Biodiversity Data Journal :
Research Article
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Corresponding author: Olga Schmidt (olga.schmidt@snsb.de)
Academic editor: Yasen Mutafchiev
Received: 03 Aug 2017 | Accepted: 30 Oct 2017 | Published: 31 Oct 2017
© 2017 Olga Schmidt, Axel Hausmann, Bruno Cancian de Araujo, Hari Sutrisno, Djunijanti Peggie, Stefan Schmidt
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Schmidt O, Hausmann A, Cancian de Araujo B, Sutrisno H, Peggie D, Schmidt S (2017) A streamlined collecting and preparation protocol for DNA barcoding of Lepidoptera as part of large-scale rapid biodiversity assessment projects, exemplified by the Indonesian Biodiversity Discovery and Information System (IndoBioSys). Biodiversity Data Journal 5: e20006. https://doi.org/10.3897/BDJ.5.e20006
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Here we present a general collecting and preparation protocol for DNA barcoding of Lepidoptera as part of large-scale rapid biodiversity assessment projects, and a comparison with alternative preserving and vouchering methods. About 98% of the sequenced specimens processed using the present collecting and preparation protocol yielded sequences with more than 500 base pairs. The study is based on the first outcomes of the Indonesian Biodiversity Discovery and Information System (IndoBioSys). IndoBioSys is a German-Indonesian research project that is conducted by the
Collecting methods, DNA barcoding, light trap, monitoring, moths, rapid biodiversity assessment, sampling protocol
Large-scale biodiversity inventory projects are becoming increasingly popular (see
The present paper introduces the ongoing large-scale biodiversity discovery project IndoBioSys (Indonesian Biodiversity Discovery and Information System). The project is focusing on establishing a vertebrate and invertebrate diversity discovery pipeline and a biodiversity information system in Indonesia. Samples are processed through an integrated sorting pipeline that has been set up and optimized at the
A targeted, biodiversity knowledge-based screening approach for the identification of novel active biological compounds is a central aspect of the project. For this purpose, the initiative aims at providing a comprehensive biodiversity inventory, including descriptions of new species with sequence data that are publicly available in the Barcode of Life Data Systems (BOLD) and also integrated in the Indonesian Biodiversity Information System (IBIS), aimed at providing access to existing and new information on Indonesia’s biological resources. The present paper focuses on the insect order Lepidoptera that is among the largest and economically most important groups of insects.
Although general collection protocols compiled for molecular studies are available (e.g. http://lepbarcoding.org/protocols.php,
The highly diverse biota of Indonesia comprise both Oriental and Australian elements, with a high proportion of endemic species (see
Map of Western Java showing the Halimun-Salak national Park (hatched). The study area is located in the Western Javan montane rain forest ecozone (dark green). Red dot in inset map shows the location of the study area in the Sundaland region. Map created with SimpleMappr (http://www.simplemappr.net).
Moths of Indonesia are poorly studied (see
Sampling of Lepidoptera has been conducted at three plots in the study area at different elevations during one dry and one wet season. Material has been collected during the day using a sweep net and at night using standard light sources having a strong emission in the ultraviolet range of the spectrum. At each plot, two light traps at a distance of about 30-40 meters from each other were operated simultaneously: (1) a UV light trap with two light sources, including black light lamps and fluorescent light tubes (8 W each) running from rechargeable 12 V batteries, (2) a mercury vapour light trap (125 W) running from a generator. The light sources were placed in front of a white sheet and protected from the rain by an umbrella. Active sampling (no killing traps) was preferred. Moths were one of the major target groups for the project. Collection- and general preparation methods, as well as digital imaging of specimens, have been described in numerous studies (e.g.
All the vouchers should be deposited in a public insect collection and stored in insect drawers in entomological cabinets under proper conditions to protect them from climatic conditions and insect pest attacks as the voucher specimens are linked to the DNA barcode reference library and establish a base for testing and verification of the results.
The present collecting and preparation protocol is a manual used by the students, technical staff and researchers involved in the study. Here, we provide responses to some of the more common issues raised.
The workflow. The collection protocol presented in this article has been successfully employed for field- and pre-lab activities that were part of the IndoBioSys project in the years 2015 and 2016. The workflow is presented in Fig.
Success rates. About 98% of the sequenced specimens of the Geometridae processed using the present collecting and preparation protocol yielded sequences with more than 500 base pairs, meeting the length requirement for DNA barcode status (see
Treatment of specimen with ethyl acetate. Sequencing for inventory projects like IndoBioSys requires killing and preserving the specimens in a DNA-friendly way. The Lepidoptera were killed with ethyl acetate vapours and in most cases removed from the killing jars after about 20-30 minutes (but not longer than 40 minutes) to make sure that the DNA is not damaged. Our results confirm the findings by
Remarks to a recently publised alternative preserving and vouchering method. In a recent publication by
Additionally, reliable storage methods were described and discussed by
Towards future outcomes. The ultimate objective of our study is to present a methodological pipeline assisting in successful sampling, preparation, preservation, morphological and molecular analyses and secure storage of high-quality material for a biodiversity assessment which combines the expertise gained through the DNA barcoding and the taxonomist’s knowledge.
The protocol has been prepared as part of the IndoBioSys project funded by the German Federal Ministry of Education and Research (BMBF) and supported by the