Silencing of JHEH and EcR genes of Plutella xylostella (Lepidoptera: Plutellidae) through double stranded RNA oral delivery

다국어 초록 (Multilingual Abstract)

Plutella xylostella (L.), Diamondback moth (DBM), is a notorious pest disrupting cruciferous crops across the world. Overuse of chemically synthesized insecticides as well as application of bio-pesticide like Bacillus thuringiensis has gained progressive resistance against them, resulting in DBM management failures. To overcome this, a more potential, non-chemical and safe technology like Ribonucleic acid interference (RNAi) is to be implemented. RNAi, is a reverse genetic tool with high sequence specificity, elicited by double-stranded RNA (dsRNA) for silencing the target genes. The objective of this study was to silence two candidate genes fromDBMviz., Juvenile Hormone Epoxide hydrolase (PxJHEH) and Ecdysteroid receptor (PxEcR) involved in regulating metamorphosis and molting respectively. In this regard, we have custom designed the dsRNAs (500 bp) by cloning and sequencing the candidate genes and delivered it orally (non-invasive mode). Further, we have assessed the extent of down regulation of target genes with five concentrations of dsRNA (1, 2, 5, 10 and 20 μg/μl) at four time points (24, 48, 72 and 96 h). The extent of gene silencing and mortality recorded for both genes were proportional to the dsRNA concentration. Our study projects that PxJHEH and PxEcR genes would be good target for dsRNA mediated gene silencing in management of insect pests such as DBM in near future.

참고문헌 (Reference)

1 Tsubota, T., 40 : 100-112, 2010

2 Keiser, K. C. L., 50 : 191-206, 2002

3 Johnston, L. A., "Wingless and Notch regulate cell-cycle arrest in the developing Drosophila wing" 394 : 82-84, 1998

4 Severson, T. F., "Urea and amide-based inhibitors of the juvenile hormone epoxide hydrolase of the tobacco hornworm (Manduca sexta: Sphingidae)" 32 : 1741-1756, 2002

5 Blankson W. Amoabeng, "Tri-Trophic Insecticidal Effects of African Plants against Cabbage Pests" Public Library of Science (PLoS) 8 (8): e78651-, 2013

6 Suzuki, Y., "The role of Broad in the development of Tribolium castaneum: implications for the evolution of the holometabolous insect pupa" 135 : 569-577, 2008

7 Zhaojiang Guo, "The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management" Springer Nature 5 : 13728-, 2015

8 Herbert, M., "The importance of RT-qPCR primer design for the detection of SiRNA-mediated mRNA silencing" 4 : 148-, 2011

9 Endersby, N. M., "The effects of local selection versus dispersal on insecticide resistance patterns; longitudinal evidence fromdiamondback moth (Plutella xylostella (Lepidoptera: Plutellidae)) in Australia evolving resistance to signallings" 98 : 145-157, 2008

10 Bustin, S. A., "The MIQE guidelines—minimum information for publication of quantitative real-time PCR experiments" 55 : 611-622, 2009

11 Mitchell, N., "The Ecdysone-inducible zinc-finger transcription factor Crol regulates Wg transcription and cell cycle progression in Drosophila" 135 : 2707-2716, 2008

12 Koelle, M. R., "The Drosophila EcR gene encodes an ecdysone receptor, a newmember of the steroid receptor superfamily" 67 : 59-77, 1991

13 Shirai, Y., "Temperature tolerance of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae) in tropical and temperate regions of Asia" 90 : 357-364, 2000

14 Sampurna Sattar, "Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles" Frontiers Media SA 7 : 1241-, 2016

15 Rajagopal, R., "Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor" 277 : 46849-46851, 2002

16 Pitino, M., "Silencing of aphid genes by dsRNA feeding from plants" 6 : 25709-, 2011

17 Mao, Y. B., "Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol" 25 : 1307-1313, 2007

18 Naito, Y., "Signal: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference" 33 : W589-W591, 2005

19 Blandin, S., "Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene" 3 : 852-856, 2002

20 Schluns, H., "Relish regulates expression of antimicrobial peptide genes in the honeybee, Apis mellifera, shown by RNA interference" 16 : 753-759, 2007

21 Anspaugh, D. D., "Regulation of JH epoxide hydrolase versus JH esterase activity in the cabbage looper, Trichoplusia ni, by juvenile hormone and xenobiotics" 51 : 523-535, 2005

22 Marco Antonio, D. S., "RNAi-mediated silencing of vitellogenin gene function turns honeybee (Apis mellifera) workers into extremely precocious foragers" 95 : 952-961, 2008

