Gene functional analysis of Harmonia axyridis by in vitro transcription

  • Park, Sang-Eun (Department of Agricultural Biology, College of Agriculture and Life Sciences, Chungnam National University) ;
  • Youn, Young-Nam (Department of Agricultural Biology, College of Agriculture and Life Sciences, Chungnam National University)
  • Received : 2019.04.11
  • Accepted : 2019.06.17
  • Published : 2019.09.01


Random genes were screened in two transforming ways to investigate the new genes of a ladybug using the Harmonia axyridis cDNA library stock cell cloned in the LITMUS 28i vector in a previous study. Phenotypic variation was observed after injection of the synthesized double-stranded RNA through the in vitro transcription process. The cDNA library of H. axyridis was transformed into E. coli $DH5{\alpha}$ and 10B competent cells by heat shock. Analysis of the nucleotide sequences of the 42 clones with the insert DNAs revealed that 21 clones were homologous with the genes of insects, and only one clone had a gene from H. axyridis. Thirteen of the 21 insect genes were homologous with genes from coleopteran insects. Fourteen genes were selected, which were identified by the gene screening results, and were synthesized as double-stranded RNA through in vitro transcription. One microgram of the synthesized double-stranded RNA between segments T1 and T2 were injected using a syringe into each anesthetized fourth larvae which were under 2 days old. As a result, a phenotypic variation appeared in the larva injected with the two genes. While the eggs of H. axyridis injected with distilled water hatched out three days after oviposition, the eggs of H. axyridis injected with dsHma 06 did not hatch but become shrivel a week after oviposition. Most of the H. axyridis injected with dsHma 08 died and were unable to complete the pupation or eclosion during ecdysis.


Supported by : 충남대학교


  1. Amdam GV, Simoes ZL, Guidugli KR, Norberg K, Omholt SW. 2003. Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA. BMC Biotechnology 3:1.
  2. Antonio DSM, Guidugli-Lazzarini KR, Do Nascimento AM, Simoes ZLP, Hartfelder K. 2008. RNAi-mediated silencing of vitellogenin gene function turns honeybee (Apis mellifera ) workers into extremely precocious foragers. Naturwissenschaften 95:953-961.
  3. Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, Johnson S, Plaetinck G, Munyikwa T, Pleau M, Vaughn T, Roberts J. 2007. Control of coleopteran insect pests through RNA interference. Nature Biotechnology 25:1322-1326.
  4. Belicek J. 1976. Coccinellidae of Western Canada and Alaska with analyses of the transmontane zoogeographic relationship between the fauna of British Columbia and Alberta. Questiones Entomologicaes 12:283-409
  5. Belles X. 2010. Beyond Drosophila : RNAi In vivo and functional genomics in insects. Annual Review of Entomology 55:111-128.
  6. Birnboim HC, Doly J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7:1513-1523.
  7. Bolognesi R, Ramaseshadri P, Anderson J, Bachman P, Clinton W, Flannagan R, Ilagan O, Lawrence C, Levine S, Moar W. 2012. Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte). PLoS ONE 7:e47534.
  8. Boo KS. 2001. Insect hormones and their actions. Korean Journal of Applied Entomology 40:155-196. [in Korean]
  9. Brown PMJ, Adriaens T, Bathon H, Cuppen J, Goldarazena A, Hagg T, Kenis M, Klausnitzer BEM, Kovar I, Loomans AJM, Majerus MEN, Nedved O, Pederson J, Rabitsch W, Roy HE, Ternois V, Zakharov IA, Roy DB. 2007. Harmonia axyridis in Europe: Spread and distribution of a non-native coccinellid. In From Biological Control to Invation: The Ladybird Harmonia axyridis as a Model Species. Springer, Dordrecht53:5-21.
  10. Chapin JB, Brou V. 1991. Harmonia axyridis (Pallas), the third species of the genus to be found in the United States (Coleoptera: Coccinellidae). Proceedings of the Entomological Society of Washington 93:630-635.
  11. Choi SY, Kim GH. 1985. Aphidivorous activity of a coccinellid beetle, Harmonia axyridis Pallas (Coleoptera: Coccinellidae). Korean Journal of Applied Entomology 24:11-14. [in Korean]
  12. Ciudad L, Piulachs MD, Belles X. 2006. Systemic RNAi of the cockroach vitellogenin receptor results in a phenotype similar to that of the Drosophila yolkless mutant. FEBS Journal 35:273-325.
