DOI: 10.5061/dryad.98sf7m0mw
title: Environmental data from: Feeding strategy and dietary preference shape the microbiome of epipelagic copepods in a warm nutrient-impoverished ecosystem

description: Copepods provide a rich organic microenvironment allowing the settlement
 and proliferation of microorganisms, forming dynamic microbial hotspots in
 the oceans. Such symbiotic associations in the plankton were previously
 hypothesized to be especially developed in warm oligotrophic seas, as they
 may serve as alternative sources of nutrients in biologically-poor waters.
 Aiming to better understand how copepod microbiomes are shaped in an
 oligotrophic sea, we characterized microbiota associated with three
 dominant coastal epipelagic copepod species in the ultra-oligotrophic
 Eastern Mediterranean Sea using amplicon sequencing of the 16S rRNA gene.
 Our results show that copepod-associated microbial communities were
 host-specific rather than determined by seasonal environmental changes. In
 the filter-feeding copepod with a tendency to herbivory, Temora stylifera,
 microbial diversity was low and relatively stable throughout the year. In
 contrast, omnivorous copepods, the ambush-feeding Oithona nana, and the
 mixed-feeding Centropages ponticus harbored more diverse microbiomes
 dominated by transient taxa. We suggest that filter-feeding strategy and
 narrow food spectrum can limit copepod-microbe interactions, while the
 ambush and mixed feeding strategies combined with omnivory confer higher
 microbial diversity. Filter feeders may reduce the recruitment of
 opportunistic microbes by maintaining high fidelity associations, as
 indicated by the large number of core taxa in T. stylifera. We underline
 the importance of the copepod-microbe associations in
 nutrient-impoverished ecosystems, based on predicted enrichment of
 nitrogen metabolism in the core microbiome, mostly during summer when the
 shallow coastal waters are nitrogen-depleted.  
description: Surface seawater samples were collected at a
 depth of 0.5-1 m in the nearshore coastal waters (bottom depth 15-30 m) of
 the Israeli Mediterranean Sea, Hadera station (32.4700° N, 34.6930° E).
 The seawater samples were seasonally collected in February (winter), April
 (spring), July (summer), and October (autumn) in 2020.
  Samples were
 collected for measurements of NH4 (ammonium), chlorophyll-a (Chla),
 bacterial abundance (BA), pico-, and nano-eukaryotic algae abundance
 (PNEA), heterotrophic (bacterial) productivity (BP) and primary
 productivity (PP). Sea Surface Temperature (SST) was measured using CTD
 (SeaBird, USA). Sampling campaigns were conducted as part of the National
 Monitoring Program of the IMS performed by the Israel
 Oceanographic and Limnological Research Institute (IOLR).
 Seawater samples (15 mL) were
 collected in acid-washed plastic scintillation vials and were kept at −20
 °C for Ammonium (NH<sub>4</sub>) analysis.
 NH<sub>4</sub> was determined using a segmented flow Seal
 Analytical AA-3 system following the methods described by
 (Kress & Herut
 2001) with a limit of
 detection of 0.04 μM. Additional samples (350 ml) were filtered through
 GF/F filters (Whatman) to determine Chlorophyll-<em>a</em>
 (Chla). Chla was extracted from the filters in cold 90% acetone for 24 h
 and determined by the non-acidification method (Welschmeyer 1994), using a Turner Designs (Trilogy) Fluorometer at 436 nm excitation
 filter, and a 680 nm emission filter.
 Pico- and nano-eukaryotic
 algae (PNEA) and
 heterotrophic bacteria abundances were determined using an Attune®
 Acoustic Focusing Flow Cytometer (Applied Biosystems). Seawater samples
 (1.8 ml) were fixed with 50% glutaraldehyde (Sigma G-7651, final
 concentration 0.02% v:v), kept at 4°C, and were analyzed within the 2-4
 days. Pico- and nano-eukaryotic algae were enumerated by discrimination
 based on red fluorescence (Chla, 630 nm), forward and side scatters. To
 determine Bacteria abundance, the samples were enumerated and stained with
 SYBR Green
 fluorescent nucleic acid
 stain and identified by discrimination based on green fluorescence (530
 nm), forward and side scatters. Bacterial production (BP) was estimated using
 the <sup>3</sup>H-leucine incorporation method following the
 micro-centrifugation technique (Smith & Azam 1992). Triplicate samples were spiked with 100nM
 leucine (15 nM of <sup>3</sup>H-leucine and 85 nM of ‘cold’
 leucine), incubated in the dark for 3-4 hours, with time zero
 killed-controls. Leucine incorporation was converted to BP using a factor
 of 1.5 kg C mol<sup>-1</sup> with an isotope dilution factor
 of 2.0 (Simon &
 Azam 1989). Primary
 productivity (PP) was estimated using the 14C incorporation method
 (Nielsen, 1952). Water
 samples were spiked with 5 µCi of
 NaH<sup>14</sup>CO<sub>3</sub> (Perkin Elmer,
 specific activity 56 mCi mmol<sup>−1</sup>) and incubated for
 4 h under in situ natural illumination. The incubations were terminated by
 filtering the spiked seawater through GF/F filters (Whatman, 0.7µm pore
 size) at low pressure (∼50 mmHg). The filters were placed overnight in 5
 mL scintillation vials containing 50 µl of 32% HCl to remove excess
 inorganic <sup>14</sup>C. Radioactivity was measured using a
 TRI-CARB 2100 TR (Packard) liquid scintillation
 counter.