Clockwise and counterclockwise hysteresis in the S. purpurea micro ecosystem

Replicate S. purpurea pitchers were enriched with 5.0 (high concentration) or 0.5 (low concentration) mg of bovine serum albumi (BSA) per mL of pitcher fluid in 2015 and 2.0 mg/mL (intermediate concentration) in 2016. For each experiment, a set of control pitchers received no organic matter. These controls were used to determine when oxygen levels in experimental pitchers had recovered. Experimental pitchers were loaded with organic matter (between 9:00 am and 9:45 am) following pitcher-fluid sampling and DO measurement for the first four days of the experiment. We measured DO twice a day each day from day 20 to day 28, and once on Days 30, 31, 33, and 35. DO was measured using a D-166MT-1S microelectrode (Lazar Research Laboratories). The microelectrode was calibrated prior to each sampling event according to the manufacturer’s instructions. A single microelectrode was used to take readings from each pitcher and was rinsed twice with reverse osmosis water between readings. The order of readings from different replicates was randomized so that changes in temperature and sunlight over the sampling period were not confounding factors. During readings, the microelectrode was placed 2.5 cm below the surface of the pitcher fluid and swirled so that the more oxygen-rich pitcher fluid at the top of the pitcher was mixed and readings reflected average DO. Due to the sensitivity of the microelectrode, readings were taken as soon as the reader settled on a value for more than 10 s. Pitcher fluid was sampled following each DO measurement for spectrophotometric analysis using a Bradford assay. Using sterile pipette tips, a 300 µL aliquot was taken from 2.5 cm below the surface of each pitcher and placed in a sterile 1-mL microfuge tube. Sample tubes were immediately transported to the lab where they were centrifuged at 13,000 xg for two minutes. The supernatant containing soluble BSA was removed, placed in a sterile 1-mL microfuge tube, and stored at -80°C until analyzed. The data were analyzed using the following R scripts: HYS_vFinal_removedreplicates, hysteresis_functions.R, reviewer3figure.R

2015年，我们向每毫升紫瓶子草（S. purpurea）捕虫笼液体中分别添加5.0 mg（高浓度组）与0.5 mg（低浓度组）牛血清白蛋白（bovine serum albumin, BSA）以富集有机质；2016年则设置2.0 mg/mL的中等浓度组。每个实验均设置一组空白对照捕虫笼，不添加任何有机质，该对照用于判断实验组捕虫笼的溶解氧（dissolved oxygen, DO）水平何时恢复至基准状态。实验前四天，我们在完成捕虫笼液体采样与溶解氧测量后，于每日上午9:00至9:45之间向实验组捕虫笼中加入有机质。自实验第20天至第28天，我们每日进行两次溶解氧测量；第30、31、33及35天则各测量一次。溶解氧测量采用D-166MT-1S型微电极（Lazar Research Laboratories公司），每次采样前均按照制造商说明书对该微电极进行校准。我们使用同一支微电极完成所有捕虫笼的读数采集，且每次读数后均用反渗透水冲洗两次。不同重复样本的读数顺序被随机化，以消除采样时段内温度与光照变化带来的混杂影响。读数时，将微电极置于捕虫笼液体表面下2.5 cm处并轻轻搅动，使捕虫笼顶部的富氧液体充分混合，确保读数反映液体的平均溶解氧浓度；鉴于微电极的高灵敏度，需待读数稳定超过10秒后再记录数值。每次完成溶解氧测量后，均采集捕虫笼液体样本用于布拉德福德法分光光度分析：使用无菌吸头，从每个捕虫笼液体表面下2.5 cm处吸取300 μL的液体样本，转移至无菌1 mL微量离心管中。样本管立即被运送至实验室，以13,000×g的转速离心2分钟，收集含有可溶性牛血清白蛋白的上清液，转移至另一支无菌1 mL微量离心管中，于-80℃下保存直至后续分析。本实验数据通过以下R脚本完成分析：HYS_vFinal_removedreplicates、hysteresis_functions.R、reviewer3figure.R