An approach to the study of copepod egg banks based on efficient DNA extraction from individual copepod eggs

A method of extracting DNA from individual copepod eggs was introduced for this study which included a modified proteinase K procedure and an efficient DNA sedimentation process. DNA was extracted from egg samples including freshly spawned eggs from the three copepod species Apocyclops borneoensis, Centropages tenuiremis and Calanus sinicus, together with 18 resting eggs separated from different sediment layers. A short fragment of the 28S rDNA (~300 bp) sequence that varied between copepod species was amplified and sequenced. These sequences were used to construct a UPGMA tree which helped to assess species composition and the distribution of copepods buried in the sediments. The results showed that C. tenuiremis and Acartia pacifica were closely clustered in the tree with egg samples from deeper sediment layers, whereas A. borneoensis and C. sinicus were grouped with surface egg samples. Species composition in the sediments varied between sediment layers and sampling locations. The DNA extraction method was valid for analysing individual copepod eggs with different egg-spawning types and sizes and the results helped us to reconstruct the copepod egg composition and distribution in the sediments. We believe that the technique has a wide usage in analysing the copepod egg bank in sediments, and possibly even for other zooplankton.