General microbiology and science, September 15, 2014

Biodiversity and evolution in microbial communities, metabolomics, swabbing, DNA extraction, ITS approaches, marine viruses, and Bik’s Picks.

Microbial Ecology

The cryptic role of biodiversity in the emergence of host–microbial mutualisms – Pradeep Pillai – Ecology Letters

“Using a metacommunity model, we show that competition between multiple cheaters within the host’s microbiome, when combined with the spatial structure of host–microbial interactions, can have a constructive rather than a disruptive effect by allowing the emergence and maintenance of mutualistic microorganisms within the host.”

Microbial communities evolve faster in extreme environments – Sheng-Jin Li – Nature Scientific Reports

“Here we report a relative evolutionary rates (rERs) analysis of microbial communities from six diverse natural environments based on 40 metagenomic samples. “

Metabolomics

Mapping the Inner Workings of the Microbiome: Genomic- and Metagenomic-Based Study of Metabolism and Metabolic Interactions in the Human Microbiome – Ohad Manor, Roie Levy, Elhanan Borenstein – Cell Metabolism

“Here, we discuss recent efforts to study the metabolic inner workings of this complex ecosystem. “

Techniques

Comparison of seven methods for extraction of bacterial DNA from fecal and cecal samples of mice – Janina Ferrand – Journal of Microbiological Methods

“DNA quantity and quality were assessed by fluorometry, spectrophotometry, gel electrophoresis and qPCR….For both feces and intestinal contents, the most efficient extraction method was the FastDNA® SPIN Kit for Soil.”

Swab Sample Transfer for Point-Of-Care Diagnostics: Characterization of Swab Types and Manual Agitation Methods – Nuttada Panpradist – PLOS ONE

“In this study, we compared bacterial release efficiency of seven swab types using manual-agitation methods typical of POC devices.”

Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer – Paul M. Ruegger – Journal of Microbiological Methods

“Comparisons of the ITS region and two SSU regions using annotated bacterial genomes in GenBank showed much greater resolving power is possible with the ITS region. “

Pyroprinting: A rapid and flexible genotypic fingerprinting method for typing bacterial strains – Michael W. Black – Journal of Microbiological Methods

“Pyroprinting relies on the simultaneous pyrosequencing of polymorphic multicopy loci, such as the intergenic transcribed spacer regions of rRNA operons in bacterial genomes.”

Bioinformatics

OrthoClust: an orthology-based network framework for clustering data across multiple species – Koon-Kiu Yan – Genome Biology

“We demonstrate the application of OrthoClust using the RNA-Seq expression profiles of Caenorhabditis elegans and Drosophila melanogaster from the modENCODE consortium.”

Viruses and Phages

Review: Factors affecting virus dynamics and microbial host–virus interactions in marine environments – Kristina D.A. Mojica and Corina P.D. Brussaard – FEMS Microbiology Ecology

“The goal of the present minireview was to contribute to the evolution of marine viral ecology, through the assimilation of available data regarding the manner and degree to which environmental factors affect viral decay and infectivity as well as influence latent period and production.”

More Microbes

Dead or Alive: Molecular Assessment of Microbial Viability – Gerard A. Cangelosi and John S. Meschke – Applied and Environmental Microbiology

“This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology.”

Bacteria in the news

Is your body mostly microbes? Actually, we have no idea – Peter Andrey Smith – The Boston Globe

“There’s only one problem: The oft-cited 10-to-1 figure is almost certainly inaccurate.”

The Death Microbiome Could Inform Forensic Science And Medicine – Randall Mayes – Design & Trend

“For pathologists working out the time of death, research has focused on the way that insects and microbes from a corpse’ environment take up residence in the flesh.”

Tiny hitchhikers, big health impact: Studying the microbiome to learn about disease – Krista Conger – ScopeBlog Stanford

Got microbes? Meet some of the tiny life forms that call you home and may make antibiotics for you (with annoying 5 questions if you want to read the whole article) – Susannah Bodman – Oregon Live

Fighting Poisons With Bacteria – Going Inside the Rice Microbiome – Carina Storrs – NY Times

“Dr. Bais is working to develop rice plants that take up less arsenic, a common contaminant in the fields of his native India and other Asian countries.”

