Comparative analysis of eight DNA extraction methods for molecular research in mealybugs

Autoři: Yu-Sheng Wang aff001;  Tian-Mei Dai aff001;  Hu Tian aff001;  Fang-Hao Wan aff001;  Gui-Fen Zhang aff001
Působiště autorů: State Key Laboratory for Biology of Plant Diseases and Insect Pests / Key Laboratory of Integrated Pest Management of Crop, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Institute of Plant Protection, Chinese Academy of Agri aff001;  Hunan Provincial Key Laboratory for Control of Forest Diseases and Pests, College of Forestry, Central South University of Forestry and Technology, Changsha, China aff002;  Caofeidian Sub-Center of Hebei Entry-Exit Inspection and Quarantine Technical Center, Tangshan, China aff003;  Center for Management of Invasive Alien Species, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Beijing, China aff004
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


For molecular research, the quality and integrity of DNA obtained will affect the reliability of subsequent results. Extracting quality DNA from scale insects, including mealybugs, can be difficult due to their small body size and waxy coating. In this study, we evaluate eight commonly used DNA extraction methods to determine their efficacy in PCR analysis across life stages and preservation times. We find that fresh samples, immediately upon collection or after 2 wks, resulted in the most effective DNA extraction. Methods using the DNeasy Blood & Tissue kit, NaCl, SDS-RNase A, and SDS isolated DNA of sufficient quality DNA. The SDS method gave high DNA yield, while the NaCl and SDS-RNase A methods gave lower yield. NaCl, SDS-RNase A, SDS, chloroform-isopentyl alcohol, and the salting-out methods all resulted in sufficient DNA for PCR, and performed equal to or better than that of the DNeasy Blood & Tissue kit. When time and cost per extraction were considered, the SDS method was most efficient, especially for later life stages of mealybug, regardless of preservation duration. DNA extracted from a single fresh sample of a female adult mealybug was adequate for more than 10,000 PCR reactions. For earlier stages, including the egg and 1st instar nymph samples, DNA was most effectively extracted by the Rapid method. Our results provide guidelines for the choice of effective DNA extraction method for mealybug or other small insects across different life stages and preservation status.

Klíčová slova:

DNA – DNA electrophoresis – DNA extraction – Gene amplification – Nymphs – Polymerase chain reaction – Ribonucleases – RNA extraction


1. Hardy NB, Gullan PJ, Hodgson CJ. A subfamily-level classification of mealybugs (Hemiptera: Pseudococcidae) based on integrated molecular and morphological data. Syst Entomol. 2008;33: 51–71.

2. Abd-Rabou S, Shalaby H, Germain JF, Ris N, Kreiter P, Malausa T. Identification of mealybug pest species (Hemiptera: Pseudococcidae) in Egypt and France, using a DNA barcoding approach. Bull Entomol Res. 2012;102: 515–523. doi: 10.1017/S0007485312000041 22360997

3. ScaleNet. Pseudococcidae; 2016. [cited 26 April 2019].

4. Zeddies J, Schaab RP, Neuenschwander P, Herren HR. Economics of biological control of cassava mealybug in Africa. Agr Econ. 2001;24: 209–219.

5. Miller DR, Miller GL, Watson GW. Invasive species of mealybugs (Hemiptera: Pseudococcidae) and their threat to US agriculture. Proc Entomol Soc Wash. 2002;104: 825–836.

6. Hodgson CJ, Abbas G, Arif MJ, Saeed S, Karar H. Phenacoccus solenopsis Tinsley (Sternorrhyncha: Coccoidea: Pseudococcidae), an invasive mealybug damaging cotton in Pakistan and India, with a discussion on seasonal morphological variation. Zootaxa. 2008;1913: 1–35.

7. Ahmed MZ, He RR, Wu MT, Gu YJ, Ren JM, Liang F, et al. First report of the papaya mealybug, Paracoccus marginatus (Hemiptera: Pseudococcidae), in China and genetic record for its recent invasion in Asia and Africa. Fla Entomol. 2015;98: 1157–1162.

