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Title: Rapid molecular identification of Scolytinae (Coleoptera: Curculionidae)

item ALBO, JONATHON - US Department Of Agriculture (USDA)
item MARELLI, JEAN-PHILIPPE - M & M Mars Company - United States
item Puig, Alina

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/21/2019
Publication Date: 11/26/2019
Citation: Albo, J.E., Marelli, J., Puig, A.S. 2019. Rapid molecular identification of Scolytinae (Coleoptera: Curculionidae). International Journal of Molecular Sciences. 20, 5944.

Interpretive Summary: Ambrosia and bark beetles are wood-boring pests often associated with diseases affecting forests and fruit production worldwide. Despite their importance to US agriculture and frequent interception at ports of entry, their identification requires specialized taxonomic knowledge that may take weeks to months to obtain. To enable more rapid screening for high priority insects and detect new invasions, scientists at the USDA-ARS (Miami, FL) collaborated with Mars Inc. to develop a molecular identification approach that requires only basic molecular biology skills and equipment. A sequence-based identification protocol was created that makes it possible to identify specimens to the species level in one day. This protocol provides faster identification of ambrosia and bark beetles, and new introductions can be detected within days of capture. It can be used by researchers in various fields, contributing to increased knowledge and improved monitoring of ambrosia and bark beetles.

Technical Abstract: Routine identification of bark and ambrosia beetles is done using morphology. For people lacking the necessary taxonomic knowledge, proper identification of a novel specimen can be challenging and time consuming. This study compares the usefulness of four genetic markers (28S, EF-1a, ITS2, and COI) and five primer pairs (D2F1/D3R2, eflafor1/eflarev1, ets149/efa754, ITS2F/ITS2R, and LCO1490/HCO2198) to identify Scolytinae beetles, and outlines a molecular identification strategy, with results possible in two days. Markers COI and EF-1a were selected based on the ability of the respective primers to amplify DNA from multiple genera (Coptoborus, Xyleborus, Hypothenemus, Theoborus, and Araptus) and the ability of the resulting sequences to provide accurate and unambiguous matches in GenBank. BLASTn analysis of EF-1a sequences (both primer pairs) correctly identified four out of the five genera and COI sequences identified at least one sample of every genus tested and was the only primer pair to correctly identify Araptus specimens. Further, 28S sequences successfully identified Coptoborus, Xyleborus, and Theoborus but not Hypothenemus or Araptus. The low number of EF-1a (1), 28S (7), and ITS2 (0) sequences from Araptus individuals present in GenBank compared with COI (137) is likely the reason that only the latter marker was capable of identifying members of this genus. ITS2 sequences were insufficient to identify any of the samples tested. This study also determined the minimum quantity of DNA that could be used for molecular identification. Primers D2F1 and D3R2, which had the highest rate of amplification in all genera tested, could yield an informative sequence with as little as 0.00048 ng of DNA, however, at least 0.0024 ng was needed for reliable amplification.