The M. aeruginosa strain used in this study was isolated from shrimp breeding ponds with cyanobacteria blooms in Guangdong province, China, and stored in the South China Sea Fisheries Research Institute (SCSFRI). Cyanobacteria were cultured in BG11 medium at pH 8.0 and 28 ˚C, with illumination at a proton flux density of 1000 lux under 12 h light/12 h dark cycles.
Healthy L. vannamei, with a mean body weight of 3.5 ± 0.5 g, were obtained from shrimp ponds of the Shenzhen Experimental Station of the SCSFRI, Shenzhen, China. The shrimp were acclimated in tanks containing aerated brackish water which was filtrated with sand (salinity 4, pH 8.2, dissolved oxygen 5.5-6.5 mg/L) at 28 ± 1 ˚Cfor one week prior to the experiment. During the acclimation period, one-half of the water in each tank was replaced daily, and the shrimp were fed with specific shrimp feed three times daily. Only apparently healthy shrimp were used for the study.
Preparation of the fractions
Algae cells and cultured medium were separated by centrifugation (10 min, 4000 ×g), then the cell-free medium was collected, while the cells were rinsed 3 times with phosphate buffer solution (PBS) and dissolved in PBS (cell density 1 ×108 cells/mL). Cell sample was subjected to ultrasonication, with 60% pulse and 30% power, at 0-4 ℃ for 20 min. After ultrasonication, the supernatant (crude extract) was collected by centrifugation (10 min, 8000×g). Finally, both the medium and cell extract were passed through a 0.22-µm pore filter for subsequent experiments. The samples were stored at 4 ˚C before the toxicity test.
The remaining aliquot of M. aeruginosa cells, weighing 0.54 g, was diluted to 10 mL with 70% methanol, homogenized for one min, and then subjected to ultrasonication as above. Cell breakage was confirmed by microscopic observation, and the supernatant was collected by centrifugation (10 min, 8000× g), and then loaded into a C18 cartridge that had been previously activated with 100% methanol and deionized water. The cartridge was rinsed with 3 mL of 20% methanol and eluted with 3 mL of 100% methanol, and the eluates were dried by using a Zymark LV TurboVap® evaporator (Biotage, Charlotte NC) at 60 ˚C, with N2. The samples were reconstituted with 5 mL of deionized water and filtered with 0.45-µm PVDF membranes prior to analysis.
Acute toxicity assays in L. vannamei
A total of 360 L. vannamei were randomly divided into four groups, including one control group and three treatment groups, and every group had 90 shrimps in triplicates. The shrimps were kept in 400-L tanks at 28 ± 1 ˚C, pH 8.2, at a concentration of dissolved oxygen of 5.5-6.5 mg/L. Three treatment groups of shrimps were injected intramuscularly with 50 µL of cultured medium, M. aeruginosa cells, and cell extract (cell density 1 ×108 cells/mL), respectively, while control shrimps were injected with 50 µL of PBS. The intra-muscular injection into the shrimp was performed with an insulin syringe in the lateral region of the animal’s body between the second and third abdominal segment and the collection of shrimp. During the trial, the shrimps were fed three times daily, and uneaten food was removed one hour later, while water was replaced daily. The shrimps were observed in situ, and the death rate was recorded.
HPLC analysis of microcystins
Microcystins in cyanobacterial extract were analyzed by HPLC with diode-array detection (HPLC-DAD). Specifically, we used an Agilent 1100 Series HPLC system with Supelcosil ABZ + Plus 150 mm × 4.6 mm 5-µm columns (Supelco) and a mobile phase consisting of (A) 0.1% (v/v) TFA, (B) acetonitrile with 0.1% (v/v) TFA (gradient elution: 20-59% B in 0-30 min), at a flow rate of 1 mL/min, a temperature of 30 ◦C, with an injection volume of 25 µL. UV-spectra between 200 and 300 nm were collected and chromatograms evaluated at 238 nm. Identification of the MCs peaks was based on the comparison of retention times and UV spectra with the standard MCs. The concentration of MCs in extract was calculated by the standard curve equations, and the standard curves were draw according to the MC-RR/YR/LR standards (USA, Sigma) dissolved in methanol, the concentration of which were 1, 0.5, 0.2, 0.1 and 0.05 µg/mL, respectively (Vesna et al., 2007).
