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Article Phylogeny and Toxin Profile of Freshwater Pufferfish ( ) Collected from 2 Different Regions in Cambodia

Hongchen Zhu 1, Akinori Yamada 1 , Yui Goto 2, Linan Horn 3, Laymithuna Ngy 3, Minoru Wada 1, Hiroyuki Doi 4, Jong Soo Lee 5, Tomohiro Takatani 1 and Osamu Arakawa 1,* 1 Graduate School of Fisheries and Environmental Sciences, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan; [email protected] (H.Z.); [email protected] (A.Y.); [email protected] (M.W.); [email protected] (T.T.) 2 Faculty of Fisheries, Nagasaki University. 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan; [email protected] 3 Faculty of Agronomy, University of Kratie, National Road No. 7, Orussey District, Kratie City, Kratie Province, Cambodia; [email protected] (L.H.); [email protected] (L.N.) 4 Nifrel, Osaka Aquarium Kaiyukan. 2-1, Senribanpakukoen, Suita, Osaka 565-0826, Japan; [email protected] 5 College of Marine Science, Gyeongsang National University, 2, Tongyeonghaean-ro, Tongyeong, Kyungnam 53064, ; [email protected] * Correspondence: [email protected]; Tel.: +81-95-819-2844

 Received: 15 October 2020; Accepted: 28 October 2020; Published: 30 October 2020 

Abstract: The classification of Cambodian freshwater pufferfish is incomplete and confusing, and scientific information on their and toxin profile is limited. In the present study, to accumulate information on the phylogeny and toxin profile of freshwater pufferfish, and to contribute to food safety in Cambodia, we conducted simultaneous genetic-based phylogenetic and toxin analyses using freshwater pufferfish individuals collected from Phnom Penh and Kratie (designated PNH and KTI, respectively). Phylogenetic analysis of partial sequences of three mitochondrial genes (cytochrome b, 16S rRNA, and cytochrome c oxidase subunit I) determined for each fish revealed that PNH and KTI are different species in the genus Pao (designated Pao sp. A and Pao sp. B, respectively). A partial sequence of the nuclear tributyltin-binding protein type 2 (TBT-bp2) gene differentiated the species at the amino acid level. Instrumental analysis of the toxin profile revealed that both Pao sp. A and Pao sp. B possess (STXs), comprising STX as the main component. In Pao sp. A, the toxin concentration in each tissue was extremely high, far exceeding the regulatory limit for STXs set by the Codex Committee, whereas in Pao sp. B, only the contained high toxin concentrations. The difference in the STX accumulation ability between the two species with different TBT-bp2 sequences suggests that TBT-bp2 is involved in STX accumulation in freshwater pufferfish.

Keywords: Cambodia; pufferfish; Pao; phylogenetic analysis; tributyltin-binding protein type 2 (TBT-bp2); (STX)

Key Contribution: Werevealed a gene-based phylogeny linked to the toxicity profile of the Cambodian freshwater pufferfish, and suggest for the first time that tributyltin-binding protein type 2 is involved in saxitoxin accumulation in freshwater pufferfish.

1. Introduction Pufferfish of the family contain (TTX) and/or saxitoxins (STXs), but the toxin ratio differs depending on the genus or species. Marine pufferfish of the genus inhabiting

Toxins 2020, 12, 689; doi:10.3390/toxins12110689 www.mdpi.com/journal/toxins Toxins 2020, 12, 689 2 of 15 the coastal waters of Japan and Dichtomyctere (formerly known as ) living in the brackish waters of have TTX as their main toxin component [1–3], whereas freshwater pufferfish of the genera Pao and Leiodon (both formerly known as Tetraodon), which inhabit Southeast Asian countries, generally have only STXs [4–7]. On the other hand, pufferfish from Florida, and and pufferfish from the , Japanese coastal waters, or the have both TTX and STXs [8–12]. The toxin ratio varies depending on the species, but STXs are dominant in many cases. Marine pufferfish of the genus Takifugu may have small amounts of STXs in addition to TTX [13–15]. Many studies examining the toxification mechanism of pufferfish with TTX revealed that pufferfish do not biosynthesize TTX, but accumulate it through the food chain that starts with TTX-producing [1]. The accumulated TTX is gradually eliminated if supply via toxic food is not continued [16]. We recently conducted various in vivo TTX administration experiments using nontoxic pufferfish artificially raised with nontoxic diets, and found that the internal TTX kinetics in pufferfish are unique: intestine skin/ovary, and change as the pufferfish grows and matures [17–22]. → → The pufferfish STX- and TTX-binding protein (PSTBP) isolated from the plasma of Takifugu pardalis by Yotsu-Yamashita et al. [23] is thought to be involved in the internal TTX kinetics, i.e., the absorption, transportation, and accumulation of TTX inside the pufferfish body [24]. PSTBP homologous proteins are widely distributed in TTX-bearing toxic pufferfish of the genera Takifugu and Arothron, but have not been detected in common fish or in nontoxic species of pufferfish [25–27]. The accumulation mechanism of STXs in pufferfish, however, remains unclear, but is presumed to be exogenous via the food chain that starts with STX-producing dinoflagellates in marine environments and STX-producing cyanobacteria in freshwater environments [6,9,11,28]. In a recent in vivo TTX/STX administration experiment using nontoxic cultured pufferfish, Gao et al. [29] found that T. pardalis, which naturally contains TTX, selectively accumulates TTX, and Pao suvattii, which naturally contains STXs, selectively accumulates STXs. In other words, the ratio of TTX/STX in pufferfish appears to depend more strongly on the inherent TTX/STX selectivity of the pufferfish than the prevalence of TTX/STX in prey organisms, but the molecular mechanisms underlying the toxin selectivity remain to be elucidated. Our ongoing and transcriptome analyses indicate that the three freshwater pufferfish species, Pao abei, P. suvattii, and , do not have the PSTBP gene (unpublished data), suggesting that PSTBP is not involved in STX accumulation or selectivity of the genus Pao. Several species of freshwater pufferfish inhabit the rivers and lakes of Cambodia. These pufferfish are considered delicious and there have been many cases of poisoning due to the ingestion of pufferfish caught with other generally edible freshwater fish. According to the Ministry of Health in Cambodia, at least seven poisoning incidents occurred due to freshwater pufferfish consumption from 2017 to 2019, with more than 40 people poisoned and 5 deaths. The Codex Committee on and Fishery Products has set the regulatory limit for STXs as 0.8 mg STX diHCl eq/kg edible tissue [30], · but there is no strict food safety standard for the edible use of freshwater pufferfish in Cambodia. This may be partly due to the fact that the species classification of Cambodian indigenous pufferfish is incomplete and quite confusing, and the limited scientific information on the toxicity and toxin profile. In the present study, to accumulate genetic information on the of freshwater pufferfish and the TTX/STX distribution profiles in pufferfish of the family Tetraodontidae, and to contribute to food safety in Cambodia, we conducted simultaneous genetic-based phylogenetic and toxin analyses using freshwater pufferfish specimens collected from two different regions in Cambodia. Moreover, to explore candidate molecules other than PSTBP involved in the absorption, transportation, and accumulation of STXs in freshwater pufferfish, a partial sequence of a putative origin of the molecular evolution of PSTBP [31,32], tributyltin-binding protein type 2 (TBT-bp2), and four commonly used genes (cytochrome b [CYTB], 16S rRNA, cytochrome c oxidase subunit I [COI], and rhodopsin) were used as targets in the phylogenetic analysis. ToxinsToxins 2020, 12, x 689 FOR PEER REVIEW 33 of of 15 15

