An Alternative Method to Niskin Sampling for Molecular Analysis of the Marine Environment

: The development of low-cost, open-source Remotely Operated Vehicle (ROV) systems has


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we describe a versatile, easily-replicated platform which achieves in situ mRNA preservation, via 22 the addition of RNAlater to filtered microbial cells, to enhance ROV or CTD functionality.

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Based on the modified Nansen bottle (invented in 1894); the Niskin bottle (1967), invented just 27 a few years after the discovery and characterisation of mRNA, was developed for the retrieval of 28 seawater samples to the surface (Hill, 1900;Niskin, 1966;Cobb, 1990) [1][2][3]. Traditional Niskin 29 sampling still dominates oceanic analysis, while metatranscriptomic (whole community mRNA 30 profiling) based techniques have revolutionised our understanding of the function of mixed 31 community assemblages at the molecular level (Gilbert et al, 2011) [4]. Together, they have provided 32 a much needed insight on the fundamental workings of global biogeochemical cycling. However, 33 while metatranscriptomics suffers from a necessity to reduce technical variation as much as possible 34 to allow meaningful interpretation of results, it is stifled by the inaccuracy and variability that is 35 irrevocably associated with current Niskin-based sampling methods. Whilst cellular mRNA profiles 36 can respond to environmental insults within milliseconds, the mandatory transcriptional alterations 37 induced by Niskin sampling, which subjects samples to unavoidable exposure to differences in 38 pressure, temperature and light, in addition to the inherent temporal delay, is difficult to circumvent.

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This irreconcilable observation has stimulated the development of many in situ profiling technologies 40 for the marine environment (Feike et al, 2012;Taylor et al, 2015;McQuillan and Robidart, 2017) [5-7], 41 however these solutions have not gained dominance or widespread use as yet, primarily due to cost 42 restrictions.

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In tandem to the dawning realisation that the majority of current marine transcriptomic and 44 metatranscriptomic analyses are inherently inaccurate, the development of low cost open source ROV 45 systems has provided easy access (to the top 100 metres of the ocean at the very least) for researchers 46 looking to monitor, and sample, the marine environment in ever greater resolution. Whilst utilising an ROV mounted Niskin system to study metatranscriptomic profiles, we were struck by the contrast 48 between the antiquated nature of this traditional and inaccurate sampling technique, and the low 49 cost, high-performance simplicity of the ROV system upon which is was mounted. To this end, we 50 looked to develop a versatile, easily replicated RNA sampling platform ("RNA Automated 51 Preservation in situ Device, RAPID") inspired by low-cost, high-performance and simplicity. It is 52 well established that in situ mRNA preservation can be achieved rapidly and simply through the 53 addition of RNAlater to microbial cells (Ottesen et al, 2011) [8].

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With this premise in mind, we looked to design a system that could both concentrate and 55 preserve samples in a rapid, simple and low cost manner. Utilising off the shelf components we 56 assembled and tested an Arduino (Leonardo) controlled dual pump system [9], capable of pushing 57 seawater through a suitable filter unit, prior to the delivery of RNAlater (Figure 1). With motors and 58 electronics encased and powered from 12V supply (4 × AA batteries) within a permanently sealed assembly. Initial trials with centrifugal pumps (adapted from a NERF Electrostorm water pistol) 65 revealed rapid degradation of internal components exposed to seawater and RNAlater, so we 66 favoured a peristaltic pump option (Model: A518, ZJchao). Any filter assembly (and filter type) 67 capable of withstanding pressure can be used (we have utilised 25 mm and 47 mm filter assemblies, 68 as well as the Sterivex system). The Arduino was mounted on the lid of the box, so that in the situation 69 of structural integrity being lost, water damage to the circuit would be minimised (total immersion 70 in silicon oil is another simple way to reduce pressure effects). Nevertheless, replacement of the 71 junction box with a more robust structure may be necessary to go beyond 100 m depth. Whilst we 72 developed here a single filter sample system, the addition of simple controlled distribution valves 73 will provide the opportunity for numerous samples to be taken and preserved in procession.

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Following activation of pump 1, seawater is pushed though the filter assembly at a rate of ~2.5 ml/s 75 (we achieved filtration of ~500 ml through a 0.22 µm Sterivex Filter in 4 minutes), applying different 76 filters varies the rate of flow, as does biomass accumulation on the filter, until pump 2 is engaged for 77 a 10 s flooding with ~27 ml of RNAlater. Although not instantaneous, the sample is not subjected to 78 any temperature, pressure and/or light variation (unless the ROV is operated to specifically induce 79 such conditions) and filtration/preservation is performed rapidly in situ. This potentially represents 80 a significant improvement in both accuracy of transcript profiles and rapidity in comparison with 81 current sampling procedures which usually rely on a delay for filtering on board ship following 82 sample retrieval.

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For samples where it is crucial to preserve the transcriptional profile immediately, pumps 1 and 84 2 can be run simultaneously to bring RNAlater into contact with the seawater immediately prior to 85 filtration or bag collection. Following retrieval of the ROV to the surface and RNA extraction in the 86 laboratory, no difference was observed in quality or quantity of total RNA obtained by Niskin or the 87 on board system (Figure 3), thereby proving the principle that sampling via systems of this type can 88 provide sufficient and suitable RNA, which is by virtue of its processing more representative of the 89 natural environment from which it is taken. In addition to costing less than £50 to build and being 90 small enough to mount on low-cost, entry-level ROV systems (which provide visualisation, easy 91 maneuverability, and often accurate depth and temperature data, in real time and therefore with the 92 opportunity for responsive action), such a system can also be utilised in conjunction with more