Detecting in situ copepod diet diversity using molecular technique: development of a copepod/symbiotic ciliate-excluding eukaryote-inclusive PCR protocol

Knowledge of in situ copepod diet diversity is crucial for accurately describing pelagic food web structure but is challenging
to achieve due to lack of an easily applicable methodology. To enable analysis with whole copepod-derived DNAs, we
developed a copepod-excluding 18S rDNA-based PCR protocol. Although it is effective in depressing amplification of
copepod 18S rDNA, its applicability to detect diverse eukaryotes in both mono- and mixed-species has not been
demonstrated. Besides, the protocol suffers from the problem that sequences from symbiotic ciliates are overrepresented in
the retrieved 18S rDNA libraries. In this study, we designed a blocking primer to make a combined primer set (copepod/
symbiotic ciliate-excluding eukaryote-common: CEEC) to depress PCR amplification of symbiotic ciliate sequences while
maximizing the range of eukaryotes amplified. We firstly examined the specificity and efficacy of CEEC by PCR-amplifying
DNAs from 16 copepod species, 37 representative organisms that are potential prey of copepods and a natural
microplankton sample, and then evaluated the efficiency in reconstructing diet composition by detecting the food of both
lab-reared and field-collected copepods. Our results showed that the CEEC primer set can successfully amplify 18S rDNA
from a wide range of isolated species and mixed-species samples while depressing amplification of that from copepod and
targeted symbiotic ciliate, indicating the universality of CEEC in specifically detecting prey of copepods. All the
predetermined food offered to copepods in the laboratory were successfully retrieved, suggesting that the CEEC-based
protocol can accurately reconstruct the diets of copepods without interference of copepods and their associated ciliates
present in the DNA samples. Our initial application to analyzing the food composition of field-collected copepods
uncovered diverse prey species, including those currently known, and those that are unsuspected, as copepod prey. While
testing is required, this protocol provides a useful strategy for depicting in situ dietary composition of copepods.