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Toxic Takifugu pardalis eggs found in Takifugu niphobles gut: Implications for TTX accumulation in the pufferfish
Toxicon 108 (2015) 141e146
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Toxic Takifugu pardalis eggs found in Takifugu niphobles gut: Implications for TTX accumulation in the pufferfish Shiro Itoi a, *, Ao Kozaki a, Keitaro Komori a, Tadasuke Tsunashima a, Shunsuke Noguchi a, 1, Mitsuo Kawane b, Haruo Sugita a a b
Department of Marine Science and Resources, Nihon University, Fujisawa, Kanagawa 252-0880, Japan Department of Sea-Farming, Aichi Fish Farming Institute, Tahara, Aichi 441-3618, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 July 2015 Received in revised form 30 September 2015 Accepted 14 October 2015 Available online 19 October 2015
Pufferfish (Takifugu spp.) possess a potent neurotoxin, tetrodotoxin (TTX). TTX has been detected in various organisms including food animals of pufferfish, and TTX-producing bacteria have been isolated from these animals. TTX in marine pufferfish accumulates in the pufferfish via the food web starting with marine bacteria. However, such accumulation is unlikely to account for the amount of TTX in the pufferfish body because of the minute amounts of TTX produced by marine bacteria. Therefore, the toxification process in pufferfish still remains unclear. In this article we report the presence of numerous Takifugu pardalis eggs in the intestinal contents of another pufferfish, Takifugu niphobles. The identity of T. pardalis being determined by direct sequencing for mitochondrial DNA. LC-MS/MS analysis revealed that the peak detected in the egg samples corresponded to TTX. Toxification experiments in recirculating aquaria demonstrated that cultured Takifugu rubripes quickly became toxic upon being fed toxic (TTXcontaining) T. rubripes eggs. These results suggest that T. niphobles ingested the toxic eggs of another pufferfish T. pardalis to toxify themselves more efficiently via a TTX loop consisting of TTX-bearing organisms at a higher trophic level in the food web. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Congeneric eggs Food chain Pufferfish Takifugu niphobles Takifugu pardalis Tetrodotoxin
1. Introduction Tetrodotoxin (TTX) is known to be the substance of pufferfish toxin and one type of potent neurotoxin specific to voltage-gated sodium channels of excitable membranes of muscle and nerve tissues (Colquhon et al., 1972; Narahashi, 2001; Noguchi et al., 2006a). TTX was believed to occur only in pufferfish (Tetraodontidae) until 1960's, when it was detected in Californian newt Taricha torosa (Mosher et al., 1964). Subsequently, TTX (along with some analogs) was also detected from potential pufferfish food organisms belonging to various disparate groups, including starfish (e.g., Astropecten spp.; Maruyama et al., 1984, 1985), gastropods (e.g., Babylonia japonica; Noguchi et al., 1981), crustaceans (e.g., the xanthid crab, Atergatis floridus; Noguchi et al., 1983), flatworms and ribbonworms (e.g., Cephalothrix simula; Asakawa et al., 2013), apart from several species of bacteria that are symbiotic with the
* Corresponding author. E-mail address: [email protected] (S. Itoi). 1 Present address: Kyoto Institute of Oceanic and Fishery Science, Miyazu, Kyoto 626-0052, Japan.
pufferfish and the potential food organisms (Noguchi et al., 1987; Wu et al., 2005; Noguchi and Arakawa, 2008). In addition, TTX has been detected in some free-living bacteria, including those in deep sea sediments (Simidu et al., 1987; Do et al., 1990), although it is not clear if these bacteria form part of the food chain leading to pufferfish. In any case, it appears plausible that the TTX in pufferfish is a result of accumulation through the food chain, which consists of several steps, starting with bacteria, as suggested by several reports (Noguchi et al., 2006a; Noguchi and Arakawa, 2008). These speculations have actually been supported by several studies: non-toxic pufferfish have been produced when grown from hatching with a non-toxic diet, and furthermore, these cultured non-toxic pufferfish have become toxic when administered orally with TTX (Matsui et al., 1981, 1982; Noguchi et al., 2006b; Saito et al., 1984; Yamamori et al., 2004; Honda et al., 2005). TTX has been detected not only in pufferfish and their prey, but also in organisms ecologically unrelated to pufferfish, such as the Costa Rican frogs of the genus Atelopus (Kim et al., 1975) and some land planarians (Stokes et al., 2014), besides Californian newt T. torosa (Mosher et al., 1964). It has also been suggested that TTX in the rough-skin newt, Taricha granulosa, is obtained endogenously
http://dx.doi.org/10.1016/j.toxicon.2015.10.009 0041-0101/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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(Hanifin et al., 2002; Cardall et al., 2004). These findings suggest that TTX accumulates in pufferfish by means other than through the classical food chain; since in vivo cultured TTX-producing bacteria are unable to produce enough quantities of TTX to account for the amount of TTX in wild pufferfish (Miyazawa and Noguchi, 2001; Food Safety Commission of Japan, 2005; Wu et al., 2005; Wang et al., 2008; Yang et al., 2010). This also indicates that TTX-bearing pufferfish prey are abundant. In any case, the origin of TTX and the acquisition process are both likely to vary across different animal species, and it remains unclear exactly where pufferfish acquire the large quantities of TTX that they possess. Recently, our lab unexpectedly found numerous eggs among the intestinal contents of the pufferfish, Takifugu niphobles, and partial mitochondrial DNA sequences from these eggs identified them to be those of another pufferfish species, namely, Takifugu pardalis. We have demonstrated the toxification process using cultured pufferfish Takifugu rubripes in this study by means of experimentally reproducing the serendipitous finding of eggs in the pufferfish gut, thus indicating that pufferfish toxification manifests from the accumulated TTX at relatively higher trophic levels in the food chain.
extraction and the remaining eggs were stored at 30 C until TTX extraction. 2.2. DNA extraction and PCR amplification Total genomic DNA was extracted from each egg by the method of Sezaki et al. (1999). The fragments of partial mitochondrial DNA were amplified by PCR using two primer sets, 16S AR-L (forward: 50 CGCCT GTTTA TCAAA AACAT-30 ) and 16S BR-H (reverse: 50 -CCGGT CTGAA CTCAG ATCAC GT-30 ) for the 16S rRNA gene (Palumbi et al., 1991), and TCytb-F1 (forward: 50 -ACCTR TGGCG TGAAA AACCA YCGTT GT-30 ) and TCytb-R1 (reverse: 50 -CATYC GGTTT ACAAG ACCGR CGCTC TG-30 ) for the cytochrome b gene. Primers for cytochrome b gene were designed based on the mitochondrial DNA sequences from multiple species of the family Tetraodontidae. PCR amplification was performed in a 20 ml reaction mixture containing genomic DNA as a template, 1 unit ExTaq DNA polymerase (Takara Bio, Shiga, Japan), 1.6 ml of 2.5 mM deoxynucleotide triphosphates (dNTP), 5 ml of 5 mM primers and 2 ml of 10 ExTaq DNA polymerase buffer (Takara Bio). The thermal cycling program for the PCR consisted of an initial denaturation at 95 C for 1 min followed by 35 cycles of denaturation at 95 C for 10 s, annealing at 55 C for 30 s and extension at 72 C for 45 s.
2. Materials and methods 2.3. Direct sequencing and phylogenetic analyses 2.1. The intestinal contents of wild pufferfish Wild specimens of the pufferfish, T. niphobles (body weight: 60.3 ± 25.7 g and 48.9 ± 21.2 g for females and males, respectively; detailed in Table 1) were collected from coastal waters in Nagai, Kanagawa, Japan (35120 N, 139 360 E), on 13 March 2012 (water temperature: 13.9 C; salinity: 31.4 practical salinity unit, psu), 08 March 2013 (16.0 C; 32.4 psu) and 14 March 2013 (14.2 C; 30.6 psu), 07 March 2014 (12.5 C; 32.3 psu) and 13 March 2014 (12.5 C; 31.8 psu), and 09 March 2015 (11.7 C; 34.3 psu), 16 March 2015 (12.3 C; 34.8 psu) and 19 March 2015 (14.6 C; 35.0 psu). The gonadosomatic index (GSI) was 2.2 ± 0.9 for females and 2.0 ± 1.4 for males (detailed in Table 1). The unexpected eggs that were found among the gut contents of the pufferfish were also collected. Five eggs from each fish were immediately subjected to DNA
Prior to the direct sequencing of the amplified product, the DNA fragment was purified by chloroform extraction, followed by polyethylene glycol (PEG) 8000 precipitation and ethanol precipitation. Sequencing was performed for both strands using a 3130xl genetic analyzer (Applied Biosystems, Foster, CA, USA) and a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems). The concatenated nucleotide sequences of the 16S rRNA gene and cytochrome b gene of eggs were aligned using CLUSTAL W (Thompson et al., 1994) with those in the DDBJ/EMBL/ GenBank databases obtained using a BLAST search (Altschul et al., 1997). The alignment was then subjected to phylogenetic inference by means of the maximum likelihood method using MEGA ver. 6.0.6 (Tamura et al., 2013), with the corresponding concatenated sequences from the yellow-stripe toadfish, Torquigener brevipennis
Table 1 Characteristics of the Takifugu niphobles specimens used in this studya. Sampling date 2012 13 Mar 2013 08 Mar 14 Mar 2014 07 Mar 13 Mar 2015 09 Mar 16 Mar 19 Mar a b c d
Sex
No. of specimen
Female n ¼ 8 Male n ¼ 2 Female Male Female Male
n n n n
¼ ¼ ¼ ¼
9 13 6 8
Male n ¼ 2 Female n ¼ 10 Male n ¼ 6 Female Male Female Male Female Male
n n n n n n
¼ ¼ ¼ ¼ ¼ ¼
13 10 11 4 4 4
No. of egg-fed specimen
Intestinal egg content (g)
Toxicity of the intestinal eggs (MU)
Standard length (mm)
Body weight (g)
GSIb
n¼8 n¼2
N/Dc N/D
N/D N/D
119.4 ± 3.8 111.0 ± 2.0
64.1 ± 9.4 54.5 ± 1.5
2.6 ± 0.5 1.5 ± 0.2
¼ ¼ ¼ ¼
0.28 0.05 0.72 0.26
2.63 ± 4.42 0.25 ± 0.74 10.38 ± 12.94 4.10 ± 6.48
112.7 108.4 122.2 110.8
0.44 ± 0.30 0.38 ± 0.62 0.09 ± 0.12
0.29 ± 0.17 0.49 ± 0.66 0.06 ± 0.13
128.0 ± 1.0 121.7 ± 17.2 111.3 ± 12.5
N/Ad 0.05 ± 0.38 ± 0.08 ± 0.47 ± N/A
N/A 2.72 ± 7.04 15.84 ± 30.37 1.63 ± 2.32 3.30 ± 2.46 N/A
118.7 115.6 164.6 140.0 130.0 133.0
n n n n
8 7 6 8
n¼2 n¼7 n¼3 n n n n n n
¼ ¼ ¼ ¼ ¼ ¼
0 3 8 2 4 0
± ± ± ±
0.25 0.09 0.48 0.36
0.08 0.57 0.08 0.37
Data are represented by mean ± standard deviation. GSI represents gonadosomatic index: gonad weight/body weight 100. N/D, not determined. N/A, not applicated.
