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.
Highlights
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]
polymerase chain reaction (PCR) with the universal 18S rDNA primer set all produced an amplicon with an expected product of,1.8 kb, indicating that the negative results with CEEC primers were not due to poor DNA quality, and cloning and sequencing results showed that they were all copepods or symbiotic ciliates
We systematically evaluated the utility of this PCR assay for detecting diverse diets of copepods in natural environments by testing it against individual species of eukaryotic organisms, mixed species, natural assemblage, laboratory fed copepods, and field-caught copepods
Summary
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 demonstrate remarkable versatility in their prey, they 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 [5,6,7,8]. 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 [9,10]. Copepods can discriminate between individuals of the same species with different properties, Station*
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