Rapid Molecular Identification of Scolytinae (Coleoptera: Curculionidae).

Jonathan Eric Albo,Jean-Philippe Marelli,Alina Sandra Puig

doi:10.3390/ijms20235944

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.

Highlights

Diseases associated with wood-boring ambrosia and bark beetles (Curculionidae: Scolytinae and Platypodinae) are a rapidly increasing threat to forests and fruit production around the world [1,2]
Over half of all journal articles on this taxonomic group have been published within the past ten years [1]. Some of these pathosystems, such as Dutch elm disease [5], have been the focus of research for many years, but others such as laurel wilt vectored by the redbay ambrosia beetle (Xyleborus glabratus) [6,7], thousand cankers disease caused by the walnut twig beetle (Pityophthorus juglandis) [8], and Fusarium dieback spread by shot hole borers in the Euwallacea fornicatus species complex [9,10] are the result of pest introductions that have prompted more recent investigations
Markers COI and EF-1a are recommended for initial molecular identification attempts 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

Summary

Introduction

Diseases associated with wood-boring ambrosia and bark beetles (Curculionidae: Scolytinae and Platypodinae) are a rapidly increasing threat to forests and fruit production around the world [1,2]. Over half of all journal articles on this taxonomic group have been published within the past ten years [1]. Some of these pathosystems, such as Dutch elm disease [5], have been the focus of research for many years, but others such as laurel wilt vectored by the redbay ambrosia beetle (Xyleborus glabratus) [6,7], thousand cankers disease caused by the walnut twig beetle (Pityophthorus juglandis) [8], and Fusarium dieback spread by shot hole borers in the Euwallacea fornicatus species complex [9,10] are the result of pest introductions that have prompted more recent investigations.

Methods

Findings

Discussion

Conclusion