± ± ± ±
± ± ± ± ± ±
18.3 17.7 16.7 11.1
12.8 17.4 17.6 26.0 18.1 22.5
60.3 55.2 56.2 55.4
± ± ± ±
22.8 22.0 24.5 12.2
73.3 ± 3.6 63.9 ± 23.8 50.0 ± 17.4 33.0 30.8 87.3 59.0 43.5 45.5
± ± ± ± ± ±
9.7 15.4 20.9 28.0 16.0 18.1
2.1 1.9 2.0 2.7
± ± ± ±
0.8 1.1 0.4 0.8
2.6 ± 0.3 2.4 ± 1.3 3.0 ± 2.1 1.9 1.3 2.5 2.1 2.5 2.0
± ± ± ± ± ±
0.5 1.3 0.7 1.1 1.6 1.2
S. Itoi et al. / Toxicon 108 (2015) 141e146
(DNA database accession number AP009537) as the outgroup. The nucleotide sequences of the partial 16S rRNA gene and cytochrome b gene obtained from the eggs were submitted to the DDBJ/EMBL/ GenBank databases with accession numbers LC060064 and LC060065, respectively. 2.4. Toxification experiment Toxic eggs for the toxification experiments were obtained from the pufferfish T. rubripes, which had been collected at Ise Bay, Aichi, Japan, and maintained in the aquarium at the Aichi Sea Farming Institute with flowing water (5.2 t/h). The non-toxic juveniles of T. rubripes (body weight: approximately 3.5 g) were purchased from Marinetech (Aichi, Japan), and were maintained in recirculating aquariums at 20 C, and fed with commercial food pellets (nontoxic). In the toxification experiment, juvenile T. rubripes (n ¼ 52, body weight: 3.1e49.6 g, 19.0 ± 12.7 g) were fed with the toxic eggs. After more than two days of feeding with toxic eggs, liver, skin, muscle and other organ samples of egg-fed T. rubripes and commercial pellet-fed control specimens were subjected to the TTX extraction process followed by LC-MS/MS analysis. In advance before the toxification experiment, preliminary experiment was carried out: after two, four and nine days of feeding with toxic eggs, egg-fed T. rubripes specimens (n ¼ 27, body weight: 14.8 ± 3.6 g) were also subjected to the TTX extraction followed by LC-MS/MS analysis. 2.5. LC-MS/MS analysis Following Tsuruda et al. (2002) with some modifications, the extract with 0.1% acetic acid was filtered through a 0.45-mm filter membrane (Dismic-13CP, Advantec, Japan), and TTX in the extract was analyzed using High Performance Liquid Chromatography (HPLC) equipment ACQUITY system (Alliance 2695), coupled to a Quattro Premier XE mass spectrometer from Waters (Milford, MA, USA), based on the method of Shinno et al. (2007) with some modifications. Chromatographic separation and detection of TTX was achieved with a Waters Atlantis HILIC silica column (2.1 150 mm, 5 mm), equipped with a Waters Atlantis HILIC silica guard column (2.1 10 mm, 5 mm) and column oven at 40 C. The HPLC was operated with 0.1% formic acid (Merck, Darmstadt, Germany) and acetonitrile (Merck) as eluents. The gradient protocol used to elute the toxins was 95% acetonitrile, mobile phase in the beginning, decreasing to 40% acetonitrile after 0.1 min, then kept for 7.9 min and back to 95% acetonitrile and finally kept 95% acetonitrile for 2.0 min before the next injection. Flow rate was 0.3 ml/min and injection volume was 5 ml. The Quattro Premier XE mass spectrometer was operated with the following optimized source-dependent parameters (ESI source): capillary potential 0.5 kV, cone voltage 42 V, desolvation temperature 400 C, desolvation gas flow 600 l/h N2, cone gas flow 50 l/h N2, source temperature 120 C, collision energy 38 V. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode, with detection in the positive mode, analyzing two product ions at m/z 162 for quantification of TTX and m/z 302 for confirmation of the compound from the precursor ion at m/z 320. The ionized molecules were monitored through a MassLynx NT operation system. TTX was quantified using their peak areas to calculate amounts and using the calibration curve obtained from 1 to 100 ng/ml TTX standard (Wako Pure Chemicals, Osaka, Japan). One mouse unit (MU) is equivalent to 0.22 mg of TTX, based on the specific toxicity of TTX. 2.6. Statistical analysis The statistical significance of differences in the amount of toxin
143
was analyzed by means of a Student's t-test and a one-way ANOVA followed by the Tukey Honestly Significant Difference (HSD) test. In order to evaluate statistical support values for the linear regression, results of toxicity from toxification experiments were logtransformed and subjected to ANOVA. 3. Results 3.1. Eggs from the intestinal contents of the pufferfish T. niphobles A number of eggs, with a diameter of approximately 1 mm, were detected in the gut contents of the pufferfish T. niphobles (Fig. S1) captured in March 2012e2015 (Table 1). The eggs were seen in the 08 March 2013 sample in 8 and 7 specimens of 9 females and 13 males, respectively. The eggs were detected in all the specimens in the 14 March 2013 sample. The eggs were seen in 7 and 5 specimens of 10 females and 8 males, respectively, in 2014, and in 12 and 5 specimens of 28 females and 18 males, respectively, in 2015 (Table 1). In a few fish, the eggs were yet to be digested in the anterior parts of the intestine that interestingly, hatched after incubation at 20 C in artificial seawater for 9 days (March 2013). Direct sequencing and phylogenetic analysis of egg samples from the intestinal contents of T. niphobles demonstrated that the sequence of 16S rRNA gene and cytochrome b gene were highly similar to and clustered with the corresponding sequences of T. pardalis (Fig. 1). 3.2. Toxicity of the eggs in the intestinal contents of T. niphobles and tissue-distribution of TTX in T. niphobles fed T. pardalis-eggs Eggs were collected from the intestines of T. niphobles, subjected to TTX extraction, and analyzed using LC-MS/MS. Mass chromatogram of the LC-MS/MS was obtained under the MRM mode, and the MRM patterns of the egg samples were found to be identical to those of the TTX standards (Fig. 2). The calibration curve generated with 1e100 ng/ml of TTX standard shows good linearity and
100 Eggs 100
Takifugu pardalis (AP009528)
67
Takifugu chrysops (AP009525) Takifugu xanthopterus (AP009533)
67
Takifugu ocellatus (AP009536) Takifugu niphobles (AP009526) 88
Takifugu snyderi (AP009531)
79 100
Takifugu stictonotus (AP009530)
Takifugu porphyreus (AP009529) Takifugu rubripes (AP006045)
63 100
Takifugu chinensis (AP009534) Takifugu oblongus (AP009535) Torquigener brevipennis (AP009537)
0.02 Fig. 1. Phylogenetic tree inferred using the maximum likelihood method based on the nucleotide sequence of the concatenation of the complete cytochrome b gene (1137 bp) and the partial 16S rRNA gene (572 bp), obtained from the eggs removed from the intestinal tract of Takifugu niphobles and those of other Takifugu pufferfish species. Numbers at branches denote the bootstrap percentages out of 1000 replicates. The concatenated cytochrome b and 16S rRNA genes in Torquigener brevipennis (AP009537) was used as the outgroup. Only bootstrap values exceeding 50% are presented. The scale at the bottom of the tree indicates the number of substitutions per nucleotide site. “Eggs” represent the corresponding concatenation of the two gene sequences of the eggs taken from the intestinal tract of T. niphobles. The accession numbers for reference sequences are shown in parentheses.