8. Ashfaq M, Noor AR, Mansoor S. DNA-based characterization of an invasive mealybug (Hemiptera: Pseudococcidae) species damaging cotton in Pakistan. Appl Entomol Zool. 2010;45: 395–404.

9. Pieterse W, Muller DL, van Vuuren BJ. A molecular identification approach for five species of mealybug (Hemiptera: Pseudococcidae) on citrus fruit exported from South Africa. Afr Entomol. 2010;18: 23–28.

10. Singh S, Sharma R, Kumar R, Gupta VK, Dilawari VK. Molecular typing of mealybug Phenacoccus solenopsis populations from different hosts and locations in Punjab, India. J Environ Biol. 2012;33: 539–543. 23029900

11. Moritz C, Cicero C. DNA barcoding: promise and pitfalls. PLoS Biol. 2004;2: e354. doi: 10.1371/journal.pbio.0020354 15486587

12. Schill RO. Comparison of different protocols for DNA preparation and PCR amplification of mitochondrial genes of tardigrades. J Limnol. 2007;66: 164–170.

13. Miller NJ, Guillemaud T, Giordano R, Siegfried BD, Gray ME, Meinke LJ, et al. Genes, gene flow and adaptation of Diabrotica virgifera virgifera. Agr Forest Entomol. 2009;11: 47–60.

14. Lagisz M, Port G, Wolff K. A cost-effective, simple and high-throughput method for DNA extraction from insects. Insect Sci. 2010;17: 465–470.

15. Allen GC, Flores-Vergara MA, Krasynanski S, Kumar S, Thompson WF. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc. 2006;1: 2320–2325. doi: 10.1038/nprot.2006.384 17406474

16. Dittrich-Schröder G, Wingfield MJ, Klein H, Slippers B. DNA extraction techniques for DNA barcoding of minute gall-inhabiting wasps. Mol Ecol Resour. 2012;12: 109–115. doi: 10.1111/j.1755-0998.2011.03074.x 21951690

17. Athanasio CG, Chipman JK, Viant MR, Mirbahai L. Optimisation of DNA extraction from the crustacean Daphnia. PeerJ. 2016;4: e2004. doi: 10.7717/peerj.2004 27190714

18. Chen H, Rangasamy M, Tan SY, Wang HC, Siegfried BD. Evaluation of five methods for total DNA extraction from western corn rootworm beetles. PLoS One. 2010;5: e11963. doi: 10.1371/journal.pone.0011963 20730102

19. Garagna S, Rebecchi L, Guidi A. Genome size variation in Tardigrada. Zool J Linn Soc. 1996;116: 115–121.

20. Williams DJ. Mealybugs of Southern Asia. London: The Natural History Museum and Kuala Lumpur: Southdene; 2004.

21. Vink CJ, Thomas SM, Paquin P, Hayashi CY, Hedin M. The effects of preservatives and temperatures on arachnid DNA. Invertebr Syst. 2005;19: 99–104.

22. Ball SL, Armstrong KF. Rapid, one-step DNA extraction for insect pest identification by using DNA barcodes. J Econ Entomol. 2008;101: 523–532. doi: 10.1603/0022-0493(2008)101[523:rodefi];2 18459420

23. Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1991;362: 709–715.

24. Dillon N, Austin AD, Bartowsky E. Comparison of preservation techniques for DNA extraction from hymenopterous insects. Insect Mol Biol. 1996;5: 21–24. doi: 10.1111/j.1365-2583.1996.tb00036.x 8630531

25. Gilbert MTP, Moore W, Melchior L, Worobey M. DNA extraction from dry museum beetles without conferring external morphological damage. PLoS One. 2007;2: e272. doi: 10.1371/journal.pone.0000272 17342206

26. Demeke T, Jenkins GR. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem. 2010;396: 1977–1990. doi: 10.1007/s00216-009-3150-9 19789856

27. Kilpatrick CW. Noncryogenic preservation of mammalian tissues for DNA extraction: an assessment of storage methods. Biochem Genet. 2002;40: 53–62. doi: 10.1023/a:1014541222816 11989787

28. Wehausen JD, Ramey RR, Epps CW. Experiments in DNA extraction and PCR amplification from bighorn sheep feces: the importance of DNA extraction method. J Hered. 2004;96: 503–509.