2. ResultsToxins 2020, 12, x FOR PEER REVIEW 3 of 15 2. Results 2. Results 2.1.2.1. Phylogeny Phylogeny 2.1. Phylogeny AA total total of of 16 16 freshwater freshwater pufferfish pufferfish were were sampled sampled from from small small lakes lakes near near Phnom Phnom Penh Penh (PNH) (PNH) and and KratieKratie (KTI) (KTI)A total (Figures (Figures of 16 freshwater 11 andand2 ,2, and pufferfishand Table Table wereA1 A1).). sampled PartialPartial from sequencessequences small lakes ofof the thenear threethree Phnom mitochondrial mitochondrial Penh (PNH) and genes,genes, CYTB,CYTB,Kratie 16S 16S (KTI)rRNA, rRNA, (Figures and and COI, COI, 1 and were were 2, and determined determined Table A1). forPa forrtial each each sequences sample ofand and the phylogenetically three mitochondrial analyzed analyzed genes, for for taxonomictaxonomicCYTB, identification. identification.16S rRNA, and Phylogenetic COI, Phylogenetic were determined analysis analysis offor 870-bp of each 870-bp sample CYTB CYTB sequencesand sequencesphylogenetically showed showed that analyzed thatthe PNH the for PNH and taxonomic identification. Phylogenetic analysis of 870-bp CYTB sequences showed that the PNH and KTIand sequences KTI sequences each formed each formed a tight cluster a tight together cluster togetherwith Pao withcochinchinensisPao cochinchinensis (AB741980.1)(AB741980.1) or Pao baileyi or KTI sequences each formed a tight cluster together with Pao cochinchinensis (AB741980.1) or Pao baileyi (AB741978.1)Pao baileyi (AB741978.1) (Figure 3). Similar (Figure 3results). Similar were results obtained were from obtained analyses from of analysesconcatenated of concatenated datasets of (AB741978.1) (Figure 3). Similar results were obtained from analyses of concatenated datasets of CYTB+16Sdatasets of rRNA CYTB +(55516S rRNAbp) and (555 CYTB+COI bp) and CYTB (621 +bp)COI (F (621igures bp) A1 (Figures and A2). A1 andConsidering A2). Considering the genetic the variationCYTB+16S of P. rRNAsuvattii (555 in bp)Figure and CYTB+COI3, each cluster (621 bp) seems (Figures to represent A1 and A2). a singleConsidering species, the thoughgenetic the geneticvariation variation of P. of suvattiiP. suvattii in Figurein Figure 3, each3, each cluster cluster seems seems to represent to represent a single a single species, species, though though the the intraspecific variation in the genus Pao might be unexpectedly large (see Pao leiurus and P. intraspecificintraspecific variation variation in the in genus the genusPao might Pao bemight unexpectedly be unexpectedly large (see largePao (see leiurus Paoand leiurusP.cochinchinensis and P. cochinchinensis in Figures 3, A1, and A2). Hereafter, we thus refer to the samples from PNH and KTI in Figurecochinchinensis3, Figure A1in Figures, and Figure 3, A1, andA2). A2). Hereafter, Hereafter, we we thus thus refer refer to to the the samplessamples from from PNH PNH and and KTI KTI as asPao Paosp.as sp. Pao A A and sp. and APao andPaosp. Paosp. B, sp.B, respectively. respectively.B, respectively.

Figure 1. Map showing the pufferfish sampling locations in Cambodia. Figure 1. Map showing the pufferfish sampling locations in Cambodia. Figure 1. Map showing the pufferfish sampling locations in Cambodia.

Figure 2. Typical pufferfish collected from Phnom Penh (A, PNH-1) and Kratie (B, KTI-2). Figure 2. Typical pufferfish collected from Phnom Penh (A, PNH-1) and Kratie (B, KTI-2). A 492 to 778 bp region of the nuclear rhodopsin gene was directly sequenced from the polymerase chain reaction (PCR) product of each sample. As no double peaks were observed in the Sanger sequence electropherograms,Figure 2. Typical the sequenced pufferfish collected regions from were Phnom considered Penh (A to, PNH-1) be homogeneous and Kratie (B in, KTI-2). all the samples. Furthermore, there was no sequence variation between any pair of samples. Compared with sequences of other Pao species in the database, all the mutations found were synonymous, and the amino acid sequence was identical among the Pao species examined.

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KTI-1, 2, 4, 5 Pao sp. B KTI-3 97/96 Pao baileyi (AB741978.1) 69/58 Pao abei (AB741977.1) 90/89 Pao abei (LC586270.1) 98/99 Pao leiurus (KF667490.1) Pao leiurus (GU057266.1) Pao leiurus (AB741985.1) 100/99 Pao cambodgiensis (JQ681921 1) 83/94 Pao suvattii (AB741991.1) 100/100 93/98 Pao suvattii (LC586271.1) Pao suvattii (JQ681934.1) 77/65 Pao cochinchinensis (JQ681922.1) Pao palembangensis (JQ681932.1)

100/100 Pao turgidus (AB741992.1) Pao leiurus (JQ681927.1) 54/55 61/93 Pao cochinchinensis (AP011925.1) PNH-1, 2, 4, 5, 6, 8, 9, 10, 11 100/100 PNH-7 Pao sp. A PNH-3 86/67 Pao cochinchinensis (AB741980.1) Pao palembangensis (AP011920.1) 100/100 Arothron manilensis (AP011929.1) Arothron hispidus (AP011930.1)

