Materials and Methods
Chemicals. The reference materials L -BMAA hydrochloride (B107, 10 mg) and DL -2,4-diaminobutyric acid dihydrochloride (DAB, D3758, 1 g) were purchased from Sigma-Aldrich (Oakville, ON, Canada), and N -2(aminoethyl) glycine (AEG, A608975, 1 g) standard was purchased from Toronto Research Chemicals Inc. (Canada). The reagents methanol, acetonitrile and formic acid (FA) were HPLC grade (Merck, Germany). Ammonium formate (NH4COOH) and trichloroacetic acid (TCA) were purchased from Sigma-Aldrich. AccQ-Fluor Reagent Kit was purchased from Waters (Taunton, MA, USA). The pure water was obtained by a MilliQ water purification system (Millipore Ltd., SAS. Mosheim, France) to 18.2 MΩ cm resistivity or better.
Field sample collection. Samples of diatom and zooplankton were collected using HydroBios net from bottom to surface at 12 sampling positions (Fig. 1) in Jiaozhou Bay in January, March, June, and September 2019. Zooplankton samples were retained by a shallow water type Ⅰ plankton net (505-μm mesh, 50-cm diameter, and net length 145 cm), and phytoplankton samples were concentrated by a shallow water type Ⅲ plankton net (20-μm mesh, 37-cm diameter, and net length 140 cm) and filtered by a 200-μm sieve to remove larger particles. A portion of plankton sub-samples from each station were spiked with a final concentration of 5% formaldehyde solution and stored for species identification. A phytoplankton sample (0.25 mL) was transferred to a Palmer-Maloney counting box and observed using light microscopy (BX53, Olympus) for species identification and cell density counting. The Leica S8APO stereo microscope was used to observe and count zooplankton species, and all species presenting in the samples were identified and counted. During the counting process, according to the density of zooplankton in the original sample, a Folsom’s Plankton Sample Divider was used (generally, zooplankton is divided into specific levels in 1/4, 1/16, 1/64, and 1/256), to ensure that the number of zooplanktons in each sample was above 200. Meanwhile, aquatic animals of mollusks and crustacea were purchased from fishermen or local seafood markets, all of which are important seafood products for daily consumption by residents.
Culture of isolated diatom strains. A total of 56 strains of diatoms isolated from Chinese coasts and provided by the diatom collection of South China Normal University were cultured in our laboratory using f/2 medium. All cultures were kept at 20℃ with a cycle of 12-h light: 12-h dark, at a light intensity of 6000 lx, which were usually lightly shaken three times daily. In the first batch culture, all 56 strains were cultured and scanned for BMAA. In the second batch culture, 11 of 21 strains containing BMAA were cultured in triplicate and collected at the stationary growth phase for the second analysis of BMAA and its isomers.
Culture and identification of symbiotic bacteria. The guts of healthy gastropods Neverita didyma (n> 10) were dissected and homogenized by a tissue homogenizer. The gut debris was diluted using different gradients of normal saline. The spread plate method was used to isolate and culture microorganisms. Part of 100 μL of gut solution were evenly spread on the surface of beef extract peptone medium and 2216E medium, respectively, and subsequently transferred to a biochemical incubator at 26 ℃ in order to culture bacterial colonies. Finally, the bacterial colonies were purified using a suitable gradient medium. Monoclonal colonies with different characteristics were picked by streak plate method. A total of 9 strains were isolated and cultured for toxin analysis. The isolated strains were stored in a refrigerator at 4 ℃ for other experiments. Molecular 16S rDNA analysis was adopted to identify the DAB-producing bacteria. The 16S rDNA was amplified by PCR with general primers (27F and 1492R), which were sent directly to BGI (Beijing Genomics Institute) for sequencing. For identification of bacteria, DNA sequences were analyzed by the Basic Local Alignment Search Tool at the NCBI (National Center for Biotechnology Information) website. Finally, two bacterial strains were identified as the same species, and one strain was not successfully sequenced.
Pretreatment for biological samples . Field plankton samples were filtered by a 2.7 μm glass fiber membrane. Mollusks and crustacea samples were dissected by a scalpel and all soft tissues were homogenized for toxin extraction. All laboratory cultures of diatom strains were collected by centrifugation at 6577 × g when they entered the stationary growth phase before toxin extraction. All above samples were stored at -20 ℃ immediately after processing.