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precision (r2 ¼ 0.99991). To estimate the effect of the matrix, LCMS/MS analysis was carried out using the samples, where the TTX standard was added into various tissue extracts from the nontoxic T. rubripes following the procedure mentioned above. TTX was recovered from the samples with >100-fold dilution except for gallbladder (>1000-fold dilutions) (Fig. S2). Therefore, quantification of TTX was carried out using the data for samples with >100fold dilution. The concentrations of TTX in the eggs from intestinal contents of the samples in 2012 were calculated to be 6.58 ± 0.88 mg/g, which correspond to 29.9 ± 4.0 MU/g; however, the contents of the eggs were not quantified because all eggs were not collected (Table 1). The concentration of TTX in the eggs collected from the gut of the 2013 specimens of T. niphobles was estimated to be 2.22 ± 4.51 mg/g (0.97 ± 1.83 mg/intestinal contents per fish), which corresponds to 10.1 ± 20.5 MU/g (4.4 ± 8.3 MU/ intestinal contents per fish). Similar patterns of the concentration of TTX in the eggs from intestinal contents of the T. niphobles were observed in the samples from each year (2013e2015; Table S1). Additionally, the TTX amount in the intestinal materials from eggfed T. niphobles specimens (4139 ± 6023 ng) were significantly higher than those from non-egg-fed counterparts (216 ± 374 ng) in 2015 (Table S2).
150000
100000
(A) Standard (25 ng/ml)
50000
0 (B) Intestinal eggs
Signal intensity
50000
The whole-body toxicity of the T. niphobles specimens fed with T. pardalis eggs was 2803 ± 10,361 MU (617 ± 2279 mg) for females and 1901 ± 1856 MU (418 ± 408 mg) for males (Table S1). The tissuespecific toxicity in females was significantly higher in the skin and ovaries than in the liver (P < 0.01), whereas in the males it was significantly higher in the skin than in other tissues (P < 0.01; Fig. S3). Similar patterns of toxin distribution were observed in the samples from each year (2013e2015; Fig. S3). 3.3. Intoxication of the pufferfish T. rubripes fed toxic eggs In preliminary toxification experiment, no significant difference in the toxicity of whole body was observed among the T. rubripes specimens fed with TTX-containing eggs after being fed toxic eggs for two days (98 ± 109 ng/g), four days (139 ± 296 ng/g) and nine days (154 ± 270 ng/g), suggesting that the pufferfish keeps the toxin level for a while (at least nine days; Fig. S4). Therefore, toxification experiments were performed after being fed toxic eggs for more than two days. Non-toxic T. rubripes (n ¼ 31, body weight: 3.1e49.6 g, 21.9 ± 12.8 g) became toxic after being fed toxic eggs for more than two days, although some specimens (n ¼ 21, body weight: 3.1e42.0 g, 14.7 ± 11.1 g) continued to remain non-toxic. The MRM patterns of the toxified tissues from the intestine, liver and skin were identical to those from the TTX extracted from the toxic eggs and the TTX standards (Fig. 2). The amount of toxin detected from several organs of toxified specimens was as follows: skin, 47.5 ± 38.1 mg/g; liver, 31.8 ± 31.6 mg/g; and intestine, 19.9 ± 29.0 mg/ g (Fig. S5). The amount of TTX in toxified fish was dependent on its body weight in an exponential relationship (y ¼ 0.9566e0.1307x, R2 ¼ 0.6402; Fig. 3) with high statistical support values for the linear regression (P ¼ 5.46 107, F statistics ¼ 44.49). On the other hand, none of the control fish fed with commercial feed were toxified (control; n ¼ 38, body weight: 3.1e57.9 g, 15.6 ± 15.6 g). Furthermore, the fish that did not ingest the toxic eggs even when given were not toxified either. 4. Discussion
0 (C) Eggs of T. rubripes
50000
0 (D) Skin of intoxicated T. rubripes
50000
Non-toxic T. rubripes were rapidly toxified after feeding on eggs containing TTX. Similar profiles of TTX transfer were observed in the cultured T. rubripes when TTX was administrated intramuscularly (Ikeda et al., 2009). All species of the genus Takifugu accumulate TTX in the ovaries, with the ovaries of T. pardalis showing the maximum toxicity of more than 1000 MU/g tissue, which corresponds to approximately 220 mg TTX/g tissue (classified as “strongly toxic”, Noguchi et al., 2006a). These suggest that
0 (E) Liver of non-toxic T. rubripes
50000
0 0
2
4
6
8
10
Retention time (min) Fig. 2. Typical mass chromatograms of the LC-MS/MS obtained under MRM mode (m/z 320 > 302). MRM patterns of 25 ng/ml TTX standard (A), the extract from eggs found in the intestinal tract of T. niphobles (B), the extract from the toxic eggs of T. rubripes used for the intoxication experiment (C), the extract from the skin of T. rubripes after the intoxication experiment (D), and the extract from the liver of non-toxic T. rubripes cultured with only commercial feed (E).
Total amount of TTX (MU)
10000 y = 0.9566e0.1307x R2 = 0.6402
1000 100 10 1 0.1
nd 0
10
20
30 Body weight (g)
40
50
60
Fig. 3. The relationship between the total amount of TTX in a specimen and the body weight of T. rubripes after the experiment with toxic eggs. The amount of TTX was calculated from the skin, liver, intestine and other tissues. nd: not detected.
S. Itoi et al. / Toxicon 108 (2015) 141e146
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TTX
Pufferfish
TTX
TTX
TTX
TTX
TTX
Food animals for pufferfish
TTX
Marine bacteria
Fig. 4. TTX accumulation process in pufferfish on the TTX loop. Upward arrows represent general TTX accumulation process in pufferfish via food chain consisting of several steps starting with marine bacteria (Noguchi et al., 2006a). A circular arrow represents novel TTX accumulation process in Takifugu niphobles (colored fish) at the spawning season of Takifugu pardalis (grey color fish) observed in this study.
T. niphobles effectively ingest TTX along with the nutrients in the toxic eggs of other pufferfish (T. pardalis in this study): our present study also demonstrated that the TTX amount in the intestinal materials from toxic egg-fed T. niphobles was significantly higher than that from non-egg-fed specimens. We noted this ingestion of eggs in four consecutive years (2012e2015), leading us to believe that this is part of the normal diet of these fish; indeed, it is possible that other Takifugu pufferfish species also adopt a similar strategy for toxification. The findings in this study perhaps remove one of several objections to postulated pathways of TTX accumulation in pufferfish via the marine food web (Miyazawa and Noguchi, 2001; Food Safety Commission of Japan, 2005; Wu et al., 2005; Wang et al., 2008; Yang et al., 2010). In TTX-bearing newts in the genus Taricha, adults prey on both conspecific eggs and larvae (Kats et al., 1992; Elliott et al., 1993), suggesting that these cannibalistic egg/ larva consumptions may also play a role in their toxification. Interestingly, the eggs removed from the intestines of T. niphobles hatched after incubation for 9 days at 20 C. This suggests that the pufferfish T. niphobles ingested the T. pardalis eggs within one week of spawning, implying that they ingest highly concentrated TTX via the eggs, because the toxicity of the eggs is known to be highest immediately after spawning (Itoi et al., 2014). Nevertheless, the TTX content in the intestinal eggs from T. niphobles were relatively low, suggesting that the TTX was immediately absorbed in intestine and transfer to other tissues such as liver, skin and ovary: the speculation was supported by the results of toxification experiment using T. rubripes in the present study. The spawning period of T. niphobles begins in May, suggesting that the pufferfish obtain TTX and nutrients required for their forthcoming spawning, when they feed on T. pardalis eggs (in the spring). Toxic organisms such as pufferfish use TTX in two ways: as a defense mechanism against predators by releasing it from the toxin gland in the skin (Kodama et al., 1985), and protecting the larvae by accumulating it in the ovaries (Hanifin et al., 2003; Itoi et al., 2014), and in predation on non-toxic organisms, such as the blue-ringed octopus Hapalochlaena maculosa (Sheumack et al., 1978) and in polyclad flatworms (Ritson-Williams et al., 2006). On the other hand, presentation of TTX against TTX-bearing predators appears to induce the opposite effect, where TTX has been reported as an attractant in trumpet snails (Hwang et al., 2004) and T. rubripes (Saito et al., 1997; Okita et al., 2013). Thus, concentrated TTX can serve a dual function: one as a chemical defense against non-toxic
predators, and the other as an attractant for TTX-bearing organisms. The latter relationship would result in the accumulation of TTX at higher trophic levels in the food chain (Fig. 4). Additionally, we speculate that the biomass of TTX-bearing organisms might be controlled (limited) by the amount of TTX in the marine environment, because the toxin content of TTX-bearing organisms could still be limited by available resources (Williams, 2010). In this study, the amount of TTX in the toxified T. rubripes was found to be a function of the size (surface area) of young fish, suggesting that the largest young fish were also the ones with the greatest amount of TTX. On the other hand, some specimens of T. niphobles, the smallest species in the genus, contained a large amount of TTX (>100,000 MU in one individual; Tani, 1945). It has also been suggested that the maximum toxin content of the pufferfish might be affected by the various physiological factors including sexual development (Ikeda et al., 2010; Itoi et al., 2012). In the toxification experiments, no TTX was detected from several specimens, suggesting that these specimens did not feed upon the toxic eggs in the aquaria. Differences in the feeding behavior among pufferfish should affect the distribution of toxicity in wild pufferfish specimens; in fact, the toxicity of pufferfish does show remarkable individual and regional variation (Miyazawa and Noguchi, 2001). In conclusion, predation on toxic eggs of related species should enable Takifugu pufferfish to accumulate TTX more efficiently than if dependent only on the classical food chain. Our results also suggest that concentrated TTX is pooled in the “TTX loop” among the TTX-bearing organisms, which are the predators at higher trophic levels in the food web.