0.200 0.150 0.100 0.050 0.000 Figure 3. Phylogenetic tree of Pao species constructed by Bayesian inference (BI) and maximum likelihoodFigure (ML) 3. Phylogenetic based on the tree CYTB of Pao gene. species The constructed BI tree is shown. by Bayesian Node numbersinference (BI) are Bayesianand maximum posterior likelihood (ML) based on the CYTB gene. The BI tree is shown. Node numbers are Bayesian posterior probabilities (left) and ML bootstrap values (right). probabilities (left) and ML bootstrap values (right). A 298 to 536 bp region of the nuclear TBT-bp2 gene was determined for all the samples except A 492 to 778 bp region of the nuclear rhodopsin gene was directly sequenced from the KTI-3polymerase by direct sequencingchain reaction of (PCR) the PCR product products. of each sample. We observed As no double double peaks peaks were at severalobserved nucleotide in the positionsSanger in sequence the Sanger electropherograms, sequence electropherograms the sequenced regions of almost were allconsidered the samples, to be homogeneous indicating that in the TBT-bp2all genethe samples. had multiple Furthermore, alleles (Tablethere A2was). Alleleno sequ typesence werevariation apparently between similar any pair between of samples. samples of Pao sp.Compared A and Pao withsp. sequences B. Compared of other with Pao the species allele typesin the found database, in Pao allsp. theA mutations and Pao sp.found B, therewere was no samplesynonymous, in which and the theallele amino types acid sequence could be was represented identical among as a the hybridization Pao species examined. of Pao sp. A and Pao sp. B. AnA ML 298 phylogenetic to 536 bp region analysis of the nuclear clearly TBT-bp2 separated gene the was samples determined into for PNH all the and samples KTI, namely except Pao sp. AKTI-3 and Pao by directsp. B, sequencing as in the case of the of thePCR mitochondrial products. We observed genes (Figure double4 ).peaks Amino at several acid sequences nucleotide also positions in the Sanger sequence electropherograms of almost all the samples, indicating that the differed between Pao sp. A and Pao sp. B. Note that although we tried to obtain longer sequences, TBT-bp2 gene had multiple alleles (Table A2). Allele types were apparently similar between samples PCR amplifications were unsuccessful, probably due to the presence of microsatellite-like sequences in of Pao sp. A and Pao sp. B. Compared with the allele types found in Pao sp. A and Pao sp. B, there was neighboringno sample introns. in which the allele types could be represented as a hybridization of Pao sp. A and Pao sp. B. An ML phylogenetic analysis clearly separated the samples into PNH and KTI, namely Pao sp. A 2.2. Toxin Profile and Pao sp. B, as in the case of the mitochondrial genes (Figure 4). Amino acid sequences also differed Toxinsbetween were Pao extractedsp. A and fromPao sp. the B. skin,Note muscle,that although liver, we and trie gonadsd to obtain of each longer freshwater sequences, pu PCRfferfish. Analysisamplifications of the extracts were byunsuccessful, high-performance probably liquiddue to chromatographythe presence of microsatellite-like with post-column sequences fluorescence in neighboring introns. derivatization (HPLC-FLD) for STXs [12,33] revealed that the tissues of each individual, except for one or two tissues of KTI-1 and KTI-2, contained STXs comprising STX as the main component and decarbamoylSTX (dcSTX) as a minor component (typical chromatograms are shown in Figure A3). NeoSTX was also contained in some tissues as a minor component, but no other known STX components, such as C toxins and gonyautoxins (GTXs), were detected. When the same extracts were analyzed for TTX by liquid chromatography tandem mass spectrometry (LC-MS/MS) [12,34], no TTX was detected at all. Toxins 2020, 12, 689 5 of 15 Toxins 2020, 12, x FOR PEER REVIEW 5 of 15

99 KTI-5 KTI-4 Pao sp. B KTI-2 86 KTI-1 PNH-1 PNH-9 PNH-2, 3, 10 83 PNH-5, 6 Pao sp. A PNH-7 82 PNH-4 59 PNH-8

0.0200 0.0150 0.0100 0.0050 0.0000 Figure 4. Figure 4.Inter-sample Inter-sample relationships relationships of theof TBT-bp2the TBT-bp gene2 usinggene maximumusing maximum likelihood likelihood (ML). A midpoint(ML). A rooted tree is shown. Node numbers are ML bootstrap values. KTI-3 and PNH-11 were not included midpoint rooted tree is shown. Node numbers are ML bootstrap values. KTI-3 and PNH-11 were not due to their shorter sequences. included due to their shorter sequences. The toxin concentration (converted value to mg STX diHCl eq/kg) in each tissue from the pufferfish 2.2. Toxin Profile · is shown in Figure5 and Table A1. The toxin concentrations of Pao sp. A were generally high and far aboveToxins the regulatory were extracted limit for from STX (0.8 the mgskin, STX muscle,diHCl eqliver,/kg) and in all gonads tissues. of The each mean freshwater toxin concentration pufferfish. · inAnalysis the ovary of (58.7the extracts mg STX bydiHCl high-performance eq/kg) was the liqu highest,id chromatography followed by the skinwith (40.8 post-column mg STX diHCl fluorescence eq/kg). · · Thederivatization mean toxin (HPLC-FLD) concentration for in STXs the liver [12,33] (23.1 revealed mg STX thatdiHCl the eq tissues/kg) was of lowereach individual, than that in except the skin, for · althoughone or two in sometissues individuals, of KTI-1 and the KTI-2, toxin concentrationcontained STXs in thecomprising liver exceeded STX as that the inmain the component skin. The mean and toxindecarbamoylSTX concentration (dcSTX) in the muscleas a minor (22.3 component mg STX diHCl (typical eq/kg) chromatograms was the lowest, are but shown still almostin Figure 28-fold A3). · higherNeoSTX than was the also regulatory contained limit. in In some contrast, tissues the toxinas a concentrationsminor component, of Pao butsp. Bno were other generally known much STX lowercomponents, than those such of asPao C toxinssp. A. and The gonyautoxins mean toxin concentration(GTXs), were detected. in the skin When (6.0 the mg same STX diHClextracts eq were/kg) · wasanalyzed the highest, for TTX followed by liquid by chromatography the ovary (1.3 mg tandem STX diHCl mass spectrometry eq/kg). The mean (LC-MS/MS) toxin concentrations [12,34], no TTX in · thewas testis, detected liver, at and all. muscle were below the regulatory limit (0.5–0.6 mg STX diHCl eq/kg), but some · individualsThe toxin had concentration concentrations (converted that slightly value exceeded to mg the STX·diHCl regulatory limit.eq/kg) in each tissue from the pufferfishThe total is shown amount in ofFigure toxin 5 perand individual Table A1. isThe shown toxin inconcentrations Figure6. As describedof Pao sp. above,A were the generally toxin concentrationhigh and far above in each the tissue regulatory was much limit higher for STX in (0.8Pao mgsp. STX·diHCl A than in Pao eq/kg)sp. B,in butall tissues. due to theThe largermean individualtoxin concentration size of Pao insp. the B ovary over Pao (58.7sp. mg A (TableSTX·diHCl A1), the eq/kg) total was toxin the amount highest, of followed KTI-3–5 (172–417 by the skinµg STX(40.8diHCL mg STX·diHCl eq/individual) eq/kg). was The generallymean toxin comparable concentration to thatin the in liverPao sp.(23.1 A mg individuals STX·diHCl (122–463 eq/kg) wasµg · STXlowerdiHCL than that eq/ individual)in the skin, although other than in some PNH-1, indivi induals, which the the toxin toxin concentration concentration in the in liver the skinexceeded was · exceptionallythat in the skin. low. The The mean toxin distributiontoxin concentration profile in in the the body, muscle however, (22.3 dimgffered STX·diHCl between eq/kg)Pao sp. was A and the Paolowest,sp.B. but In stillPao almostsp. A,in 28-fold addition higher to the than skin, the the regu amountlatory oflimit. toxin In in contrast, the muscle the andtoxin ovary concentrations was high, whereasof Pao sp. in BPao weresp. generally B, most of much the toxin lower amount than those in the of body Pao sp. was A. accounted The mean for toxin by the concentration skin, irrespective in the ofskin the (6.0 sex. mg STX·diHCl eq/kg) was the highest, followed by the ovary (1.3 mg STX·diHCl eq/kg). The meanThe toxin toxin concentrations composition in in the each testis, tissue liver, per and species muscle is shownwere below in Figure the regu7. InlatoryPao limitsp. B, (0.5–0.6 the toxin mg concentrationsSTX·diHCl eq/kg), in the but muscle, some liver, individuals and gonads had wereconcentr veryations low, and that neoSTX slightly concentrations exceeded the were regulatory below thelimit. limit of quantification. Other than this, however, there was no considerable difference between Pao sp. A and Pao sp. B. In each tissue, most of the toxins were accounted for by STX. In the whole body, both Pao sp. A and Pao sp. B contained 6% to 7% neoSTX and 3% to 4% dcSTX as the minor components, in addition to the main component STX (~90%).

