Anindya Safita Ningtias, Istiana Prihatini, Maryatul Qiptiyah


Identification of insect species using molecular approach is one of the first steps in managing Ceratocystis. Proper species identification of the vectorsof Ceratocystiswill provide an effective way in limiting the distribution of this pathogenic fungi. The isolation of insect DNA is a crucial step in species identification using molecular characters. The aim of this research was to obtain the most effective method for isolating the COI gene and to confirm the insect DNA using amplification of the DNA through polymerase chain reaction (PCR). Twelve unidentified Coleoptera specimen collected from Acacia spp. plantation in Pelalawan Riau were randomly selected for this study. The insect DNA were isolated using the CTAB buffer with four different preincubation treatments, involved a non destructive method, i.e. soaking (R), destructive methods, i.e. crushing (H), soaking-crushing (RH) and freezing-crushing (FH). Two sets of primer, LCO1490/HCO2196 and LCO1490/HCO2198 were used to amplify the DNA of COI gene. The results shows, the COI gene was isolated from all pre-incubation treatments, except in the non-destructive treatment. The isolated DNA of COI gene was successfully amplified using both primer sets used in this study.


DNA extraction; insect; DNA amplification; CTAB buffer; pre-incubation


Albo, J. E., Marelli, J. P., & Puig, A. S. (2019). Rapid molecular identification of Scolytinae (Coleoptera: Curculionidae). International Journal of Molecular Sciences, 20(23).

Asghar, U., Malik, M. F., Anwar, F., Javed, A., & Raza, A. (2015). DNA Extraction from insects by using different techniques: A review. Advances in Entomology, 03(04), 132–138.

Ball, S. L., & Armstrong, K. F. (2008). Rapid, one-step DNA extraction for insect pest identification by using DNA barcodes. Journal of Economic Entomology, 101(2), 523–532.[523:RODEFI]2.0.CO;2

Cabral, H., Ruiz, M. T., Carareto, C. M. A., & Bonilla-Rodriguez, G. O. (2000). A plant proteinase, extracted from Bromelia fastuosa, as an alternative to proteinase K for DNA extraction. Drosophila Information Service, 83, 178–185.

Castalanelli, M. A., Severtson, D. L., Brumley, C. J., Szito, A., Foottit, R. G., Grimm, M., … Groth, D. M. (2010). A rapid non-destructive DNA extraction method for insects and other arthropods. Journal of Asia-Pacific Entomology, 13(3), 243–248.

Chen, H., Rangasamy, M., Tan, S. Y., Wang, H., & Siegfried, B. D. (2010). Evaluation of five methods for total DNA extraction from western corn rootworm beetles. PLoS ONE, 5(8).

Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19(1), 11–15.

Edde, P. A. (2012). A review of the biology and control of Rhyzopertha dominica (F.) the lesser grain borer. Journal of Stored Products Research, 48, 1–18.

Favret, C. (2005). A new non-destructive DNA extraction and specimen clearing technique for aphids (Hemiptera). Proceedings of the Entomological Society of Washington, 107(2), 469–470.

Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3(5), 294–299.

Gilbert, M. T. P., Moore, W., Melchior, L., & Worebey, M. (2007). DNA extraction from dry museum beetles without conferring external morphological damage. PLoS ONE, 2(3), 1–4.

Greco, M., Sáez, C. A., Brown, M. T., & Bitonti, M. B. (2014). A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga. PLoS ONE, 9(5).

Halos, L., Jamal, T., Vial, L., Maillard, R., Suau, A., Menach, A. L., … Vayssier-Taussat, M. (2004). Determination of an efficient and reliable method for DNA extraction from ticks. Veterinary Research, 35(6), 709–713.

Hara, A. H., & Beardsley, J. W. J. (1979). The biology of the black twig borer, Xylosandrus compactus (Eichhoff), in Hawaii. Proceedings of the Hawaiian Entomological Society, XIII(1), 55–70.

Heath, R. N., Wingfield, M. J., Van Wyk, M., & Roux, J. (2009). Insect associates of Ceratocystis albifundus and patterns of association in a native savanna ecosystem in South Africa. Environmental Entomology, 38(2), 356–364.

Li, Y., Gu, X., Kasson, M. T., Bateman, C. C., Guo, J., Huang, Y., … Hulcr, J. (2016). Distribution, host records, and symbiotic fungi of Euwallacea fornicatus (Coleoptera: Curculionidae: Scolytinae) in China. Florida Entomologist, 99(4), 801–804.

Lienhard, A., & Schäffer, S. (2019). Extracting the invisible: Obtaining high quality DNA is a challenging task in small arthropods. PeerJ, 2019(4).

Liu, S., Sun, J., Yu, L., Zhang, C., Bi, J., Zhu, F., … Yang, Q. (2012). Extraction and characterization of chitin from the beetle Holotrichia parallela Motschulsky. Molecules, 17(4), 4604–4611.

Lobo, J., Costa, P. M., Teixeira, M. A. L., Ferreira, M. S. G., Costa, M. H., & Costa, F. O. (2013). Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans. BMC Ecology, 13, 1–8.

Miura, K., Higashiura, Y., & Maeto, K. (2017). Evaluation of easy, non-destructive methods of DNA extraction from minute insects. Applied Entomology and Zoology, 52(2), 349–352.

Nair, K. S. S. (2000). Insect pests and diseases in Indonesian forest: an assessment of the major threats, research efforts and literature. (K. S. S. Nair, Ed.), Insect pests and diseases in Indonesian forest: an assessment of the major threats, research efforts and literature. Bogor.

Phillips, A. J., & Simon, C. (1995). Simple, efficient, and nondestructive DNA extraction protocol for arthropods. Annals of the Entomological Society of America, 88(3), 281–283.

Qamar, W., Khan, M. R., & Arafah, A. (2017). Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method. Saudi Journal of Biological Sciences, 24(7), 1465–1469.

Rahayu, S., Nurjanto, H. H., & Pratama, R. G. (2015). Karakter jamur Ceratocystis sp. penyebab penyakit busuk batang padaAcacia decurrens dan status penyakitnya di Taman Nasional Gunung Merapi. Jurnal Ilmu Kehutanan, 9(2), 94–104.

Rohland, N., & Hofreiter, M. (2007). Comparison and optimization of ancient DNA extraction. BioTechniques, 42(3), 343–352.

Roux, J., Van Wyk, M., Hatting, H., & Wingfield, M. J. (2004). Ceratocystis species infecting stem wounds on Eucalyptus grandis in South Africa. Plant Pathology, 53(4), 414–421.

Roux, J., & Wingfield, M. J. (2009). Ceratocystis species: Emerging pathogens of non-native plantation Eucalyptus and Acacia species. Southern Forests, 71(2), 115–120.

Setiawan, Y., Rachmawati, R., & Tarno, H. (2018). Diversity of ambrosia beetles (Coleoptera: Scolytidae) on teak forest in Malang District, East Java, Indonesia. Biodiversitas Journal of Biological Diversity, 19(5), 1791–1797.

Tarigan, M., Roux, J., Wyk, M. Van, Tjahjono, B., & Wingfield, M. J. (2011). A new wilt and die-back disease of Acacia mangium associated with a new wilt and die-back disease of Acacia mangium associated with Ceratocystis manginecans and C . acaciivora sp . nov . in Indonesia. South African Journal of Botany, 77, 292–304.

Tarno, H., Septia, E. D., & Aini, L. Q. (2016). Microbial community associated with ambrosia beetle, Euplatypus parallelus on sonokembang, Pterocarpus indicus in Malang. Agrivita, 38(3), 312–320.



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