Acknowledgments We express sincere thanks to the staff of Kanagawa Prefectural Marine Science High School for help in collecting samples. We also express sincere thanks to K.I., N.T., R.M., N.Y., S.T., S.D. and T.O. of Nihon University College of Bioresource Sciences for help in collecting samples and rearing the pufferfish. This study was supported in part by Research Grants for 2010 and 2013 from the Nihon University College of Bioresource Sciences (S.I.), Grant-in-Aid for Young Scientists (A) from Japan Society for the Promotion of Science (JSPS) (23688023, S.I.), Grant-in-Aid for challenging Exploratory Research from JSPS (26660177, S.I.), and Grant-in-Aid for Scientific Research (B) from JSPS (15H04552, S.I.).
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Conflict of interest The authors declare that there are no conflicts of interest. Ethical statement The authors declare that this manuscript complies with the Elsevier Ethical Guidelines for Journal Publication. All animal procedures comply with the Japanese Government Animal Protection and Management Law (No. 105) and Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2015.10.009. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.toxicon.2015.10.009. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389e3402. Asakawa, M., Ito, K., Kajihara, H., 2013. Highly toxic ribbon worm Cephalothrix simula containing tetrodotoxin in Hiroshima Bay, Hiroshima Prefecture, Japan. Toxin 5, 376e395. Cardall, B.L., Brodie, E.D., Hanifin, C.T., 2004. Secretion and regeneration of tetrodotoxin in the rough-skin newt (Taricha granulosa). Toxicon 44, 933e938. Colquhon, D., Henderson, R., Ritchie, J.M., 1972. The binding of labeled tetrodotoxin to non-myelineated nerve fibres. J. Physiol. 227, 95e126. Do, H.K., Kogure, K., Simidu, U., 1990. Identification of deep-sea-sediment bacteria which produce tetrodotoxin. Appl. Environ. Microbiol. 56, 1162e1163. Elliott, S.A., Kats, L.B., Breeding, J.A., 1993. The use of conspecific chemical cues for cannibal avoidance in California newts (Taricha torosa). Ethology 95, 186e192. Food Safety Commission of Japan, 2005. Food Safety Risk Assessment Concerning “Liver of Takifugu rubripes Cultivated by the Method Proposed by Saga Prefecture and Ureshinocho in the Prefecture under the Law on Special Zones for Structural Reform (Law No. 189, 2002)” (Risk Assessment Reports e Natural toxins/mycotoxins). Hanifin, C.T., Brodie III, E.D., Brodie Jr., E.D., 2002. Tetrodotoxin levels of the roughskin newt, Taricha granulosa, increase in long-term captivity. Toxicon 40, 1149e1153. Hanifin, C.T., Brodie III, E.D., Brodie Jr., E.D., 2003. Tetrodotoxin levels in eggs of the rough-skin newt, Taricha granulosa, are correlated with female toxicity. J. Chem. Ecol. 29, 1729e1739. Honda, S., Arakawa, O., Takatani, T., Tachibana, K., Yagi, M., Tanigawa, A., Noguchi, T., 2005. Toxification of cultured puffer fish Takifugu rubripes by feeding on tetrodotoxin-containing diet. Nippon. Suisan Gakkaishi 71, 815e820. Hwang, P.A., Noguchi, T., Hwang, D.F., 2004. Neurotoxin tetrodotoxin as attractant for toxic snails. Fish. Sci. 70, 1106e1112. Ikeda, K., Murakami, Y., Emoto, Y., Ngy, L., Taniyama, S., Yagi, M., Takatani, T., Arakawa, O., 2009. Transfer profile of intramuscularly administrated tetrodotoxin to non-toxic cultured specimens of the pufferfish Takifugu rubripes. Toxicon 53, 99e103. Ikeda, K., Emoto, Y., Tatsuno, R., Wang, J.J., Ngy, L., Taniyama, S., Takatani, T., Arakawa, O., 2010. Maturation-associated changes in toxicity of the pufferfish Takifugu poecilonotus. Toxicon 55, 289e297. Itoi, S., Yoshikawa, S., Tatsuno, R., Suzuki, M., Asahina, K., Yamamoto, S., Takanashi, S., Takatani, T., Arakawa, O., Sakakura, Y., Sugita, H., 2012. Difference in the localization of tetrodotoxin between the female and male pufferfish Takifugu niphobles, during spawning. Toxicon 60, 1000e1004. Itoi, S., Yoshikawa, S., Asahina, K., Suzuki, M., Ishizuka, K., Takimoto, N., Mitsuoka, R., Yokoyama, N., Detake, A., Takayanagi, C., Eguchi, M., Tatsuno, R., Kawane, M., Kokubo, S., Takanashi, S., Miura, A., Suitoh, K., Takatani, T., Arakawa, O., Sakakura, Y., Sugita, H., 2014. Larval pufferfish protected by maternal tetrodotoxin. Toxicon 78, 35e40. Kats, L.B., Elliott, S.A., Currens, J., 1992. Intraspecific oophagy in stream-breeding California newts Taricha torosa. Herpetol. Rev. 23, 7e8. Kim, Y.H., Brown, G.B., Mosher, H.S., Fuhrman, F.A., 1975. Tetrodotoxin: occurrence in atelopid frogs of costa rica. Science 189, 151e152. Kodama, M., Ogata, T., Sato, S., 1985. External secretion of tetrodotoxin from puffer fishes stimulated by electric shock. Mar. Biol. 87, 199e202. Maruyama, J., Noguchi, T., Jeon, J.K., Harada, T., Hashimoto, K., 1984. Occurrence of
tetrodotoxin in the starfish Astropecten latespinosus. Experimentia 40, 1395e1396. Maruyama, J., Noguchi, T., Narita, H., Jeon, J.K., Otsuka, M., Hashimoto, K., 1985. Occurrence of tetrodotoxin in a starfish, Astropecten scoparius. Agric. Biol. Chem. 49, 3069e3070. Matsui, T., Hamada, S., Konosu, S., 1981. Difference in accumulation of puffer fish toxin and crystalline tetrodotoxin in the puffer fish, Fugu rubripes rubripes. Bull. Jpn. Soc. Sci. Fish. 47, 535e537. Matsui, T., Sato, H., Hamada, S., Shimizu, C., 1982. Comparison of toxicity of the cultured and wild puffer fish Fugu niphobles. Bull. Jpn. Soc. Sci. Fish. 48, 253. Miyazawa, K., Noguchi, T., 2001. Distribution and origin of tetrodotoxin. J. Toxicol. Toxin Rev. 20, 11e33. Mosher, H.S., Fuhrman, G.J., Fuhrman, F.A., Fischer, H.G., 1964. Tarichatoxin-tetrodotoxin, a potent neurotoxin. Science 144, 1100e1110. Narahashi, T., 2001. Pharmacology of tetrodotoxin. J. Toxicol. Toxin Rev. 20, 67e84. Noguchi, T., Arakawa, O., 2008. Tetrodotoxin e distribution and accumulation in aquatic organisms, and cases of human intoxication. Mar. Drugs 6, 220e242. Noguchi, T., Arakawa, O., Takatani, T., 2006a. TTX accumulation in pufferfish. Comp. Biochem. Physiol. D 1, 145e152. Noguchi, T., Arakawa, O., Takatani, T., 2006b. Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land. Comp. Biochem. Physiol. D 1, 153e157. Noguchi, T., Hwang, D.F., Arakawa, O., Sugita, H., Deguchi, Y., Shida, Y., Hashimoto, K., 1987. Vibrio alginolyticus, a tetrodotoxin-producing bacterium, in the intestine of the fish Fugu vermicularis vermicularis. Mar. Biol. 94, 625e630. Noguchi, T., Maruyama, J., Ueda, Y., Hashimoto, K., Harada, T., 1981. Occurrence of tetrodotoxin in the Japanese ivory shell Babylonia japonica. Bull. Jpn. Soc. Sci. Fish. 47, 901e913. Noguchi, T., Uzu, A., Koyama, K., Hashimoto, K., 1983. Occurrence of tetrodotoxin as the major toxin in the xanthid crab Atergatis floridus. Bull. Jpn. Soc. Sci. Fish. 49, 1887e1892. Okita, K., Yamazaki, H., Sakiyama, K., Yamane, H., Niina, S., Takatani, T., Arakawa, O., Sakakura, Y., 2013. Puffer smells tetrodotoxin. Ichthyol. Res. 60, 386e389. Palumbi, S., Martin, A., Romano, S., McMillan, W.O., Stice, L., Grabowski, G., 1991. The Simple Fool's Guide to PCR, Version 2. Department of Zoology and Kewalo Marine Laboratory, University of Hawaii, Honolulu. Ritson-Williams, R., Yotsu-Yamashita, M., Paul, V.J., 2006. Ecological functions of tetrodotoxin in a deadly polyclad flatworm. Proc. Natl. Acad. Sci. U. S. A. 103, 3176e3179. Saito, T., Kageyu, K., Goto, H., Murakami, K., Noguchi, T., 1997. Tetrodotoxin attracts pufferfish (‘torafugu’, Takifugu rubripes). Toxicon 35, 489. Saito, T., Maruyama, J., Kanoh, S., Jeon, J.-K., Noguchi, T., Harada, T., Murata, O., Hashimoto, K., 1984. Toxicity of the cultured pufferfish Fugu rubripes rubripes along with their resistibility against tetrodotoxin. Bull. Jpn. Soc. Sci. Fish. 50, 1573e1575. Sezaki, K., Begum, R.A., Wongrat, P., Srivastava, M.P., SriKantha, S., Kikuchi, K., Ishihara, H., Tanaka, S., Taniuchi, T., Watabe, S., 1999. Molecular phylogeny of Asian freshwater and marine stingrays based on the DNA nucleotide and deduced amino acid sequence of the cytochrome b gene. Fish. Sci. 65, 563e570. Sheumack, D.D., Howden, M.E., Spence, I., Quinn, R.J., 1978. Maculotoxin: a neurotoxin from the venom glands of the octopus Hapalochlaena maculosa identified as tetrodotoxin. Science 188e189. Simidu, U., Noguchi, T., Hwang, D.F., Shida, Y., Hashimoto, K., 1987. Marine bacteria which produce tetrodotoxin. Appl. Environ. Microbiol. 53, 1714e1715. Shinno, M., Nabae, Y., Higashi, T., Oka, Y., Takechi, T., Ogasawara, M., Oseto, M., Inouye, H., 2007. Determination of tetrodotoxin by liquid chromatography with tandem mass spectrometry in the case of puffer fish poisoning. Annu. Rep. Ehime Pref. Inst. Publ. Health Environ. Sci. 10, 14e17. Stokes, A.N., Ducey, P.K., Neuman-Lee, L., Hanifin, C.T., French, S.S., Pfrender, M.E., Brodie III, E.D., Brodie Jr., E.D., 2014. Confirmation and distribution of tetrodotoxin for the first time in terrestrial invertebrates: two terrestrial flatworm species (Bipalium adventitium and Bipalium kewense). PLoS One 9, e100718. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725e2729. Tani, I., 1945. Toxicological Studies on Japanese Puffers. Teikoku Tosho, Tokyo, p. 103. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673e4680. Tsuruda, K., Arakawa, O., Kawatsu, K., Hamano, Y., Takatani, T., Noguchi, T., 2002. Secretory glands of tetrodotoxin in the skin of the Japanese newt Cynops pyrrhogaster. Toxicon 40, 131e136. Williams, B.L., 2010. Behavioral and chemical ecology of marine organisms with respect to tetrodotoxin. Mar. Drugs 8, 381e398. Wu, Z., Xie, L., Xia, G., Zhang, J., Nie, Y., Hu, J., Wang, S., Zhang, R., 2005. A new tetrodotoxin-producing actinomycete, Nocardiopsis dassonvillei, isolated from the ovaries of puffer fish Fugu rubripes. Toxicon 45, 851e859. Wang, X.J., Yu, R.C., Luo, X., Zhou, M.J., Lin, X.T., 2008. Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus. Toxicon 52, 55e61. Yamamori, K., Kono, M., Furukawa, K., Matsui, T., 2004. The toxification of juvenile cultured kusafugu Takifugu niphobles by oral administration of crystalline tetrodotoxin. J. Food Hyg. Soc. Jpn. 45, 73e75. Yang, G., Xu, J., Liang, S., Ren, D., Yan, X., Bao, B., 2010. A novel TTX-producing Aeromonas isolated from the ovary of Takifugu obscurus. Toxicon 56, 324e329.
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Toxic Takifugu pardalis eggs found in Takifugu niphobles gut: Implications for TTX accumulation in the pufferfish Shiro Itoi a, *, Ao Kozaki a, Keitaro Komori a, Tadasuke Tsunashima a, Shunsuke Noguchi a, 1, Mitsuo Kawane b, Haruo Sugita a a b
Department of Marine Science and Resources, Nihon University, Fujisawa, Kanagawa 252-0880, Japan Department of Sea-Farming, Aichi Fish Farming Institute, Tahara, Aichi 441-3618, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 July 2015 Received in revised form 30 September 2015 Accepted 14 October 2015 Available online 19 October 2015
Pufferfish (Takifugu spp.) possess a potent neurotoxin, tetrodotoxin (TTX). TTX has been detected in various organisms including food animals of pufferfish, and TTX-producing bacteria have been isolated from these animals. TTX in marine pufferfish accumulates in the pufferfish via the food web starting with marine bacteria. However, such accumulation is unlikely to account for the amount of TTX in the pufferfish body because of the minute amounts of TTX produced by marine bacteria. Therefore, the toxification process in pufferfish still remains unclear. In this article we report the presence of numerous Takifugu pardalis eggs in the intestinal contents of another pufferfish, Takifugu niphobles. The identity of T. pardalis being determined by direct sequencing for mitochondrial DNA. LC-MS/MS analysis revealed that the peak detected in the egg samples corresponded to TTX. Toxification experiments in recirculating aquaria demonstrated that cultured Takifugu rubripes quickly became toxic upon being fed toxic (TTXcontaining) T. rubripes eggs. These results suggest that T. niphobles ingested the toxic eggs of another pufferfish T. pardalis to toxify themselves more efficiently via a TTX loop consisting of TTX-bearing organisms at a higher trophic level in the food web. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Congeneric eggs Food chain Pufferfish Takifugu niphobles Takifugu pardalis Tetrodotoxin
1. Introduction Tetrodotoxin (TTX) is known to be the substance of pufferfish toxin and one type of potent neurotoxin specific to voltage-gated sodium channels of excitable membranes of muscle and nerve tissues (Colquhon et al., 1972; Narahashi, 2001; Noguchi et al., 2006a). TTX was believed to occur only in pufferfish (Tetraodontidae) until 1960's, when it was detected in Californian newt Taricha torosa (Mosher et al., 1964). Subsequently, TTX (along with some analogs) was also detected from potential pufferfish food organisms belonging to various disparate groups, including starfish (e.g., Astropecten spp.; Maruyama et al., 1984, 1985), gastropods (e.g., Babylonia japonica; Noguchi et al., 1981), crustaceans (e.g., the xanthid crab, Atergatis floridus; Noguchi et al., 1983), flatworms and ribbonworms (e.g., Cephalothrix simula; Asakawa et al., 2013), apart from several species of bacteria that are symbiotic with the
* Corresponding author. E-mail address: [email protected] (S. Itoi). 1 Present address: Kyoto Institute of Oceanic and Fishery Science, Miyazu, Kyoto 626-0052, Japan.
pufferfish and the potential food organisms (Noguchi et al., 1987; Wu et al., 2005; Noguchi and Arakawa, 2008). In addition, TTX has been detected in some free-living bacteria, including those in deep sea sediments (Simidu et al., 1987; Do et al., 1990), although it is not clear if these bacteria form part of the food chain leading to pufferfish. In any case, it appears plausible that the TTX in pufferfish is a result of accumulation through the food chain, which consists of several steps, starting with bacteria, as suggested by several reports (Noguchi et al., 2006a; Noguchi and Arakawa, 2008). These speculations have actually been supported by several studies: non-toxic pufferfish have been produced when grown from hatching with a non-toxic diet, and furthermore, these cultured non-toxic pufferfish have become toxic when administered orally with TTX (Matsui et al., 1981, 1982; Noguchi et al., 2006b; Saito et al., 1984; Yamamori et al., 2004; Honda et al., 2005). TTX has been detected not only in pufferfish and their prey, but also in organisms ecologically unrelated to pufferfish, such as the Costa Rican frogs of the genus Atelopus (Kim et al., 1975) and some land planarians (Stokes et al., 2014), besides Californian newt T. torosa (Mosher et al., 1964). It has also been suggested that TTX in the rough-skin newt, Taricha granulosa, is obtained endogenously
http://dx.doi.org/10.1016/j.toxicon.2015.10.009 0041-0101/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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(Hanifin et al., 2002; Cardall et al., 2004). These findings suggest that TTX accumulates in pufferfish by means other than through the classical food chain; since in vivo cultured TTX-producing bacteria are unable to produce enough quantities of TTX to account for the amount of TTX in wild pufferfish (Miyazawa and Noguchi, 2001; Food Safety Commission of Japan, 2005; Wu et al., 2005; Wang et al., 2008; Yang et al., 2010). This also indicates that TTX-bearing pufferfish prey are abundant. In any case, the origin of TTX and the acquisition process are both likely to vary across different animal species, and it remains unclear exactly where pufferfish acquire the large quantities of TTX that they possess. Recently, our lab unexpectedly found numerous eggs among the intestinal contents of the pufferfish, Takifugu niphobles, and partial mitochondrial DNA sequences from these eggs identified them to be those of another pufferfish species, namely, Takifugu pardalis. We have demonstrated the toxification process using cultured pufferfish Takifugu rubripes in this study by means of experimentally reproducing the serendipitous finding of eggs in the pufferfish gut, thus indicating that pufferfish toxification manifests from the accumulated TTX at relatively higher trophic levels in the food chain.
extraction and the remaining eggs were stored at 30 C until TTX extraction. 2.2. DNA extraction and PCR amplification Total genomic DNA was extracted from each egg by the method of Sezaki et al. (1999). The fragments of partial mitochondrial DNA were amplified by PCR using two primer sets, 16S AR-L (forward: 50 CGCCT GTTTA TCAAA AACAT-30 ) and 16S BR-H (reverse: 50 -CCGGT CTGAA CTCAG ATCAC GT-30 ) for the 16S rRNA gene (Palumbi et al., 1991), and TCytb-F1 (forward: 50 -ACCTR TGGCG TGAAA AACCA YCGTT GT-30 ) and TCytb-R1 (reverse: 50 -CATYC GGTTT ACAAG ACCGR CGCTC TG-30 ) for the cytochrome b gene. Primers for cytochrome b gene were designed based on the mitochondrial DNA sequences from multiple species of the family Tetraodontidae. PCR amplification was performed in a 20 ml reaction mixture containing genomic DNA as a template, 1 unit ExTaq DNA polymerase (Takara Bio, Shiga, Japan), 1.6 ml of 2.5 mM deoxynucleotide triphosphates (dNTP), 5 ml of 5 mM primers and 2 ml of 10 ExTaq DNA polymerase buffer (Takara Bio). The thermal cycling program for the PCR consisted of an initial denaturation at 95 C for 1 min followed by 35 cycles of denaturation at 95 C for 10 s, annealing at 55 C for 30 s and extension at 72 C for 45 s.