Toxins 2020, 12, x FOR PEER REVIEW 6 of 15

Toxins 2020, 12, 689 6 of 15 Toxins 2020, 12, x FOR PEER REVIEW 6 of 15

Figure 5. Toxin concentration in each tissue in each pufferfish collected from Phnom Penh (PNH, Pao sp. A) and Kratie (KTI, Pao sp. B). * The ovary was too small to analyze.

The total amount of toxin per individual is shown in Figure 6. As described above, the toxin concentration in each tissue was much higher in Pao sp. A than in Pao sp. B, but due to the larger individual size of Pao sp. B over Pao sp. A (Table A1), the total toxin amount of KTI-3–5 (172–417 µg STX·diHCL eq/individual) was generally comparable to that in Pao sp. A individuals (122–463 µg STX·diHCL eq/individual) other than PNH-1, in which the toxin concentration in the skin was exceptionally low. The toxin distribution profile in the body, however, differed between Pao sp. A and Pao sp. B. In Pao sp. A, in addition to the skin, the amount of toxin in the muscle and ovary was Figure 5. Toxin concentration in each tissue in each pufferfish collected from Phnom Penh (PNH, high, whereasFigure 5. in Toxin Pao concentration sp. B, most in ofeach the tissue toxin in each amount pufferfish in collectedthe body from was Phnom accounted Penh (PNH, for Pao by the skin, Pao sp. A) and Kratie (KTI, Pao sp. B). * The ovary was too small to analyze. irrespectivesp. A) of and the Kratie sex. (KTI, Pao sp. B). * The ovary was too small to analyze.

The total amount of toxin per individual is shown in Figure 6. As described above, the toxin concentration in each tissue was much higher in Pao sp. A than in Pao sp. B, but due to the larger individual size of Pao sp. B over Pao sp. A (Table A1), the total toxin amount of KTI-3–5 (172–417 µg STX·diHCL eq/individual) was generally comparable to that in Pao sp. A individuals (122–463 µg STX·diHCL eq/individual) other than PNH-1, in which the toxin concentration in the skin was exceptionally low. The toxin distribution profile in the body, however, differed between Pao sp. A and Pao sp. B. In Pao sp. A, in addition to the skin, the amount of toxin in the muscle and ovary was high, whereas in Pao sp. B, most of the toxin amount in the body was accounted for by the skin, irrespective of the sex.

Figure 6. Total toxin amount per individual pufferfish collected from Phnom Penh (PNH, Pao sp. A) Figure 6. Total toxin amount per individual pufferfish collected from Phnom Penh (PNH, Pao sp. A) and Kratie (KTI, Pao sp. B). * The ovary was too small to analyze. and Kratie (KTI, Pao sp. B). * The ovary was too small to analyze.

Figure 6. Total toxin amount per individual pufferfish collected from Phnom Penh (PNH, Pao sp. A) and Kratie (KTI, Pao sp. B). * The ovary was too small to analyze.

Toxins 2020, 12, x FOR PEER REVIEW 7 of 15

The toxin composition in each tissue per species is shown in Figure 7. In Pao sp. B, the toxin concentrations in the muscle, liver, and gonads were very low, and neoSTX concentrations were below the limit of quantification. Other than this, however, there was no considerable difference between Pao sp. A and Pao sp. B. In each tissue, most of the toxins were accounted for by STX. In the whole body, both Pao sp. A and Pao sp. B contained 6% to 7% neoSTX and 3% to 4% dcSTX as the Toxinsminor2020 components,, 12, 689 in addition to the main component STX (~90%). 7 of 15

