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Saxitoxin Puffer Fish Poisoning in the United States, with the First Report of Pyrodinium bahamense as the Putative Toxin Source

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From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002. We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL. We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 microg STX equivalents (eq)/100 g tissue (action level, 80 microg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida's marine waters and implicate the dinoflagellate P. bahamense as the putative toxin source. Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused by STXs in shellfish.
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1502
VOLUME 114 | NUMBER 10 | October 2006
Environmental Health Perspectives
Research
Puffer fish poisoning (PFP) is usually caused
by ingestion of tetrodotoxins (TTXs) found
naturally in certain species of puffer fish
(Halstead 1967; Mosher and Fuhrmann
1984). In Japan, 20–100 people die annually
from PFP, in spite of stringent controls by
authorities (Ogura 1971). TTXs can cause
fatal human poisoning, which is similar to
paralytic shellfish poisoning (PSP) caused by
saxitoxins (STXs). PSP is caused by the con-
sumption of toxic shellfish (Shumway 1990)
and rarely by fish that have have become
toxic after feeding on STX-producing
microalgae (Maclean 1979). As well as
TTXs, STXs have also been found in at least
12 marine and freshwater puffer fish species
in Asia (Ahmed et al. 2001; Kodama et al.
1983; Kungsuwan et al. 1997; Nakamura
et al. 1984; Nakashima et al. 2004; Sato
et al. 1997, 2000; Zaman et al. 1997), but
their bioorigin has not been identified.
TTXs are chemically distinct from STXs,
but both neurotoxins produce similar symp-
toms in mammals because they act on site 1
of the voltage-dependent sodium channel,
blocking the influx of sodium into excitable
cells and restricting signal transmission along
nerve and muscle membranes (Ahmed
1991). The symptoms of traditional PFP
from TTXs and of PSP from STXs include
tingling and numbness of the mouth, lips,
tongue, face, and fingers; paralysis of the
extremities; nausea; vomiting; ataxia; drowsi-
ness; difficulty in speaking; progressively
decreasing ventilatory efficiency; and finally
in extreme cases, death by asphyxiation
caused by respiratory paralysis (Ahmed 1991;
Catterall 1985; Kao 1993).
PFP cases in Europe (Kao 1993) and
Mexico (Nuñez-Vazquez et al. 2000) have
occasionally been reported. In the United
States, PFP has been associated with imports
of puffer fish [Centers for Disease Control
and Prevention (CDC) 1996]; rarely have
fatalities occurred after the consumption of
indigenous puffer fish. In Hawaii, white-spot-
ted puffer fish, Arothron hispidus, were impli-
cated in seven deaths (Ahmed 1991). Until
1974, seven PFP cases in Florida, outside of
the Indian River Lagoon (IRL), were caused
by the consumption of locally caught “blow-
fish” or puffer fish (Ahmed 1991; Benson
1956; Bigler 1999; Hemmert 1974; Mosher
and Fuhrmann 1984). These cases included
three fatalities, likely from TTX; for example,
one woman died 45 min after consuming
toxic liver from a checkered puffer fish
(Sphoeroides testudineus) (Benson 1956). The
toxins involved in the previous Florida PFP
cases were not characterized, but because PFP
is usually associated with TTX, investigators
likely assumed that TTX was the cause
(Benson 1956; Bigler 1999; Hemmert 1974).
Address correspondence to J.H. Landsberg, Fish and
Wildlife Research Institute, Florida Fish and
Wildlife Conservation Commission, 100 Eighth
Ave. SE, St. Petersburg, FL 33701 USA. Telephone:
(727) 896-8626. Fax: (727) 893-9840. E-mail:
jan.landsberg@myfwc.com
We thank L. Sebastian, R. Paperno, D. Adams,
D. Tremain, S. Fisk, S. Stahl, S. Cook, J. D’Urso, and
A. Shurtleff, Florida Fish and Wildlife Conservation
Commission (FWC), for technical assistance and
D. Bodager and G. Jackow, Florida Department of
Health (FDOH), for specimen collection.
Funding or support for this research was provided
by the FWC, FDOH, U.S. Food and Drug
Administration, Centers for Disease Control and
Prevention, and the National Oceanic and
Atmospheric Administration (NOAA). This article is
a result of research partially funded by the NOAA
Coastal Ocean ECOHAB Program under award
#NA03NOS4780196 to the FWC (ECOHAB con-
tribution #152).
The authors declare they have no competing
financial interests.
Received 11 January 2006; accepted 5 July 2006.
Saxitoxin Puffer Fish Poisoning in the United States, with the First Report
of
Pyrodinium bahamense
as the Putative Toxin Source
Jan H. Landsberg,
1
Sherwood Hall,
2
Jan N. Johannessen,
3
Kevin D. White,
4
Stephen M. Conrad,
2
Jay P. Abbott,
1
Leanne J. Flewelling,
1
R. William Richardson,
1
Robert W. Dickey,
5
Edward L.E. Jester,
5
Stacey M. Etheridge,
2
Jonathan R. Deeds,
2
Frances M. Van Dolah,
6
Tod A. Leighfield,
6
Yinglin Zou,
7
Clarke G. Beaudry,
4
Ronald A. Benner,
2
Patricia L. Rogers,
2
Paula S. Scott,
1
Kenji Kawabata,
1
Jennifer L. Wolny,
1,8
and
Karen A. Steidinger
1,8
1
Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, St. Petersburg, Florida, USA;
2
Food and Drug
Administration, Center for Food Safety and Applied Nutrition, Laurel, Maryland, USA;
3
Food and Drug Administration, Office of the
Commissioner, Rockville, Maryland, USA;
4
Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park,
Maryland, USA;
5
Food and Drug Administration, Center for Food Safety and Applied Nutrition, Gulf Coast Seafood Laboratory, Dauphin
Island, Alabama, USA;
6
National Oceanic and Atmospheric Administration, National Ocean Service, Center for Coastal Environmental
Health and Biomolecular Research, Charleston, South Carolina, USA;
7
Key Laboratory of Science and Engineering for Marine Ecology
and Environment, First Institute of Oceanography, State Oceanic Administration, Qingdao, China;
8
Florida Institute of Oceanography,
University of South Florida, St. Petersburg, Florida, USA
BACKGROUND: From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida,
New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida.
Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP
case in 2002.
M
ETHODS: We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse
neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liq-
uid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl
STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural
bloom samples of Pyrodinium bahamense from the IRL.
R
ESULTS: We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides
testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we
detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 µg STX equivalents
(eq)/100 g tissue (action level, 80 µg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cul-
tures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA,
MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by
HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic
compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous
reservoir of STXs in Florida’s marine waters and implicate the dinoflagellate P. bahamense as the
putative toxin source.
C
ONCLUSIONS: Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not
known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We
propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to
distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused
by STXs in shellfish.
K
EY WORDS: dinoflagellate, Florida, harmful algae, puffer fish, Pyrodinium bahamense, saxitoxin
puffer fish poisoning, saxitoxins, Sphoeroides spp. Environ Health Perspect 114:1502–1507 (2006).
doi:10.1289/ehp.8998 available via http://dx.doi.org/ [Online 6 July 2006]
Tissues from Florida bandtail (Sphoeroides
spengleri), checkered, and southern puffer fish
(Sphoeroides nephelus) were found to be lethal
in the mouse bioassay (MBA) (Burklew and
Morton 1971; Lalone et al. 1963), but, again,
the toxins were not determined.
Until January 2002 the harvest and con-
sumption of puffer fish from the IRL was not
a risk to public health. Since then (until May
2004), however, 28 PFP cases occurring in
Florida (n = 21), New Jersey (n = 3), Virginia
(n = 2), and New York (n = 2) caused by
puffer fish originating from the IRL were
reported (Bodager 2002; CDC 2002a,
2002b). Analyses of toxins from unidentified
puffer fish fillet remnants from one of the
early 2002 PFP cases in New Jersey revealed
STXs (Quilliam et al. 2004), not TTXs, a dis-
tinction that alone could not be made on the
basis of consumer symptoms or traditional
screening methods (i.e., MBA).
During 2002–2004, all PFP cases were
linked to puffer fish originating from the
northern IRL and the Banana River on
Florida’s east coast (Figure 1). Except for one
case, where puffer fish were commercially har-
vested and reached a New Jersey fish market,
puffer fish were caught recreationally [Bodager
2002; Florida Fish and Wildlife Conservation
Commission (FWC) 2004]. In April 2002,
state and federal officials issued health advi-
sories, and the FWC banned puffer fish har-
vesting in the IRL, a ban that currently
remains in effect. In New York on 14 October
2002, two PFP cases were caused from fish
caught near Titusville, Florida, but frozen in
March 2002 before the harvesting ban
(Bodager D, personal communication). This
case demonstrated the stability of toxins in
puffer fish frozen for almost 9 months.
Because STXs had not previously been
identified in Florida’s marine waters and their
distribution, source, and origin were unknown
in April 2002 (Abbott et al. 2003; Landsberg
et al. 2002), we initiated an intensive survey of
biota in the IRL. In this article we present a
summary from 3 years of monitoring, as well
as the first report of the putative toxin source.
Materials and Methods
Field collections. From April 2002 through
April 2005, southern, checkered, and bandtail
puffer fish (n = 516) were harvested via a range
of fishing gear from the original source loca-
tions of the PFP incidents in the northern and
central IRL (Figure 1). The fish were shipped
biweekly or monthly on ice to the FWC’s Fish
and Wildlife Research Institute (FWRI) or to
the Food and Drug Administration’s (FDA)
Center for Food Safety and Applied Nutrition
Washington Seafood Laboratory and frozen in
individual sealable plastic bags until required
for toxicity testing.
Live phytoplankton samples were collected
routinely with a 62-µm mesh plankton net at
multiple locations along the IRL; also, a 1-L
water bottle was used to directly sample a
phytoplankton bloom. Water samples were
transported to FWRI at ambient temperature.
Live puffer fish. To determine if puffer fish
maintained toxicity once they were removed
from the putative toxin source, we kept puffer
fish in captivity. We obtained southern puffer
fish by rod and line or by seine net from the
IRL near Titusville (Atlantic coast) (n = 2) and
from Tampa Bay (Gulf coast), Florida (n = 2),
and transported them live in ambient seawater
to the wet laboratory at FWRI. Southern
puffer fish were individually held in covered,
80-L aquaria in 25 psu (practical salinity
units) artificial sea water (Instant Ocean;
Aquarium Systems, Inc., Mentor, OH) and
fed shrimp or squid that originated from non-
toxic locations. We measured water quality
daily and routinely carried out 30% water
exchanges. After several weeks acclimation,
we tested fish skin mucus bimonthly by
lightly anesthetizing the fish [100 ppm tri-
caine methanesulfonate (MS-222; Sandoz
Pharmaceuticals Corp., Basel, Switzerland) in
4 L], placing the fish on a dissection board,
and collecting the mucus on a preweighed
47-mm–diameter, glass-fiber filter (Whatman,
Clifton, NJ) by gently rubbing the paper
along both sides of the body.
Fish care. We conducted research in com-
pliance with the Animal Welfare Act and
other federal statutes and regulations relating
to animals and experiments involving ani-
mals. All fish were treated humanely and with
regard for alleviation of suffering, according
to the Guide for Care and Use of Laboratory
Animals (Institute of Laboratory Animal
Resources 1996).
Preparation of tissues. Within several weeks
of collection, we thawed frozen puffer fish,
measured standard lengths and wet weights,
and removed skin, liver, stomach, intestinal
tract, muscle, and gonads.
Pyrodinium bahamense cultures. We
established 11 clonal nonaxenic cultures of
P. bahamense from IRL samples, using the
micropipette technique to isolate single cells.
We maintained batch cultures in environ-
mental chambers at near-ambient light and
temperature conditions (35 microEinsteins/
m
2
/sec, 25°C) and at salinities of 20–36 psu.