Possum faeces may cause flesh-eating ulcers in humans, Victorian researchers say – ABC Australia

“Infectious disease researchers at Victoria’s Barwon Health have found that possums can carry the buruli bacteria, which can cause major skin ulcers in humans.”

Scientific publishing

Ten Simple Rules for Better Figures – Nicolas P. Rougier – PLOS Computational Biology

“we aim to provide a basic set of rules to improve figure design and to explain some of the common pitfalls.”

The digital toolbox – Nature Editorial

“A new section of Nature examines the software and websites that make research easier.”

Scientific career

There is life after academia – Nature Editorial

“With high numbers of postdocs emerging from universities, prospective PhD students must be prepared for the fact that they will probably not end up with a career in research.”

Bik’s Picks

PBS: Video on the science behind brewing beer hops to it – CNET

“PBS series “It’s Okay To Be Smart” takes a look at how beer is made, and what external factors influence the final product.”

The coffee genome provides insight into the convergent evolution of caffeine biosynthesis -France Denoeud – Science

“We generated a high-quality draft genome of the species Coffea canephora, which displays a conserved chromosomal gene order among asterid angiosperms. “

On the Other Hand – Bob Grant – The Scientist

“Handedness, a conspicuous but enigmatic human trait, may be shared by other animals. What does it mean for evolution and brain function?”

[hr]

Non-human microbiome, August 19, 2014

A very diverse mix today: kangaroo, rumen, Daphnia, ticks, corals, rice, switchgrass, drinking water, acid mine drainage, rocks, soil microbiomes.

Mammal microbiome

Investigation of the microbial metabolism of carbon dioxide and hydrogen in the kangaroo foregut by stable isotope probing – Scott Godwin – ISME Journal

“Our results clearly demonstrate that the activity of bacterial reductive acetogens is a key factor in the reduced methane output of kangaroos”

Temporal dynamics of fibrolytic and methanogenic rumen microorganisms during in situ incubation of switchgrass determined by 16S rRNA gene profiling – Hailan Piao – Frontiers in Microbiology

“Here we investigated the temporal succession of microbial taxa and its effect on fiber composition during rumen incubation using 16S rRNA amplicon sequencing.”

Arthropod microbiome

Bacterial epibionts of Daphnia: a potential route for the transfer of dissolved organic carbon in freshwater food webs – Ester M Eckert and Jakob Pernthaler – ISME Journal

“We analysed the activity and identity of bacterial epibionts in cultures of Daphnia galeata and of natural daphnid populations.”

Evaluation of microbial communities and symbionts in Ixodes ricinus and ungulate hosts (Cervus elaphus and Ovis aries) from shared habitats on the west coast of Norway
Erik G. Granquist – Ticks and Tick-borne Diseases

“Five different species; M. mitochondrii, W. pipientis, Borrelia spp., Rickettia spp. and A. phagocytophilum, were analyzed on a 7500 Fast Real-time PCR system (Life Technologies, Carlsbad, CA), and carried out in separate 96-wells reaction plates. “

Coral microbiome

New insights into Oculina patagonica coral diseases and their associated Vibrio spp. communities – Esther Rubio-Portillo – ISME Journal

“….we have analysed the seasonal patterns of the culturable Vibrio spp. assemblages associated with healthy and diseased O. patagonica colonies. “

Bloom of the cyanobacterium Moorea bouillonii on the gorgonian coral Annella reticulata in Japan – Hideyuki Yamashiro – Nature Scientific Reports

“In addition to the cyanobacterium–shrimp interaction, the new trait of anchoring by the cyanobacterium into gorgonian coral may contribute to persistence of this bloom.”

Not just who, but how many: the importance of partner abundance in reef coral symbioses – Ross Cunning and Andrew C. Baker – Frontiers in Microbiology

“…we generate testable hypotheses regarding the importance of symbiont abundance by first discussing different metrics and their potential links to symbiosis performance and breakdown, and then describing how natural variability and dynamics of symbiont communities may help explain ecological patterns on coral reefs and predict responses to environmental change.”

Plant microbiome

Phylogeny and Functions of Bacterial Communities Associated with Field-Grown Rice Shoots – Takashi Okubo – Microbes and Environments

“Metagenomic analysis was applied to bacterial communities associated with the shoots of two field-grown rice cultivars, Nipponbare and Kasalath.”