  13. Coderre D, Lucas E, Gagne I. 1995. The occurrence of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) in Canada. Canadian Entomologist 127:609-611.
  14. David SR, Watkinsa NL, Serafina RB, Gilbertb LI. 1998. Ecdysteroids regulate yolk protein uptake by Drosophila melanogaster oocytes. Journal of Insect Physiology 44:637-644.
  15. Day WH, Prokrym DR, Ellis DR, Chianese RJ. 1994. The known distribution of the predator Propylea quatuordecimpunctata (Coleoptera: Coccinellidae) in the United States, and thoughts on the origin of this species and five other exotic lady beetles in eastern North America. Entomological News 105:244-256.
  16. Dobzhansky T. 1933. Geographical variation in lady-beetles. The American Naturalist 67:97-126.
  17. ESK and KSAE. 1994. Check list of insects from Korea (eds). Kon-Kuk University Press, Seoul, Korea.
  18. Ferran A, Iperti G, Kreiter S, Quilicci S, Shanderl H. 1986. Preliminary results of a study of the potentials of some aphidophagous coccinellids for use in biological control. Ecology of Aphidophaga, Vol. 2. I. Hodek (ed.). pp. 479-484. Academia, Praha, Czech.
  19. Ferran A, Niknam H, Kabiri F, Picart J-L, De Herce C, Brun J, Iperti G, Lapchin L. 1996. The use of Harmonia axyridis larvae (Coleoptera: Coccinellidae) against Macrosiphum rosae (Hemiptera: Sternorrhyncha: Aphididae) on rose bushes. European Journal of Entomology 93:59-67
  20. Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE, Mello CC. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811.
  21. Fjose A, Ellingsen S, Wargelius A, Seo HC. 2001. RNA interference: Mechanisms and applications. Biotechnology Annual Review 7:31-57.
  22. Freier B, Triltsch H. 1995. Harmonia axyridis (Pallas) - An interesiting ladybird for biological plant protection. Gesund Pflanzen 47:269-271.
  23. Glisovic T, Bachorik JL, Yong J, Dreyfuss G. 2008. RNA-binding proteins and post-transcriptional gene regulation. FEBS Letters 582:1977-1986.
  24. Gordon RD. 1985. The Coccinellidae (Coleoptera) of America north of Mexico. Journal of the New York Entomological Society 93:1-912.
  25. Grill CP, Moore AJ. 1998. Effects of a larval antipredator response and larval diet on adult phenotype in an aposematic ladybird beetle. Oecologia 114:274-282.
  26. Hagedorn H, Kunkel JG. 1979. Vitellogenin and vitellin in insects. Annual Review of Entomology 24:475-505.
  27. Hodek I, Honek A. 1996. Ecology of Coccinellidae. Kluwer Academic Publishers, London, UK.
  28. Hogan DJ, Riordan DP, Gerber AP, Herschlag D, Brown PO. 2008. Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system. PLoS Biology 6:e255.
  29. Huvenne H, Smagghe G. 2010. Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: Review. Journal of Insect Physiology 56:216-225.
  30. Jung YB. 2016. cDNA library construction and application of RNA interference for gene functional analysis in Harmonia axyridis. M.S. dissertation, Chungnam National Univ., Daejeon, Korea. [in Korean]
  31. Kang EJ, Jo CW, Park CR, Yoon KS, Kang MA, Kwon HR, Seo MJ, Yu YM, Youn YN. 2009. Effects of environmental factors on elytra colored patterns of multicolored Asian lady beetles, Harmonia axyridis (Coleoptera: Coccinellidae). Koran Journal of Applied Entomology 48:459-466. [in Korean]
  32. Kauffman WC, Schwalbe CP. 1991. Plant growth respomses to Aphis fabae injury: Importance of predation by Coccinella septempunctata (Coleoptera: Coccinellidae). Behaviour and Impact of Ahidophaga. Polgar L, Chambers RJ, Dixon AFG, Hodek I. (eds.). SPB Academic Publishing, Netherlands
  33. Kenis M, Roy HE, Zindel R, Majerus MEN. 2007. Current and potential management strategies against Harmonia axyridis. BioControl 53:235-252.
  34. Kennerdell JR, Carthew RW. 1998. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95:1017-1026.
  35. Kidd K, Nalepa C, Day E, Waldvogel M. 1995. Distribution of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) in North Carolina and Virginia. Proceedings of Entomological Society of Washington 97:729-731.