Growth media consisted of filtered, auto-
claved natural offshore seawater enriched to
ES-DK (enriched natural seawater medium
modified by D. Kulis) (Kulis D, personal
communication; Kokinos and Anderson
1995) levels with the addition of 10
-7
M sele-
nium (as sodium selenite).
Toxin detection. At various stages of
this survey, we tested puffer fish tissues for
STX bioactivity using the standard MBA,
Ridascreen ELISA (R-Biopharm GmBH,
Darmstadt, Germany), MIST Alert (Jellet
Biotek, Dartmouth, Canada) PSP kit, and
mouse neuroblastoma cytotoxicity assay
(MNCA, Neuro-2A) and receptor-binding
assay (RBA) [Association of Official Analytical
Chemists (AOAC) 1990; Cembella et al.
2003; Jellett et al. 2002; Luckas et al. 2003;
Powell and Doucette 1999; Ruberu et al.
2003; Usleber et al. 1991]. We also prepared
selected samples for toxin characterization and
confirmation by HPLC (Thermo Electron
Corporation, San Jose, CA), with postcolumn
oxidation and fluorescence detection and liq-
uid chromatography-mass spectrometry
(LC-MS) (Waters Corporation, Milford, MA)
(Negri et al. 2003; Oshima 1995) using in-
house FDA reference standards. We split tissue
samples for interlaboratory calibrations and
then extracted them by one of two methods.
For bioactivity assays, tissue samples were
homogenized and weighed (wet weight) into
glass test tubes. Samples were extracted using
0.1 N HCl, adjusted to pH 2.5–4, boiled for
5 min in a boiling water bath, centrifuged at
3,000 × g for 10 min, and the supernatant
retained for toxin testing. For toxin characteri-
zation by HPLC and LC-MS, tissue splits
were extracted with 0.1 M aqueous acetic acid,
centrifuged, and the supernatants filtered
(0.22 µm).
After the initial 2002 saxitoxin puffer fish
poisoning (SPFP) events, 11 southern puffer
fish were divided into the tissue compartments
(listed above), and tissue samples were extracted
by boiling in 0.1 N HCl (Washington Seafood
Laboratory) and analyzed for toxic activity
using three independent methods. MBAs
were performed at the Washington Seafood
Laboratory; MNCAs were performed at the
FDA Gulf Coast Seafood Laboratory; and
RBAs were performed at the National
Oceanic and Atmospheric Administration
National Ocean Service Center for Coastal
Environmental Health and Biomolecular
Research.
Saxitoxins in Pyrodinium bahamense in the United States
Environmental Health Perspectives
VOLUME 114 | NUMBER 10 | October 2006
1503
SEMINOLE
ORANGE
OSCEOLA
Atlantic
Ocean
Atlantic
Ocean
Gulf of
Mexico
Area
shown
Pineda
Causeway
Cocoa
B
REVARD
N
10 0 10 miles
Titusville
Figure 1. Map showing locations (circles) in the
Indian River Lagoon, Florida, where toxic puffer
fish in the SPFP incidents originated (FWC 2004).
Sample collections of puffer fish and
Pyrodinium
bahamense
were conducted throughout this area
and further south to the St. Lucie River (not shown).
Clonal cultures and natural bloom sam-
ples of P. bahamense were filtered onto
25-mm glass-fiber filters (Whatman) or cen-
trifuged at 3,000 × g for 5 min and then
extracted. Puffer fish mucus or Pyrodinium
samples on filters were homogenized in 0.1 N
HCl using a ground-glass tissue grinder and
treated as above. Pyrodinium extracts were
tested for toxicity by ELISA and MBA and
characterized for toxin profile using HPLC
and LC-MS. Puffer fish mucus was tested for
toxicity by ELISA.
Electron microscopy. We prepared natural
field samples or clonal cultures of P. bahamense
for the scanning electron microscope (SEM)
using standard fixation methods (Truby 1997).
Pyrodinium samples were added to unacidified
Lugol’s at a dilution of 1:100 in suspension,
collected onto a 5-µm polycarbonate filter,
secondarily fixed with 4% paraformaldehyde
for 20 min, washed with water, dehydrated in
an ethanol series followed by a freon series,
critical-point-dried using carbon dioxide,
mounted onto aluminum stubs using carbon
adhesive tape, sputter-coated with gold/palla-
dium, and photographed with a Cambridge
Stereoscan 240 SEM (Cambridge Instruments,
Cambridge, UK).
Results
Puffer fish toxin analyses. During 36 months
of continuous monitoring after the initial
SPFP events, STXs were routinely detected in
the skin, muscle, viscera, and gonads of
516 puffer fish (southern, n = 402; checkered,
n = 105; bandtail, n = 9), which tested toxic in
MBA, RBA, Ridascreen ELISA, and MIST
Alert assays. By ELISA, maximum STX levels
in the muscle fillet were well above the action
level [80 µg STX equivalents (eq)/100 g tissue]
in southern puffer fish (maximum, 14,571 µg
STX eq/100 g tissue, mean = 938.4) and just
over the action limit in bandtail (maximum,
364.5 µg STX eq/100 g tissue; mean, 121.7)
and checkered puffer fish (maximum, 104.3 µg
STX eq/100 g tissue; mean, 6.9) (Table 1).
Maximum STX concentrations in the liver of
southern and checkered puffer fish were
1,443 and 51.1 µg STX eq/100 g tissue,
respectively. The highest tissue concentration,
22,104 µg STX eq/100 g tissue, was measured
in the ovaries of a southern puffer fish (data
not shown).
All three assays (MBA, MNCA, and
RBA) confirmed elevated concentrations of
toxic activity in the muscle compared to the
liver (5- to 20-fold) of 11 southern puffer fish
(Table 2). By MBA, MNCA, and RBA,
ranges of STX concentrations in muscle were
197–5,264 (mean ± SD, 2,302.3 ± 1,539.3),
120–2,294 (957.7 ± 659.5), and 198–6,091
(2,439 ± 1,995.3) µg STX eq/100 g tissue,
Landsberg et al.
1504
VOLUME 114 | NUMBER 10 | October 2006
Environmental Health Perspectives
Table 1. Comparison of saxitoxin concentrations (µg STX eq/100 g tissue) in muscle and liver of IRL puffer
fish species by ELISA.