Figure 7. Toxin composition in each tissue in Pao sp. A (left) and Pao sp. B (right). S = skin, M = muscle, LFigure= liver, 7. GToxin= gonads, composition and WB in= eachwhole tissue body. in Pao sp. A (left) and Pao sp. B (right). S = skin, M = muscle, L = liver, G = gonads, and WB = whole body. 3. Discussion 3. DiscussionThe present study revealed that freshwater pufferfish collected from two regions of Cambodia, PhnomThe Penh present and study Kratie, revealed are diff erentthat freshwater species belonging pufferfish to collected the genus fromPao two(Pao regionssp. A and of Cambodia,Pao sp. B, respectively),Phnom Penh and Kratie, both possess are different STXs, species with Pao belongingsp. A having to the highgenus toxicity Pao (Pao and sp.Pao A andsp. BPao having sp. B, lowrespectively), toxicity. and both possess STXs, with Pao sp. A having high toxicity and Pao sp. B having low toxicity.The CYTB analysis suggests that Pao sp. A is the same species as P. cochinchinensis (AB741980.1), whereasThe CYTB CYTB sequences analysis suggests of other thatP. cochinchinensis Pao sp. A is thespecimens same species (AP011925.1, as P. cochinchinensis JQ681922.1) (AB741980.1), are distantly relatedwhereas to CYTBPao sp. sequences A (Figure of other3), which P. cochinchinensis makes it di specimensfficult to identify (AP011925.1, species JQ681922.1) with DNA are barcoding distantly methods.related to AccordingPao sp. A (Figure to Igarashi 3), which et al. [makes35], at it least difficult one ofto theidentify CYTB species sequences with is DNA likely barcoding an error resultingmethods. fromAccording the misidentification to Igarashi et ofal. specimens.[35], at least Likewise, one of thePao CYTBsp. B issequences closely related is likely to P.an baileyi error asresulting well as from to P. the abei misidentificationand P. leiurus (KF667490.1,of specimens. GU057266.1)Likewise, Pao (Figuresp. B is 3closely), and related could beto P. the baileyi same as specieswell as to as P. one abei of and these. P. leiurus The (KF667490.1, phylogenetic GU057266.1) positions of (FigureP. leiurus 3),, and however, could be largely the same differ species among as specimensone of these. (Figure The 3phylogenetic). As discussed positions in Igarashi of P. etleiurus al. [ ,35 however,], these situations largely differ are mostamong likely specimens due to similarities(Figure 3). inAs the discussed morphologic in Igarashi characteristics, et al. [35], geographic these situations distribution, are most and likely ecology due of to the similaritiesPao species. in Ourthe mitochondrialmorphologic characteristics, gene analyses indicated geographic the presencedistribution, of actual and andecology/or potential of the genetic Pao species. boundaries Our ofmitochondrial species (i.e., gene species-level analyses clustersindicated in the the presence phylogenetic of actual trees), and/or suggesting potential thatgenetic an accumulationboundaries of ofspecies genetic (i.e., information species-level could clusters contribute in the to phylogen resolve theetic taxonomictrees), suggesting status and that relationships an accumulation among of Paogeneticspecies. information On the other could hand, contribute if crossbreeding to resolve occurredthe taxonomic in the statusPao species, and relationships an introgression among of thePao mitochondrialspecies. On the genome other mighthand, have if crossbreeding caused such discordance occurred in between the Pao morphologic species, an speciesintrogression identification of the andmitochondrial mitochondrial genome DNA phylogeny.might have Crossbreedingcaused such isdiscordance frequently observedbetween inmorphologic the marine Takifuguspecies puidentificationfferfish species and (e.g., mitochondrial Takahashi et DNA al., 2017 phylogeny. [36]), and Crossbreeding an introgression is offrequently the mitochondrial observed genome in the frommarine the Takifugu freshwater pufferfishRhinogobius speciesgoby (e.g., species Takahashi was reported et al., [37 ].2017 As demonstrated[36]), and an byintrogression the analysis of of thethe TBT-bp2mitochondrial gene, thisgenome possibility from should the freshwater be carefully Rhinogobius considered ingoby parallel species to taxonomic was reported reexamination, [37]. As thoughdemonstrated no evidence by the was analysis found of for the the TBT-bp2 presence gene, of crossbreeding this possibility between shouldPao be carefullysp. A and consideredPao sp. B. in parallelThe to toxin taxonomic concentration reexamination, in each though tissue di noffered evidence greatly was between found forPao thesp. presence A and Pao of crossbreedingsp. B: it was generallybetween Pao much sp. higherA and Pao in Pao sp. sp.B. A than in Pao sp. B. Ngy et al. [7] examined the toxicity of two Cambodian freshwater pufferfish species, Pao turgidus and Pao sp., during the rainy and dry seasons, and reported that only the skin and ovary of P. turgidus were toxic in both seasons. The highest toxicity scores of the skin and ovary were 37 mouse unit (MU)/g (5.5 mg STX diHCl eq/kg) and 27 MU/g · (4.0 mg STX diHCl eq/kg), respectively, which indicate low levels of toxicity, similar to Pao sp. B. · Ngy et al. [7] did not conduct a genetic analysis, and while there is some uncertainty in species identification, it is assumed that several species of low-toxicity freshwater pufferfish inhabit Cambodia. The Bangladeshi L. cutcutia (highest toxicity score of the most toxic tissue, the skin, 20 MU/g (3.0 mg Toxins 2020, 12, 689 8 of 15

STX diHCl eq/kg)) [6] could also be considered a low-toxicity species, similar to Pao sp. B. According to · Kungsuwan et al. [4], the Thai freshwater pufferfish P. leiurus and P. suvattii are both highly toxic, with the highest mean toxicity scores of the skin, muscle, liver, and ovary ranging from 61 to 109 MU/g (9.1–16 mg STX diHCL eq/kg) in P. leiurus and 42 to 200 MU/g (6.3–30 mg STX diHCL eq/kg) in P. suvattii. · · The toxin concentration of Pao sp. A is comparable to or even higher than that of P. leiurus/P. suvattii, and this species is one of the most highly toxic species among freshwater pufferfish. The total toxin amount per fish was generally similar between Pao sp. A and Pao sp. B, with the highest score exceeding 400 µg STX diHCL eq/individual in both species. As the minimum lethal dose · for humans is approximately 400–1000 µg STX diHCL eq [38,39], ingestion of one–two whole bodies of · these freshwater pufferfish can cause death by poisoning. Therefore, both species should be considered extremely dangerous to eat. In Pao sp. B, however, the majority of the harbored toxin was distributed in the skin, and the toxin concentrations of the other tissues were below or only slightly above the regulatory limit. Therefore, at least with the individuals examined in this study, removing the skin may decrease the possibility of poisoning. In addition to individual differences, regional and seasonal variations are observed in pufferfish toxicity [1,40]. Careful accumulation of data on the phylogeny and toxin profile of freshwater pufferfish, with such considerations in mind, may help to establish a taxonomic system linked with toxicity profiles and the presentation of species that can be consumed by removing toxic parts and other treatments. In both Pao sp. A and Pao sp. B, the main toxin was STXs comprising STX as the main component, and neoSTX and dcSTX as the minor components, and TTX was not detected at all. The toxin of P. turgidus from Cambodia and Pao freshwater pufferfish from Thailand is similarly composed of STX-dominated STXs and does not contain TTX [4,5,7]. There are some exceptional cases, such as the case of the Bangladeshi L. cutcutia in which a unique STX derivative (carbamoyl-N-methylSTX) is the main component [6,41], but even in the case of marine pufferfish that simultaneously possess STXs and TTX, the main component of STXs is often STX [9,11,13–15]. The origins of STXs in Floridian Sphoeroides and Philippine Arothron marine pufferfish are considered to be STX-producing dinoflagellates Pyrodinium bahamense and P. bahamense var. compressum, respectively, based on similarities in the toxin composition [8,9]. Although the origin of STXs in freshwater pufferfish is presumed to be STX-producing cyanobacteria [6], little is known about the toxicity of cyanobacteria in the freshwater environments of Cambodia, and the origin of STXs in Cambodian freshwater pufferfish remains to be elucidated. The STX distribution profiles clearly differed between Pao sp. A and Pao sp. B. In contrast to TTX, of which the absorption, transportation, and accumulation in marine pufferfish have been studied with respect to candidate molecules involved in these processes (i.e., PSTBP), such molecules for STXs remain unknown regardless of the marine or freshwater pufferfish species. PSTBP genes in marine pufferfish obviously evolved as a result of the fusion of two TBT-bp2 genes at the sixth and first exons, and the sequences remain quite similar to TBT-bp2 genes on the same genome (Hashiguchi et al. 2015 [32]; Yamada et al., unpublished). TBT-bp2 was originally identified as a protein that binds the environmental toxin TBT in the fish body [31]. These findings together suggest that the TBT-bp2 gene could be one of the molecules involved in the kinetics of STX in the pufferfish body. As expected from the observed difference in the STX distribution profiles between Pao sp. A and Pao sp. B, we found different amino acid sequences of TBT-bp2 between Pao sp. A and Pao sp. B, which could result in interspecific differences in the structure and function of TBT-bp2. Given the identical amino acid sequence of the rhodopsin gene in Pao species, including Pao sp. A and Pao sp. B, the TBT-bp2 gene might be one of the genes that determines the physiologic and ecologic characteristics of the host. A more comprehensive analysis of the expression, distribution, and function of PSTBP/TBT-bp2 isoforms coupled with TTX/STX distribution profiles could provide a better understanding of the molecular mechanisms and evolutionary processes of TTX/STX accumulation in Tetraodontidae. Toxins 2020, 12, 689 9 of 15