23 Price, D. R., "RNAi-mediated crop protection against insects" 26 : 393-400, 2008

24 Yojana R. Chikate, "RNAi of selected candidate genes interrupts growth and development of Helicoverpa armigera" Elsevier BV 133 : 44-51, 2016

25 Agrawal, N., "RNA interference: biology, mechanism, and applications" 67 : 657-685, 2003

26 Griebler, M., "RNA interference with the allatoregulating neuropeptide genes from the fall armyworm Spodoptera frugiperda and its effects on the JH titer in the hemolymph" 54 : 997-1007, 2008

27 Araujo, R., "RNA interference of the salivary gland nitrophorin 2 in the triatomine bug, Rhodnius prolixus (Hemiptera:Reduviidae) by dsRNA ingestion or injection" 36 : 683-693, 2006

28 Li, X., "RNA interference of four genes in adult Bactrocera dorsalis by feeding their dsRNAs" 6 : e17788-, 2011

29 Bautista, M. A., "RNA interference mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval resistance to permethrin" 39 : 38-46, 2009

30 Zhou, X., "RNA interference in the termite Reticulitermes flavipes through ingestion of double-stranded RNA" 38 : 805-881, 2008

31 Turner, C. T., "RNA interference in the light brown applemoth, Epiphyas postvittana (Walker)induced by double-stranded RNA feeding" 15 : 383-391, 2006

32 Terenius, O., "RNA interference in Lepidoptera:an overview of successful and unsuccessful studies and implications for experimental design" 57 : 231-245, 2011

33 Wan, P. J., "RNA interference depletion of the Halloween gene disembodied implies its potential application for management of plant hopper Sogatella furcifera and Laodelphax striatellus" 9 (9): e86675-, 2014

34 Hannon, G. J., "RNA interference" 418 : 244-251, 2002

35 K. B. Rebijith, "RNA Interference of Odorant-Binding Protein 2 (OBP2) of the Cotton Aphid, Aphis gossypii (Glover), Resulted in Altered Electrophysiological Responses" Springer Nature 178 (178): 251-266, 2016

36 Walshe, D. P., "Prolonged gene knockdown in the tsetse fly Glossina by feeding double stranded RNA" 18 : 11-19, 2009

37 Fire, A., "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans" 391 : 806-811, 1998

38 Bucher, G., "Parental RNAi in Tribolium (Coleoptera)" 12 : R85-R86, 2002

39 Singh, A. D., "Oral delivery of double stranded RNA in larvae of the yellow fever mosquito, Aedes aegypti: implications for pest mosquito control" 13 : 69-, 2013

40 King-Jones, K., "Nuclear receptors—a perspective from Drosophila" 6 : 311-323, 2005

41 Baxter, S. W., "Novel genetic basis of field-evolved resistance to Bt toxins in Plutella xylostella" 14 (14): 327-334, 2005

42 Zhao, J. Z., "Monitoring of diamondback moth resistance to spinosad, indoxacarb and emamectin benzoate" 99 : 176-181, 2006

43 Nakagawa, Y., "Molecular cloning of the ecdysone receptor and the retinoid X receptor from the scorpion Liocheles australasiae" 274 : 6191-6203, 2007

44 Zhang, Qi-Rui, "Molecular and biochemical characterization of juvenile hormone epoxide hydrolase from the silkworm, Bombyx mori" 35 : 153-164, 2005

45 Sambrook, J., "Molecular Cloning" Cold Spring Harbor Laboratory Press 2001

46 Huvenne, H., "Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review" 56 : 227-235, 2010

47 Tamura, K., "MEGA6: molecular evolutionary genetics analysis version 6.0" 30 : 2725-2729, 2013

48 Tomoyasu, Y., "Larval RNAi in Tribolium(Coleoptera) for analysing adult development" 214 : 575-578, 2004

49 Dykxhoorn, D. M., "Killing the messenger: short RNAs that silence gene expression" 4 : 457-467, 2003

50 Shizuo G. Kamita, "Juvenile hormone esterase: biochemistry and structure" Pesticide Science Society of Japan 35 (35): 265-274, 2010

51 Zhou, M., "Insilco detection and characteristics of novel microRNA genes in the Equus caballus genome using an integrated ab initio and comparative genomic approach" 94 (94): 125-131, 2009

52 Santos, V. C., "Insecticide resistance in populations of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), from the state of Pernambuco, Brazil" 40 (40): 264-270, 2011

53 Whyard, S., "Ingested double-stranded RNAs can act as speciesspecific insecticides" 39 : 824-832, 2009

54 Lum, L., "Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells" 299 : 2039-2045, 2003

55 Liu, P., "Hunchback is required for suppression of abdominal identity, and for proper germband growth and segmentation in the intermediate germband insect Oncopeltus fasciatus" 131 : 1515-1527, 2004