Muscle Liver
Puffer fish species No. Mean ± SD Maximum No. Mean ± SD Maximum
Southern 402 938.4 ± 1,418 14,571 55 265.6 ± 393 1,443
Checkered 105 6.9 ± 11.4 104.3 3 20.3 ± 27.1 51.1
Bandtail 9 121.7 ± 117.9 364.5 0
Table 2. Comparison of saxitoxin-like activity levels (µg STX dihydrochloride eq/100 g tissue) by LC-MS
in muscle and liver of southern puffer fish (
S. nephelus
) collected from the IRL after the first SPFP cases
in 2002.
MBA RBA MNCA
Fish Muscle Liver Fold diff Muscle Liver Fold diff Muscle Liver Fold diff
1 5,264 1,034 5.1 4,136 711 5.8 2,294 420 5.5
2 4,697 376 12.5 6,091 304 20.0 1,230 280 4.4
3 2,986 242 12.3 2,433 280 8.7 1,947 160 12.2
4 2,804 203 13.8 1,423 147 9.7 1,100 120 9.2
5 2,564 149 17.2 5,253 297 17.7 844 110 7.7
6 2,153 135 15.9 2,911 173 16.8 790 240 3.3
7 1,970 263 7.5 2,257 142 15.9 750 150 5.0
8 1,216 254 4.8 805 154 5.2 350 140 2.5
9 1,098 221 5.0 1,089 180 6.1 480 110 4.4
10 376 83 4.5 198 16 12.4 630 70 9.0
11 197 149 1.3 231 50 4.6 120 60 2.0
Fold diff indicates fold difference of muscle compared with liver.
Figure 2. Toxin analysis of southern puffer fish muscle. HPLC chromatograms showing (
A
) dc-STX (7%) and STX (92%), and (
B
) B1 (1%). LC-MS ion chromatograms
(
C
,
E
) and mass spectra (
D
,
F
) of STX in reference standard (
C
,
D
) and Titusville puffer fish muscle (
E
,
F
).
5
4
3
2
1
0
0.20
0.15
0.10
0.05
0
0
510152025
0 5 10 15 20 25
Relative fluorescence (mV)Relative fluorescence (mV)
Time (min)
Time (min)
15.00 25.00
Time (min)
15.00 25.00
Time (min)
m/z
260 300 340 380
m/z
260 300 340 380
IntensityIntensity
Relative intensity Relative intensity
314 100
152
100
A
B
C
D
E
F
STX
dc-STX
B1
STX
standard
20.79
300
301
282
300
301
282
20.77
STX
in Titusville
puffer muscle
respectively. By MBA, MNCA, and RBA,
STX concentrations in liver were 83–1,034
(mean ± SD, 282.6 ± 261.5), 60–420 (169.1
± 106.2) and 16–711 (223.1 ± 186.5) µg
STX eq/100 g tissue, respectively (Table 2).
Skin mucus of IRL southern puffer fish
held captive for 1 year was highly toxic
(2,407–9,039 µg STX eq/100 g) compared
with that of Florida Gulf Coast southern
puffer fish (6.25–140 µg STX eq/100 g).
Over a period of at least 6 months, STX levels
in the IRL southern puffer fish fluctuated but
remained at highly toxic concentrations.
Toxin profiles in unconsumed puffer fish
fillets (n = 4) from a 2004 PFP event were con-
firmed by HPLC (Figure 2A,B) and LC-MS
(Figure 2C–F) to be STX (92.4% ± 3.1),
decarbamoyl saxitoxin (dcSTX; 6.9% ± 2.4),
and N-sulfocarbamoyl B1 toxin (B1; 0.7% ±
0.7) as originally found in a 2002 PFP case
(Quilliam et al. 2004).
We also detected TTX (quantified by MBA
and confirmed by LC-MS) in IRL checkered
puffer fish (n = 3) at concentrations of 1,553 ±
919 and 53,700 ± 19,212 µg TTX/100 g in the
muscle and liver, respectively (Figure 3A–D).
Pyrodinium bahamense toxin analyses.
All clonal cultures (n = 11) and natural bloom
samples (n = 2) (> 3 million cells/L) of
P. bahamense (Figure 4) obtained from the IRL
tested positive for STX by HPLC, LC-MS,
Ridascreen ELISA, MIST Alert, and RBA
assays. Toxin concentrations for P. bahamense
isolates (n = 11) ranged from 1.68 to 25.57 pg
STX eq/cell (as determined by ELISA).
Further analysis of five of these isolates using
HPLC determined that the toxin profile was
composed of B1 (91.1% ± 2.2, mean ± SD)
and STX (8.9% ± 2.2), with integrated toxicity
values ranging from 2.02 to 12.74 pg STX
eq/cell. The HPLC toxin profile of a 2002
bloom sample at 3.28 pg STX eq/cell was
composed of STX (26%), B1 (73%), and
dcSTX (1%) (Figure 5A,B).
Discussion
PFP cases have been associated with STXs in
Asia (Ahmed et al. 2001), but the IRL incidents
are the first in which STX poisoning has been
confirmed in puffer fish originating in the
United States (Quilliam et al. 2004). The high
and low concentrations of STXs and TTX,
respectively, in the muscle of IRL puffer fish are
similar to those found in Philippine (Sato et al.
2000) and Japanese (Kodama et al. 1984)
puffer fish, although in the latter, visceral toxic-
ity from TTX is high and fish-poisoning inci-
dents usually occur after consumption of
fillet(s) contaminated due to improper prepara-
tion. Unlike the tissue distribution of TTXs
reported previously in various puffer fish species
(Kodama et al. 1984), STXs in IRL southern
puffer fish have been consistently much higher
in the muscle than in the liver and, in many
individual fish, were more than two orders of
magnitude above the action limit. Therefore,
even careful preparation of IRL puffer fish fillets
would not prevent intoxication in consumers.
Interestingly, the confirmation of extremely
high concentrations of TTX in the liver of
checkered puffer fish suggests that the earlier-
reported fatality from the consumption of this
species in south Florida (Benson 1956) was
likely caused by this toxin and not STX.