Reviving of the endophytic bacterial community as a putative mechanism of plant resistance – Olga Podolich – Plant and Soil

“Based on the published results, we suggest that the endophyte-mediated stress tolerance or disease resistance can develop, if the plant hosts a sufficient diversity of ‘protective’ endophytes. “

The rhizosphere microbiota of plant invaders: an overview of recent advances in the microbiomics of invasive plants – Vanessa C. Coats, Mary E. Rumpho – Frontiers in Microbiology

“This review discusses recent advances in invasive plant biology that have resulted from microbiome analyses as well as the microbial factors that direct plant fitness and adaptability in natural systems.”

Kelp forest size alters microbial community structure and function on Vancouver Island, Canada – Jessica Liz Clasen and Jonathan B. Shurin – Ecology

“We investigated the response of microbial communities to variation in kelp abundance between regions with and without sea otter populations along the west coast of Vancouver Island, British Columbia, Canada”

Soil, sediments, rocks

* Impact of fire on active layer and permafrost microbial communities and metagenomes in an upland Alaskan boreal forest – Neslihan Taş – ISME Journal

“We measured geochemistry, GHG fluxes, potential soil enzyme activities and microbial community structure via 16SrRNA gene and metagenome sequencing.”

Integrated Metagenomics and Metatranscriptomics Analyses of Root-Associated Soil from Transgenic Switchgrass – Archana Chauhan – Genome Announcements

“Here we report metagenomic and metatranscriptomic analyses of root-associated soil from COMT switchgrass compared with nontransgenic counterparts.”

Abiotic stress tolerance and competition-related traits underlie phylogenetic clustering in soil bacterial communities – Marta Goberna – Ecology Letters

“We examined the distribution of 15 traits and 3290 bacterial taxa in 28 plots. Communities showed a marked functional response to the environment. “

Elevated nitrate enriches microbial functional genes for potential bioremediation of complexly contaminated sediments – Meiying Xu – ISME Journal

“Here we analyzed sediment microbial communities from a field-scale in situ bioremediation site, a creek in Pearl River Delta containing a variety of contaminants “

Occurrence and expression of bacterial human virulence gene homologues in natural soil bacteria – Ditte A. Søborg – FEMS Microbiology Ecology

“The presence and in vitro expression of homologues to 22 bacterial human virulence determinants amongst culturable soil bacteria was investigated.”

Endolithic bacterial communities in rock coatings from Kärkevagge, Swedish Lapland – Cassandra L. Marnocha – FEMS Microbiology Ecology

“Using 454 Roche pyrosequencing of the 16S rDNA gene, nine rock coating samples from three different coating mineralogies were sequenced.”

Biodegradation Potential of Organically Enriched Sediments under Sulfate- and Iron-Reducing Conditions as Revealed by the 16S rRNA Deep Sequencing – Tomoyuki Hori – Journal of Water and Environment Technology

“The effects of supplementation with sulfate and lepidocrocite (a crystalline Fe[III] oxide) on the structure and activity of the slurry microorganisms were examined by the combined physicochemical analyses and 16S rRNA deep sequencing. “

Soil bacterial diversity in degraded and restored lands of Northeast Brazil – Ademir Sérgio Ferreira Araújo – Antonie van Leeuwenhoek

“In this study we compare the structure and diversity of bacterial communities in degraded and restored lands in Northeast Brazil and determine the soil biological and chemical properties influencing bacterial communities. “

Biogeochemical environments of streambed-sediment pore waters with and without arsenic enrichment in a sedimentary rock terrain, New Jersey Piedmont, USA
A.C. Mumford – Science of the Total Environment

“The 16S rRNA gene and the As(V) respiratory reductase gene, arrA, were amplified from DNA extracted from streambed pore water at both sites and analyzed, revealing that distinct bacterial communities that corresponded to the redox conditions were present at each site. “

Water, wastewater, sludge microbiome

* A comprehensive insight into bacterial virulence in drinking water using 454 pyrosequencing and Illumina high-throughput sequencing – Kailong Huang – Ecotoxicology and Environmental Safety

“In order to comprehensively investigate bacterial virulence in drinking water, 454 pyrosequencing and Illumina high-throughput sequencing were used to detect potential pathogenic bacteria and virulence factors (VFs) in a full-scale drinking water treatment and distribution system. “

A high-throughput sequencing ecotoxicology study of freshwater bacterial communities and their responses to tebuconazole – Pascault Noémie – FEMS Microbiology Ecology

“Pyrosequencing revealed that bacterial diversity was higher in the river than in the lakes and in previously-exposed sites than in pristine sites.”