2. Materials and methods 2.3. Direct sequencing and phylogenetic analyses 2.1. The intestinal contents of wild pufferfish Wild specimens of the pufferfish, T. niphobles (body weight: 60.3 ± 25.7 g and 48.9 ± 21.2 g for females and males, respectively; detailed in Table 1) were collected from coastal waters in Nagai, Kanagawa, Japan (35120 N, 139 360 E), on 13 March 2012 (water temperature: 13.9 C; salinity: 31.4 practical salinity unit, psu), 08 March 2013 (16.0 C; 32.4 psu) and 14 March 2013 (14.2 C; 30.6 psu), 07 March 2014 (12.5 C; 32.3 psu) and 13 March 2014 (12.5 C; 31.8 psu), and 09 March 2015 (11.7 C; 34.3 psu), 16 March 2015 (12.3 C; 34.8 psu) and 19 March 2015 (14.6 C; 35.0 psu). The gonadosomatic index (GSI) was 2.2 ± 0.9 for females and 2.0 ± 1.4 for males (detailed in Table 1). The unexpected eggs that were found among the gut contents of the pufferfish were also collected. Five eggs from each fish were immediately subjected to DNA
Prior to the direct sequencing of the amplified product, the DNA fragment was purified by chloroform extraction, followed by polyethylene glycol (PEG) 8000 precipitation and ethanol precipitation. Sequencing was performed for both strands using a 3130xl genetic analyzer (Applied Biosystems, Foster, CA, USA) and a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems). The concatenated nucleotide sequences of the 16S rRNA gene and cytochrome b gene of eggs were aligned using CLUSTAL W (Thompson et al., 1994) with those in the DDBJ/EMBL/ GenBank databases obtained using a BLAST search (Altschul et al., 1997). The alignment was then subjected to phylogenetic inference by means of the maximum likelihood method using MEGA ver. 6.0.6 (Tamura et al., 2013), with the corresponding concatenated sequences from the yellow-stripe toadfish, Torquigener brevipennis
Table 1 Characteristics of the Takifugu niphobles specimens used in this studya. Sampling date 2012 13 Mar 2013 08 Mar 14 Mar 2014 07 Mar 13 Mar 2015 09 Mar 16 Mar 19 Mar a b c d
Sex
No. of specimen
Female n ¼ 8 Male n ¼ 2 Female Male Female Male
n n n n
¼ ¼ ¼ ¼
9 13 6 8
Male n ¼ 2 Female n ¼ 10 Male n ¼ 6 Female Male Female Male Female Male
n n n n n n
¼ ¼ ¼ ¼ ¼ ¼
13 10 11 4 4 4
No. of egg-fed specimen
Intestinal egg content (g)
Toxicity of the intestinal eggs (MU)
Standard length (mm)
Body weight (g)
GSIb
n¼8 n¼2
N/Dc N/D
N/D N/D
119.4 ± 3.8 111.0 ± 2.0
64.1 ± 9.4 54.5 ± 1.5
2.6 ± 0.5 1.5 ± 0.2
¼ ¼ ¼ ¼
0.28 0.05 0.72 0.26
2.63 ± 4.42 0.25 ± 0.74 10.38 ± 12.94 4.10 ± 6.48
112.7 108.4 122.2 110.8
0.44 ± 0.30 0.38 ± 0.62 0.09 ± 0.12
0.29 ± 0.17 0.49 ± 0.66 0.06 ± 0.13
128.0 ± 1.0 121.7 ± 17.2 111.3 ± 12.5
N/Ad 0.05 ± 0.38 ± 0.08 ± 0.47 ± N/A
N/A 2.72 ± 7.04 15.84 ± 30.37 1.63 ± 2.32 3.30 ± 2.46 N/A
118.7 115.6 164.6 140.0 130.0 133.0
n n n n
8 7 6 8
n¼2 n¼7 n¼3 n n n n n n
¼ ¼ ¼ ¼ ¼ ¼
0 3 8 2 4 0
± ± ± ±
0.25 0.09 0.48 0.36
0.08 0.57 0.08 0.37
Data are represented by mean ± standard deviation. GSI represents gonadosomatic index: gonad weight/body weight 100. N/D, not determined. N/A, not applicated.
± ± ± ±
± ± ± ± ± ±
18.3 17.7 16.7 11.1
12.8 17.4 17.6 26.0 18.1 22.5
60.3 55.2 56.2 55.4
± ± ± ±
22.8 22.0 24.5 12.2
73.3 ± 3.6 63.9 ± 23.8 50.0 ± 17.4 33.0 30.8 87.3 59.0 43.5 45.5
± ± ± ± ± ±
9.7 15.4 20.9 28.0 16.0 18.1
2.1 1.9 2.0 2.7
± ± ± ±
0.8 1.1 0.4 0.8
2.6 ± 0.3 2.4 ± 1.3 3.0 ± 2.1 1.9 1.3 2.5 2.1 2.5 2.0
± ± ± ± ± ±
0.5 1.3 0.7 1.1 1.6 1.2
S. Itoi et al. / Toxicon 108 (2015) 141e146
(DNA database accession number AP009537) as the outgroup. The nucleotide sequences of the partial 16S rRNA gene and cytochrome b gene obtained from the eggs were submitted to the DDBJ/EMBL/ GenBank databases with accession numbers LC060064 and LC060065, respectively. 2.4. Toxification experiment Toxic eggs for the toxification experiments were obtained from the pufferfish T. rubripes, which had been collected at Ise Bay, Aichi, Japan, and maintained in the aquarium at the Aichi Sea Farming Institute with flowing water (5.2 t/h). The non-toxic juveniles of T. rubripes (body weight: approximately 3.5 g) were purchased from Marinetech (Aichi, Japan), and were maintained in recirculating aquariums at 20 C, and fed with commercial food pellets (nontoxic). In the toxification experiment, juvenile T. rubripes (n ¼ 52, body weight: 3.1e49.6 g, 19.0 ± 12.7 g) were fed with the toxic eggs. After more than two days of feeding with toxic eggs, liver, skin, muscle and other organ samples of egg-fed T. rubripes and commercial pellet-fed control specimens were subjected to the TTX extraction process followed by LC-MS/MS analysis. In advance before the toxification experiment, preliminary experiment was carried out: after two, four and nine days of feeding with toxic eggs, egg-fed T. rubripes specimens (n ¼ 27, body weight: 14.8 ± 3.6 g) were also subjected to the TTX extraction followed by LC-MS/MS analysis. 2.5. LC-MS/MS analysis Following Tsuruda et al. (2002) with some modifications, the extract with 0.1% acetic acid was filtered through a 0.45-mm filter membrane (Dismic-13CP, Advantec, Japan), and TTX in the extract was analyzed using High Performance Liquid Chromatography (HPLC) equipment ACQUITY system (Alliance 2695), coupled to a Quattro Premier XE mass spectrometer from Waters (Milford, MA, USA), based on the method of Shinno et al. (2007) with some modifications. Chromatographic separation and detection of TTX was achieved with a Waters Atlantis HILIC silica column (2.1 150 mm, 5 mm), equipped with a Waters Atlantis HILIC silica guard column (2.1 10 mm, 5 mm) and column oven at 40 C. The HPLC was operated with 0.1% formic acid (Merck, Darmstadt, Germany) and acetonitrile (Merck) as eluents. The gradient protocol used to elute the toxins was 95% acetonitrile, mobile phase in the beginning, decreasing to 40% acetonitrile after 0.1 min, then kept for 7.9 min and back to 95% acetonitrile and finally kept 95% acetonitrile for 2.0 min before the next injection. Flow rate was 0.3 ml/min and injection volume was 5 ml. The Quattro Premier XE mass spectrometer was operated with the following optimized source-dependent parameters (ESI source): capillary potential 0.5 kV, cone voltage 42 V, desolvation temperature 400 C, desolvation gas flow 600 l/h N2, cone gas flow 50 l/h N2, source temperature 120 C, collision energy 38 V. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode, with detection in the positive mode, analyzing two product ions at m/z 162 for quantification of TTX and m/z 302 for confirmation of the compound from the precursor ion at m/z 320. The ionized molecules were monitored through a MassLynx NT operation system. TTX was quantified using their peak areas to calculate amounts and using the calibration curve obtained from 1 to 100 ng/ml TTX standard (Wako Pure Chemicals, Osaka, Japan). One mouse unit (MU) is equivalent to 0.22 mg of TTX, based on the specific toxicity of TTX. 2.6. Statistical analysis The statistical significance of differences in the amount of toxin
143
was analyzed by means of a Student's t-test and a one-way ANOVA followed by the Tukey Honestly Significant Difference (HSD) test. In order to evaluate statistical support values for the linear regression, results of toxicity from toxification experiments were logtransformed and subjected to ANOVA. 3. Results 3.1. Eggs from the intestinal contents of the pufferfish T. niphobles A number of eggs, with a diameter of approximately 1 mm, were detected in the gut contents of the pufferfish T. niphobles (Fig. S1) captured in March 2012e2015 (Table 1). The eggs were seen in the 08 March 2013 sample in 8 and 7 specimens of 9 females and 13 males, respectively. The eggs were detected in all the specimens in the 14 March 2013 sample. The eggs were seen in 7 and 5 specimens of 10 females and 8 males, respectively, in 2014, and in 12 and 5 specimens of 28 females and 18 males, respectively, in 2015 (Table 1). In a few fish, the eggs were yet to be digested in the anterior parts of the intestine that interestingly, hatched after incubation at 20 C in artificial seawater for 9 days (March 2013). Direct sequencing and phylogenetic analysis of egg samples from the intestinal contents of T. niphobles demonstrated that the sequence of 16S rRNA gene and cytochrome b gene were highly similar to and clustered with the corresponding sequences of T. pardalis (Fig. 1). 3.2. Toxicity of the eggs in the intestinal contents of T. niphobles and tissue-distribution of TTX in T. niphobles fed T. pardalis-eggs Eggs were collected from the intestines of T. niphobles, subjected to TTX extraction, and analyzed using LC-MS/MS. Mass chromatogram of the LC-MS/MS was obtained under the MRM mode, and the MRM patterns of the egg samples were found to be identical to those of the TTX standards (Fig. 2). The calibration curve generated with 1e100 ng/ml of TTX standard shows good linearity and