The MBA, the traditional screening
method for PFP, does not distinguish between
STXs and TTXs. New reports in Asia (Ahmed
et al. 2001; Nakashima et al. 2004; Sato et al.
2000) have found both toxin groups co-occur-
ring in puffer fish species previously thought to
contain only TTX. Both our results and these
reports suggest that STXs in puffer fish may be
more widespread than previously thought;
therefore, comprehensive analytical assessments
of PFP incidents are needed to distinguish
TTX from STX. We propose that the food-
poisoning syndrome caused by intoxication
from STX exposure from fish should be char-
acterized as SPFP to distinguish it from PFP,
which is caused by—but not always verified to
be from—TTX, and to distinguish SPFP from
PSP associated with STXs in shellfish.
In a 1960s toxicity study of IRL southern
puffer fish [erroneously identified by Lalone
et al. (1963) as northern puffer fish, Sphoeroides
maculatus, which are not found in the IRL
and occur only as far south as Jacksonville, FL
(Shipp and Yerger 1969a, 1969b; Tremain
and Adams, 1995)], muscle was demonstrated
to be toxic to mice by intraperitoneal injec-
tion. However, the toxins in these puffer fish
Saxitoxins in Pyrodinium bahamense in the United States
Environmental Health Perspectives
VOLUME 114 | NUMBER 10 | October 2006
1505
Figure 3. LC-MS ion chromatograms (
A
,
C
) and mass spectra (
B
,
D
) of TTX in reference standard (
A
,
B
) and
checkered puffer fish liver (
C
,
D
).
10.00 20.00
Time (min)
Intensity
139
A
TTX
standard
14.68
30.00
10.00 20.00 30.00
Time (min)
14.72
320
321
302
320
321
302
TTX in
checkered
puffer liver
Intensity
1,030
B
C
D
m/z
240 290 330 380
m/z
240 290 330 380
100
100
Relative intensity Relative intensity
Figure 4. Scanning electron micrograph of two
P. bahamense
cells isolated from the IRL (FWC
2004). Left cell, posterior-lateral view; right cell,
dorsal view. Bar = 20 µm.
Figure 5. Toxin analysis of
P. bahamense
by HPLC
chromatograms showing (
A
) dc-STX (1%) and STX
(26%), and (
B
) B1 (73%).
0
Relative fluorescence (mV)
1.5
1.0
0.5
0
A
STX
Time (min)
510152025
dc-STX
B
1.5
1.0
0.5
0
0 5 10 15 20 25
Time (min)
B1
Relative fluorescence (mV)
samples were not characterized. Of the tissues
investigated in that study, including skin,
liver, muscle, and testes or ovary, the muscle
was the most lethal to mice, similar to the pat-
tern seen today. Although this anecdotal evi-
dence suggests that southern puffer fish may
have been mildly toxic from STX in the IRL
for the past 45 years, there has been no indica-
tion that toxin levels were even close to the
order of magnitude observed since 2002 nor
was the FDOH informed of any poisoning
incidents from this area prior to this time.
Globally, human food-poisoning incidents
from STX exposure are usually caused by toxic
marine shellfish (Kao 1993) that filter-feed on
STX-producing microalgae. PSP can be fatal
(Kao 1993), but the successful implementa-
tion of programs monitoring STX-producing
microalgae and STXs in shellfish has helped
minimize the risk of toxin exposure to
humans. In marine waters, PSP is caused by
toxic dinoflagellates, where STXs are produced
by more temperate Alexandrium species and
Gymnodinium catenatum and by tropical
Pyrodinium bahamense var. compressum (Kao
1993). PSP in the United States has been lim-
ited to New England and the Pacific West
Coast, including Alaska, and has only been
associated with STXs produced by temperate
Alexandrium spp. in these areas (Gessner
2000).
The epidemiology of PSP incidents is
related to the global distribution of the various
STX-producing species and their toxigenic
strains. PSP outbreaks due to P. bahamense
have caused more fatalities than any other
microalgal species known (Usup and Azanza
1998). In 1987, PSP associated with P.
bahamense var. compressum in Champerico,
Guatemala, hospitalized at least 187 individu-
als and resulted in 26 fatalities (Rodrigue
et al. 1990). Before 1996, 1,768 cases of PSP
with 107 deaths had been reported in the
Philippines, mostly attributable to P.
bahamense var. compressum (Babaran et al.
1998). These fatalities were largely due to the
sudden appearance of P. bahamense in areas
previously unknown to contain toxic species,
because monitoring activities were not in
place or because hospital facilities had not
treated people in these previously unaffected
areas (Kao 1993).
In the present study we confirm unequivo-
cally that puffer fish are a primary reservoir of
STXs in marine waters in Florida, and we
implicate for the first time the tropical western
Atlantic dinoflagellate P. bahamense as the
source of toxicity. We found the STX profile of
P. bahamense isolates from Florida to be similar
to, but proportionately different from, the toxin
profile of southern puffer fish fillet (Etheridge
et al. 2006; Quilliam et al. 2004), and we iden-
tified P. bahamense as the putative source of the
STXs. Confirmatory toxin-transfer studies from
Pyrodinium via shellfish to puffer fish are in
progress. Although many temperate marine
Alexandrium species, Gymnodinium catenatum,
and a few freshwater cyanobacteria species pro-
duce STXs (Kodama 2000), these organisms
have not been found in the IRL.
In addition to the Caribbean and Gulf
coasts of Mexico, bioluminescent P. bahamense
blooms are found only along Florida’s Atlantic
and Gulf coasts (Badylak et al. 2004; Phlips
et al. 2004; Steidinger et al. 1980). However,
until the IRL SPFP incidents, the Atlantic/
Caribbean P. bahamense var. bahamense was not
known to be toxic (Steidinger et al. 1980),
unlike the Pacific P. bahamense var. compressum
found in Asia and the Pacific Coast of Central
America (Rodrigue et al. 1990; Usup and
Azanza 1998; Vargas-Montero and Freer
2004). The Atlantic P. bahamense var.
bahamense was separated from the Pacific
P. bahamense var. compressum based on mor-
phologic criteria and evident lack of toxicity in
the former variety (Steidinger et al. 1980).
