2.2 Antibiotic experiment and physiological monitoring ofA. erythraea (Expt1.)
Copepods were categorized into five groups: the first two groups were fed P. brasiliana and P. cuspidata , respectively; the second two groups received the same algal diets with the addition of antibiotics; and the final group served as a control with no supplements. The survival rate of copepods in each group was monitored over five days by culturing them in 2-liter beakers, with 50 individuals per beaker and three replicates per group. Additionally, ingestion and respiration rates were measured for the first two groups (Fig. 1).
Three commercially available antibiotics—ciprofloxacin, sulfamethoxazole, and trimethoprim—were used at low concentrations of 5 mg/L, as recommended by Edlund et al. (2012). These three antibiotics, which possess different antibacterial mechanisms, were combined to maximize the elimination of gut microbiota. To evaluate the effectiveness of the antibiotic treatment, we employed a BD FACSCelesta flow cytometer to measure the absolute abundance of copepod gut microbiota before and after antibiotic application (24 hours post treatment). Copepod guts were dissected and homogenized using a 40 μm cell strainer (#93040; SPL Life Sciences). The filtrates were carefully collected, diluted to a final volume of 10 mL, and fixed with paraformaldehyde (PFA) to achieve a final concentration of 0.5%. Samples were stained with SYBRTM-Green I nucleic acid gel stain (1:10,000 final dilution) and analyzed based on their PE-CF594 fluorescence and forward scatter (FSC) properties.
Before conducting the grazing experiment, we performed a preliminary study to determine the optimal algal concentrations. Five concentrations were tested: 500, 1000, 2000, 4000, and 6000 cells mL⁻¹ for P. brasiliana , and 300, 600, 1200, 2400, and 3600 cells mL⁻¹ for P. cuspidata . Each concentration was added to experimental bottles (200 mL) containing 10 copepods, with control bottles containing no copepods. All experimental setups were replicated three times. The ingestion rate was calculated based on the method described by Yang et al. (2024) after determining the concentration at which ingestion was maximal. Ingestion rates were measured at the start (D0) and daily thereafter (D1-D5).
Respiration rates were measured using a SensorDish®reader (24-channel oxygen meter, PreSens, Germany) (Lelong et al. 2012). Copepods were washed with 0.2 μm filtered sterilized seawater and transferred to 2.5 mL sensor vials (PreSens, Germany). Each group contained six vials with copepods, and three other vials served as blank controls. These vials were filled with 0.2 μm-filtered sterilized seawater and placed in the SensorDish® reader to monitor O₂ concentration. O₂ concentration was recorded every 15 seconds for one hour. The respiration rate for each vial was determined by calculating the slope of the regression line of O₂ concentration versus time. The copepod respiration rate was obtained by subtracting the control vial readings from those of the vials containing copepods.
2.3 Gut dissection experiment of A. erythraea on different diets (Expt2.)
To obtain DNA and RNA from the gut microbiota of A. erythraea , gut dissections were performed on days 1 and 2 (D1 and D2) for both diets (Fig. 1). Guts were extracted using sterilized dissection tweezers (Regine 5, Switzerland) in a sterile Petri dish under a stereomicroscope (Stemi 305). The extracted guts were then transferred to 0.5 mL tubes containing 0.2 μm-filtered sterile seawater and kept on ice. Subsequently, the samples were filtered through a 0.2 μm polycarbonate membrane (IsoporeTM, 47 mm) and stored in 1 mL of Trizol Reagent (Sigma, USA). A total of 90 copepod guts were dissected per sample to ensure sufficient RNA yield. Three skilled experimenters conducted the dissections simultaneously, completing each sample within 30 minutes. These samples were designated for subsequent metatranscriptome analyses of gut microbiota and transcriptome analyses of copepod. Additionally, 12 guts were dissected from copepods on toxic and non-toxic, respectively, on D1 and D2, with each gut placed in separate 0.5 mL tubes for 16S V4 amplicon analysis. All samples were stored at −80°C for subsequent DNA and RNA extraction.