Sampling of organisms and bioassay experiments
Shrimp post-larvae (PL5) of Litopenaeus vannamei were collected from the aquaculture farm Parque acuícola Cruz de Piedra, Guaymas, Sonora, Mexico (27° 51′ 05.9″ N 110° 31′ 57.0″ W). Afterward, post-larval shrimp were transported in aerated tanks with the same pond water as the farm to the Departamento de Investigaciones Científicas y Tecnológicas de la Universidad de Sonora (DICTUS), where a lab-scale system was previously tested and used. The bioassay was conducted for 80 days with healthy shrimp, each weighing 0.5 ± 0.1 g, and post-larvae were randomly distributed in the lab-scale system. The system consisted of nine 80-L culture units linked to a recirculation aquaculture system (RAS), and sterile seawater was used to fill the units to an operating volume of 60 L. Influent seawater was filtrated and flowed through a UV lamp for sterilization, and the seawater was equally dispensed to all culture units, as depicted in the supplementary material (Fig. S2). The culture units were maintained under similar indoor conditions with artificial aeration (2000F heat bonded silica; pore size, 140 µm), and the salinity was maintained at around 35‰ with the addition of sterile freshwater (MilliQ grade, Millipore) to avoid the incorporation of outside bacteria and to compensate evaporation. Finally, the effluent generated by the system flowed through a biofilter containing nitrifying bacteria to control toxic nitrogen compounds in the recirculation system (Fig. S2). The unconsumed feed, feces, moults, and dead organisms (if any) were removed daily.
The salinity, dissolved oxygen (DO), pH, and temperature were measured twice per day (07:00 and 18:00 h) using a YSI multiprobe system 556 (YSI Incorporated).
The bioassay started with PL5 on day 0. At this point, 40 organisms were randomly introduced into each culture unit, and the experiment lasted 80 days. Throughout the experiment, shrimp were fed twice a day at a rate of 4% wet biomass day–1 using feeding trays with the same formulated feed consisting of commercial grow-out pelletized feed with 25% crude protein, 5% lipids, and 4% fiber.
Water quality and productive response
The water quality was monitored daily throughout the bioassay, and samples were collected weekly from each culture unit using sterile falcon tubes by filtering the water through 0.45 μm membranes (Millipore). Nitrite (NO2–N), nitrate (NO3–N), ammonia (NH3–NH4), and phosphate (P–PO4) concentrations were measured using commercial Hanna reagent kits HI 93707-01, HI 93728-01, HI 93700-01, and HI 93717-01, respectively (Hanna Instruments, Romania).
Biometry analyses were performed at the four different developmental stages, denominated as I, II, III, and IV, corresponding to 0–20, 20–40, 40–60, and 60–80 culture days, respectively, and the productive response was calculated15.
Collection of intestine and water samples and DNA and RNA extraction
To discard the transitory microbiota, the shrimp were fasted for 6 h before sampling. Once the intestines were empty, these were dissected on the corresponding dates using a sterile dissection kit, placed in sterile cryogenic tubes, and stored at – 80 °C until nucleic acid extraction. Each sample date belongs to an experimental unit with three culture tanks. The intestine samples from culture tanks were pooled and considered as a replicate, giving three replicates per sampling time. At the same sampling points, 1 L samples of water (W) from each culture unit corresponding to an experimental replicate were collected and pooled for filtration through 0.22 µm sterile filters of mixed cellulose ester membrane (Whatman, Sigma, St Louis, USA) and placed in sterile, 50 mL falcon tubes for storage at – 80 °C until nucleic acid extraction.
Total DNA and RNA were extracted and purified from the membrane filters previously used to filter seawater samples and from intestines that were also previously sampled, both of them with the FastDNA Spin Kit for Soil15, and the FastRNA Pro Blue Kit (MP-Bio, Santa Ana, CA, USA) in combination with mechanical lysis using the FastPrep Systems (MP-BIO, Santa Ana, CA, USA). The obtained RNA samples were digested according to the TURBO DNA protocol (Ambion, Life Technologies Corporation, Carlsbad, CA, USA) and EDTA to stop the DNase activity and to ensure that any DNA residuals were presented. Finally, samples were purified according to the RNA Cleanup protocol from the RNeasy Mini Kit (Qiagen, Hamburg, Germany). The quality and concentration of nucleic acids were tested as previously described Maza-Márquez et al.39.
