title: 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.
authors.affiliation: Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, California 95039, USA. methane@mbari.org
authors.forename: William
authors.initials: W
authors.lastname: Ussler
authors.forename: Christina
authors.initials: C
authors.lastname: Preston
authors.forename: Patricia
authors.initials: P
authors.lastname: Tavormina
authors.forename: Doug
authors.initials: D
authors.lastname: Pargett
authors.forename: Scott
authors.initials: S
authors.lastname: Jensen
authors.forename: Brent
authors.initials: B
authors.lastname: Roman
authors.forename: Roman
authors.initials: R
authors.lastname: Marin
authors.forename: Sunita R
authors.initials: SR
authors.lastname: Shah
authors.forename: Peter R
authors.initials: PR
authors.lastname: Girguis
authors.forename: James M
authors.initials: JM
authors.lastname: Birch
authors.forename: Victoria
authors.initials: V
authors.lastname: Orphan
authors.forename: Christopher
authors.initials: C
authors.lastname: Scholin
mesh_terms: D059001:Aquatic Organisms / Q000235:genetics; D004275:DNA, Ribosomal / Q000302:isolation & purification*; D005798:Genes, Bacterial; D008697:Methane / Q000378:metabolism; D008772:Methylococcaceae / Q000235:genetics / Q000302:isolation & purification*; D010140:Pacific Ocean; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
chemical_list: D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D008697:Methane