2.1. Sample collection
We collected freshwater zooplankton samples from multiple ponds and stagnant water bodies in and around the Cauvery River in Tamil Nadu, India (Table 2). Prior to transportation to the laboratory for further processing, the samples were filtered using a 70 µm mesh. The collection took place during the morning hours, around 6 a.m., before the surface water temperature began to rise.
2.2. Probiotic
Bacillus subtilis KAF061, Bacillus cereus KAF124, Bacillus thuringiensis KAF135, and a commercial probiotic called Bacillus subtilis were used in this study to find possible probiotic strains that would help increase the number of copepods. Husain et al. [9] revealed the powerful probiotics KAF124 and KAF135. B. subtilis, a commercially available probiotic, served as a positive control in this investigation.
2.3. Morphology & Anatomy of Acanthocyclops sp.
Witty [10] described the morphological analysis using stereomicroscopy. Copepods were the most numerous groups, which were isolated zooplanktons, followed by rotifers, Cladocera, protozoa, and Ostracoda (Table 3).
2.4. Copepod analysis in water samples: isolation, identification, and hemolysis
Nylon mesh with a 300 µm hole size was used to isolate the copepods from surface water samples. An inverted microscope at 40x magnification evaluated the samples after 24 h of stabilization. Copepods were the most prevalent zooplankton and had sophisticated eyes, sexual dimorphism, colouring, and viable female egg sacs [11]. For research purposes, copepods were mass-produced in the lab.
Husain et al. (2022) provided instructions for the hemolysis test, a method for identifying potentially harmful microorganisms on the outside and inside of copepods. We homogenized the copepods and plated them on nutrient agar at 37 °C for 24 h. We formed separate colonies on blood agar plates and examined blood cell lysis in cultures after 72 h [12].
2.5. Probiotic survival at varied temperatures with commercial fish feed
To investigate the shelf life, we blended autoclaved commercial fish feed (0.5 g) with KAF061, KAF124, and KAF135 (6.5 x 107 CFU/mL) probiotic isolates and incubated them at 4, 27, and 37 °C. After 5, 10, 15, 20, 30, and 45 days, we tested the bacterial isolate's survival using plate counts at different storage temperatures. We quantified the logarithmic colony-forming unit (CFU) values [13].
2.6. Mass Culture of Copepods using Spirulinasp. and Chlorellasp. as feed: Cultivation and feeding strategies
We used Spirulina sp. for copepod mass culture, and we cultivated pure Spirulina sp. culture in Zarrouk's medium [14] with the following contents: (g/L) NaHCO3, 16.8; K2HPO4 0.5; NaNO3, 2.5; K2SO4, 1.0; NaCl, 1.0; MgSO4-7H20, 0.2; CaCl2, 0.04; FeSO4-7H2O, 0.01; EDTA, 0.08; Solution A5, (g/L) H3BO3, 2.86; MnCl2-4H2O, 1.81; ZnSO4-7H2O, 0.222; CuSO4 5—1 ml/L; Solution B6, (mg/L, NH4VO3, 22.96; KCr (SO4)2 - 12H2O, 192.0; NiSO4-6H2O, 44.8; Na2PO4-2H2O, 17.94; TiOSO4H2SO4 - 8H2O, 61.1; and Co(NO3)2 - 6H2O, 43.98) optimized for copepod feed. The culture broth was incubated at 27 °C with a 10 mg/L inoculWe measured the optical density (OD) of the culture broth at 450 nm every 24 h for 10 days. every 24 h for 10 days. We produced the growth curve using the OD values of the broth culture.
Similarly, copepod mass culture used Chlorella sp. as food. A BG11 medium [15] was used to grow Chlorella sp. It had 1.5 g of NaNO3, 0.04 g of K2HPO4∙2H2O, 0.075 g of MgSO4∙7H2O, 0.036 g of CaCl2∙2H2O, 0.006 g of citric acid, 0.02 g of Na2CO3, 0.006 g of ferric ammonium citrate, 0.001 g of Na-EDTA, and 1 mL of trace metal A5. The trace metal A5 solution had 2.86 H3BO3, 1.81 MnCl2 ∙ 4H2O, 0.22 ZnSO4∙7H2O, 0.39 NaMoO4∙2H2O, 0.079 CuSO4∙5H2O, and 0.05 CoCl2∙6H2O (g/L). The solution was incubated at 27 °C with a 10 mg/L inoculum in BG11 media. We measured the optical density (OD) of the Chlorella sp. culture broth at 450 nm every 24 h for 10 days. We plotted the growth curve using the OD values of the broth culture.
We used Whatman No. 1 paper to isolate the cultures of Spirulina sp. and Chlorella sp. To make it more suitable for copepods, we diluted it in sterile water.
We mixed Spirulina sp. and Chlorella sp. to feed copepods. Once we collected the pure cultures of Spirulina sp. and Chlorella sp., we diluted each culture to 10 mg/L and blended them at a 1:1 ratio.
2.7. Copepod culture, media composition and optimisation
We optimized the culture media composition of copepods in 20 L culture tanks using various diets. Group 1: water alone (10 L) (W), Group 2: water + fish feces matter (0.5 g/L) (W+F), Group 3: water (10 L) + Chlorella sp. (10 mg/L) (W+S+C) + Spirulina sp. (10 mg/L), Group 4: water + Spirulina sp. + fish feces, Group 5: water + Chlorella sp.+ fish feces, and Group 6: water + Spirulina sp. + Chlorella sp. + fish feces (W+S+C+F). I inoculated each tank with 25 copepods. We aerated the tank for 1 hour at 8 a.m. and 7 p.m. during the experiment. The light and dark conditions lasted 12 h. Room temperature (27 ± 1 °C). We manually counted copepods under a stereomicroscope from the culture broth every 5 days [16].
2.8. Optimization and visualization of copepod bio enrichment with probiotic bacteria
Copepods were washed in sterilized water (1.5 mL) in groups of 6 to optimize bio-enrichment. Before adding the probiotic culture, they were kept at room temperature for 12 h. We cultured probiotic bacteria KAF061, 124, 135 and commercial probiotics in nutritional broth for 12 h, and then cleaned it twice with sterilized water. Approximately 3 µL of bacterial strains (≈2.4 × 107 CFU/mL) were introduced and maintained at room temperature. We visualized the bioenriched copepods using a phase-contrast microscope (Nikon, Inverted Microscope, Japan) after 0, 30, 60, 90, and 180 min and took images.
2.9. Statistical analysis
The investigations in this study were performed in triplicate and analysed using GraphPad Prism 8 software's one-way analysis of variance (ANOVA) and student's t-test. The dataset was presented as mean and standard deviation (SD) values for triplicate measurements. We evaluated the statistical significance at three different levels: P = 0.05, P = 0.01, and P = 0.001.