Disturbance and temporal partitioning of the activated sludge metacommunity – David C Vuono – ISME Journal

“Bacterial small subunit (SSU) rRNA genes were surveyed from sludge samples resulting in a sequence library of >417 000 SSU rRNA genes. “

Review: Environmental bacteriophages: viruses of microbes in aquatic ecosystems – Télesphore Sime-Ngando – Frontiers in Microbiology

“The present chapter sought to review the literature on the diversity and functional roles of viruses of microbes in environmental microbiology,”

* Inter-species interconnections in acid mine drainage microbial communities
Luis R. Comolli and Jill F. Banfield – Frontiers in Microbiology

Here, we present cryogenic transmission electron microscope (cryo-TEM) 2D images and 3D tomographic datasets for archaeal species from natural acid mine drainage (AMD) microbial communities. “

A study on the microbial community structure in oil reservoirs developed by water flooding – Lin Junzhang – Journal of Petroleum Science and Engineering

“This study was performed to identify the microbial community structures in 10 different types of water-flooded oil reservoirs on Sinopec Shengli Oil Field. “

Microbes of the built environment

* Microbial Existence in Controlled Habitats and Their Resistance to Space Conditions – Kasthuri Venkateswaran – Microbes and Environments

“In this review, we discussed the presence of microbes in space research-related closed habitats and the resistance of some microbial species to the extreme environmental conditions of space.”

Microbial Monitoring of Crewed Habitats in Space—Current Status and Future Perspectives – Nobuyasu Yamaguchi – Microbes and Environments

“In this review, the current status of microbial monitoring conducted in the International Space Station (ISS) as well as the results of recent microbial spaceflight experiments have been summarized and future perspectives are discussed.”

Food microbiology

Investigation of bacterial and fungal diversity in tarag using high-throughput sequencing
Zhihong Sun – Journal of Dairy Science

“This is the first study on the bacterial and fungal community diversity in 17 tarag samples (naturally fermented dairy products) through a metagenomic approach involving high-throughput pyrosequencing. “

[hr]

General microbiome digest, July 1

Diet and gut microbiome (again), Campylobacter in chicken is not a commensal, microbiomes of fish, insects, corals, plant, and hospitals, and women in science.

Human gut microbiome

Screen shot 2014-07-01 at 06.07PM, Jul 01Correlation network analysis reveals relationships between diet-induced changes in human gut microbiota and metabolic health – T Kelder – Nutrition & Diabetes

“We assessed fecal microbiota composition and host response patterns of metabolic and inflammatory markers in 10 apparently healthy men subjected to a high-fat high-caloric diet (HFHC, 1300 kcal/day extra) for 4 weeks. “

Pregnancy and birth

Screen shot 2014-07-01 at 06.08PM, Jul 01 1Gestational Age and Age at Sampling Influence Metabolic Profiles in Premature Infants
Reese H. Clark – Pediatrics

“Metabolic profiles (15 amino acids and 35 acylcarnitines) were obtained by using standard newborn techniques on infants born between 23 and 31 completed weeks of gestation.”

Screen shot 2014-07-01 at 06.08PM, Jul 01Non-invasive analysis of intestinal development in preterm and term infants using RNA-Sequencing – Jason M. Knight – Nature Scientific Reports

“we have developed a novel, noninvasive, molecular approach that utilizes next generation RNA sequencing on stool samples containing intact epithelial cells for the purpose of quantifying intestinal gene expression.”

Screen shot 2014-07-01 at 06.09PM, Jul 01Cesarean Section and Rate of Subsequent Stillbirth, Miscarriage, and Ectopic Pregnancy: A Danish Register-Based Cohort Study – Sinéad M. O’Neill – PLOS Medicine

“This study found that cesarean section is associated with a small increased rate of subsequent stillbirth and ectopic pregnancy.”

