Environmental data from: Feeding strategy and dietary preference shape the microbiome of epipelagic copepods in a warm nutrient-impoverished ecosystem

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.  

Methods
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.

桡足类（Copepods）可提供富含有机质的微环境，支持微生物定殖与增殖，在海洋中形成动态的微生物热点。此前学界曾假设，这种浮游生物间的共生关系在温暖寡营养海域（oligotrophic seas）尤为发达，因为它们可在生物匮乏的水域中作为营养的替代来源。为进一步阐明超寡营养东地中海（ultra-oligotrophic Eastern Mediterranean Sea）中桡足类微生物组的构建模式，本研究依托16S rRNA基因（16S rRNA gene）扩增子测序（amplicon sequencing）技术，对东地中海近岸海域3种优势表层浮游桡足类物种的共生菌群进行了表征。结果显示，桡足类关联的微生物群落具有宿主特异性，而非受季节性环境变化驱动。在滤食性（filter-feeding）且偏植食性的哲水蚤（Temora stylifera）中，微生物多样性全年处于较低水平且相对稳定。与之相反，杂食性（omnivorous）的伏击型捕食者小胸剑水蚤（Oithona nana）以及混合摄食（mixed-feeding）的Pontic角水蚤（Centropages ponticus）所携带的微生物组多样性更高，且以暂时性类群为主导。本研究推测，滤食策略与狭窄的食物谱可限制桡足类与微生物的互作，而伏击、混合摄食策略结合杂食性特征则赋予更高的微生物多样性。哲水蚤拥有大量核心类群（core taxa），表明其可通过维持高度专一的共生关系，减少机会性微生物的定植。基于核心菌群（core microbiome）中氮代谢通路的预测富集结果（尤其在浅海沿岸水域氮匮乏的夏季），本研究强调了桡足类-微生物共生关系在营养匮乏生态系统中的重要性。

方法
本研究于以色列地中海哈代拉站位（32.4700° N，34.6930° E）的近岸海域（底质水深15~30 m），在0.5~1 m水层采集表层海水样本，采样覆盖2020年的四季：2月（冬季）、4月（春季）、7月（夏季）与10月（秋季）。本研究采集海水样本用于测定铵态氮（NH₄）、叶绿素a（Chla）、细菌丰度（BA）、微微型及纳型真核藻类丰度（PNEA）、异养细菌生产力（BP）与初级生产力（PP）。海水表层温度（SST）通过CTD（SeaBird, USA）测定。本采样工作隶属于以色列海洋与湖沼研究所（IOLR）实施的以色列海洋调查国家监测计划（IMS）。

取15 mL海水样本至经酸洗的塑料闪烁瓶中，于-20℃保存。参照Kress与Herut（2001）的方法，采用分段流动式Seal Analytical AA-3系统测定铵态氮，检出限为0.04 μM。另取350 mL海水样本通过GF/F滤膜（Whatman）过滤，将滤膜置于90%冷丙酮中萃取24 h，采用非酸化法（Welschmeyer 1994），通过Turner Designs（Trilogy）荧光光度计在激发波长436 nm、发射波长680 nm条件下测定叶绿素a含量。

采用Attune®声波聚焦流式细胞仪（Applied Biosystems）测定微微型/纳型真核藻类（PNEA）与异养细菌丰度。取1.8 mL海水样本，以终浓度0.02%（v:v）的50%戊二醛（Sigma G-7651）固定，置于4℃保存并在2~4 d内完成分析。基于红色荧光（叶绿素a，630 nm）、前向散射与侧向散射区分并计数微微型与纳型真核藻类；对细菌样本则采用SYBR Green荧光核酸染料染色，基于绿色荧光（530 nm）、前向散射与侧向散射区分并计数细菌丰度。

异养细菌生产力（BP）采用3H-亮氨酸掺入法结合微量离心技术（Smith & Azam 1992）估算。设置三个平行样本，加入100 nM亮氨酸（其中15 nM为3H-亮氨酸，85 nM为“冷”亮氨酸），于黑暗中孵育3~4 h，同时设置零时刻灭活对照组。参照Simon与Azam（1989）的方法，以1.5 kg C mol⁻¹的转换系数结合2.0的同位素稀释因子，将亮氨酸掺入量转换为细菌生产力。

初级生产力（PP）采用14C掺入法（Nielsen, 1952）估算。向海水样本中加入5 μCi的NaH¹⁴CO₃（Perkin Elmer，比活度56 mCi mmol⁻¹），于原位自然光照条件下孵育4 h。通过低压（约50 mmHg）过滤将孵育后的海水通过GF/F滤膜（Whatman，孔径0.7 μm）终止反应，将滤膜置于含50 μL 32% HCl的5 mL闪烁瓶中过夜，以去除过量的无机¹⁴C。采用TRI-CARB 2100 TR（Packard）液体闪烁计数器测定放射性活度。