100 Eggs 100
Takifugu pardalis (AP009528)
67
Takifugu chrysops (AP009525) Takifugu xanthopterus (AP009533)
67
Takifugu ocellatus (AP009536) Takifugu niphobles (AP009526) 88
Takifugu snyderi (AP009531)
79 100
Takifugu stictonotus (AP009530)
Takifugu porphyreus (AP009529) Takifugu rubripes (AP006045)
63 100
Takifugu chinensis (AP009534) Takifugu oblongus (AP009535) Torquigener brevipennis (AP009537)
0.02 Fig. 1. Phylogenetic tree inferred using the maximum likelihood method based on the nucleotide sequence of the concatenation of the complete cytochrome b gene (1137 bp) and the partial 16S rRNA gene (572 bp), obtained from the eggs removed from the intestinal tract of Takifugu niphobles and those of other Takifugu pufferfish species. Numbers at branches denote the bootstrap percentages out of 1000 replicates. The concatenated cytochrome b and 16S rRNA genes in Torquigener brevipennis (AP009537) was used as the outgroup. Only bootstrap values exceeding 50% are presented. The scale at the bottom of the tree indicates the number of substitutions per nucleotide site. “Eggs” represent the corresponding concatenation of the two gene sequences of the eggs taken from the intestinal tract of T. niphobles. The accession numbers for reference sequences are shown in parentheses.
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precision (r2 ¼ 0.99991). To estimate the effect of the matrix, LCMS/MS analysis was carried out using the samples, where the TTX standard was added into various tissue extracts from the nontoxic T. rubripes following the procedure mentioned above. TTX was recovered from the samples with >100-fold dilution except for gallbladder (>1000-fold dilutions) (Fig. S2). Therefore, quantification of TTX was carried out using the data for samples with >100fold dilution. The concentrations of TTX in the eggs from intestinal contents of the samples in 2012 were calculated to be 6.58 ± 0.88 mg/g, which correspond to 29.9 ± 4.0 MU/g; however, the contents of the eggs were not quantified because all eggs were not collected (Table 1). The concentration of TTX in the eggs collected from the gut of the 2013 specimens of T. niphobles was estimated to be 2.22 ± 4.51 mg/g (0.97 ± 1.83 mg/intestinal contents per fish), which corresponds to 10.1 ± 20.5 MU/g (4.4 ± 8.3 MU/ intestinal contents per fish). Similar patterns of the concentration of TTX in the eggs from intestinal contents of the T. niphobles were observed in the samples from each year (2013e2015; Table S1). Additionally, the TTX amount in the intestinal materials from eggfed T. niphobles specimens (4139 ± 6023 ng) were significantly higher than those from non-egg-fed counterparts (216 ± 374 ng) in 2015 (Table S2).
150000
100000
(A) Standard (25 ng/ml)
50000
0 (B) Intestinal eggs
Signal intensity
50000
The whole-body toxicity of the T. niphobles specimens fed with T. pardalis eggs was 2803 ± 10,361 MU (617 ± 2279 mg) for females and 1901 ± 1856 MU (418 ± 408 mg) for males (Table S1). The tissuespecific toxicity in females was significantly higher in the skin and ovaries than in the liver (P < 0.01), whereas in the males it was significantly higher in the skin than in other tissues (P < 0.01; Fig. S3). Similar patterns of toxin distribution were observed in the samples from each year (2013e2015; Fig. S3). 3.3. Intoxication of the pufferfish T. rubripes fed toxic eggs In preliminary toxification experiment, no significant difference in the toxicity of whole body was observed among the T. rubripes specimens fed with TTX-containing eggs after being fed toxic eggs for two days (98 ± 109 ng/g), four days (139 ± 296 ng/g) and nine days (154 ± 270 ng/g), suggesting that the pufferfish keeps the toxin level for a while (at least nine days; Fig. S4). Therefore, toxification experiments were performed after being fed toxic eggs for more than two days. Non-toxic T. rubripes (n ¼ 31, body weight: 3.1e49.6 g, 21.9 ± 12.8 g) became toxic after being fed toxic eggs for more than two days, although some specimens (n ¼ 21, body weight: 3.1e42.0 g, 14.7 ± 11.1 g) continued to remain non-toxic. The MRM patterns of the toxified tissues from the intestine, liver and skin were identical to those from the TTX extracted from the toxic eggs and the TTX standards (Fig. 2). The amount of toxin detected from several organs of toxified specimens was as follows: skin, 47.5 ± 38.1 mg/g; liver, 31.8 ± 31.6 mg/g; and intestine, 19.9 ± 29.0 mg/ g (Fig. S5). The amount of TTX in toxified fish was dependent on its body weight in an exponential relationship (y ¼ 0.9566e0.1307x, R2 ¼ 0.6402; Fig. 3) with high statistical support values for the linear regression (P ¼ 5.46 107, F statistics ¼ 44.49). On the other hand, none of the control fish fed with commercial feed were toxified (control; n ¼ 38, body weight: 3.1e57.9 g, 15.6 ± 15.6 g). Furthermore, the fish that did not ingest the toxic eggs even when given were not toxified either. 4. Discussion
0 (C) Eggs of T. rubripes
50000
0 (D) Skin of intoxicated T. rubripes
50000
Non-toxic T. rubripes were rapidly toxified after feeding on eggs containing TTX. Similar profiles of TTX transfer were observed in the cultured T. rubripes when TTX was administrated intramuscularly (Ikeda et al., 2009). All species of the genus Takifugu accumulate TTX in the ovaries, with the ovaries of T. pardalis showing the maximum toxicity of more than 1000 MU/g tissue, which corresponds to approximately 220 mg TTX/g tissue (classified as “strongly toxic”, Noguchi et al., 2006a). These suggest that
0 (E) Liver of non-toxic T. rubripes
50000
0 0
2
4
6
8
10
Retention time (min) Fig. 2. Typical mass chromatograms of the LC-MS/MS obtained under MRM mode (m/z 320 > 302). MRM patterns of 25 ng/ml TTX standard (A), the extract from eggs found in the intestinal tract of T. niphobles (B), the extract from the toxic eggs of T. rubripes used for the intoxication experiment (C), the extract from the skin of T. rubripes after the intoxication experiment (D), and the extract from the liver of non-toxic T. rubripes cultured with only commercial feed (E).
Total amount of TTX (MU)
10000 y = 0.9566e0.1307x R2 = 0.6402
1000 100 10 1 0.1
nd 0
10
20
30 Body weight (g)
40
50
60
Fig. 3. The relationship between the total amount of TTX in a specimen and the body weight of T. rubripes after the experiment with toxic eggs. The amount of TTX was calculated from the skin, liver, intestine and other tissues. nd: not detected.
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145
TTX
Pufferfish
TTX
TTX
TTX
TTX
TTX
Food animals for pufferfish
TTX
Marine bacteria
Fig. 4. TTX accumulation process in pufferfish on the TTX loop. Upward arrows represent general TTX accumulation process in pufferfish via food chain consisting of several steps starting with marine bacteria (Noguchi et al., 2006a). A circular arrow represents novel TTX accumulation process in Takifugu niphobles (colored fish) at the spawning season of Takifugu pardalis (grey color fish) observed in this study.
