Molecular Analysis Method And Its Application For The Detection Of In Situ Diet Information Of Calanus Sinicus In Bohai Sea And Yellow Sea, China

Marine pelagic copepods are the most abundant metazoans and importantpredators in marine ecology system. It is essential to study the grazing behavior ofcopepods to reveal the interaction with phytoplankton and explore the importance infood webs of marine ecosystem. The study of methods to analyze copepods in situfood composition is also a key point in feeding ecology aeras. The emerging PCRbased molecular analysis tools has advantages over the traditional methods, such asthe isotope tracer method and gut content analysis method. As a new method, it ispromising with powerful emphasis on the acurate diversity of prey.Two parts are included in this study. The first part is to design the ciliateblocking primers and acquire the optimal blocking primer and PCR blockingcondition. In the other part, the food information of Calanus sinicus in Bohai Sea andYellow Sea, China was discussed. Apostome ciliates were the common protozoansymbionts on copepods, they could infect wide variety marine copepods. Smallsubunit ribosomal RNA gene(18S rDNA) of the ciliates as symbionts on the copepodwas amplified,which effected the results of the analysis in the copepod grazingexperiment. In the study, toward amplifying prey18S rDNA genes of the planktoniccopepods more efficiently, reflecting the bait composition more realistically,clarifying the relationship of copepods and phytoplankton in the natural sea andproviding a reliable scientific basis for building food web structure, three ciliateblocking primers were designed.Molecular analysis method based on PCR reaction was applied in the ciliateblocking experiment. The blocking efficiency of three apostome ciliates blockingprimers was tested and compared, after series experiments, best blocking primers waschosen, along with the optimal PCR condation. Phaeodactylum tricornutum and apostome ciliate18S rDNA were obtained, and then mixing the diatom gene as areference gene, ciliate gene and different blocking primer proportionally. Finally, theoptimal combination was determined by the semi-quantitative PCR method, theoptimal blocking primer was Ciliate18Sblk1and the best reaction condition was3min at94°C,5cycles of10sec at95°C,60sec at68°C and followed by30cycles of95°C for10sec,54.2°C for30sec,72°C for40sec, and finally10min at72°C bycomparison the strength of gel band in the gel electrophoresis figure, the amplificationof the ciliate gene was blocked more efficiently in the PCR progress under thiscondition.18S non-copepod primer set and Ciliate18Sblk1primer were applied in theamplification of calanus cinicus prey18S rDNA. The diversity of prey was discussedby analyzing the gene sequence of the prey.103sequences were screened andanalyzed. The results show that the bait food composition of Calanus cinicus in Juneinclude dinoflagellates、diatoms、chrysophyte、Katablepharid、Katablepharidophyta,and maybe also contain egg、larva、organic particles of some marine animals such asjellyfish class、Sagittidae、Echinoidea、Appendicularia.