  36. Kim JH, Seo EY, Kim JK, Lim HS, Yu YM, Youn YN. 2015. Gene expression in plant according to RNAi treatment of the tobacco Whitefly. CNU Journal of Agricultural Science 42:81-86. [in Korean]
  37. Kim SH, Seo MJ, Park MW, Yu YM, Youn YN. 2012. Molecular cloning of prophenoloxidase (PPO) gene related to melanin formation of elytra of Harmonia axyridis. CNU Journal of Agricultural Science 39:23-28. [in Korean]
  38. Ko NY, Youn YN. 2015. Change of population density of tobacco whitefly (Bemisia tabaci, Aleyrodidae, Hemiptera) by RNAi. CNU Journal of Agricultural Science 42:7-13.
  39. Koch RL. 2003. The multicolored Asian lady beetle, Harmonia axyridis: A review of its biology, uses in biological control, and non-target impacts. Journal of Insect Science 3:1-16.
  40. Koch RL, Burkness EC, Burkness SJW, Huschison WD. 2004. Phytophagous preferences of the multicolored Asian lady beetle (Coleoptera: Coccinellidae) to autumn ripening fruits. Journal of Economic Entomology 97:539-544.
  41. Koch RL, Galvan TL. 2008. Bad side of a good beetle: The North American experience with Harmonia axyridis. Biocon 53:23-35.
  42. Koch RL, Venette RC, Hutchison WD. 2006. Invasions by Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) in the western hemisphere: Implications for South America. Neotropical Entomology 35:421-434.
  43. Komai T. 1956. Genetics of ladybeetles. Advances in Genetics 8:155-189.
  44. Kozhanova NI. 2000. Hormonal regulation of gametogenesis in insect. Tsitologiia 42:115-127.
  45. Kuwayama H, Yaginuma T, Yamashita O, Niimi T. 2006. Germ-line transformation and RNAi of the ladybird beetle, Harmonia axyridis. Insect Molecular Biology 15:507-512.
  46. Lamana ML, Miller JC. 1998. Temperature-dependent development in an Oregon population of Harmonia axyridis (Coleoptera: Coccinellidae). Environmental Entomology 27:1001-1005.
  47. Lee DH, Seo MJ, Kang EJ, Park CR, Jo CW, Hwang IC, Yu YM, Youn YN. 2011. Molecular identification of AFLP fragments associated with elytra color variation of the Asian ladybird beetle, Harmonia axyridis (Coleoptera: Coccinellidae). Journal of Asia-Pacific Entomology 14:99-105.
  48. Liu E, Page JE. 2008. Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus. Plant Methods 4:1-13.
  49. Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X. 2003. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301:1921-1925.
  50. Majerus MEN. 1994. Ladybirds. Harper Collins Publishers, Toronto, Canada.
  51. Mao YB, Cai WJ, Wang JW, Hong GJ, Tao WY, Wang LJ, Huang YP, Chen XY. 2007. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nature Biotechnology 25:1307-1313.
  52. Markkula M, Tiittanen K. 1980. Biological control of pests in glasshouses in Finland - the situation today and in the future. Bulletin SROP/WPRS 3:127-134.
  53. Michie LJ, Mallard F, Majerus MEN, Jiggins FM. 2010. Melanic through nature or nurture: Genetic polymorphism and phenotypic plasticity in Harmonia axyridis . Journal of Evolutionary Biology 23:1699-1707.
  54. Mitchell III RD, Ross E, Osgood D, Sonenshine DE, Donohue KV, Khalil SM, Thompson DM, Michael RR. 2007. Molecular characterization, tissue-specific expression and RNAi knockdown of the first vitellogenin receptor from a tick. Insect Biochemistry and Molecular Biology 37:375-388.
  55. Niimi T, Kuwayama H, Yaginuma T. 2005. Larval RNAi applied to the analysis of postembryonic development in the ladybird beetle, Harmonia axyridis . Journal of Insect Biotechnology and Sericology 74:95-102.
  56. Ohde T, Masumoto M, Morita-Miwa M, Matsuura H, Yoshioka H, Yaginuma T, Niimi T. 2009. Vestigial and scalloped in the ladybird beetle: A conserved function in wing development and a novel function in pupal ecdysis. Insect Molecular Biology 18:571-581.