Based on our initial findings, we are testing
the hypothesis that this varietal distinction
may no longer be valid and that P. bahamense
is all one species.
Florida has many toxigenic marine algal
species, but none were known to produce
STXs (Steidinger et al. 1999). It is conceivable
that STXs might have appeared in the IRL
because of one of several scenarios: a) toxigenic
populations of Pyrodinium have been intro-
duced; b) ecologic conditions have changed
and have induced toxicity in a variety that was
previously nontoxic; c
) toxic Pyrodinium was
present but produced toxins at undetectable
concentrations; or d) ecologic conditions have
changed and increased the food-web exposure
of susceptible biota to toxins. We believe that c
is the most likely scenario. In the IRL there is a
history of Pyrodinium (Badylak et al. 2004;
Phlips et al. 2004), and as mentioned previ-
ously, there is a historical precedent for low-
level toxicity in puffer fish.
In the past few years, the northern IRL has
experienced a number of unusual events: dol-
phin, manatee, fish, and horseshoe crab mor-
talities; increased tumor incidence in hard
clams; diseased shrimp; and reductions in the
natural recruitment of and increases in the
hatchery losses of hard clams (Bossart et al.
2003; Landsberg et al. 2002; Landsberg and
Kiryu 2005). To what extent, if at all, these
events are linked to the emerging issue of toxic
P. bahamense blooms remains undetermined.
The significant risk of SPFP and PSP from
saxitoxins in the IRL has been assessed and
management plans implemented accordingly.
Thus far, routine monitoring by Florida state
agencies has determined that STX levels in
shellfish, principally hard clams (Mercenaria
spp.), are not a significant risk to public health
(Landsberg et al. 2005). The extreme toxicity
of puffer fish fillet, well above the action level,
emphasizes the danger that puffer fish pose to
the public and supports the permanent ban on
their harvest in this area (FWC 2004).
The widespread implications for public
health incidents from the tropical western
Atlantic P. bahamense remain unknown.
Public health officials and natural resource
managers should be aware of these new find-
ings and remain vigilant to examine any poten-
tial association between the co-occurrence of
this species throughout its range and the
appearance of toxic food-poisoning incidents.
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... These blooms are the cause of both neurotoxic shellfish poisoning (NSP) and respiratory irritation ( Table 1) and are the focus of intensive state monitoring and management (Heil and Steidinger, 2009). Several Florida coastal regions (Indian River Lagoon, Tampa Bay, Charlotte Harbor) are also subject to blooms of the dinoflagellate Pyrodinium bahamense, which can contain saxitoxins that are responsible for both paralytic shellfish poisoning (PSP) and saxitoxin puffer fish poisoning (SPFP) (Landsberg et al., 2006;Abbott et al., 2009). The detection of domoic acid, a toxic, water soluble (Falk et al., 1991) amino acid produced by some species in the diatom genus Pseudo-nitzschia, has resulted in shellfish closures in St. ...
... Besides the taxonomic differences between the two varieties, Steidinger et al. (1980) also proposed that only the Pacific variety was toxic, while the Atlantic variety was not. In 2002, however, Landsberg et al. (2006) demonstrated that P. bahamense var. bahamense can produce paralytic shellfish toxins (PST) including saxitoxin (STX), decarbamoyl STX and M-sulfocarbamoyl toxins, in clonal cultures and in natural bloom samples from the Indian River Lagoon (IRL). ...
... bahamense. This species grows better in culture medium enriched in natural organic acids such as humic acids and soil extract supplements (McLaughlin and Zahl, 1961;Usup, 1996;Landsberg et al., 2006) and selenium (Usup, 1996;Landsberg et al., 2006). Usup et al. (2012) observed that P. bahamense is often found in areas adjacent to mangrove forests, which are a rich source of dissolved organic matter in Florida coastal waters (Jaffé et al., 2004). ...
... Nevertheless, the isolates from Guatemala contained STX, neoSTX, B1, GTX2, GTX3 and GTX4 [81] (Table 3); in Florida, P bahamense most likely produced STX, dc-STX and B1 as detected in puffer fish and HAB of P. bahamense [22] (Table 3), and one strain from Isla San José in the Gulf of California produced only STX [72] (Table 3). ...
... This was observed by Núñez-Vázquez et al. [42,54] indirectly in puffer fish, probably due to the toxins transmitted via bivalves and other invertebrates that regularly make up part of the diet of these fish in Campeche coastal waters [44]. Previous studies by [22,86] demonstrated the presence of high concentrations of PSTs in puffer fish from Florida, resulting from the P bahamense HAB event. ...
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Pyrodinium bahamense var. compressum, a saxitoxin-producing dinoflagellate, frequently blooms on the west coast of Sabah. According to previous studies, saxitoxin from cyanobacteria and dinoflagellates is manufactured from similar precursors (three arginines, one methionine via S-adenosylmethionine and one acetate) through identical biochemical pathways. The saxitoxin biosynthetic starting gene, sxtA is essential for synthesizing the final compound. The genes associated with saxitoxin synthesis in Alexandrium spp. and cyanobacteria have been previously identified; in spite of this, limited information is known about P. bahamense var. compressum, the principal tropical saxitoxin-producing dinoflagellate. In this study, the exclusive starting gene for saxitoxin biosynthesis, sxtA, specifically the SAM-dependent methyltransferase, sxtA1 and the class II aminotransferase coding gene, sxtA4 of P. bahamense was studied. Comparative sequence analysis revealed that sxtA1 and sxtA4 genes in P. bahamense exhibited high sequence similarity with other toxic dinoflagellates such as Alexandrium fundyense and Alexandrium tamarense. This study provides a genetic insight into saxitoxin biosynthesis in P. bahamense, which will be helpful in future investigations such as the development of genetic markers to study the expression of the sxtA gene and the identification of potential molecular targets for bloom characterization.
