PCR-based In Situ Dietary Analysis Of Two Common Copepods In Bohai Sea And Yellow Sea Coastal Waters

Marine pelagic copepods are conventionally considered as the key linkagebetween primary production and higher trophic levels in marine food webs.Information of copepods in situ diets has pivotal impact on the understanding ofstructure and function of the marine food web, and yet remains obscure. PCR-baseddietary analysis, in which prey DNA as biomarkers, has advantages with highuniversality, specificity and stability. Unrestricted to the morphology of prey, DNAand PCR based method could get more accurate results efficiently.In this study, we applied molecular analysis to determine the in situ diets ofCalanus sinicus and Acartia pacifica along coastal waters of Bohai Sea and YellowSea. Besides, laboratory-controlled feeding experiments were conducted for somepotential prey to verify molecular method-based results. Results showed that the twocopepods had very diverse in situ diets, including some groups that never suspectedto be copepods prey before. As new potential prey detected, what we found enlargedour knowledge about copepods in situ dietary compostion. Moreover, our researchinformed alternative and promising way to better understanding the strcture andfunction of marine food webs.1. Molecular detection of copepods in situ dietsTo explore the application of PCR based method in copepod feeding research, asa case study, natural diets of two common and dominant copepods, Calanus sinicusBrodsky and Acartia pacifica Steuer in Jiaozhou Bay, Qingdao coastal waters, andYellow River estuary adjacent waters were investigated. Using a18S rDNA basedmolecular approach, as many lineages of eukaryotes as possible were amplified, butC. sinicus, A. pacifica and related copepods were excluded. In situ diet compositionand occurance of some species/lineages were discussed. The main results and conclusions are listed below.(1) Abundant and diverse potential diets: non-copepod18S rDNA-basedanalysis of all in situ fixed copepod samples showed that there are totally near20lineages were discovered. They are Diatoms, Dinoflagellates, Choanoflagellida,Cryptophyta, Chrysophyceae, Chlorophyta, Phaeophyta, Streptophyta, Stramenopiles,Marine fungi, Rhizaria, Hydrozoa, Ctenophora, Mollusca, Chaetognatha, Tunicata,Coniferopsida, Liliopsida. Besides, several sequences were clustered to unkowngroups, which indicated the unidentified species/lineages were included in the dietsof copepods. Discovery of the highly diverse plankton community resulted fromintrinsic advantages of PCR based analysis and the non-copepod primers.(2) Difference in feeding diatoms and dinoflagellates: diatoms are minorityin all in situ fixed copepod samples. For C. sinicus, in QD-I_Jan, only1sequencebelonged to Thalassiosiraceae was detected, the proportion of diatom was a littlehigher in YRE-I_May (12.0%). For A. pacifica, proportion of diatom in JZB-I_Oct ismuch smaller (3.8%) than that (21.7%) in corresponding water sample. Diatomoriginated sequences even didn’t detected in QD-I_Jun while the proportion in watersample is10.4%. All the results showed that, maybe diatoms are not favorite foodfor both C. sinicus and A. pacifica. On the contrary, dinoflagellates were abundant inQD-I_Jun and JZB-I_Oct, especially in JZB-I_Oct, much more abundant (26.9%)than that in corresponding water sample (13.0%). These results maybe showed apositive feeding selectivity of the2copepods to dinoflagellates.(3) Universality and dominace of some lineages in Hydrozoa andCtenophora: some lineages of Hydrozoa and Ctenophora emerged in all in situfixed copepod samples with much abundant proportion in each samples.Anthomedusae (Rathkea octopunctata, most likely) dominanted QD-I_Jan by aproportion higher than40%, similar proportion of Leptomedusae (Clytiahemisphaerica, most likely) dominated QD-I_Jun. Both Ctenophora and Hydrozoaoriginated sequences were discovered in JZB-I_Oct and YRE-I_May respectively,and Ctenophora was the first dominated jellyfish, represented by lineages inTentaculata, Cydippida (Pleurobrachia globosa, most likely). Hydrozoa was the second dominated lineages, represented by Rathkea octopunctata and Eirene. In all,jellyfish related sequences are very abundant in these2samples, with a proportion of65.4%and52.0%respectively. Occurance of some lineages in Hydrozoa andCtenophora were discussed, we think feeding of the2copepods to jellyfish is highlyrelated to their distribution and abundance in seas. Based on the results, we inferredthat the eggs or planula larva fmighte be captured by C. snicus and A. pacifica.(4) Occurance and abundant of marine fungi and Syndiniales (parasiticdinoflagellates) in detected diets: among all samples, abundant marine fungi weremostly discovered in QD-I_Jun, represented by Phoma, Filobasidium andMalassezia, they account for more than20%of the sample. Sequences belonged toSyndiniales were detected in QD-I_Jan and YRE-I_May, they are main part ofdinoflagellates in these two samples, represented by Euduboscquella, Hematodiniumand Ichthyodinium. Compared to traditional methods, application of PCR-basedmethod facilitated the discovery of more copepods-related organisms. Marine fungiand Syndiniales are two lineages of them. Copepods are always connected withfungi in the form of copepods-fungi symbiont system, where complex trophicrelationship existed. Similar condition may existed in the Syndiniales-copepodssystems. Although we discovered these two lineages by non-copepod primers, itwould be arbitrary to designate their relationship as predator-prey system. Newmethods should be jointed to further study their complicated trophic relationship.(5) Feeding to some scarce lineages in ambient waters: some lineages whichnever suspected to be prey of copepods were discovered in diets by molecularanalysis. Among these lineages, some even had substaintial proportion. Taketerrestrial plants Pinus and Lolium for example, they account for7.4%in QD-I_Jun.Besides, macroalage were detected in diets of C. sinicus, Scytosiphon (Phaeophyceae)and Ulva (Chlorophyta) were detected in QD-I_Jan and YRE-I_May respectively,although they were scarce in ambient waters. We speculated that the feeding of C.sinicus to these seemingly ambient water-scarce species most likely in the form oftheir pollens and swarmers.2. Experiments to verify the feeding of some organisms Besides the in situ fixed copepod samples, live A. pacifica and C. sinicus werecollected to conduct gut evacuation processing and the following feedingexperiments in laboratory to verify the feeding of some organisms. According towhat we found in in situ fixed copepod samples, an attempt was made to verify thefeeding of copepods to some unexpected species.4lab-cultured algae (Isochrysisgalbana Parke8701, Platymonas helgolandica Kylin var. tsingtaoensis, Pavlovaviridis and Phaeodactylum tricornutum), eggs of Sagitta crassa and larva of Artemiasalina were supplied as food in two series laboratory-controlled feeding experiments,for C. sinicus and A. pacifica respectively. Molecular detection showed that Artemia.salina was detected in diet of A. pacifica, while S. crassa wasn’t detected in the dietof C. sinicus after feeding experiments. The mostly discovered dietary species werePhaeodactylum tricornutum and Pavlova viridis. To some degree, the results showedthe bias of laboratory-controlled feeding experiments: when only one or limitedkinds of food were supplied, the real feeding habit of copepods would be masked bythe man-supplied food.