Metatranscriptomic analysis of autonomously collected and preserved marine 2 bacterioplankton 3 4

32 Planktonic microbial activity and community structure is dynamic, and can change on 33 time scales of hours to days. Yet for logistical reasons, this temporal scale is typically under34 sampled in the marine environment. In order to facilitate higher-resolution, long-term 35 observation of microbial diversity and activity, we developed a protocol for automated collection 36 and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The 37 protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and 38 preservation of total cellular RNA. Microbial samples preserved using this protocol yielded 39 high-quality RNA after thirty days of storage at room temperature, or onboard the ESP at in situ 40 temperatures. Pyrosequencing of cDNA libraries generated from ESP-collected and preserved 41 samples yielded transcript abundance profiles nearly indistinguishable from those derived from 42 conventionally-treated replicate samples. To demonstrate the utility of the method, we used a 43 moored ESP to remotely and autonomously collect Monterey Bay seawater for 44 metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples 45 collected at four time points over the course of a single day. In all four samples, the oxygenic 46 photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated 47 by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity 48 with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct 49 species abundance and gene transcript profiles. These laboratory and field tests confirmed that 50 autonomous collection and preservation is a feasible and useful approach for characterizing the 51 expressed genes and environmental responses of marine microbial communities. 52 53