Detecting In Situ Copepod Diet Diversity Using Molecular Technique: Development of a Copepod/Symbiotic Ciliate-Excluding Eukaryote-Inclusive PCR Protocol

et al. (2014) Detecting In Situ Copepod Diet Diversity Using Molecular Technique: Development of a Copepod/ Symbiotic Ciliate-Excluding Eukaryote-Inclusive PCR Protocol. PLoS ONE 9(7): e103528. doi:10.1371/journal.pone.0103528 Detecting In Situ Copepod Diet Diversity Using Molecular Technique: Development of a Copepod/ Symbiotic Ciliate-Excluding Eukaryote-Inclusive PCR Protocol Simin Hu 0 Zhiling Guo 0 Tao Li 0 Edward J. Carpenter 0 Sheng Liu 0 Senjie Lin 0 John F. Valentine, Dauphin Island Sea Lab, United States of America 0 1 Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences , Guangzhou , China , 2 University of Chinese Academy of Sciences , Beijing , China , 3 Tropical Marine Biological Research Station in Hainan, Chinese Academy of Sciences , Sanya , China , 4 Romberg Tiburon Center, San Francisco State University , San Francisco , California, United States of America, 5 Marine Biodiversity and Global Change Research Center, Xiamen University , Xiamen , China , 6 Department of Marine Sciences, University of Connecticut , Groton, Connecticut , United States of America 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. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The partial 18S rDNA gene sequences obtained in this study had been deposited at GenBank under accession numbers KF733525-KF733602 and KJ569308. Funding: This work was supported by Chinese Academy of Sciences (CAS) for Key Topics in Innovation Engineering (KZCX2-YW-JS206) (http://www.cas.cn/), and Natural Science Foundation of China (41076096, 41276160 and 40828006) (http://www.nsfc.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. As the most numerous animals in marine ecosystem, copepods are critical link of primary production to higher trophic levels, and important driver of the marine biological pump [1]. Copepods can be herbivores, carnivores and omnivores, or can switch their trophic mode according to the relative abundances of the prey species. They can feed on a variety of prey belonging to diverse taxa and size categories, including phytoplankton, protozoans, eggs and larva of aquatic organisms, including those of copepods, and detritus [24]. Although copepods demonstrate remarkable versatility in their prey, they also exhibit specific feeding preferences among different prey species based on the traits of prey, such as motility, cell size and shape, nutritional value, dissolved chemical cues, and cell surface properties [58]. For example, both laboratory and field incubation studies have shown that copepods preferentially graze on ciliates and dinoflagellates when diverse foods are offered because they have higher nutritional quality than other prey species and are rich in polyunsaturated fatty acids (PUFA) and eicosapentaenoic acid (EPA) that influence the growth, survival and fecundity of copepods [910]. In addition, copepods can also discriminate between individuals of the same species with different properties, Fixed water sample Gut evacuation and fixed Gut evacuation and fixed Gut evacuation and fixed Gut evacuation and fixed *: AV, Avery Point (41u18.9179N, 72u3.819W), Connecticut, USA; DYB, Daya Bay (22u36.2749N, 114u34.09E), South China Sea, China; SYB, Sanya Bay (18u12.7949N, 114u34.09E), South China Sea, China; PRE, Pearl river estuary (22u7.0229N, 113u52.1759E), South China Sea, China. doi:10.1371/journal.pone.0103528.t001 including biochemical composition, growth stage and nutritional quality [11]. Current knowledge of copepod feeding is largely derived from incubation experiments, which do not necessarily provide information on true diet composition of copepods at sea. Although natural dietary information can be obtained from gut content analysis of wild-caught copepods, the currently available microscopic technique is not only time-consuming but also challenging because partially digested prey can be extremely difficult to identify. Pigment analysis has also been used to investigate prey diversity, but it is limited to phytoplankton and has low taxonresolving power [12]. Stable isotope analysis of organic materials and fatty acid analysis are helpful in tracing sources of carbon or nitrogen and can provide information of diet categories or trophic level, but still could not reconstruct species composition of the diet [1314]. Molecular techniques have been widely used for detecting the prey composition of aquatic predators in the natural environment due to its sensitivity, specificity and rapidness [15], among which polymerase chain reaction (PCR) assay has been successfully (...truncated)