Extraction for the total soluble form of BMAA . The membranes containing plankton samples were removed from the refrigerator and allowed to reach room temperature. The samples were then placed in an oven at 55 ℃ until their weight did not change and the dry weight of samples was calculated through subtracting the average weight of blank membranes. Subsequently, the membranes were completely shredded and transferred into a 10 mL centrifuge tube. Five mL of 0.1 mol L-1 trichloroacetic acid (TCA) were added to the tube. The mixture was then frozen in liquid nitrogen for 10 min and thawed at room temperature, which was repeated three times. The thawed samples were ultrasonically broken for 20 min with an ice-water bath. Finally, 2 mL of the supernatant was removed by centrifugation and dried under N2 gas at 55 °C. The residual materials were re-dissolved in 6 mol L-1 HCl and hydrolyzed at 110 °C for 24 h to get the soluble-bound form of BMAA. The hydrolyzed solution was dried again and reconstituted with 20 mmol L-1HCl. Prior to analysis for BMAA using HILIC-MS/MS, the extract was purified using solid-phase extraction (SPE)56 with minor modifications. The Oasis-MCX SPE cartridges (3 cc, 60 mg) were activated by 3 mL of methanol and 3 mL of 5% NH4OH were used to elute toxins.
Cultures of the isolated diatom strains were lyophilized in a vacuum freeze dryer for 24 h and then transferred to a 10 mL centrifuge tube. The toxin extraction steps followed the same procedure as that used for the field plankton samples described above.
Mollusk or crustacea tissues (0.5 g) were mixed with 3 mL of 0.1 mol L-1 TCA and homogenized by a tissue homogenizer. Supernatant was transferred to a 10 mL volumetric flask after centrifugation at 6577 ×g for 10 min. The above extraction steps were repeated three times. The combined supernatant was finally made up to 10 mL. An aliquot of the supernatant (1 mL) was dried under N2 gas at 55 °C and the residual material was re-dissolved in 6 mol L-1 HCl and hydrolyzed at 110 °C for 24 h. The hydrolysis solution was dried again and reconstituted in 20 mmol L-1 HCl before purification using the above SPE treatment.
Extraction for the precipitated bound BMAA . The precipitated bound BMAA was also analyzed in the cultures of isolated diatom strains. In the process for extracting soluble BMAA, the microalgal deposit was mixed with 3 mL of 6 mol L-1 HCl, vortexed and transferred to a 4 mL glass bottle. The mixture was hydrolyzed at 110 °C for 24 h and then transferred to a 10 mL centrifuge tube. The glass vial was rinsed with 1 mL of 6 mol L-1 HCl and then combined into the 10 mL centrifuge tube follow by centrifugation at 6577 ×g for 10 min. Two mL of supernatant were dried under N2 gas at 55 °C and the residue was reconstructed in 1 mL of 20 mmol L-1 HCl before purification using SPE treatment described above.
HILIC–MS/MS analysis. Analysis for BMAA and its isomers was conducted on a Thermo Ultimate 3000 HPLC (Thermo Fisher Scientific, Bremen, Germany) coupled with an AB-Sciex Qtrap 4500 mass spectrometer (AB Sciex Pte. Ltd, Singapore) with an electrospray ionization source. A binary mobile phase system composed of water (solvent A) and acetonitrile (solvent B) each with 0.1% formic acid was used to separate chemical targets on a SeQuant® ZIC-HILIC® column (150 mm × 2.1 mm, 5 µm) maintained at 30 ℃. The gradient was linear from 95% to 60% solvent B over 19 min and then decreased to 40% B at 25 min, and increased to 95% B at 27.01 min, held for 2.99 min before re-equilibration. The flow rate was set at 350 µL min-1 and injection volume was set at 5 µL. The electro-spray voltage was set to 5500 V with a source temperature of 350 ℃. Nitrogen was used for the nebulizer and curtain gases. The selective reaction monitoring (SRM) mode was used to quantify BMAA and its isomers. Five transitions m/z 119 -> 102, 101, 88, 56, and 44 (CE: 13, 11, 17, 24, and 24 eV) were used for BMAA and its isomers. BMAA and DAB was quantified by the m/z 119 -> 88 and 119 -> 101, respectively, and 119 -> 102 was used to semi-quantify BAMA.
Statistical analysis. Trophic magnification factor (TMF) was calculated by the ratio of BMAA concentrations in organisms within different trophic levels to the average concentration of BMAA in phytoplankton samples. The average concentration of BMAA in phytoplankton as well as zooplankton was taken from the average value of the total concentration of BMAA in biological samples from 12 sampling stations. The average concentrations of BMAA in bivalve and gastropod mollusks and crustacea animals were taken from the average concentrations of BMAA in different species of animals within each trophic level. Data processing was performed using the Microsoft Excel 2016 software (Microsoft Corporation, Redmond, USA). Figures of data and sampling station map were performed using the Origin 2018 (OriginLab Corporation, Northampton, USA) and ArcGIS Desktop 10.2 software (ESRI Corporation, Redlands, CA), respectively.