T. niphobles effectively ingest TTX along with the nutrients in the toxic eggs of other pufferfish (T. pardalis in this study): our present study also demonstrated that the TTX amount in the intestinal materials from toxic egg-fed T. niphobles was significantly higher than that from non-egg-fed specimens. We noted this ingestion of eggs in four consecutive years (2012e2015), leading us to believe that this is part of the normal diet of these fish; indeed, it is possible that other Takifugu pufferfish species also adopt a similar strategy for toxification. The findings in this study perhaps remove one of several objections to postulated pathways of TTX accumulation in pufferfish via the marine food web (Miyazawa and Noguchi, 2001; Food Safety Commission of Japan, 2005; Wu et al., 2005; Wang et al., 2008; Yang et al., 2010). In TTX-bearing newts in the genus Taricha, adults prey on both conspecific eggs and larvae (Kats et al., 1992; Elliott et al., 1993), suggesting that these cannibalistic egg/ larva consumptions may also play a role in their toxification. Interestingly, the eggs removed from the intestines of T. niphobles hatched after incubation for 9 days at 20 C. This suggests that the pufferfish T. niphobles ingested the T. pardalis eggs within one week of spawning, implying that they ingest highly concentrated TTX via the eggs, because the toxicity of the eggs is known to be highest immediately after spawning (Itoi et al., 2014). Nevertheless, the TTX content in the intestinal eggs from T. niphobles were relatively low, suggesting that the TTX was immediately absorbed in intestine and transfer to other tissues such as liver, skin and ovary: the speculation was supported by the results of toxification experiment using T. rubripes in the present study. The spawning period of T. niphobles begins in May, suggesting that the pufferfish obtain TTX and nutrients required for their forthcoming spawning, when they feed on T. pardalis eggs (in the spring). Toxic organisms such as pufferfish use TTX in two ways: as a defense mechanism against predators by releasing it from the toxin gland in the skin (Kodama et al., 1985), and protecting the larvae by accumulating it in the ovaries (Hanifin et al., 2003; Itoi et al., 2014), and in predation on non-toxic organisms, such as the blue-ringed octopus Hapalochlaena maculosa (Sheumack et al., 1978) and in polyclad flatworms (Ritson-Williams et al., 2006). On the other hand, presentation of TTX against TTX-bearing predators appears to induce the opposite effect, where TTX has been reported as an attractant in trumpet snails (Hwang et al., 2004) and T. rubripes (Saito et al., 1997; Okita et al., 2013). Thus, concentrated TTX can serve a dual function: one as a chemical defense against non-toxic
predators, and the other as an attractant for TTX-bearing organisms. The latter relationship would result in the accumulation of TTX at higher trophic levels in the food chain (Fig. 4). Additionally, we speculate that the biomass of TTX-bearing organisms might be controlled (limited) by the amount of TTX in the marine environment, because the toxin content of TTX-bearing organisms could still be limited by available resources (Williams, 2010). In this study, the amount of TTX in the toxified T. rubripes was found to be a function of the size (surface area) of young fish, suggesting that the largest young fish were also the ones with the greatest amount of TTX. On the other hand, some specimens of T. niphobles, the smallest species in the genus, contained a large amount of TTX (>100,000 MU in one individual; Tani, 1945). It has also been suggested that the maximum toxin content of the pufferfish might be affected by the various physiological factors including sexual development (Ikeda et al., 2010; Itoi et al., 2012). In the toxification experiments, no TTX was detected from several specimens, suggesting that these specimens did not feed upon the toxic eggs in the aquaria. Differences in the feeding behavior among pufferfish should affect the distribution of toxicity in wild pufferfish specimens; in fact, the toxicity of pufferfish does show remarkable individual and regional variation (Miyazawa and Noguchi, 2001). In conclusion, predation on toxic eggs of related species should enable Takifugu pufferfish to accumulate TTX more efficiently than if dependent only on the classical food chain. Our results also suggest that concentrated TTX is pooled in the “TTX loop” among the TTX-bearing organisms, which are the predators at higher trophic levels in the food web.
Acknowledgments We express sincere thanks to the staff of Kanagawa Prefectural Marine Science High School for help in collecting samples. We also express sincere thanks to K.I., N.T., R.M., N.Y., S.T., S.D. and T.O. of Nihon University College of Bioresource Sciences for help in collecting samples and rearing the pufferfish. This study was supported in part by Research Grants for 2010 and 2013 from the Nihon University College of Bioresource Sciences (S.I.), Grant-in-Aid for Young Scientists (A) from Japan Society for the Promotion of Science (JSPS) (23688023, S.I.), Grant-in-Aid for challenging Exploratory Research from JSPS (26660177, S.I.), and Grant-in-Aid for Scientific Research (B) from JSPS (15H04552, S.I.).
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Conflict of interest The authors declare that there are no conflicts of interest. Ethical statement The authors declare that this manuscript complies with the Elsevier Ethical Guidelines for Journal Publication. All animal procedures comply with the Japanese Government Animal Protection and Management Law (No. 105) and Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2015.10.009. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.toxicon.2015.10.009. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389e3402. Asakawa, M., Ito, K., Kajihara, H., 2013. Highly toxic ribbon worm Cephalothrix simula containing tetrodotoxin in Hiroshima Bay, Hiroshima Prefecture, Japan. Toxin 5, 376e395. Cardall, B.L., Brodie, E.D., Hanifin, C.T., 2004. Secretion and regeneration of tetrodotoxin in the rough-skin newt (Taricha granulosa). Toxicon 44, 933e938. Colquhon, D., Henderson, R., Ritchie, J.M., 1972. The binding of labeled tetrodotoxin to non-myelineated nerve fibres. J. Physiol. 227, 95e126. Do, H.K., Kogure, K., Simidu, U., 1990. Identification of deep-sea-sediment bacteria which produce tetrodotoxin. Appl. Environ. Microbiol. 56, 1162e1163. Elliott, S.A., Kats, L.B., Breeding, J.A., 1993. The use of conspecific chemical cues for cannibal avoidance in California newts (Taricha torosa). Ethology 95, 186e192. Food Safety Commission of Japan, 2005. Food Safety Risk Assessment Concerning “Liver of Takifugu rubripes Cultivated by the Method Proposed by Saga Prefecture and Ureshinocho in the Prefecture under the Law on Special Zones for Structural Reform (Law No. 189, 2002)” (Risk Assessment Reports e Natural toxins/mycotoxins). Hanifin, C.T., Brodie III, E.D., Brodie Jr., E.D., 2002. Tetrodotoxin levels of the roughskin newt, Taricha granulosa, increase in long-term captivity. Toxicon 40, 1149e1153. Hanifin, C.T., Brodie III, E.D., Brodie Jr., E.D., 2003. Tetrodotoxin levels in eggs of the rough-skin newt, Taricha granulosa, are correlated with female toxicity. J. Chem. Ecol. 29, 1729e1739. Honda, S., Arakawa, O., Takatani, T., Tachibana, K., Yagi, M., Tanigawa, A., Noguchi, T., 2005. Toxification of cultured puffer fish Takifugu rubripes by feeding on tetrodotoxin-containing diet. Nippon. Suisan Gakkaishi 71, 815e820. Hwang, P.A., Noguchi, T., Hwang, D.F., 2004. Neurotoxin tetrodotoxin as attractant for toxic snails. Fish. Sci. 70, 1106e1112. Ikeda, K., Murakami, Y., Emoto, Y., Ngy, L., Taniyama, S., Yagi, M., Takatani, T., Arakawa, O., 2009. Transfer profile of intramuscularly administrated tetrodotoxin to non-toxic cultured specimens of the pufferfish Takifugu rubripes. Toxicon 53, 99e103. Ikeda, K., Emoto, Y., Tatsuno, R., Wang, J.J., Ngy, L., Taniyama, S., Takatani, T., Arakawa, O., 2010. Maturation-associated changes in toxicity of the pufferfish Takifugu poecilonotus. Toxicon 55, 289e297. Itoi, S., Yoshikawa, S., Tatsuno, R., Suzuki, M., Asahina, K., Yamamoto, S., Takanashi, S., Takatani, T., Arakawa, O., Sakakura, Y., Sugita, H., 2012. Difference in the localization of tetrodotoxin between the female and male pufferfish Takifugu niphobles, during spawning. Toxicon 60, 1000e1004. Itoi, S., Yoshikawa, S., Asahina, K., Suzuki, M., Ishizuka, K., Takimoto, N., Mitsuoka, R., Yokoyama, N., Detake, A., Takayanagi, C., Eguchi, M., Tatsuno, R., Kawane, M., Kokubo, S., Takanashi, S., Miura, A., Suitoh, K., Takatani, T., Arakawa, O., Sakakura, Y., Sugita, H., 2014. Larval pufferfish protected by maternal tetrodotoxin. Toxicon 78, 35e40. Kats, L.B., Elliott, S.A., Currens, J., 1992. Intraspecific oophagy in stream-breeding California newts Taricha torosa. Herpetol. Rev. 23, 7e8. Kim, Y.H., Brown, G.B., Mosher, H.S., Fuhrman, F.A., 1975. Tetrodotoxin: occurrence in atelopid frogs of costa rica. Science 189, 151e152. Kodama, M., Ogata, T., Sato, S., 1985. External secretion of tetrodotoxin from puffer fishes stimulated by electric shock. Mar. Biol. 87, 199e202. Maruyama, J., Noguchi, T., Jeon, J.K., Harada, T., Hashimoto, K., 1984. Occurrence of
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