Including planocerid flatworms in the diet effectively toxifies the pufferfish, Takifugu niphobles

Abstract

Beginning with the larval stages, marine pufferfish such as Takifugu niphobles contain tetrodotoxin (TTX), an extremely potent neurotoxin. Although highly concentrated TTX has been detected in adults and juveniles of these fish, the source of the toxin has remained unclear. Here we show that TTX in the flatworm Planocera multitentaculata contributes to the toxification of the pufferfish throughout the life cycle of the flatworm. A species-specific PCR method was developed for the flatworm, and the specific DNA fragment was detected in the digesta of wild pufferfish adults. Predation experiments showed that flatworm larvae were eaten by the pufferfish juveniles, and that the two-day postprandial TTX content in these pufferfish was 20–50 μg/g. Predation experiments additionally showed flatworm adults were also eaten by pufferfish young, and after two days of feeding, TTX accumulated in the skin, liver and intestine of the pufferfish.

Toxic Flatworm Egg Plates Serve as a Possible Source of Tetrodotoxin for Pufferfish

“…Previously, we had reported that T. niphobles (at present Takifugu alboplumbeus ) juveniles and adults/young became toxic by feeding on P. multitentaculata larvae and adults, respectively [29]. Our findings in this study now show that the pufferfish also feed on P. multitentaculata egg plates, and thus, the flatworm egg plates appear to be an additional source of TTX.…”

“…600 bp) were amplified by PCR using pairs of primers HRNT-F2 (5′-AGTTC AAGAG TACGT GAAAC C-3′) / HRNT-R2 (5′-AACAC CTTTT GTGGT ATCTG ATGA-3′) and HRpra2 (5′-AATAA GTATC ATGTA RACTD ATRTC T-3′) / HRprb2-2 (5′-GDGGV TTTGG DAATT GAYTA ATACC TT-3′), respectively [32]. PCR amplification and sequencing of the PCR products were conducted following the protocol of our previous report [29]. The flatworm 28S rRNA and COI gene sequences have been submitted to the DDBJ/EMBL/GenBank databases under the accession numbers LC489235–LC489238.…”

“…TTX extraction and LC-MS/MS analysis were done following the method used in a previous study [29]. The calibration curve was generated with 1–100 ng/mL of TTX standard (FUJIFILM Wako Pure Chemicals, Osaka, Osaka, Japan), which showed good linearity and precision ( y = 28.7128 x + 21.1116, r 2 = 0.9911).…”

“…The life histories of the flatworm Planocera multitentaculata and the pufferfish Takifugu niphobles (at present Takifugu alboplumbeus ), connected by the TTX present in the flatworm. The pufferfish ingests TTX by feeding on the flatworm egg plates (present study) and adults/young [29]. Red and black arrows indicate transfer of TTX and hatching, respectively.…”

“…In another paper, our lab recently reported that the pufferfish T. niphobles (at present Takifugu alboplumbeus ) effectively toxified itself by feeding on the flatworm P. multitentaculata [29], suggesting that one of the major sources of TTX in pufferfish was planocerid flatworms. In addition, the flatworm P. multitentaculata spawns toxic egg plates on rock and stone surfaces in the intertidal zone from spring to early summer [30,31].…”