Results
BMAA and its isomers in the field phytoplankton and zooplankton. The observed results for the field phytoplankton samples showed that diatoms dominated the phytoplankton community in Jiaozhou Bay, China (Supplementary Fig. 2). The dominant species in phytoplankton and zooplankton samples in different seasons are listed in Supplementary Table 1. Concentrations of total soluble BMAA in these samples were determined by LC-MS/MS (Fig. 2, Supplementary Table 2). Results showed that concentrations of BMAA varied in plankton samples in different seasons and were basically higher in zooplankton than phytoplankton except for January. Interestingly, BMAA concentrations in phytoplankton were higher than that in zooplankton in some sampling stations in January. The highest concentration of BMAA in all seasons occurred in the zooplankton at station C3 in January andOikopleura dioica dominated the zooplankton community at this station. Average concentration of BMAA in phytoplankton samples collected in January (~0.47 μg g-1 DW) was significantly higher than that in other seasons (March ~0.03 μg g-1 DW, June ~0.02 μg g-1 DW, and September ~0.05 μg g-1 DW). However, average concentrations of BMAA in zooplankton were similar in samples collected in January (~0.44 μg g-1 DW) and June (~0.43 μg g-1 DW), which was followed by March (~0.11 μg g-1 DW) and September (~0.23 μg g-1 DW) (Supplementary Fig. 3).
An isomer of BMAA, BAMA, was widely detected in the field plankton samples collected in the four seasons with a high detection rate 97.9% (47/48). All samples were analyzed early by ZIC-HILIC column, then both BMAA and BAMA were separated and confirmed by C18 column using AQC-derivatization method (Supplementary Fig. 1). Another isomer of BMAA, DAB, was also commonly detected in plankton samples in this study, and the detection rates of DAB in phytoplankton and zooplankton were 83.3% (40/48) and 75% (36/48), respectively. Concentrations of DAB in phytoplankton and zooplankton samples varied in the ranges of 0.01 ~ 0.48 μg g-1 DW and 0.10 ~ 5.73 μg g-1 DW, respectively, of which the average concentration of DAB in zooplankton (0.43 μg g-1 DW) was higher than that in phytoplankton (0.05 μg g-1 DW). Comparing the detection rates of BAMA, DAB, and BMAA in plankton samples showed that BAMA is more universal than other compounds, in which the detection rates ranked in an order of BAMA (97.9%) > DAB (83.3%) > BMAA (79.2%) and BAMA (100%) > BMAA (95.8%) > DAB (75.0%) in phytoplankton and zooplankton samples, respectively. No AEG was detected in any plankton samples.
BMAA and its isomers in mollusk and crustacea animals. Bivalve and gastropod mollusks and crustacea animals including Portunus trituberculatus , Oratosquilla oratoria and Penaeus chinensis were collected in four different seasons (Supplementary Table 3). Quantity of total soluble BMAA in these samples was determined by two different LC-MS/MS methods and characterized by wet weight (WW) (Supplementary Table 4). BMAA was commonly detected in bivalve mollusks including Atrina pectinate , Chlamys farreri ,Ruditapes philippinarum , Mytilus galloprovincialis ,Sinonovacula constricta , Crassostrea sp., Arca inflata , and Scapharca kagoshimensis . The larger individuals ofAtrina pectinata contained higher concentrations of BMAA than other bivalves, with an average BMAA concentration of 2.50 μg g-1 WW. Average concentrations of BMAA inChlamys farreri and Scapharca kagoshimensis were 1.17 and 0.94 μg g-1 WW, respectively, which were above the average levels of BMAA in bivalve mollusks (0.84 μg g-1 WW). Obviously, the range of concentrations (0.11~4.29 μg g-1 WW) varied in different gastropod mollusks including Neverita didyma ,Glossaulax reiniana , Rapana venosa , Chlorostoma rustica , Planaxis sulcatus , Abalone sp., due to different dietary habits. The highest concentration BMAA was detected in N. didyma at 4.29 μg g-1 WW. The ranges of BMAA concentrations in marine animals were shown in Fig. 3. Totally the BMAA concentrations gradually decreased in the order of gastropod, crustacea and bivalves. However, it is remarkable that the concentration of BMAA accumulated in gastropod mollusks in March because some gastropod mollusks collected in this month were in the infancy life stage.
Another isomer of BMAA, DAB, was detected in all mollusk samples, and the concentrations of DAB in bivalve and gastropod mollusks varied in the ranges of 0.16 ~ 1.13 μg g-1 WW and 0.06 ~ 1.45 μg g-1 WW, respectively. The detection rate of DAB was 89.5% (17/19) in arthropods with concentration ranging from 0.02 to 0.99 μg g-1WW. The detection rates of BMAA and its isomers BAMA and DAB in mollusks were 100%, while in arthropods, BMAA (94.7%) > DAB (89.5%) > BAMA (84.2%). No AEG was detected in any animal samples.
BMAA and its isomers inthe cultures of isolated diatom strains. A total of 56 strains of diatoms attaching to five genera including Pseudo-nitzschia ,Chaetoceros , Thalassiosira , Planktoniella andMinidiscus , were cultured and analyzed for total soluble BMAA and precipitated bound BMAA. Concentrations of BMAA in diatom strains are shown in Supplementary Table 5. The neurotoxin BMAA detected in diatoms was mainly in the form of precipitated-bound (19/21), and only 5/21 strains contained total soluble BMAA, in which only Thalassiosira minima ,Thalassiosira allenii and Thalassiosira gravida contained both forms of BMAA. The concentrations of BMAA decreased in order of four genera including Thalassiosira , Pseudo-nitzschia ,Chaetoceros , and Planktoniella (Fig. 4).
The detection rate of total soluble DAB was 83.9% (47/56) in the 56 strains of diatom cultures with the concentration ranging from 0.24 to 407.0 μg g-1 DW, while the detection rate of precipitated bound DAB was 44.6% (25/56) with concentration ranging from 0.02 to 13.42 μg g-1 DW (Supplementary Table 5). Among the 56 cultures of diatom strains, Pseudo-nitzschia americana had the highest DAB production ability of 407.0 μg g-1 DW. Comparing the average concentrations of DAB in diatoms, these diatoms ranked in an order of Pseudo-nitzschia(37.4 μg g-1 DW) > Chaetoceros(8.96 μg g-1 DW) > Thalassiosira(5.75 μg g-1 DW) > Minidiscua(0.68 μg g-1 DW) > Planktoniella(0.27 μg g-1 DW). No AEG was detected in any diatom cultures.
Only DAB detected in symbiotic bacteria isolated from the gut ofN. didyma. A total of 7 strains of symbiotic bacteria were isolated and identified from the gut of N. didyma in the laboratory. The biological phylogenetic tree of these bacteria is shown in Fig. 5. Analytical results showed that these symbiotic bacteria did not produce BMAA or BAMA, under these experimental conditions, but DAB was detected in all strains. Interestingly there are also some unknown compounds suspected as analogues of DAB because they have the same product ions but different retention times.