Kantima THONGJUED, Karina GARCIA, Delia SCOTT, David J. GONTHIER, Julian R. DUPUIS
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We evaluated the efficacy of three DNA extraction kits, three primer sets for plant identification (targeting <i>rbcL</i>, <i>trnL</i>, and internal transcribed spacer 2 [ITS2]), and three primer sets for arthropod identification (targeting cytochrome oxidase subunit I [<i>COI</i>]). The detection success rate of our positive control food items was highly variable, ranging from 2.04% to 93.88% for all kit/primer combinations and averaging 37.35% and 43.57% for the most effective kit/primer combination for plants and insects, respectively. Extraction kits using bead-based homogenization positively affected the recovery proportion of plant and insect DNA in excreta samples. The minimum time to detect known food items was 44 min post-feeding. Two <i>COI</i> primer sets significantly outperformed the third, and both recovery proportion and taxonomic resolution from ITS2 were significantly higher than those from <i>rbc</i>L and <i>trn</i>L. Taken together, these results display the potential variability that can be inherently present in DNA-based diet analyses and highlight the utility of experimental feeding trials in validating such approaches, particularly for omnivores with diverse diets.</p>","PeriodicalId":13654,"journal":{"name":"Integrative zoology","volume":null,"pages":null},"PeriodicalIF":3.5000,"publicationDate":"2024-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"DNA metabarcoding diet analysis in a generalist omnivore: feeding trials reveal the efficacy of extraction kits and a multi-locus approach for identifying diverse diets\",\"authors\":\"Kantima THONGJUED, Karina GARCIA, Delia SCOTT, David J. GONTHIER, Julian R. 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引用次数: 0
摘要
基于代谢条形码的食物分析是了解多种物种摄食行为的重要工具。然而,许多针对野生动物使用这些方法的研究都假定了准确性和精确性,而没有使用已知的阳性对照食物进行实验评估。在此,我们对牧场饲养的鸡进行了阳性对照群体的饲喂试验,并评估了几种常用 DNA 提取试剂盒和引物组的功效。我们给六只散养蛋鸡人工喂食了 22 种已知食物,包括昆虫和植物,并收集了它们八小时的排泄物。我们评估了三种 DNA 提取试剂盒、三种植物鉴定引物组(针对 rbcL、trnL 和内部转录间隔 2 [ITS2])和三种节肢动物鉴定引物组(针对细胞色素氧化酶亚单位 I [COI])的功效。阳性对照食品的检测成功率差异很大,所有试剂盒/引物组合的检测成功率从 2.04% 到 93.88% 不等,植物和昆虫的最有效试剂盒/引物组合的平均检测成功率分别为 37.35% 和 43.57%。使用珠状均质化提取试剂盒对排泄物样本中植物和昆虫 DNA 的回收率有积极影响。检测已知食物的最短时间为进食后 44 分钟。两个 COI 引物组的性能明显优于第三个引物组,ITS2 的回收率和分类分辨率都明显高于 rbcL 和 trnL。总之,这些结果显示了基于 DNA 的食性分析中可能存在的潜在变异性,并强调了实验性喂食试验在验证此类方法中的作用,尤其是对于食性多样化的杂食动物。
DNA metabarcoding diet analysis in a generalist omnivore: feeding trials reveal the efficacy of extraction kits and a multi-locus approach for identifying diverse diets
Metabarcoding-based diet analysis is a valuable tool for understanding the feeding behavior of a wide range of species. However, many studies using these methods for wild animals assume accuracy and precision without experimental evaluation with known positive control food items. Here, we conducted a feeding trial experiment with a positive control community in pasture-raised chickens and assessed the efficacy of several commonly used DNA extraction kits and primer sets. We hand-fed 22 known food items, including insects and plants, to six backyard laying hens and collected their excreta for eight h. We evaluated the efficacy of three DNA extraction kits, three primer sets for plant identification (targeting rbcL, trnL, and internal transcribed spacer 2 [ITS2]), and three primer sets for arthropod identification (targeting cytochrome oxidase subunit I [COI]). The detection success rate of our positive control food items was highly variable, ranging from 2.04% to 93.88% for all kit/primer combinations and averaging 37.35% and 43.57% for the most effective kit/primer combination for plants and insects, respectively. Extraction kits using bead-based homogenization positively affected the recovery proportion of plant and insect DNA in excreta samples. The minimum time to detect known food items was 44 min post-feeding. Two COI primer sets significantly outperformed the third, and both recovery proportion and taxonomic resolution from ITS2 were significantly higher than those from rbcL and trnL. Taken together, these results display the potential variability that can be inherently present in DNA-based diet analyses and highlight the utility of experimental feeding trials in validating such approaches, particularly for omnivores with diverse diets.
期刊介绍:
The official journal of the International Society of Zoological Sciences focuses on zoology as an integrative discipline encompassing all aspects of animal life. It presents a broader perspective of many levels of zoological inquiry, both spatial and temporal, and encourages cooperation between zoology and other disciplines including, but not limited to, physics, computer science, social science, ethics, teaching, paleontology, molecular biology, physiology, behavior, ecology and the built environment. It also looks at the animal-human interaction through exploring animal-plant interactions, microbe/pathogen effects and global changes on the environment and human society.
Integrative topics of greatest interest to INZ include:
(1) Animals & climate change
(2) Animals & pollution
(3) Animals & infectious diseases
(4) Animals & biological invasions
(5) Animal-plant interactions
(6) Zoogeography & paleontology
(7) Neurons, genes & behavior
(8) Molecular ecology & evolution
(9) Physiological adaptations