Underwater Application of Quantitative PCR on an Ocean Mooring

Abstract

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.

The ecology of environmental DNA and implications for conservation genetics

“…Already, vast oceanic distances and depths have led marine microbiologists to develop robotic systems for collection of eDNA samples (Scholin 2010). The most sophisticated platform autonomously conducts water filtration, DNA extraction, and genetic assays while deployed and transmits data via satellite (Preston et al 2011). Surveillance of airborne biological hazards has motivated similar developments (Hindson et al 2005), and engineers have also demonstrated use of unmanned aerial systems (i.e.…”

Seasonal Synechococcus and Thaumarchaeal population dynamics examined with high resolution with remote in situ instrumentation

“…The Thaumarchaeota (or group I Archaea, formerly the Crenarchaeota (Brochier- Armanet et al, 2008;Spang et al, 2010)) also have an important role in the nitrogen cycle in coastal marine systems by oxidizing ammonia to nitrite (Kö nneke et al, 2005), thus altering the form of nitrogen available for photosynthesis and other processes in upwelled waters and deeper in the photic zone where they reside (Mincer et al, 2007;Santoro et al, 2010;Preston et al, 2011). The Thaumarchaeota have widespread global distributions, and have been estimated to comprise over 20% of marine picoplankton (Karner et al, 2001).…”

“…The utility of the ESP for quantifying microbial population dynamics in Monterey Bay has been demonstrated by Preston et al (2011), where microbes with predictable dynamics were quantified during the upwelling season, for technical validation of the ESP technology. Here it was shown that Thaumarchaeota abundances increase with upwelling, as expected based on their deeper distributions.…”

Microalgae: Current Research and Applications

“…The limit of detection achieved was 100 cells per litre. An additional module, the MicroFludics Block (MFB) was later developed for in situ solid-phase nucleic acid extraction and quantitative PCR (Preston et al, 2011). The DNA was extracted and purified from 100-1000 mL samples down to 50 µL eluent aliquots.…”