Bird microbiome

Screen shot 2014-07-01 at 06.10PM, Jul 01Campylobacter jejuni Is Not Merely a Commensal in Commercial Broiler Chickens and Affects Bird Welfare – Suzanne Humphrey – mBio

“Through experimental infection of four commercial breeds of broiler chickens, we show that breed has a significant effect on C. jejuni infection and the immune response of the animals, although these factors have limited impact on the number of bacteria in chicken ceca. “

Fish microbiome

Screen shot 2014-07-01 at 06.10PM, Jul 01 1Quorum quenching bacteria Bacillus sp. QSI-1 protect zebrafish (Danio rerio) from Aeromonas hydrophila infection – Weihua Chu – Nature Scientific Reports

“In this study, the effect of Bacillus sp. QSI-1 as an efficient quorum quencher on virulence factors production and biofilm formation of fish pathogen Aeromonas hydrophila was investigated. ”

Insect microbiome

Screen shot 2014-07-01 at 06.11PM, Jul 01Evolutionary origin of insect–Wolbachia nutritional mutualism – Naruo Nikoh – PNAS

“In the bedbug’s Wolbachia genome, we identified a gene cluster encoding the complete synthetic pathway for biotin (vitamin B7), which is not present in other Wolbachia genomes and is presumably acquired via lateral transfer from a coinfecting endosymbiont. “

Corals and sponges microbiome

Screen shot 2014-07-01 at 06.12PM, Jul 01Local genomic adaptation of coral reef-associated microbiomes to gradients of natural variability and anthropogenic stressors – Linda W. Kelly – PNAS

“The present study investigated the community structure and metabolic potential of microbes inhabiting coral reefs located across an extensive area in the central Pacific. “

Plant microbiome

Screen shot 2014-07-01 at 06.13PM, Jul 01The role of sulfur and phosphorus mobilizing bacteria in biochar induced growth promotion of Lolium perenne – A Fox – FEMS Microbiology Ecology

“16S rRNA gene based rhizobacteria community analysis revealed a significant biochar treatment effect. Abundance of nematodes feeding on bacteria was also significantly increased in the biochar treatments.”

Functional diversification within bacterial lineages promotes wide functional overlapping between taxonomic groups in a Mediterranean soil forest – J Curiel Yuste – FEMS Microbiology Ecology

“We investigated the relationship between taxonomy and functioning of soil bacterial communities in soils from a Mediterranean Holm-oak forest using a high throughput DNA pyrosequencing technique. “

Soil, sewage and sludge microbiome

Screen shot 2014-07-01 at 06.15PM, Jul 01Response of soil-associated microbial communities to intrusion of coal mine-derived acid mine drainage – Justin S Brantner and John M Senko – Environmental Science & Technology

” Evaluation of pyrosequencing-derived 16S rRNA gene sequences recovered from incubations revealed the development of microbial community characteristics that were similar to those of the mature iron mound sediment. “

Transformation Products and Human Metabolites of Triclocarban and Triclosan in Sewage Sludge Across the United States – Benny F. G. Pycke – Environmental Science & Technology

“Here, we report on the levels of eight transformation products, human metabolites, and manufacturing byproducts of TCC and TCS in raw and treated sewage sludge.”

Built Environment Microbiome

Screen shot 2014-07-01 at 06.14PM, Jul 01Rethinking Sterile: The Hospital Microbiome – Carrie Arnold – Environ Health Perspect

“Gilbert, an environmental microbiologist at Argonne National Laboratory, and his platoon of graduate students, postdocs, and research assistants descended on the hospital several times each day, even before it opened to the public. Armed with cotton swabs, they focused their efforts on the floors devoted to surgery and oncology. “

Bioinformatics

Screen shot 2014-07-01 at 06.15PM, Jul 01 1A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples – Samia N. Naccache – Genome Research

“Here we describe SURPI (“sequence-based ultrarapid pathogen identification”), a computational pipeline for pathogen identification from complex metagenomic NGS data generated from clinical samples, and demonstrate use of the pipeline in the analysis of 237 clinical samples comprising more than 1.1 billion sequences. “

Metagenomics

Metagenomic scaffolds enable combinatorial lignin transformation
Cameron R. Strachan – PNAS

