertebrate integrins are discussed with respect to models of integrin structure and function.

Molecular cloning and analysis of small optic lobes, a structural brain gene of Drosophila melanogaster.

Mutations in the small optic lobes (sol) gene of Drosophila melanogaster cause specific cells to degenerate in the developing optic lobes, resulting in the absence of certain classes of columnar neurons. These neuronal defects lead to specific alterations in behavioral characteristics, particularly during flight and walking maneuvers. We have isolated the wild-type sol locus by microcloning and chromosomal walking and have established its genetic and molecular limits. Two major transcripts of 5.8 and 5.2 kilobases are produced from this locus by alternative splicing and are present throughout the entire life cycle. Sequence analyses of cDNAs corresponding to these two classes of transcripts predict two proteins of 1597 and 395 amino acids. The first shows similarity in its carboxyl-terminal region to the catalytic domain of a vertebrate calcium-activated neutral protease (calpain), whereas its amino-terminal region contains several zinc-finger-like repeats of the form WXCX2CX10-11CX2C. The second predicted protein contains only the first two of the zinc-finger-like repeats and is missing the calpain domain. By constructing transgenic flies carrying a single wild-type copy of the sol gene in a homozygous sol mutant background, we have restored the normal neuroanatomical phenotype to individuals that would have developed mutant brains.

Sequence and expression of grasshopper antennapedia: comparison to Drosophila.

We have cloned and characterized the Antennapedia (Antp) gene from the grasshopper Schistocerca americana. The Antennapedia protein contains seven blocks of sequence, including the homeodomain, that are conserved in the homologous proteins of other insects, interspersed with (usually repetitive) sequences unique to each species. There is no similarity between 1.8 kb of 3' untranslated sequence in grasshopper and Drosophila. We examined Antennapedia protein expression in grasshopper using an antibody raised against a grasshopper fusion protein and reexamined its expression in Drosophila using several different antibodies. Early patterns of expression in the two insects are quite different, reflecting differing modes of early development. However, by the germband stage, expression patterns are quite similar, with relatively uniform epithelial expression throughout the thoracic and abdominal segments which later retracts to the thorax. Expression is observed in muscle pioneers, the peripheral nervous system, and the central nervous system (CNS). In the CNS expression is initially limited to a few neurons, but eventually becomes widespread. Both insects show strong expression in certain homologous identified neurons and similar temporal modulation of expression.

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

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.

Grasshopper hunchback expression reveals conserved and novel aspects of axis formation and segmentation.

While the expression patterns of segment polarity genes such as engrailed have been shown to be similar in Drosophila melanogaster and Schistocerca americana (grasshopper), the expression patterns of pair-rule genes such as even-skipped are not conserved between these species. This might suggest that the factors upstream of pair-rule gene expression are not conserved across insect species. We find that, despite this, many aspects of the expression of the Drosophila gap gene hunchback are shared with its orthologs in the grasshoppers S. americana and L. migratoria. We have analyzed both mRNA and protein expression during development, and find that the grasshopper hunchback orthologs appear to have a conserved role in early axial patterning of the germ anlagen and in the specification of gnathal and thoracic primordia. In addition, distinct stepped expression levels of hunchback in the gnathal/thoracic domains suggest that grasshopper hunchback may act in a concentration-dependent fashion (as in Drosophila), although morphogenetic activity is not set up by diffusion to form a smooth gradient. Axial patterning functions appear to be performed entirely by zygotic hunchback, a fundamental difference from Drosophila in which maternal and zygotic hunchback play redundant roles. In grasshoppers, maternal hunchback activity is provided uniformly to the embryo as protein and, we suggest, serves a distinct role in distinguishing embryonic from extra-embryonic cells along the anteroposterior axis from the outset of development - a distinction made in Drosophila along the dorsoventral axis later in development. Later hunchback expression in the abdominal segments is conserved, as are patterns in the nervous system, and in both Drosophila and grasshopper, hunchback is expressed in a subset of extra-embryonic cells. Thus, while the expected domains of hunchback expression are conserved in Schistocerca, we have found surprising and fundamental differences in axial patterning, and have identified a previously unreported domain of expression in Drosophila that suggests conservation of a function in extra-embryonic patterning.

