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.  

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 (NH₄) analysis. NH₄ 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-a (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 ³H-leucine incorporation method following the micro-centrifugation technique (Smith & Azam 1992). Triplicate samples were spiked with 100nM leucine (15 nM of ³H-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^-1 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 NaH¹⁴CO₃ (Perkin Elmer, specific activity 56 mCi mmol⁻¹) 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 ¹⁴C. Radioactivity was measured using a TRI-CARB 2100 TR (Packard) liquid scintillation counter.