A new primer for metabarcoding of spider gut contents
A new primer for metabarcoding of spider gut contents
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DOI：
10.7287/peerj.preprints.27854v1
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发表时间：
2019-07
影响因子：
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通讯作者：
D. Lafage;Vasco Elbrecht;J. Cuff;D. Steinke;P. Hambäck;A. Erlandsson
中科院分区：
文献类型：
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作者：
D. Lafage;Vasco Elbrecht;J. Cuff;D. Steinke;P. Hambäck;A. Erlandsson
蜘蛛作为一类重要的捕食者，在害虫控制、污染物迁移、跨生态系统通量等领域受到了食物网生态学家的广泛关注。研究他们的饮食遇到的困难导致了新技术的使用，比如肠道内容物的元生物编码。在不扩增蜘蛛本身的情况下扩增大范围的蜘蛛猎物是具有挑战性的，到目前为止，还没有为此目的而设计的有效的通用引物。我们开发了一种针对细胞色素C氧化酶亚基I基因的新型正向引物(NoSpi2)。该引物的设计不是为了扩增椭圆形Calamstrum分支(狼蛛科和近缘物种)的蜘蛛，而是仍然扩增大多数其他无脊椎动物。NoSpi2与反向引物BR2一起在硅胶中、在单个猎物和蜘蛛标本上、在模拟和病态陷阱群落上以及在生态应用中进行了测试。在计算机评估中，预测了椭圆形蜈蚣支蜘蛛具有较高的引物偏好性，而所有其他无脊椎动物的偏倚较低。这些结果在很大程度上得到了体外试验的证实。此外，一些蜘蛛家族并没有被放大，这与我们的预期相反。我们证明了NoSpi2/BR2的高效率，它可以恢复94%的模拟群落中的分类群和85%的已知的最好的无脊椎动物捕获群落中的分类群(BF3+BR2)。田间试验表明，NoSpi2引物不能扩增狼蛛科蜘蛛DNA。它还展示了广泛的可检测到的猎物物种。共发现猎物来自12目67科117种。NoSpi2/BR2引物组合能够可靠地扩增猎物物种，而不会扩增任何捕食者的读数，这使其成为狼蛛和近缘物种蜘蛛物种肠道内容物分析的理想选择，甚至能够在不解剖的情况下对整个蜘蛛标本进行同质化。鉴于检测到的猎物物种包括其他蜘蛛和步甲甲虫，该引物不仅可用于饮食和生物防治研究，还可用于研究行会内的捕食行为。
As a key predator group, spiders have received a lot of attention by food web ecologists in diverse fields such as pest control, pollutant transfers, and cross-ecosystem fluxes. The difficulty involved in studying their diet has led to the use of new technologies such as metabarcoding of gut contents. The amplification of a broad range of spider prey without amplifying spiders themselves is challenging and, until now, an efficient universal primer purposed for this has not existed. We developed a novel forward primer (NoSpi2) targeting the cytochrome c oxidase subunit I gene. The primer was designed not to amplify spiders of the oval calamistrum clade (Lycosidae and closely related species) while still amplifying most other invertebrates. NoSpi2 was tested together with the reverse primer BR2 in silico, in vitro on single specimens of prey and spiders, on mock and malaise trap communities, and in an ecological application. In silico evaluation predicted high primer bias for spiders of the oval calamistrum clade and low bias for all other invertebrates. These results were largely confirmed by in vitro tests. Additionally, some spider families were not amplified contrary to our expectations. We demonstrated a high efficiency for the primer pair NoSpi2/BR2 which recovered up to 94% of taxa in the mock community and 85% of the taxa detected by the best invertebrate primer pair known (BF3+BR2) for the malaise trap community. The field experiment showed that Lycosidae spider DNA is not amplified by the NoSpi2 primer set. It also demonstrated a broad range of detectable prey species. We found prey from 12 orders, 67 families and 117 species. The ability of the NoSpi2/BR2 primer combination to reliably amplify prey species, without amplifying any predator reads, makes it an ideal choice for gut-content analysis for spider species of lycosids and closely related species, even enabling the homogenization of entire spider specimens without dissection. Given that the detected prey species included other spiders and carabid beetles, this primer could be used for not only diet and biological control studies, but also to study intra-guild predation.