Some Preservation Techniques for (Deep Water) Coral Samples for Subsequent Molecular Studies: A Special Supplement from Harbor Branch Oceanographic Institute

Around the time that the thirteen original Atlantic colonies were fighting for independence from Britain, there existed little agreement among naturalists as to the nature of corals. Were they inanimate (stones), plants, animals, or intermediate between the latter two (zoophytes)? This diversity of definition and opinions undoubtedly produced considerable confusion and disagreement among naturalists interested in such things. The symbiotic nature of algal cells in the tissues of some corals was also not well understood. It was not until the Darwinian period in the nineteenth century that little doubt remained, and therefore it was generally agreed, that corals were actually animals - heterotrophic living organisms that prey on other organisms for nutrition and do not produce their own food. In the past fifty years the basic goals and tenets of deep-sea coral collection, curation, and taxonomy have changed little. On the other hand, the techniques and tools of this particular avenue of research have changed significantly. Regarding the collection of material in the field, some aspects remain fundamentally the same. The use of research vessels, bottom trawls, and naturalist\u27s dredges are still frequently used for deep water research. In shallow water collecting, improvements in SCUBA diving equipment and new innovations, such as Trimix gas and Nitrox diving, have allowed divers to work at greater depths with longer bottom times. Pressure independent dive suits have permitted researchers to attain depths not possible in traditional wet or dry suits. In the past four decades, advances in optics, electronics, and robotic technology have allowed for a rapid sophistication and broader scope of possibilities regarding manned submersibles, remotely operated vehicles (ROV\u27s), and more recently, autonomous underwater vehicles (AUV\u27s). Great strides have been made since the early 1990\u27s in the technological aspects of the collection and photography of the deep water benthos. Concerning the techniques and tools of research in the laboratory, the scanning electron microscope has for several decades provided a valuable tool for documenting surface details of sclerites. Newer electron microscopes and digital technology have negated the necessity of using photographic film and images can now be unloaded directly on to computers for processing. Relatively recently, the fields of phylogenetics, molecular biology, and natural products biochemistry have been applied to more traditional modes of research in the field of octocoral systematics. This document on deep-sea coral collection protocols stands as a valuable resource, because it provides a standardized procedure for researchers during a time of rapidly changing technology regarding exploration of the deep-sea benthos. Stated another war, the protocols give us an instruction manual for research procedures in deep-sea benthic fieldwork. Technological improvements coupled with workable field-tested procedures (such as provided by the present volume) can in the long run, only help to expand and improve our base of knowledge concerning octocoral diversity. The synthesis of results from research endeavors both old and new, and opportunities allowed by a constantly improving technological scene, provide for a positive potential for the future in the fields of octocoral diversity and systematics

Discrimination of Species in the Montastrea annularis Complex using Multiple Genetic Loci

Montastraea annularis, M. franksi and M. faveolata are a complex of recently distinguished coral species whose status remains controversial due to the lack of fixed differences. Here we report on two types of genetic analyses for these taxa: 1) DNA sequences of two nuclear genes [internal transcribed spacers of rDNA (ITS-1, ITS-2) and a ɮ-tubulin intron] and 2) a preliminary screening of the entire nuclear genome using amplified fragment length polymorphisms (AFLP). There was very little variation within or among the three species in DNA sequences. Onlly 3 of 300 nucleotide positions in ITS-1, 3 of the 350 positions in ITS-2, and 3 of 350 positions in the ɮ-tubulin intron had large amounts of polymorphism. At some of these sites there were allele frequency differences among the species, but no diagnostic substitutions. Sequences from polymorphic sites in the ɮ-tubulin intron also showed evidence of heterozygosity. Pilot experiments with AFLP yielded stronger evidence of genetic boundaries among these species. Two AFLP primers gave patterns of band presence or absence that were potentially diagnostic for M. faveolata (n=7) versus M. franksi (n=7); M. annularis (n=4) may also have diagnostic patterns, but the differences between it and M. franksi were quantitative (band intensity) rather than qualitative

Molecular Evolution of Numt, a Recent Transfer and Tandem Amplification of Mitochondrial DNA into the Nuclear Genome of the Domestic Cat (Felis catus)