4. Materials and Methods

4.1. Pufferfish Specimens In November 2019, 11 (PNH-1–11) and 5 (KTI-1–5) freshwater pufferfish were sampled from a small lake connected by a channel to the Mekong River near Phnom Penh and Kratie, Cambodia, respectively (Figures1 and2, and Table A1). The specimens were transported alive to the laboratory at the University of Kratie, where PNH-1–3 were dissected to obtain the skin (including the fins), muscle, liver, and gonads. Each tissue was placed in a plastic bag, frozen, and transported to the laboratory at Nagasaki University. The other specimens were frozen as whole bodies and transported to the laboratory at Nagasaki University, where they were similarly dissected. Each tissue was stored at 80 C until DNA extraction or toxin quantification. − ◦ 4.2. DNA Extraction, PCR Amplification, and Sequencing Total DNA was extracted from an approximately 5 5 mm piece of fin using a standard DNA × lysis solution containing proteinase K. The 3 mitochondrial genes, CYTB, 16S rRNA, and COI, and 2 nuclear genes, rhodopsin and TBT-bp2, were PCR-amplified using the following primers: cytball-Fl and cytball-Rl for CYTB [35], 16sar-L and 16sar-H for 16S rRNA [42], FF2d and FR1d for COI [43], Rh193, Rh545, and Rh1039r for rhodopsin [44], and TBTBP2EX1F: 50-AAC CAG CGC TKC TSC TGC TG-30 and TBTBP2EX4R: 50-TTC TCC TCT GTC AGG ACT CC-30 for TBT-bp2 (this study). The PCR amplification was carried out in a reaction volume of 25 µL containing 12.5 µL of 2 Ampirect Plus, × 0.125 µL of BIOTAQ DNA Polymerase (TaKaRa Bio Inc., Kusatsu, Japan), 500 nM each of forward primer and reverse primer, 10 µL of 10-fold diluted DNA, and the remaining volume made up by nuclease-free water. The PCR conditions were as follows: initial denaturation at 94 ◦C for 2 min, 35 cycles of amplification with each cycle containing 94 ◦C for 30 s, 52 ◦C for 40 s (16S rRNA, COI) or 50 ◦C for 40 s (CYTB, rhodopsin) or 60 ◦C for 40 s (TBT-bp2), 72 ◦C for 1 min (16S rRNA, COI), 2 min (CYTB, rhodopsin, TBT-bp2), and a final extension at 72 ◦C for 10 min. The amplicons were purified using ExoSAP-IT (Thermo Fisher Scientific Inc., Waltham, USA) and directly sequenced by outsourcing (FASMAC Co., Ltd., Atsugi, Japan). GenBank/EMBL accession numbers are from LC581801 to LC581879.

4.3. Phylogenetic Reconstructions The sequences determined were checked and trimmed manually. In the case of the 2 nuclear genes, the Sanger sequence electropherograms were carefully examined for clear double peaks with comparable height, which reflects allelic heterogeneity at the locus. These sites were coded with IUPAC ambiguity codes in the sequence alignments. For the mitochondrial genes, a couple of previous studies [35,45] published CYTB sequences of closely related species with 16S rRNA or COI sequences. Phylogenetic reconstructions using Bayesian inference (BI) and maximum likelihood (ML) methods were conducted for CYTB and the 2 concatenated datasets, CYTB+16S rRNA, and CYTB+COI. Best fit models of nucleotide substitution for BI analyses by MrBayes 3.2.7 [46] were inferred using Kakusan 4 [47], and the GTR+I+G model was used for ML analyses by FastTree 2 [48]. In the BI analyses, 4 runs of 5 million generations were performed with 4 chains each, trees were sampled at 1000-generation intervals, and the first 25% of the trees were discarded as burn-in. In the ML analyses, bootstrap values were calculated using 1000 trees generated by SEQBOOT in the PHYLIP Package 3.698 [49], and the consensus tree was obtained by CompareToBootstrap.pl in FastTree 2 [50]. For TBT-bp 2, as the MrBayes analysis did not provide ambiguity codes, only ML analysis was conducted as described above, but with the JC+CAT model.

4.4. Toxin Quantification Each tissue of the pufferfish was extracted with 0.1 M HCl, passed through an HLC-DISK membrane filter (0.45 µm, Kanto Chemical Co., Inc., Tokyo, Japan), and subjected to HPLC-FLD for STXs and Toxins 2020, 12, 689 10 of 15

LC-MS/MS for TTX according to the previously reported methods [12]. The reference materials of C1, C2, GTX1-4, and decarbamoylGTX2,3 were provided by the Japan Fisheries Research and Education Agency, and neoSTX, dcSTX, and STX purified from the toxic aeneus [51] and crystalline TTX (Nacalai Tesque, Inc., Kyoto, Japan) were used as external standards. The limit of detection and limit of quantification for STXs were 0.001–0.007 nmol/mL (S/N = 3) and 0.003–0.02 nmol/mL (S/N = 10), and for TTX, 0.0009 nmol/mL (S/N = 3) and 0.003 nmol/mL (S/N = 10), respectively. Based on the concentrations of STX, neoSTX, and dcSTX in each tissue (s, n, and d nmol/g, respectively), the toxin concentration in each tissue (c mg STX diHCl eq/kg) was calculated with the · toxicity equivalence factors (STX = 1, neoSTX = 2, and dcSTX = 0.5) [30] and molecular weight of STX diHCl (372.211) using the following equation; c = 372.211 (1s + 2n + 0.5d) 10 3. · × × − Author Contributions: Conceptualization, A.Y., M.W., and O.A.; funding acquisition, M.W., J.S.L., and O.A.; investigation, H.Z., A.Y., Y.G., J.S.L., and T.T.; resources, L.H., L.N., and H.D.; writing—original draft, H.Z. and A.Y.; writing—review and editing, A.Y. and O.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Japan Society for the Promotion of Science, 19H03051, and Nagasaki University Grant for New Developments in Research, and supported by the Gyeongsang National University Fund for Professors on Sabbatical Leave, 2020. Conflicts of Interest: The authors declare no conflict of interest.