... The IRL has a long history of diverse HABs (Landsberg et al., 2006;Phlips et al., 2015), which include some toxic species that pose a risk to marine mammals (Fire et al., 2015). In 2011, a series of non-toxic bloom events with direct ecosystem disruption resulted in a chain of effects that ultimately triggered a manatee UME in 2013. ...
... Harmful and toxic microalgae in the IRL include the saxitoxin-producing dinoflagellate Pyrodinium bahamense, occasional incursions by Karenia brevis (which produces brevetoxins), and low-toxicity to non-toxic Pseudo-nitszchia spp. (which produce domoic acid) (Landsberg et al., 2006;Phlips et al., 2015), yet there was no evidence that manatees had been exposed to these neurotoxins during the UME. Benthic dinoflagellates Prorocentrum spp., which produce okadaic acid and dinophysistoxins, utilize macroalgae and seagrass as substrate in the IRL (Anderson, 2002), and although these toxins can cause diarrheic shellfish poisoning (DSP) in humans (Murata et al., 1982), manatee gastrointestinal contents were negative for these toxins. ...
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The Indian River Lagoon (IRL) on Florida’s east coast is a biologically diverse estuary and an important habitat to the threatened Florida manatee (Trichechus manatus latirostris). An unusual mortality event (UME) was declared by the Working Group on Marine Mammal Unusual Mortality Events in 2013 after a marked increase in manatee deaths in the IRL of an unknown cause. This UME followed a dramatic reduction of seagrass coverage in the IRL due to chronic non-toxic phytoplankton blooms, with a resultant ecosystem shift to mixed macroalgal dominance. At least 199 manatee deaths fitting the UME case definition were documented in and adjacent to the IRL during 2012–2019; mortality was highest in 2013, when 111 of these deaths were documented. The case definition included carcasses in good nutritional condition, with multiorgan congestion or wet lungs consistent with drowning without trauma. The gastrointestinal compartments of manatee carcasses were filled with diverse macroalga species, and the contents were notably more fluid than usual. Gross intestinal findings included blebbing to segmental thickening of the wall. Microscopic lesions were primarily intestinal, including necrosis, edema, hemorrhage, mucosa-associated lymphoid changes, and inflammation, sometimes associated with Gram-positive bacterial rods. A multidisciplinary approach of environmental and carcass sampling found no causative evidence through tests for micro- and macroalgal biotoxins, trace metals, general toxin screening, or vitreum biochemistry. Microbiological, cytological, immunohistochemical, and molecular analyses of Clostridiales from intestinal samples identified Clostridioides difficile toxin A, toxins A/B and toxin A gene; Paeniclostridium sordellii lethal gene (and other potential virulence factors from a sequenced strain); and Clostridium perfringens alpha and epsilon toxin genes. The results from this 8 year-long investigation are indicative that the cause of death in this manatee UME was associated with clostridial infection, initiated by a shift to a predominantly macroalgal diet.
... For example, their mutualistic relationships with reef-building corals form the basis of a highly diverse and productive ecosystem. Many dinoflagellates are well-known producers of toxins that can be harmful to humans via syndromes like paralytic shellfish poisoning (PSP) [7][8][9][10][11], neurotoxic shellfish poisoning (NSP) [12][13][14], azaspiracid shellfish poisoning (AZP) [15,16], Diarrheic shellfish poisoning (DSP) [17] and ciguatera fish poisoning (CFP) [18,19]. These metabolites are generally detected because of their bioaccumulation through the marine food-chain into marine organisms including fish, crustaceans and mollusks, which are consumed by humans. ...
... Eutrophication and longduration harmful algal blooms have been prominent in the IRL during recent years (Lapointe et al. 2015;Phlips et al. 2015). An extensive Pyrodinium bahamense bloom in the IRL coincided with pufferfish toxicity events in 2002 (Phlips et al. 2004;Landsberg et al. 2006). Beginning in 2011, a widespread and long-duration phytoplankton bloom, termed the superbloom, occurred within the IRL system. ...
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Coastal lagoons and other estuarine habitats are increasingly exposed to the negative effects of growing human populations with associated intensifications in nutrient loading, harmful algal blooms, pollution, and habitat degradation. We examined population dynamics of Gulf pipefish Syngnathus scovelli in estuarine waters of the Indian River Lagoon, on the Atlantic coast of Florida. Substantial declines in abundance of this sentinel species were concurrent with significant losses of seagrass habitat associated with ongoing harmful algal blooms, and other perturbations during the study period spanning from 1998 to 2018. Moderate declines in S. scovelli were observed with early downward trends in seagrasses ahead of the onset of the precipitous seagrass reductions observed. The massive decline of seagrass habitats in the Indian River Lagoon in recent years had negative influences and was directly linked to population declines we observed in the S. scovelli population. Lack of seagrass habitat essential to this and related syngnathid species may reduce optimum seagrass-associated prey, increase predation by lack of appropriate cover, and increase energetic costs which may be realized through reduced growth rates and potential reproductive impairment. Identification and monitoring of population trends of S. scovelli and related sentinel fish species allow for early implementation of management actions that reduce the impact of anthropogenic pressures on the services that estuarine systems provide to the fishes and fisheries they support.
... The active toxin was identified as STX and two of its variants, with Pyrodinium bahamense being the main producer. PFP is usually linked to tetrodotoxin, whereas PSP is named after STX pufferfish poisoning (SPFP), which causes food poisoning [31]. ...
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Phytoplankton are photosynthetic microorganisms in aquatic environments that produce many bioactive substances. However, some of them are toxic to aquatic organisms via filter-feeding and are even poisonous to humans through the food chain. Human poisoning from these substances and their serious long-term consequences have resulted in several health threats, including cancer, skin disorders, and other diseases, which have been frequently documented. Seafood poisoning disorders triggered by phytoplankton toxins include paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), diarrheic shellfish poisoning (DSP), ciguatera fish poisoning (CFP), and azaspiracid shellfish poisoning (AZP). Accordingly, identifying harmful shellfish poisoning and toxin-producing species and their detrimental effects is urgently required. Although the harmful effects of these toxins are well documented, their possible modes of action are insufficiently understood in terms of clinical symptoms. In this review, we summarize the current state of knowledge regarding phytoplankton toxins and their detrimental consequences, including tumor-promoting activity. The structure, source, and clinical symptoms caused by these toxins, as well as their molecular mechanisms of action on voltage-gated ion channels, are briefly discussed. Moreover, the possible stress-associated reactive oxygen species (ROS)-related modes of action are summarized. Finally, we describe the toxic effects of phytoplankton toxins and discuss future research in the field of stress-associated ROS-related toxicity. Moreover, these toxins can also be used in different pharmacological prospects and can be established as a potent pharmacophore in the near future.
