Autonomous Application of Quantitative PCR in the Deep Sea: In Situ Surveys of Aerobic Methanotrophs Using the Deep-Sea Environmental Sample Processor

Abstract

Recent advances in ocean observing systems and genomic technologies have led to the development of the deep-sea environmental sample processor (D-ESP). The D-ESP filters particulates from seawater at depths up to 4000 m and applies a variety of molecular assays to the particulates, including quantitative PCR (qPCR), to identify particular organisms and genes in situ. Preserved samples enable laboratory-based validation of in situ results and expanded studies of genomic diversity and gene expression. Tests of the D-ESP at a methane-rich mound in the Santa Monica Basin centered on detection of 16S rRNA and particulate methane monooxygenase (pmoA) genes for two putative aerobic methanotrophs. Comparison of in situ qPCR results with laboratory-based assays of preserved samples demonstrates the D-ESP generated high-quality qPCR data while operating autonomously on the seafloor. Levels of 16S rRNA and pmoA cDNA detected in preserved samples are consistent with an active community of aerobic methanotrophs near the methane-rich mound. These findings are substantiated at low methane sites off Point Conception and in Monterey Bay where target genes are at or below detection limits. Successful deployment of the D-ESP is a major step toward developing autonomous systems to facilitate a wide range of marine microbiological investigations.

Co-registered Geochemistry and Metatranscriptomics Reveal Unexpected Distributions of Microbial Activity within a Hydrothermal Vent Field

“…To this end, we collected samples for geochemical analyses and metatranscriptomic library construction from an extensive survey of fluids within and around diffuse hydrothermal flows at the ASHES vent field (Butterfield et al, 2004) in the Axial volcano. As part of that effort, we deployed the Deep-sea Environmental Sample Processor, or D-ESP, a state-of-the-art, autonomous microbial analytical laboratory (Ottesen et al, 2011; Pargett et al, 2013; Ussler et al, 2013), to collect, filter, and preserve six RNA samples in situ for metatranscriptomics from one diffuse flow and one intra-field site. We also collected ten additional fluid and particulate samples from five adjacent sites spanning three habitat types: diffuse hydrothermal flows (in this work all <40°C) where there was visible shimmering and microbial mats (hereafter, “diffuse”), intra-field fluids found among the diffuse vents (~2.5°C), but without any visual biotic or abiotic indications of fluid flow (hereafter, “intra-field”), and background seawater collected hundreds of kilometers outside of Axial Caldera (hereafter, “background seawater”) using a Niskin water sampler.…”

A comparison of in situ vs. ex situ filtration methods on the assessment of dissolved and particulate metals at hydrothermal vents

“…Several specialized samplers have been designed and employed in hydrothermal vents to collect dissolved and particulate samples for in situ filtration for various types of analyses (Huber et al 2003;Taylor et al 2006;Preston et al 2011;Ussler et al 2013), including the analysis of dissolved and particulate Fe (Kádár et al 2005;Sarradin et al 2008;Breier et al 2009;. However, there have been no comparison of results between in situ and ex situ sample processing methods.…”

High-resolution monitoring of marine protists based on an observation strategy integrating automated on-board filtration and molecular analyses

“…The identification is based on solid phase hybridization of molecular probes, immobilized to the surface of the sensor chips that bind to the rRNA or rDNA of the target species (Diercks et al, 2008a, b;Ussler et al, 2013). Quantitative, or real-time, PCR is a PCR-based method that utilizes fluorescent dyes or fluorescently labeled molecular probes to quantify nucleic acids after each PCR cycle.…”

“…The continually growing number of available algal 18S rDNA sequences, e.g., in the Ribosomal Database Project (Quast et al, 2013), and phylogenetic analysis make it possible to design hierarchical sets of probes that specifically target the 18S rDNA of different taxa (Metfies and Medlin, 2007;Thiele et al, 2014). The probes can be used in combination with a wide variety of hybridization-based methods, such as RNA-based nucleic acid biosensors (Diercks et al, 2008a;Ussler et al, 2013), quantitative polymerase chain reaction (PCR) (Bowers et al, 2010;Toebe et al, 2013), or fluorescence in situ hybridization (FISH) (Thiele et al, 2014) to identify marine microbes. Other methods, such as molecular fingerprinting approaches and next generation sequencing (NGS), provide information on variability and composition of whole microbial communities.…”