title: Detecting in situ copepod diet diversity using molecular technique: development of a copepod/symbiotic ciliate-excluding eukaryote-inclusive PCR protocol.
abstract: 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.
authors: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China; University of Chinese Academy of Sciences, Beijing, China.
authors: Simin
authors: S
authors: Hu
authors: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China; University of Chinese Academy of Sciences, Beijing, China.
authors: Zhiling
authors: Z
authors: Guo
authors: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China; Tropical Marine Biological Research Station in Hainan, Chinese Academy of Sciences, Sanya, China.
authors: Tao
authors: T
authors: Li
authors: Romberg Tiburon Center, San Francisco State University, San Francisco, California, United States of America.
authors: Edward J
authors: EJ
authors: Carpenter
authors: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.
authors: Sheng
authors: S
authors: Liu
authors: Marine Biodiversity and Global Change Research Center, Xiamen University, Xiamen, China; Department of Marine Sciences, University of Connecticut, Groton, Connecticut, United States of America.
authors: Senjie
authors: S
authors: Lin
mesh_terms: D000818:Animals; D016798:Ciliophora / Q000145:classification / Q000235:genetics*; D033342:Copepoda / Q000145:classification / Q000235:genetics* / Q000469:parasitology; D017931:DNA Primers / Q000032:analysis*; D048028:DNA, Algal / Q000032:analysis*; D005247:Feeding Behavior; D014644:Genetic Variation; D016013:Likelihood Functions; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D013045:Species Specificity; D015048:Zooplankton / Q000235:genetics*
chemical_list: D017931:DNA Primers; D048028:DNA, Algal; D012337:RNA, Ribosomal, 18S