Mitochondrial DNA (mtDNA) are functional cytoplasmic chromosomes, tracing origins to a symbiotic infection of eukaryotic cells by bacterial progenitors. As prescribed by the Serial Endosymbiosis Theory, symbionts have gradually transferred their genes to the nuclear genome that enable functional interaction. In this dissertation, a 7.9 kb transposition of a typically 17.0 kb mitochondrial genome to a specific chromosomal position in the domestic cat is reported. The integrated mtDNA has amplified about 38-76 times and now occurs as a macrosatellite -like tandem repeat with multiple length alleles resolved by pulse field gel electrophoresis (PFGE) segregating in cat populations. To examine the tempo and mode of evolution between different organelles, characterization of the complete 7946 bp nuclear mitochondrial DNA monomer, Numt, and cytoplasmic mtDNA (17,009 bp) sequences reveals about 95% similarity, which supports recent divergence within 1.8-2.0 MYA and the radiation of four modern species in genus Felis. The motif, (ACACACGT), appears imperfectly repeated at the deletion junction of the control region (CR), and a likely target for recombination. Simple repeats are also implicated in indel generation. Most substitutions between the cat homologues are attributable to accelerated cytoplasmic mtDNA evolution, yet maintain a uniform rate of synonymous substitutions between different mitochondrial genes. Results of ribonuclease protection assays on cellular RNA verify the lack of Numt-specific transcription and the appraisal of Numt as a molecular fossil . Despite an elevated number of transversions and no increase in dA/dT content over cytoplasmic mtDNA, Numt resembles archetypal pseudogene evolution. To place the felid data in the context of functional mitochondrial genomes, pairwise similarity comparisons of all 37 mtDNA coding genes and the CR among eight complete mitochondrial genomes of five placental mammals were performed. In carnivores, the ND4L and ATPase 6 genes exhibit higher sequence conservation, while cyt B shows accelerated divergence. Lastly, the occurrence of Numt-like loci in other exotic felids deviates from current phylogenetic predictions. To confirm homology with the F. catus Numt locus, a series of experiments was conducted to isolate chromosomal sequences directly flanking Numt-like loci. These observations provide an empirical glimpse of historic genomic events that may parallel the accommodation of organelles in eukaryotes

Co-Cultures of Pseudomonas aeruginosa and Roseobacter denitrificans Reveal Shifts in Gene Expression Levels Compared to Solo Cultures

Consistent biosynthesis of desired secondary metabolites (SMs) from pure microbial cultures is often unreliable. In a proof-of-principle study to induce SM gene expression and production, we describe mixed “co-culturing” conditions and monitoring of messages via quantitative real-time PCR (qPCR). Gene expression of model bacterial strains (Pseudomonas aeruginosa PAO1 and Roseobacter denitrificans Och114) was analyzed in pure solo and mixed cocultures to infer the effects of interspecies interactions on gene expression in vitro, Two P. aeruginosa genes (PhzH coding for portions of the phenazine antibiotic pathway leading to pyocyanin (PCN) and the RhdA gene for thiosulfate: cyanide sulfurtransferase (Rhodanese)) and two R. denitrificans genes (BetaLact for metallo-beta-lactamase and the DMSP gene for dimethylpropiothetin dethiomethylase) were assessed for differential expression. Results showed that R. denitrificans DMSP and BetaLact gene expression became elevated in a mixed culture. In contrast, P. aeruginosa co-cultures with R. denitrificans or a third species did not increase target gene expression above control levels. This paper provides insight for better control of target SM gene expression in vitro and bypass complex genetic engineering manipulations

Characterising Players of a Cube Puzzle Game with a Two-level Bag of Words

Ponencia presentada en UMAP '21: Adjunct Proceedings of the 29th ACM Conference on User Modeling, Adaptation and Personalization, Utrecht (Netherlands), June 21 - 25, 2021This work explores an unsupervised approach for modelling players of a 2D cube puzzle game with the ultimate goal of customising the game for particular players based solely on their interaction data. To that end, user interactions when solving puzzles are coded as images. Then, a feature embedding is learned for each puzzle with a convolutional network trained to regress the players’ comple tion effort in terms of time and number of clicks. Next, the known bag-of-words technique is used at two levels. First, sets of puzzles are represented using the puzzle feature embeddings as the input space. Second, the resulting first-level histograms are used as input space for characterising players. As a result, new players can be characterised in terms of the resulting second-level histograms. Preliminary results indicate that the approach is effective for char acterising players in terms of performance. It is also tentatively observed that other personal perceptions and preferences, beyond performance, are somehow implicitly captured from behavioural data

Population Structure and Dispersal of the Coral-Excavating Sponge Cliona delitrix

Some excavating sponges of the genus Cliona compete with live reef corals, often killing and bioeroding entire colonies. Important aspects affecting distribution of these species, such as dispersal capability and population structure, remain largely unknown. Thus, the aim of this study was to determine levels of genetic connectivity and dispersal of Cliona delitrix across the Greater Caribbean (Caribbean Sea, Bahamas and Florida), to understand current patterns and possible future trends in their distribution and effects on coral reefs. Using ten species-specific microsatellite markers, we found high levels of genetic differentiation between six genetically distinct populations: one in the Atlantic (Florida-Bahamas), one specific to Florida and four in the South Caribbean Sea. In Florida, two independent breeding populations are likely separated by depth. Gene flow and ecological dispersal occur among other populations in the Florida reef tract, and between some Florida locations and the Bahamas. Similarly, gene flow occurs between populations in the South Caribbean Sea, but appears restricted between the Caribbean Sea and the Atlantic (Florida-Bahamas). Dispersal of C. delitrix was farther than expected for a marine sponge and favoured in areas where currents are strong enough to transport sponge eggs or larvae over longer distances. Our results support the influence of ocean current patterns on genetic connectivity, and constitute a baseline to monitor future C. delitrix trends under climate change