The structure of food webs and carbon flow in aquatic ecosystems can be better understood by studying contributing factors such as the diets of herbivorous fish. Metabarcoding using a high-throughput sequencer has recently been used to clarify prey organisms of various fish except herbivorous fish. Since sequences of predator fish have dominated in sequences obtained by metabarcoding, we investigated a method for suppressing the amplification of fish DNA by using a blocking primer or peptide nucleic acid (PNA) clamp to determine the prey organisms of herbivorous fish. We designed three blocking primers and one PNA clamp that anneal to fish-specific sequences and examined how efficient they were in suppressing DNA amplification in various herbivorous fish. The results showed that the PNA clamp completely suppressed fish DNA amplification, and one of the blocking primers suppressed fish DNA amplification but less efficiently than the PNA clamp. Finally, we conducted metabarcoding using mock community samples as templates to determine whether the blocking primer or the PNA clamp was effective in suppressing fish DNA amplification. The results showed that the PNA clamp suppressed 99.3%–99.9% of fish DNA amplification, whereas the blocking primer suppressed 3.3%–32.9%. Therefore, we propose the application of the PNA clamp for clarifying the prey organisms and food preferences of various herbivorous fish.

Ciguatera fish poisoning (CFP) is caused by toxins originating from an epiphytic/benthic dinoflagellate of the genus Gambierdiscus. In Japan, CFP cases have been increasingly reported not only in subtropical areas but also in temperate areas. It is therefore important to study Gambierdiscus cell occurrences, cell densities, and population dynamics to address CFP outbreaks in Japan. This study assessed the densities in Japanese shallow waters (0.1-3 m depths) and revealed that the densities were lower than those in tropical and subtropical areas worldwide. In the shallow waters of Tosa Bay, a Japanese temperate area, population dynamics of Gambierdiscus cells were assessed monthly between 2007 and 2013. Gambierdiscus did not show substrate preferences for macroalgal species. The cell densities in the area ranged from 0 to 232.2 cells g −1 wet weight algae. The average cell densities in spring, summer, autumn, and winter were 0.1±0.4, 0.9±2.6, 4.0±20.6, and 0.4±1.4 cells g −1 wet weight algae, respectively. The cell densities in summer and autumn were not significantly different (p>0.05), whereas those in summer and autumn were significantly higher than those in spring and winter (p<0.01). A significant positive correlation between cell densities and sea surface temperatures (SSTs) was observed (r s =0.21, p<0.001), while a significant negative correlation between cell densities and salinity was recognized (r s =−0.18, p<0.001). These results suggest that cell densities of Gambierdiscus in Japanese temperate shallow waters increase in summer and autumn when the SST is high and salinity is moderately low.

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