Appendix A

Table A1. Specification and toxin concentration in each tissue from freshwater pufferfish.

Toxin Concentration (mg STXdi HCL eq/kg) Species Sample Sex TL (cm) 1 BW (g) 2 · Skin Muscle Liver 3 Ovary 3,4 Testis Pao sp. A PNH-1 9.7 26.1 2.1 1.2 1.5 22.5 2 ♀ 8.0 15.1 70.5 13.7 2.7 19.3 3 ♀ 9.1 22.7 63.0 14.4 73.4 96.3 4 ♀ 7.0 14.4 43.1 32.1 15.8 78.5 5 ♀ 8.0 15.9 37.5 17.0 18.1 28.2 6 ♀ 6.5 12.8 39.7 45.3 63.4 63.5 7 ♀ 7.5 21.1 33.2 21.2 18.3 – 8 ♀ 7.5 22.2 60.8 45.4 34.7 97.3 9 ♀ 7.6 17.2 37.2 22.7 8.3 – 10 ♀ 8.0 17.0 33.4 16.7 14.8 – 11 ♀ 8.2 22.0 28.4 16.0 2.7 63.7 Mean SD♀ 7.9 0.9 18.8 4.2 40.8 18.9 22.3 13.6 23.1 24.4 58.7 31.9 ± ± ± ± ± ± ± Pao sp. B KTI-1 15.0 85.0 0.3 0.1 0 0 2 ♀ 16.0 90.0 0.8 0.3 0 1.2 3 ♀ 15.7 108 15.8 1.1 1.0 2.8 4 ♀ 15.5 105 6.5 0.5 1.5 0.9 5 ♂ 16.0 109 6.3 0.4 0.2 0.3 Mean SD♂ 15.6 0.4 99.4 10.5 6.0 6.2 0.5 0.4 0.6 0.7 1.3 1.4 0.6 ± ± ± ± ± ± ± 1 TL: total length. 2 BW: body weight. 3 0: less than the limit of quantification, which was considered to be 0. 4 –: not examined due to tissue scarcity. Toxins 2020, 12, x FOR PEER REVIEW 11 of 15 Toxins 2020 12 Toxins, 2020, 689, 12, x FOR PEER REVIEW 11 of 1115 of 15

KTI-3 PaoKTI-3 baileyi (AB741961.1, AB741978.1)

94/96 KTI-1,Pao baileyi 2, 4, 5(AB741961.1, AB741978.1) 71/89 94/96 KTI-1,Pao abei 2, 4,(AB741960.1, 5 AB741977.1) 95/92 71/89 Pao leiurusabei (AB741960.1, (KF667490.1) AB741977.1) 95/92 100/99 PaoPao leiurus abei (LC586270.1) (KF667490.1) 100/99 100/100 PaoPao leiurusabei (LC586270.1) (AB741968.1, AB741985.1)

100/100 85/67 PaoPNH-3 leiurus (AB741968.1, AB741985.1)

100/10085/67 PaoPNH-3 cochinchinensis (AB741963.1, AB741980.1)

100/100 PaoPNH- cochinchinensis 1, 2, 4, 5, 6, 8, 9,(AB741963.1, 10, 11 AB741980.1) PNH-PNH-7 1, 2, 4, 5, 6, 8, 9, 10, 11

99/100 PaoPNH-7 suvattii (AB741974.1, AB741991.1)

99/100 PaoPao suvattii suvattii (AB741974.1, (LC586271.1) AB741991.1) 78/67 PaoPao turgidus suvattii (LC586271.1)(AB741975.1, AB741992.1) 78/67100/100 PaoPao cochinchinensis turgidus (AB741975.1, (AP011925.1) AB741992.1) Pao100/100 palembangensisPao cochinchinensis (AP011920.1) (AP011925.1) 100/100 Pao palembangensisArothron manilensis (AP011920.1) (AP011929.1) 100/100 ArothronArothron manilensis hispidus (AP011929.1) (AP011930.1) Arothron hispidus (AP011930.1)

0.140 0.120 0.100 0.080 0.060 0.040 0.020 0.000 0.140 0.120 0.100 0.080 0.060 0.040 0.020 0.000 Figure A1. Phylogenetic tree of the Pao species constructed by Bayesian inference (BI) and maximum Figure A1. Phylogenetic tree of the Pao species constructed by Bayesian inference (BI) and maximum Figurelikelihood A1. Phylogenetic(ML) based on tree mitochondrial of the Pao species 16S rRNAconstructed and CYTB by Bayesian genes. inferenceThe BI tree (BI) is and shown. maximum Node likelihood (ML) based on mitochondrial 16S rRNA and CYTB genes. The BI tree is shown. Node likelihoodnumbers are (ML) Bayesian based posterioron mitochondrial probabilities 16S (left)rRNA and and ML CYTB bootstrap genes. values The (right).BI tree is shown. Node numbers are Bayesian posterior probabilities (left) and ML bootstrap values (right). numbers are Bayesian posterior probabilities (left) and ML bootstrap values (right). KTI-3 57/82 KTI-3KTI-4, 5 99/9957/82 KTI-4,KTI-1, 52 98/95 99/99 PaoKTI-1, leiurus 2 (KF667490.1) 98/95 98/97

98/97 PaoPao leiurus abei (LC586270.1) (KF667490.1) PaoPao cambodgiensis abei (LC586270.1) (JQ681828.1, JQ681921.1) Pao leiurusPao cambodgiensis(JQ681834.1, JQ681927.1) (JQ681828.1, JQ681921.1) 100/90 PaoPao palembangensis leiurus (JQ681834.1, (JQ681839.1, JQ681927.1) JQ681932.1) 100/90 Pao cochinchinensispalembangensis (AP011925.1)(JQ681839.1, JQ681932.1) 100/100 99/99PaoPao cochinchinensis suvattii (JQ681841.1, (AP011925.1) JQ681934.1) 83/60100/100 99/99 PaoPao suvattii suvattii (JQ681841.1, (LC586271.1) JQ681934.1) 83/60 PaoPao cochinchinensissuvattii (LC586271.1) (JQ681829.1, JQ681922.1) PaoPNH-3 cochinchinensis (JQ681829.1, JQ681922.1) 100/100 PNH-3PNH-10 100/100 PNH-10PNH- 1, 2, 4, 5, 6, 8, 9, 11 PNH-PNH-7 1, 2, 4, 5, 6, 8, 9, 11 Pao palembangensisPNH-7 (AP011920.1)

100/100 Pao palembangensisArothron (AP011920.1) manilensis (AP011929.1)