The evolution of nuclear receptors: evidence from the coral Acropora.

We have amplified and sequenced PCR products derived from 10 nuclear receptor (NR) genes from the anthozoan cnidarian Acropora millepora, including five products corresponding to genes not previously reported from the phylum Cnidaria. cDNAs corresponding to seven of these products were sequenced and at least three encode full-length proteins, increasing the number of complete cnidarian NR coding sequences from one to four. All clear orthologs of Acropora NRs either lack an activation domain or lack a known ligand, consistent with the idea that the ancestral nuclear receptor was without a ligand. Phylogenetic analyses indicate that most, and possibly all, presently identified cnidarian NRs are members of NR subfamily 2, suggesting that the common ancestor of all known nuclear receptors most resembled members of this subfamily.

The sluggish-A gene of Drosophila melanogaster is expressed in the nervous system and encodes proline oxidase, a mitochondrial enzyme involved in glutamate biosynthesis.

Certain gene mutations in Drosophila melanogaster cause sluggish motor activity. We have localized the transcription unit of the sluggish-A gene to a 14.7-kb region at the base of the X chromosome and have cloned corresponding cDNAs. The predicted protein product has significant sequence similarity to Saccharomyces cerevisiae proline oxidase (EC 1.5.99.8), a mitochondrial enzyme which catalyzes the first step in the conversion of proline to glutamate. In the mutant fly, mitochondrial proline oxidase activity is reduced and has kinetic properties different from those of the wild type, providing further evidence that the gene encodes proline oxidase. Indeed, the free proline level in mutant flies is elevated. When the mutant is rescued by transformation, the proline oxidase and free proline levels, as well as the motor and phototactic behavior, are restored to normal. During embryonic development the sluggish-A transcript is predominantly expressed in the nervous system. Significantly, it has previously been reported that a mouse mutant, PRO/Re, which has reduced proline oxidase activity and elevated free proline levels, also exhibits sluggish behavior.

Pax gene diversity in the basal cnidarian Acropora millepora (Cnidaria, Anthozoa): implications for the evolution of the Pax gene family.

Pax genes encode a family of transcription factors, many of which play key roles in animal embryonic development but whose evolutionary relationships and ancestral functions are unclear. To address these issues, we are characterizing the Pax gene complement of the coral Acropora millepora, an anthozoan cnidarian. As the simplest animals at the tissue level of organization, cnidarians occupy a key position in animal evolution, and the Anthozoa are the basal class within this diverse phylum. We have identified four Pax genes in Acropora: two (Pax-Aam and Pax-Bam) are orthologs of genes identified in other cnidarians; the others (Pax-Cam and Pax-Dam) are unique to Acropora. Pax-Aam may be orthologous with Drosophila Pox neuro, and Pax-Bam clearly belongs to the Pax-2/5/8 class. The Pax-Bam Paired domain binds specifically and preferentially to Pax-2/5/8 binding sites. The recently identified Acropora gene Pax-Dam belongs to the Pax-3/7 class. Clearly, substantial diversification of the Pax family occurred before the Cnidaria/higher Metazoa split. The fourth Acropora Pax gene, Pax-Cam, may correspond to the ancestral vertebrate Pax gene and most closely resembles Pax-6. The expression pattern of Pax-Cam, in putative neurons, is consistent with an ancestral role of the Pax family in neural differentiation and patterning. We have determined the genomic structure of each Acropora Pax gene and show that some splice sites are shared both between the coral genes and between these and Pax genes in triploblastic metazoans. Together, these data support the monophyly of the Pax family and indicate ancient origins of several introns.