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

Hongchen Zhu,Hiroyuki Doi,Laymithuna Ngy,Linan Horn,Yui Goto,Minoru Wada,Osamu Arakawa,Tomohiro Takatani,Jong Soo Lee,Akinori Yamada

doi:10.3390/toxins12110689

Hongchen Zhu, Hiroyuki Doi + Show 8 more

Open Access

https://doi.org/10.3390/toxins12110689

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Abstract

The species classification of Cambodian freshwater pufferfish is incomplete and confusing, and scientific information on their toxicity 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 saxitoxins (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 skin 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.

Highlights

Pufferfish of the family Tetraodontidae contain tetrodotoxin (TTX) and/or saxitoxins (STXs), but the toxin ratio differs depending on the genus or species
To explore candidate molecules other than pufferfish STX- and TTX-binding protein (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 were used as targets in the phylogenetic analysis
The present study revealed that freshwater pufferfish collected from two regions of Cambodia, Phnom andstudy

Summary

Introduction

Pufferfish of the family Tetraodontidae contain tetrodotoxin (TTX) and/or saxitoxins (STXs), but the toxin ratio differs depending on the genus or species. The Codex Committee on Fish 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. To accumulate genetic information on the taxonomy 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. 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

Phylogeny

Discussion

Pufferfish Specimens

Phylogenetic Reconstructions

Findings

Toxin Quantification