Genus-specific quantitative PCR of thraustochytrid protists

Abstract

Thraustochytrids have the capability to recycle refractory organic matter, with a resulting impact on carbon cycling in coastal and open seawaters. The abundance of thraustochytrids has traditionally been estimated by acriflavine direct counting. However, this technique may lead to over- or underestimation. To accurately quantify the abundance of thraustochytrids, we developed a quantitative PCR (qPCR) system using 7 genus-specific primer sets targeting 7 genera (Aurantiochytrium, Botryochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, and Ulkenia) from the family Thraustochytriaceae. The high specificity was verified in silico and with culture strains of each genus. In addition, we applied this qPCR assay to test for the presence of thraustochytrids in coastal and open seawaters around Japan. We successfully detected the presence of Aurantiochytrium (in the range of 1.12 × 104 to 1.31 × 104 cells l-1) and Oblongichytrium (in the range of 1.02 × 104 to 3.14 × 104 cells l-1) in 8 surface water samples from around Satsuma-Iwojima (western Japan) and off the Karakuwa in Sanriku (eastern Japan). We obtained higher estimates using qPCR than the traditional acriflavine method in all cases, with a weak positive correlation between the 2 methods (r 2 = 0.495). Interestingly, we quantified thraustochytrids in 104 additional samples by direct count, but not by qPCR, possibly because of inhibition of the qPCR reaction and/or the presence of novel thraustochytrid groups. Although these trials are preliminary, our approach can provide the genus-specific value of abundance in the environment. It will also promote further advances in our understanding of thraustochytrid diversity. © Inter-Research 2013.