Bio-Protection Research Centre, PO Box 84, Lincoln University, Lincoln 7647, Christchurch, New Zealand.
Mol Ecol Resour. 2012 May;12(3):389-95. doi: 10.1111/j.1755-0998.2011.03103.x. Epub 2011 Dec 22.
Despite the negative impact that many scarab larvae have on agro-ecosystems, very little attention has been paid to their taxonomy. Their often extremely similar morphological characteristics have probably contributed to this impediment, which has also meant that they are very difficult to identify in the field. Molecular methods can overcome this challenge and are particularly useful for the identification of larvae to enable management of pest species occurring sympatrically with nonpest species. However, the invasive collection of DNA samples for such molecular methods is not compatible with subsequent behavioural, developmental or fitness studies. Two noninvasive DNA sampling and DNA analysis methods suitable for the identification of larvae from closely related scarab species were developed here. Using the frass and larval exuviae as sources of DNA, field-collected larvae of Costelytra zealandica (White) and Costelytra brunneum (Broun) (Scarabaeidae: Melolonthinae) were identified by multiplex PCR based on the difference in size of the resulting PCR products. This study also showed that small quantities of frass can be used reliably even 7 days after excretion. This stability of the DNA is of major importance in ecological studies where timeframes rarely allow daily monitoring. The approach developed here is readily transferable to the study of any holometabolous insect species for which morphological identification of larval stages is difficult.
尽管许多金龟幼虫对农业生态系统有负面影响，但人们对它们的分类学关注甚少。它们通常极其相似的形态特征可能是造成这种障碍的原因之一，这也意味着它们在野外很难识别。分子方法可以克服这一挑战，对于识别幼虫以管理与非害虫物种共存的害虫物种特别有用。然而，为这种分子方法采集侵入性 DNA 样本与后续的行为、发育或适应性研究不兼容。本研究开发了两种适用于鉴定近缘金龟幼虫的非侵入性 DNA 采样和 DNA 分析方法。使用粪便和幼虫蜕壳作为 DNA 来源，通过基于 PCR 产物大小差异的多重 PCR，鉴定了从新西兰白纹花金龟（Costelytra zealandica (White)）和褐斑粗角花金龟（Costelytra brunneum (Broun)）（鞘翅目：丽金龟科）采集的田间幼虫。本研究还表明，即使在排泄后 7 天，少量的粪便也可以可靠地使用。这种 DNA 的稳定性在生态研究中非常重要，因为时间框架很少允许每天进行监测。这里开发的方法很容易转移到任何形态学上难以识别幼虫阶段的完全变态昆虫物种的研究中。