... For example, Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum have been confirmed to produce yessotoxins, which are responsible for diarrhetic shellfish poisoning (Paz et al. 2008, Howard et al. 2009, Cusick & Widder 2014. Alexandrium ostenfeldii, A. tamarense and Pyro dinium bahamense are known as saxitoxin producers that cause paralytic shellfish poisoning (Gribble et al. 2005, Landsberg et al. 2006, Hakanen et al. 2012, Le Tortorec et al. 2014, Zou et al. 2014.The presence of toxic bioluminescent dinoflagellates during the 'blue tears' season has yet to be confirmed. Therefore, regular investigations of the distribution of bioluminescent dinoflagellates would provide fundamental information to explain the source of bio luminescence along the coast. ...
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Noctiluca scintillans is considered to be a bioluminescent bloom-forming species in the coastal water around the Matsu archipelago. To identify the bioluminescent dinoflagellates and their distributions around the Matsu archipelago, metatranscriptome and luciferase (lcf) gene sequencing were conducted from June 2016 to July 2017. Metatranscriptomes retrieved lcf genes mainly from Noctiluca and other bioluminescent dinoflagellates. This result demonstrates that lcf genes were actually expressed in multiple dinoflagellate species. An analysis of the lcf composition of dinoflagellates indicated that N. scintillans was the dominant bioluminescent species during May and July. In late summer, this dominance was replaced by other bioluminescent dinoflagellate species, such as Alexandrium affine and Ceratium fusus. No lcf gene from the known toxic bioluminescent dinoflagellates was obtained during the period of investigation. Our results suggest that N. scintillans is not the only dinoflagellate species producing bioluminescence around the Matsu archipelago.
... In contrast, Guatemalan isolates produce gonyautoxin (GTX) 2, GTX3, and GTX4 [9]. The Atlantic strain of P. bahamense from the Indian River Lagoon, Florida, USA yielded STX, dcSTX, and B1 [10]. ...
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Proteins, lipids, and carbohydrates from the harmful algal bloom (HAB)-causing organism Pyrodinium bahamense were characterized to obtain insights into the biochemical processes in this environmentally relevant dinoflagellate. Shotgun proteomics using label-free quantitation followed by proteome mapping using the P. bahamense transcriptome and translated protein databases of Marinovum algicola, Alexandrium sp., Cylindrospermopsis raciborskii, and Symbiodinium kawagutii for annotation enabled the characterization of the proteins in P. bahamense. The highest number of annotated hits were obtained from M. algicola and highlighted the contribution of microorganisms associated with P. bahamense. Proteins involved in dimethylsulfoniopropionate (DMSP) degradation such as propionyl CoA synthethase and acryloyl-CoA reductase were identified, suggesting the DMSP cleavage pathway as the preferred route in this dinoflagellate. Most of the annotated proteins were involved in amino acid biosynthesis and carbohydrate degradation and metabolism, indicating the active roles of these molecules in the vegetative stage of P. bahamense. This characterization provides baseline information on the cellular machinery and the molecular basis of the ecophysiology of P. bahamense.
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Antecedentes y Objetivos: Pyrodinium es un género monotípico con dos variedades, var. bahamense en el Atlántico, no tóxica, y var. compressum en el Indo-Pacífico y tóxica. Hallazgos recientes de toxicidad por envenenamiento paralitico por mariscos (PSP), en poblaciones de P. bahamense var. bahamense, debilitaron el interés por aceptarlas como independientes, e incluso investigar la posibilidad de cripticismo. En los pocos estudios que han incorporado evidencia molecular en el tratamiento de las variedades, su independencia taxonómica sigue siendo negada, a pesar de la evidente y consistente separación genética presentada en todos los análisis. Por lo tanto, el objetivo de este estudio fue reconocer, desde la evidencia genética, la independencia taxonómica de las dos variedades de Pyrodinium: P. bahamense var. compressum y var. bahamense. Métodos: A partir de tres marcadores moleculares (Lcf, LSU, SSU) de secuencias disponibles en el Genbank, se construyeron redes de parsimonia estadística y un análisis filogenético concatenado. Resultados clave: En todos los análisis, se obtuvo de manera consistente una estructura genética para P. bahamense var. bahamense y otra para P. bahamense var. compressum. Además, la correspondencia de haplotipos y ribotipos resultantes fue siempre la misma, tanto en redes como en la filogenia; es decir, las muestras del Océano Pacífico siempre se colocaron en un grupo distinto al de las secuencias del Océano Atlántico. Conclusiones: La evidencia proporcionada en este estudio demostró que existe un aislamiento reproductivo entre ambas variedades, e incluso la posible presencia de una tercera variedad aún no descrita, por lo cual proponemos la validez e independencia taxonómica de P. bahamense var. compressum y P. bahamense var. bahamense.
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Fugu (puffer-fish) is one of many poisonous fishes. It produces a potent, toxic substance that is found in various of its organs, for example, the ovaries, liver, intestine, skin, and spawn. Although fugu is dangerous to eat, it is very interesting that there are special restaurants for cooking fugu in Japan. Such restaurants are obligated by the Japanese government to prepare the fish in such a way that fugu intoxication will not be a hazard to diners. One will naturally raise the question—what reasons have Japanese for this dangerous custom. This point-blank question is fired at us by many foreigners. Dangerous, but interesting, eating habits have been rooted among Japanese for a long time; in fact, such habits are localized to Japan. However, the white meat of fugu can please the Japanese palate without causing danger, because a very good technique was developed by our ancestors for treating organs contaminated with its toxic substance. This is a rather unique example of food-life history.