livers contained similar amounts of toxin (relative effects
ranged from 0.8 to 1.0), suggesting that there is little variation
in the amount of TTX among individuals of T. rubripes,
although the puffers were captured at various times during
Dec./2007 to March/2008 in the Sea of Japan. On the other
hand, for A. reticularis (a species not normally eaten) livers
showed some variation (relative effects ranged between 0.6
and 1.0) in the individuals, although the puffers were
captured at the same time (March/2008) along the coast of
Ishigaki Island.
In addition, the muscles excised from T. rubripes, T.
vermicularis and L. wheeleri contained much less neurotoxin
(TTX) than the livers. Fig. 4A and B summarizes the
dilution–inhibition curve for the toxin extracts from muscle
and liver of the three puffers. The strength of the toxin
extracts, estimated from IC50 values of dilution–inhibition
curves for the muscle, was in the order of T. vermicularis
(4.7 103 Dil) > L. wheeleri (1.0 103 Dil) > T. rubripes
(7.5 102 Dil), and for liver, T. vermicularis
(1.8 105 Dil) j T. rubripes (1.8 105 Dil) > L. wheeleri
(1.6 104 Dil). All IC50 values were obtained from curve
fitting by use of following equation (Eq. 2):
Y ¼ A1 þ ðA2 A1Þ=
1 þ 10ðlogX0XÞp
where Y is the measured response of the INa, A2 and A1 are
the maximum and the minimum responses respectively of
the INa obtained with a control sample, p is the slope
parameter of the dose–response curve, X is the applied
dose, and logX0 is the center of the curve that is the
concentration for half the INa response.
3.2. Effects of toxin extracts from puffers not supplied as food
The toxin extracts from muscle and liver of C. rivulata
and A. reticularis on INa were also examined. C. rivulata is
commonly found in large groups along the rocky and
shallow sea coast of the Ariake Sea. The mature C. rivulata is
small (50–70 g) and the body length is less than 15 cm.
On the other hand, A. reticularis is one of the biggest puffers
(up to 2 kg) that inhabits the warm sea coast of Ishigaki
Island in the subtropical area in Japan. However, till now
there is a report concerning the amount of toxin distributed
in the organs of C. rivulata and A. reticularis by use of mouse
bioassay (Noguchi and Arakawa, 2008). The toxin extracts
from muscle or liver of these two puffers inhibited INa
reversibly with similar time course to that observed in the
case of other puffers (Fig. 5). The inhibitory effects estimated
from IC50 values were in the order of C. rivulata
(7.9 102 Dil) T. rubripes (7.5 102 Dil) A. reticularis
(5.7 102 Dil) for muscle and T. rubripes
(1.8 105 Dil) > A. reticularis (5.3 103 Dil) C. rivulata
(3.6 103 Dil) for liver. The inhibitory effects of toxin
extracts from the liver of these two puffers were much
weaker than those of T. rubripes (Fig. 6).
3.3. Effects of toxin extracts from liver of A. reticularis on
activation and inactivation curves for INa
Fig. 7A shows a current–voltage (I–V) relationship of
INa with and without toxin extract from the liver of
F