56 Liu, S., "Gene knockdown by introthoracic injection of double-stranded RNA in the brown plant hopper, Nilaparvata lugens" 40 : 666-671, 2010

57 Jaubert-Possamai, S., "Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum" 7 : 63-, 2007

58 Lelio, I.Di., "Functional analysis of an immune gene of Spodoptera littoralis by RNAi" 64 : 90-97, 2014

59 Mao, J., "Feeding-based RNA Intereference of a gap gene is lethal to the pea aphid, Acyrthosiphon pisum" 7 (7): e48718-, 2012

60 Newman, J. W., "Epoxide hydrolases: their roles and interactions with lipid metabolism" 44 : 1-51, 2005

61 Kotwica-Rolinska, Joanna, "Effects of period RNAi on V-ATPase expression and rhythmic pH changes in the vas deferens of Spodoptera littoralis (Lepidoptera:Noctuidae)" 43 : 522-532, 2013

62 Sheikh, El-Sayed, A., "Effects of juvenile hormone (JH)analog insecticides on larval development and JH esterase activity in two spodopterans" 128 : 30-36, 2016

63 Nadeem Ahmad, M., "Effect of neemarin on life table indices of Plutella xylostella (L)" 38 : 7-14, 2012

64 Liu, X. X., "Effect of insecticides and Plutella xylostella (Lepidoptera: Plutellidae) genotype on a predator and parasitoid and implications for the evolution of insecticide resistance" 105 (105): 54-362, 2012

65 Schwedes, C. C., "Ecdysone signalling in adult Drosophila melanogaster" 58 : 293-302, 2012

66 Yamanaka, N., "Ecdysone control of developmental transitions:lessons from Drosophila research" 58 : 497-516, 2013

67 Caplen, N. J., "DsRNA-mediated gene silencing in culture Drosophila cells: a tissue culture model for analysis of RNA interference" 252 : 95-105, 2000

68 Chaitanya, B. N., "Doublestranded RNA-mediated silencing of sodium channel and ultraspiracle genes in Aphis gossypii (Hemiptera: Aphididae)" 1-7, 2015

69 Amdam, G. V., "Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA" 3 : 1-8, 2003

70 Furlong, M. J., "Differential effects of the acylurea insect growth-reuator teflubenzuron on the adults of 2 endolaval parasitoids of P. xylostella, Cotesia plutellae and Diadegma semiclausum" 41 (41): 359-364, 1994

71 Rebijith, K.B., "Diet-delivered dsRNAs for juvenile hormone-binding protein and vacuolar ATPase-H implied their potential in the management of the melon aphid (Hemiptera:Aphididae)" 1-8, 2015

72 Furlong, M. J., "Diamondback moth ecology and management:problems progress and prospects" 58 : 517-541, 2013

73 Tian, H., "Developmental control of a Lepidopteran pest Spodoptera exigua by ingestion of bacterial expressing dsRNA of a non-midgut gene" 4 : e6225-, 2009

74 Na, Yu., "Delivery of dsRNA for RNAi in insects: an overview and future directions" 20 : 4-14, 2013

75 Kang Zhou, "Crystal structure of juvenile hormone epoxide hydrolase from the silkworm Bombyx mori" Wiley-Blackwell 82 (82): 3224-3229, 2014

76 Baum, J. A., "Control of coleopteran insect pests through RNA interference" 25 : 1322-1326, 2007

77 Sayyed, A. H., "Common, but complex,mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac" 71 (71): 6863-6869, 2005

78 Harris, S. V., "Cloning and expression of a novel juvenile hormone metabolizing epoxide hydrolase during larval–pupal metamorphosis of the cabbage looper" 8 : 85-96, 1999

79 Zhang, X., "Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae)" 19 : 683-693, 2010

80 Konopova, B., "Broad-complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis" 135 : 559-568, 2008

81 Hall, T. A., "BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT" 41 : 95-98, 1999

82 Livak, K. J., "Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCT method" 25 : 402-408, 2001

83 Lynch, J. A., "Amethod for parental RNA interference in the wasp Nasonia vitripennis" 1 : 486-494, 2006

84 Yamada, H., "A simple mass rearing method for the diamondback moth. (in Japanese)" 32 : 253-256, 1978

85 Dietzl, G., "A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila" 448 : 151-156, 2007

86 Li, T. R., "A conditional rescue system reveals essential functions for the ecdysone receptor (EcR) gene duringmolting and metamorphosis in Drosophila" 127 : 2897-2905, 2000