“In this study, we devised a previously unidentified biosensor responsive to lignin transformation products. We used this biosensor in a functional screen to recover metagenomic scaffolds sourced from coal bed bacterial communities. “

Arsenic metabolism

Sealing rice field boundaries in Bangladesh: a pilot study demonstrates reductions in water use, arsenic loading to field soils, and methane emissions from irrigation water – Rebecca B Neumann – Environmental Science & Technology

“Irrigation of rice fields in Bangladesh with arsenic-contaminated and methane-rich groundwater loads arsenic into field soils and releases methane into the atmosphere. We tested the water-savings potential of sealing field bunds (raised boundaries around field edges) as a way to mitigate these negative outcomes.”

Localised Flux-Maxima of Arsenic, Lead and Iron around Root Apices in Flooded Lowland Rice – Paul Nicholas Williams – Environmental Science & Technology

“Here we use new diffusive gradients in thin-film (DGT)/planar optode sandwich sensors deployed in situ on rice roots to demonstrate a new geochemical niche of greatly enhanced As, Pb and Fe(II) mobilization into solution immediately adjacent to the root tips characterized by O2 enrichment and low pH. “

Dengue

Screen shot 2014-07-01 at 06.16PM, Jul 01Baicalin, a metabolite of baicalein with antiviral activity against dengue virus – Ehsan Moghaddam – Nature Scientific Reports

“Here, we examined the anti-DENV properties of baicalin in vitro, and described the inhibitory potentials of baicalin at different steps of DENV-2 (NGC strain) replication.”

Antibiotic resistance

Review: The expression of antibiotic resistance genes in antibiotic-producing bacteria – Stefanie Mak – Molecular Microbiology

“Antibiotic-producing bacteria encode antibiotic resistance genes that protect them from the biologically active molecules that they produce. The expression of these genes needs to occur in a timely manner: either in advance of or concomitantly with biosynthesis. It appears that there have been at least two general solutions to this problem. “

General microbiology

Screen shot 2014-07-01 at 06.20PM, Jul 01Bacterial sugar utilization gives rise to distinct single-cell behaviors – Taliman Afroz – Molecular Microbiology

“Here, we performed single-cell analyses to probe the behavior of representative pathways in the model bacterium Escherichia coli. “

Identification of essential Alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems – Patrick D. Curtis and Yves V. Brun – Molecular Microbiology

“These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism’s essential gene pool is specific to that organism.”

Science and career

Screen shot 2014-07-01 at 06.22PM, Jul 01Elite male faculty in the life sciences employ fewer women – Jason M. Sheltzer and Joan C. Smith – PNAS

“To explore the current causes of women’s underrepresentation in biology, we collected publicly accessible data from university directories and faculty websites about the composition of biology laboratories at leading academic institutions in the United States. “

Screen shot 2014-07-01 at 06.01PM, Jul 01Americans Think We Have the World’s Best Colleges. We Don’t. – Kevin Carey – New York Times

We see K-12 schools and colleges differently because we’re looking at two different yardsticks: the academic performance of the whole population of students in one case, the research performance of a small number of institutions in the other.”

Dr. Bik’s picks

Screen shot 2014-07-01 at 06.23PM, Jul 01Herbivore diet breadth mediates the cascading effects of carnivores in food webs – Michael S. Singer – PNAS

“Accounting for phylogenetic nonindependence of herbivores and plants, we show for the first time (to our knowledge) that dietary specialization of herbivore species is associated with reduced bird predation across an herbivore phylogeny, and that dietary specialization of herbivores increases the antipredator effects of camouflage and aposematism.”

Screen shot 2014-07-01 at 06.23PM, Jul 01 1Evolution of the ribosome at atomic resolution – Anton S. Petrov – PNAS

“Comparing eukaryotic and prokaryotic ribosomes allows us to identify 3D insertion fingerprints of the expansion segments. Similar fingerprints allow us to analyze the common core and detect ancestral expansion segments within it. “

Plastic mapPlastic debris in the open ocean – Andrés Cózar – PNAS

“In this work, we synthetize data collected across the world to provide a global map and a first-order approximation of the magnitude of the plastic pollution in surface waters of the open ocean.”

[hr]