29. Aime MC, Phillips-Mora W. The causal agents of witches’ broom and frosty pod rot of cacao (chocolate, Theobroma cacao) form a new lineage of Marasmiaceae. Mycologia. 2005;97: 1012–1022. 16596953

30. Goolsby JA, De Barro PJ, Makinson JR, Pemberton RW, Hartley DM, Frolich DR. Matching the origin of an invasive weed for selection of a herbivore haplotype for biological control programme. Mol Ecol. 2006;15: 287–297. doi: 10.1111/j.1365-294X.2005.02788.x 16367847

31. A'Hara S, Harling R, McKinlay R, Topping C. RAPD profiling of spider (Araneae) DNA. J Arachnol. 1998;26: 397–400.

32. Peist R, Honsel D, Twieling G, Löffert D. PCR inhibitors in plant DNA preparations. Qiagen New. 2001;3: 7–9.

33. Hoy MA. Insect molecular genetics: An introduction to principles and applications. 2nd ed. San Diego: Academic Press; 2003.

34. Xue M, Wu LY, He YY, Liang HF, Wen CQ. Biases during DNA extraction affect characterization of the microbiota associated with larvae of the Pacific white shrimp, Litopenaeus vannamei. PeerJ. 2018;6: e5257. doi: 10.7717/peerj.5257 30038871

35. Shi J, Xie YP, Xue JL, Yao GQ. Comparative study of methods for isolation of genomic DNA of scale insects. Chinese Bull Entomol. 2005;42: 207–211.

36. Phillips AJ, Simon C. Simple, efficient, and nondestructive DNA extraction protocol for arthropods. Ann Entomol Soc Am. 1995;88: 281–283.

37. Zhou ZX, Wan FH, Zhang GF, Chen B. A rapid method for extraction of genomic DNA of Bemisia tabaci. Plant Protect. 2007;33: 131–133.

38. Dai TM, Lü ZC, Wan FH. Comparison of four methods for whole genomic DNA extraction from Bemisia tabaci. Biotech Bull. 2014;8: 70–75.

39. Sunnucks P, Hales DF. Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in Aphids of the genus Sitobion (Hemiptera: Aphididae). Mol Biol Evol. 1996;13: 510–524. doi: 10.1093/oxfordjournals.molbev.a025612 8742640

40. De Barro PJ, Driver F. Use of RAPD PCR to distinguish the B biotype from other biotypes of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Aust J Entomol. 1997;36: 149–152.

41. Park DS, Suh SJ, Hebert PDN, Oh HW, Hong KJ. DNA barcodes for two scale insect families, mealybugs (Hemiptera: Pseudococcidae) and armored scales (Hemiptera: Diaspididae). Bull Entomol Res. 2011;101: 429–434. doi: 10.1017/S0007485310000714 21272395

42. Dietrich CH, Rakitov RA, Holmes JL, Black WC IV. Phylogeny of the major lineages of Membracoidea (Insecta: Hemiptera: Cicadomorpha) based on 28S rDNA sequences. Mol Phylogenet Evol. 2001;18: 293–305. doi: 10.1006/mpev.2000.0873 11161763

43. Shokere LA, Holden MJ, Jenkins GR. Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA. Food Control. 2009;20: 391–401.

44. Bujang NS, Harrison NA, Su NY. An improved method for extraction and purification of termite endo-β-1-1,4-glucanase from FTA® cards. Fla Entomol. 2011;94: 356–358.

45. You YQ, Chen Y, Yi JQ, Lin YW. Comparison of genomic DNA extracting methods for Hemiberlesia pitysophila. Entomol J East China. 2007;16: 26–29.

46. Zhu YY, Huang F, Lu YB. Bionomics of mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) on cotton. Acta Entomol Sin. 2011;54: 246–252.

47. Wei YH, Zheng SZ, Cai P, Zhan GH, Gao Y. Comparison of methods for extracting and preserving the DNA of adult Pseudococcidae specimens. Chinese J Appl Entomol. 2015;52: 925–932.

Článek vyšel v časopise


2019 Číslo 12
Nejčtenější tento týden