Department of Biology, Memorial University of Newfoundland, St. John's, NF, Canada A1B 3X9 Genomics and Proteomics Facility, CREAIT Network, Memorial University of Newfoundland, St. John's, NF, Canada A1B 3X9 Science Branch, Department of Fisheries and Oceans, Northwest Atlantic Fisheries Centre, PO Box 5667, St. John's, NF, Canada A1C 5X1 Department of Psychology, Memorial University of Newfoundland, St. John's, NF, Canada A1B 3X9.
Mol Ecol Resour. 2010 Jan;10(1):181-9. doi: 10.1111/j.1755-0998.2009.02713.x. Epub 2009 May 12.
All methods of diet analysis in marine mammals, including hard part analysis (HPA), have biases affecting the accuracy of prey-species identification and frequency in the estimated diet due to differential consumption, digestion and retention. Using PCR amplification of specific prey DNA with species-specific primers, we developed a DNA-based method that complements HPA and provides an alternative means to detect prey from stomach contents of Harp Seals (Pagophilus groenlandicus). The target size that could be reliably amplified was determined using a digestion time-series of Atlantic Cod (Gadus morhua) tissue in simulated seal stomachs. Various target lengths were trialed using general teleost primers; amplicons of approximately 800 bp or less were consistently obtained. Prey species-specific PCR primers for Atlantic Cod, Arctic Cod (Boreogadus saida) and Capelin (Mallotus villosus) were designed and tested with DNA from the stomach contents of 31 Harp Seals. Amplicons were obtained for all three species-specific primer sets. Amplification results compared with HPA revealed: (i) Atlantic Cod hard parts were found in five stomachs where no Atlantic Cod DNA amplified, suggesting that Atlantic Cod may be over-represented in the estimated diet, (ii) amplification of Arctic Cod DNA occurred for 17 stomachs, including all 12 stomachs with, and five stomachs without, Arctic Cod hard parts, and (iii) Capelin DNA amplified for four of five stomachs with Capelin hard parts and for one stomach without Capelin hard parts. We conclude that PCR amplification of specific prey DNA provides a viable means to complement Harp Seal diet analysis by HPA, but suggest that valuable information for quantitative diet analysis rests in a quantitative PCR approach.
所有的海洋哺乳动物饮食分析方法，包括硬组织分析（HPA），都存在偏差，这些偏差会影响到猎物物种识别的准确性和在估计饮食中的出现频率，这是由于不同的消耗、消化和保留。本研究使用特定猎物 DNA 的 PCR 扩增与物种特异性引物，开发了一种基于 DNA 的方法，该方法补充了 HPA，并为从环斑海豹（Pagophilus groenlandicus）的胃内容物中检测猎物提供了另一种替代方法。使用在模拟海豹胃中的大西洋鳕鱼（Gadus morhua）组织的消化时间序列来确定可以可靠扩增的目标大小。使用一般的硬骨鱼引物对各种目标长度进行了试验；始终获得约 800bp 或更小的扩增子。为大西洋鳕鱼、北极鳕鱼（Boreogadus saida）和毛鳞鱼（Mallotus villosus）设计了猎物物种特异性 PCR 引物，并使用 31 只环斑海豹胃内容物的 DNA 进行了测试。所有三种物种特异性引物组都获得了扩增子。与 HPA 的扩增结果相比：（i）在五个没有扩增到大西洋鳕鱼 DNA 的胃中发现了大西洋鳕鱼硬组织，这表明大西洋鳕鱼在估计的饮食中可能被过度代表，（ii）17 个胃中扩增到了北极鳕鱼 DNA，包括所有 12 个有北极鳕鱼硬组织的胃和 5 个没有北极鳕鱼硬组织的胃，（iii）有 5 个毛鳞鱼硬组织胃中有 4 个扩增到了毛鳞鱼 DNA，有 1 个没有毛鳞鱼硬组织的胃也扩增到了毛鳞鱼 DNA。我们得出结论，特定猎物 DNA 的 PCR 扩增为 HPA 补充环斑海豹饮食分析提供了一种可行的方法，但我们建议，定量饮食分析的有价值信息在于定量 PCR 方法。