Effect of M. aeruginosa extract on the immune response of L. vannamei
A total of 750 L. vannamei were randomly divided into three groups, including two control groups and one treatment group, and each group had 250 shrimps in 5 parallels. L. vannamei was treated with 50 µL of a median lethal concentration of extract (cell density was 1×107 cells/mL, based on the results of preliminary experiments) in treatment group，50 µL of MC-LR (the concentration was 1.69 µg/mL) in positive control group and 50 µL of PBS in negative control group, respectively. At 0, 8, 24, 48, 72, 96, and 120 h post-injection, 10 shrimps were dissected. The hepatopancreas of each shrimp was used for further assay, and a part of organs were used for analysis of enzymes activity, while the rest were stored in RNAlater (Life technology, USA) for gene transcription analysis. All samples were immediately stored at -80 ˚C until use.
Immune-related enzyme activity assay
The activity of SOD, GSH, and LZM was measured according to the manufacturer’s instructions. Briefly, hepatopancreas samples stored at -80 °C were weighed and homogenized for 30 s in phosphate buffer (0.125 M, pH 7.2, containing 0.05 M Na2EDTA, 2-4 ◦C). After centrifugation at 3000 g for 10 min at 4◦C, the supernatant was separated and analyzed by protein assay. The enzyme activity and protein assay were all determined using a relevant Detection Kit (Jian Cheng Bioengineering Ltd., Nanjing, China). SOD assay was based on the ability of SOD to inhibit WST-1 auto-reduction by superoxide anion (O2-). SOD activity was monitored by measuring the absorbance at 450 nm and expressed as unit/mg protein (U/mg protein). The GSH content was evaluated by incubation with 6 mM 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB), and measurement of the absorbance at 310 nm. Lysozyme (LZM) assay was based on the ability of LZM to lyse the Micrococcus lysodeikticus cells. During incubation of the lysozyme sample with the substrate, the reaction was followed by monitoring the decrease of the absorbance at 530 nm.
RNA extraction, reverse transcription and quantitative real-time PCR (qPCR)
Total RNA was extracted from hepatopancreas using a RNeasy Plus Mini kit (Qiagen, Germany). RNA quantity, purity, and integrity were evaluated by spectrophotometry (A260/A280) and electrophoresis on 1% agarose gels. cDNA was synthesized from 2 µg of total RNA by using the Prime-ScriptTM Real-time PCR Kit (TaKaRa, Dalian, China). The expression of target genes (sod, gst, lzm) and of the internal control gene (β-actin) was measured by qPCR. Primer premier 5.0 was used to design the fluorescent primers before gene synthesis performed (PREMIER Biosoft International, Palo Alto, CA, USA) based on the published L. vannamei mRNA sequences (Table 1), and each primer pair was designed and tested for specificity. Primer efficiency was determined by performing serial dilutions of reference cDNA. Quantitative real-time PCR (qPCR) was carried out with a LightCycler480 384-well Real-Time PCR System (Roche, Switzerland). The amplification was performed in a total volume of 10 µL, containing 5 mL of SYBR Premix Ex Taq II (Takara), 1 µL of the diluted cDNA, 0.5 µL of each primer, and 3µL ddH2O. The qPCR program was 95 ˚C for 30 s, followed by 40 cycles of 95 ˚C for 5 s and 60 ˚Cfor 30 s. The relative quantification of transcripts was performed by the 2-ΔΔCT method (Livak and Schmittgen, 2001).
Mortality rate was calculated as: [number of deaths /total number of shrimp)] ×100
All data presented are means of three independent sets of experiments ± standard error (SE). Significant differences between control and exposed shrimp were determined using a one-way ANOVA followed by multiple comparison via Dunnett’s test. p values < 0.05 were considered to be statistically significant. Statistical computations were performed with SPSS 13.0 for Windows (SPSS Inc.).