  57. Osawa N, Nishida T. 1992. Seasonal variation in elytral colour polymorphism in Harmonia axyridis (the ladybird beetle): The role of non-random mating. Heredity 69:297-307.
  58. Oshima C, Tokuchiro S, Horonori I. 1956. Studies on the mechanism of pattern formation in the elytra of lady beetles. Genetics 41:4-20.
  59. Park CR, Kim JH, Yu YM, Youn YN. 2016. Differentiation of elytra color patterns in multicolored Asian ladybird beetle, Harmonia axyridis (Coleoptera; Coccinellidae), using AFLP analyses. Korean Journal of Applied Entomology 55:245-256. [in Korean]
  60. Roy H, Wajnberg E. 2008. From biological control to invasion: The ladybird Harmonia axyridis as a model species. BioControl 53:1-4.
  61. Seo MJ, Kang EJ, Kang MK, Lee HJ, Seok HB, Lee DH, Park SN, Yu YM, Youn YN. 2007. Phenotypic variation and genetic correlation of elyta colored patterns of multicolored Asian lady beetles, Harmonia axyridis (Coleoptera: Coccinellidae) in Korea. Korean Journal of Applied Entomology 46:235-248. [in Korean]
  62. Seo MJ, Kim GH, Youn YN. 2008. Differences in biological and behavioral characteristics of Harmonia axyridis (Coleoptera: Coccinellidae) according to colour patterns of elytra. Korean Journal of Applied Entomology 132:239-247. [in Korean]
  63. Seo MJ, Youn YN. 2000. The Asian ladybird, Harmonia axyridis, as biological control agents:I. Predacious behavior and feeding ability. Korean Journal of Applied Entomology 39:59-71. [in Korean]
  64. Seo MJ, Youn YN. 2001. The Asian ladybird, Harmonia axyridis , as a biological control agent: Control effects of aphid populations in the greenhouses at different seasons. CNU Journal of Agricultural Scien 28:18-26. [in Korean]
  65. Seo MJ, Youn YN. 2002. Effective preservation methods of the Asian ladybird, Harmonia axyridis (Coleoptera: Coccinellidae), Asian application strategy for the biological control of aphids. Journal of Asia-Pacific Entomology 5:209-214.
  66. Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RH, Fire A. 2001. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465-476.
  67. Singh P. 1977. Artificial diets for insect, mite, and spiders. IFI/Plenum Data Corp., NY, USA.
  68. Soares AO, Coderre D, Schanderl H. 2001. Influence of prey quality on the fitness parameters of Harmonia axyridis Pallas (Coleoptera: Coccinellidae). European Journal of Entomology 98:287-293.
  69. Swevers L, Liu J, Huvenne H, Smagghe G. 2011. Search for limiting factors in the RNAi pathway in silk moth tissues and the Bm5 cell line: the RNA-binding proteins R2D2 and translin. PLoS ONE 6:e20250.
  70. Tan CC. 1946. Mosaic dominance in the inheritance of color patterns in the lady-bird beetle, Harmonia axyridis. Genetics 31:195-210.
  71. Tan CC, Li JC. 1932. Variations in the color patterns in the lady-bird beetle, Ptychanatis axyridis Pall. Peck National History Bulletin 7:175-193.
  72. Tan CC, Li JC. 1934. Inheritance of the elytral color patterns of the lady-bird beetle, Harmonia axyridis Pallas. The American Naturalist 68:252-265.
  73. Tedders W, Schaefer P. 1994. Release and establishment of Harmonia axyridis (Coleoptera: Coccinellidae) in the southeastern United States. Entomological News 105:228-243.
  74. Tomoyasu Y, Denell RE. 2004. Larval RNAi in Tribolium (Coleoptera) for analyzing adult development. Development Genes and Evolution 214:575-578.
  75. Yu L, Christiaens O, Liu J, Niu J, Cappelle K, Caccia S, Huvenne H, Smagghe G. 2013. Delivery of dsRNA for RNAi in insects: An overview and future directions. Insect Science 20:4-14.
  76. Zou Jr. BX, Stoffolana JG, Nordin JH, Yin CM. 1988. Subunit composition of vitellin, and concentration profiles of vitellogenin, and vitellin in Phormia regina (Meig.) following a protein meal. Comparative Biochemistry and Physiology 9:861-867.
  77. Iperti G, 1986. Les coccinellidae de France. Phytoma 14:22.