100/100 ArothronArothron hispidusmanilensis (AP011930.1) (AP011929.1) Arothron hispidus (AP011930.1)

0.200 0.150 0.100 0.050 0.000 Figure A2.0.200Phylogenetic0.150 tree of the0.100Pao species0.050 constructed0.000 by Bayesian inference (BI) and maximum Figure A2. Phylogenetic tree of the Pao species constructed by Bayesian inference (BI) and maximum likelihood (ML) based on mitochondrial COI and CYTB genes. The BI tree is shown. Node numbers Figurelikelihood A2. (ML)Phylogenetic based on tree mitochondrial of the Pao species COI and constructed CYTB genes. by Bayesian The BI tree inference is shown. (BI) Nodeand maximum numbers are Bayesianlikelihoodare Bayesian posterior (ML) posterior based probabilities onprobabilities mitochondrial (left) (left) and COIand ML andML bootstrap bootstrapCYTB genes. valuesvalues The (right). (right).BI tree is shown. Node numbers are Bayesian posterior probabilities (left) and ML bootstrap values (right).

Toxins 2020, 12, x FOR PEER REVIEW 12 of 15

Table A2. Sequence variation at 29 nucleotide sites for TBT-bp2 gene sequences.

Sample 7 23 24 33 138 169 193 225 228 231 232 286 367 429 432 448 499 505 524

ToxinsPNH-12020, 12,A 689 T G C C T C C T G C G A G C C T T 12A of 15 PNH-2 . . . Y 1 . . . . W 1 R 1 M 1 . . . S 1 Y 1 . . . PNH-3 . C . Y . . . . W R M . . . S Y . . . PNH-4 . TableY A2.. SequenceY M variation. . at 29. nucleotideW R sitesM for. TBT-bp2. gene. S sequences. . . . R PNH-5 . . . Y . . . M W R M . . . S . . . . Sample 7 23 24 33 138 169 193 225 228 231 232 286 367 429 432 448 499 505 524 PNH-6 . . . Y . . . M W R M . . . S . . . . PNH-1 A T G C C T C C T G C G A G C C T T A PNH-2PNH-7 .. .Y . . Y 1Y ....M . . . WW1 RR 1 MM 1 ...... S S 1 . Y 1 . .... G PNH-3PNH-8 .nd 1 Cnd .nd Ynd .M .. .. .M WW R R M M ...... S S. Y. .. .R . PNH-4 . Y . Y M . . . W R M . . . S . . . R PNH-5PNH-9 .nd .nd .nd Ynd ...... M. W. R R M M ...... S...... PNH-6PNH-10 .nd .nd .nd Ynd ...... M. WW R R M M ...... S SY ...... PNH-7 . Y . Y M . . . W R M . . . S . . . G PNH-8PNH-11nd 1nd ndnd ndnd ndnd Mnd .nd .nd Mnd Wnd nd R nd M ...... S...... R PNH-9 nd nd nd nd . . . . . R M . .1 ...... PNH-10KTI-1 ndnd ndC nd. nd. .. .G .Y .. W. . R. MA .W .K .G S . Y C . G . G . PNH-11KTI-2 ndG ndC nd. nd. nd. ndG ndY nd. nd. . nd. ndA .W .T .G .. .C .G .G . KTI-1 nd C . . . G Y . . . . A W K 1 G.CGG KTI-2KTI-3 G nd Cnd .nd .nd .nd Gnd Ynd .nd nd . nd . nd . ndA nd W ndT nd G nd .nd C nd G nd G KTI-3KTI-4 ndG ndC ndR nd. nd. ndG ndY nd. nd. . nd. ndR ndW ndT ndG nd. ndC ndG ndG nd KTI-4 G C R . . G Y . . . . R W T G . C G G KTI-5KTI-5 ndnd CC RR .. .. GG ...... A A. .T TG G. .C CG GG G 1 1Y Y= =C Cor orT, T, W W= =A A ororT, T, R R= =A A ororG, G,M M= = AA oror C,C, SS= = C oror G,G, KK == G or T, ndnd == not determined.

Figure A3. HPLC-FLD chromatograms of the ovary extract from PNH-4 (left), skin extract from KTI-3Figure (middle), A3. HPLC-FLD and of the chromatograms saxitoxin (STX), of decarbamoylSTXthe ovary extract from (dcSTX), PNH-4 and (left), neoSTX skin extr standardsact from KTI- (right). 3 (middle), and of the saxitoxin (STX), decarbamoylSTX (dcSTX), and neoSTX standards (right). References References 1. Noguchi, T.; Arakawa, O. Tetrodotoxin—Distribution and accumulation in aquatic organisms, and cases of 1. Noguchi, T.; Arakawa, O. Tetrodotoxin—Distribution and accumulation in aquatic organisms, and cases human intoxication. Mar. Drugs 2008, 6, 220–242. [CrossRef][PubMed] of human intoxication. Mar. Drugs 2008, 6, 220–242. 2. Mahmud, Y.; Yamamori, K.; Noguchi, T. Occurrence of TTX in a brackish water puffer ‘midorifugu’, 2. Mahmud, Y.; Yamamori, K.; Noguchi, T. Occurrence of TTX in a brackish water puffer ‘midorifugu’, TetraodonTetraodon nigroviridis nigroviridis, collected, collected from from Thailand. Thailand.J. J. Food Food Hyg.Hyg. Soc. Soc. Jpn. Jpn. 19991999, 40, ,40 363–367., 363–367. [CrossRef] 3. Mahmud,3. Mahmud, Y.; Yamamori,Y.; Yamamori, K.; K.; Noguchi, Noguchi, T. T. Toxicity Toxicity and and tetr tetrodotoxinodotoxin as the as toxic the prin toxicciple principle of a brackish of awater brackish waterpuffer, puffer, TetraodonTetraodon steindachneri steindachneri, collected, collected from Thailand. from J. Food Thailand. Hyg. Soc. Jpn.J. Food 1999, Hyg.40, 391–395. Soc. Jpn. 1999, 40, 391–395. [CrossRef] 4. Kungsuwan, A.; Arakawa, O.; Promdet, M.; Onoue, Y. Occurrence of paralytic shellfish poisons in Thai freshwater puffers. Toxicon 1997, 35, 1341–1346. [CrossRef] 5. Sato, S.; Kodama, M.; Ogata, T.; Saitanu, K.; Furuya, M.; Hirayama, K.; Kamimura, K. Saxitoxin as a toxic principle of a freshwater puffer, Tetraodon fangi, in Thailand. Toxicon 1997, 35, 137–140. [CrossRef] 6. Zaman, L.; Arakawa, O.; Shimosu, A.; Onoue, Y. Occurrence of paralytic shellfish poison in Bangladeshi freshwater puffers. Toxicon 1997, 35, 423–431. [CrossRef] Toxins 2020, 12, 689 13 of 15

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