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.