qPCR and RT-qPCR assays
Real-time polymerase chain reaction (qPCR) assays have been widely implemented for estimating total cell count based on DNA gene markers, regardless of their level of metabolic activity40,41, while reverse transcription qPCR (RT-qPCR) is a useful method for analyzing the expression of specific genes. RT-qPCR is also used because of its high sensitivity, accuracy, specificity, and rapidity in analyzing the time-specific expression of particular genes, allowing for the detection of low-abundance transcripts42. The absolute abundance of total and metabolically active populations of bacteria and Vibrio in both target samples were measured by qPCR and RT-qPCR, respectively, using a StepOne Real-Time PCR system (Applied Biosystems, USA). For RT-qPCR, the synthesis of cDNA was performed by reverse transcription of RNA with the aid of SuperScript III Reverse Transcriptase (Invitrogen, Life Technologies Corporation, Carlsbad, CA, EEUU), following the manufacturer’s specifications, in a final volume of 20 µL and using 150–200 ng of total RNA as a template (specific primers described in Table S4) (Sigma Aldrich; St. Louis, MO, USA) and dNTPs (Invitrogen; Carlsbad, USA). In addition, the cDNA quality and concentration were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific Waltham, MA USA). The number of copies (nbc) of 16S rRNA genes (16S rDNA) and 16S rRNA (16S rRNA) were evaluated in each sample using either extracted DNA or cDNA, respectively, as templates with a set of primers previously described (Table S4) based on the increasing fluorescence intensity of the SYBR Green dye during amplification. For the amplification and detection of specific fragments, the iTaq Universal SYBR Green Supermix (Biorad, USA) was used in a final volume of 15 µL for each reaction. All quantitative amplifications were performed in triplicate. The qPCR reaction mixtures contained 1.8 µL of cDNA or DNA, 250 ng of T4 gene 32 (QBiogene, Illkirch, France), 1.2 µL of each primer (10 mM), supplied by Sigma Aldrich (St. Louis, MO, USA), and 1 × SYBR Green Supermix. The amplification and detection conditions are described in Table S5.
To provide absolute quantification of the target microorganisms, standard curves were constructed with the aid of a standard plasmid that contained the inserts of the targeted genes. Amplicons of the 16S rDNA were generated from culture strains of Pseudomonas putida NCB957 (quantification of bacteria) and Vibrio parahaemolyticus ATCC17802 (quantification of Vibrio). The PCR products were cloned with the aid of the pCR2.1-TOPO plasmid vector using the TOPO TA cloning system (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA), following the manufacturer’s protocols. The calibration curves for absolute quantification in the DNA samples (16S rDNA) were generated using serial ten-fold dilutions of linearized plasmid standards, and for absolute quantification in RNA samples (16S rRNA), non-linearized plasmid standards were used as templates for in vitro transcription of the target genes into RNA39,40,43. The copy number per ng was calculated as previously described43. All calibration curves had a correlation coefficient (r2) of > 0.99 in all assays, and the efficiency of PCR amplification was always between 90 and 110%. Finally, the number of copies of the targeted genes was expressed per gram of tissue sampled, while for water samples these were expressed as the number of copies per mL.
Statistical analyses
All statistical analyses were performed in R Studio (version 3.6.0)44 using the following R packages: maggrittr45, ade446, factoextra47, vegan48, and gplots49. Analyses of variance (ANOVA) and multiple-range tests (Student’s-test) were used with a significance level of 95% (p < 0.05). Since most of the data sets did not fit the normal distribution, a nonparametric ordination method based on a multiple-comparison test, such as the Kruskall–Wallis test, followed by a Conover–Iman test was conducted for each biological assay, comparing the developmental stage or environment tested (intestine and water) between each shrimp, with a 95% significance level (p < 0.05). PCA based on Euclidean distances was used to better explain the analysis of the four sets of biotic data referred to the number of copies per gram of sample of 16S rDNA and 16S rRNA both from total Bacteria and Vibrio derived from the quantifications by qPCR/RT-qPCR in water and intestine samples. Moreover, to correlate the influence of the abiotic variables such as NO2–N, NH3–NH4, NO3–N, P–PO4, DO, salinity, pH, and temperature, with the set of biotic data from the aqueous environment, a PCA was also performed. Differences in gene copy abundance and physical–chemical parameters were evaluated using ANOVA followed by Tukey’s honestly significant difference (HSD) tests.
Source: Ecology - nature.com
