CHO, N; SUZUKI, N; MURAKAMI, K; National Resarch Institute of Far Seas Fisheries; National Resarch Institute of Far Seas Fisheries; Japan Sea-Farming Association: A molecular approach to determine natural diets of lobster phyllosoma

Despite the recent success in producing juveniles of palinurid lobsters (the family Palinuridae) in the laboratory, natural diets of the larvae (phyllosoma) are not known well. We here introduce a molecular technique which may identify eukaryotic organisms in the stomach of phyllosoma. In order to verify whether or not the technique works for detecting foreigner DNAs in the stomach, we first used laboratory-reared phyllosoma of Panulirus japonicus fed exclusively on Mytilus gonad or Artemia nauplii, and then wild phyllosoma collected in the Atlantic and Pacific were subjected to the analysis. The cephalic tissues supposedly containing stomach contents were dissected and DNA was extracted using standard phenol-chloroform method. For nested PCR amplification, we designed two primer pairs which targeted the central domain of 18SrDNA (c.a. 600 bp), and PCR products were cloned using TA vector. Clones were screened by colony direct PCR using the PCR primers, those having restriction profiles inconsistent with those of host DNAs were selected via RFLP analysis, and nucleotide sequences of the screened clones were determined. Among 91 clones from two larvae fed on Mytilus gonad, 30 had non-host derived DNAs, and Mytilus DNA was detected from 13 clones. Only 5 out of 115 clones from two larvae fed on Artemia nauplii had non-host derived DNAs, and Artemia DNA was confirmed in 1 clone. The results indicate the method to be sensitive enough to detect DNAs from stomach content. Detection of non-host DNA from the stomach of a wild phyllosoma (identified as Panulirus echinatus) caught in the Atlantic was failed, while DNAs of fish, Gastrotricha and fungi were detected in two phyllosoma larvae (identified as P. japonicus) caught around the Ryukyu Islands.