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Impairment of pupation by RNA interference-aided knockdown of Broad-Complex gene in Leptinotarsa decemlineata (Say)

  • Q.-Y. Xu (a1), Q.-W. Meng (a1), P. Deng (a1), K.-Y. Fu (a2), W.-C. Guo (a2) and G.-Q. Li (a1)...


Dietary delivery of bacterially expressed double-stranded RNA (dsRNA) has a great potential for management of Leptinotarsa decemlineata. An important first step is to discover possible RNA-interference (RNAi)-target genes effective against larvae, especially the old larvae. In the present paper, five putative Broad-Complex (BrC) cDNAs (Z1-Z4, and Z6) were identified in L. decemlineata. The expression of the five LdBrC isoforms was suppressed by juvenile hormone signaling, whereas the transcription was upregulated by 20-hydroxyecdysone signaling at the fourth (final) instar larval stage. Feeding of bacterially expressed dsBrC (derived from a common fragment of the five LdBrC variants) in the third- and fourth-instar larvae successfully knocked down the target mRNAs. For the fourth-instar LdBrC RNAi hypomorphs, they had a higher larval mortality compared with the controls. Moreover, most dsBrC-fed beetles did not pupate normally. After removal of the apolysed larval cuticle, a miniature adult was found. The adult head, compound eyes, prothorax, mesothorax, metathorax were found on the dorsal view. Distinct adult cuticle pigmentation was seen on the prothorax. The mouthparts, forelegs, midlegs, and hindlegs could be observed on the ventral view of the miniature adults. For the third-instar LdBrC RNAi specimens, around 20% moribund beetles remained as prepupae and finally died. Therefore, LdBrC is among the most attractive candidate genes for RNAi to control the fourth-instar larvae in L. decemlineata.


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Amdam, G.V., Simões, Z.L., Guidugli, K.R., Norberg, K. & Omholt, S.W. (2003) Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA. BMC Biotechnology 3, 1.
Bayer, C.A., Holley, B. & Fristrom, J.W. (1996) A switch in Broad-Complex zinc-finger isoform expression is regulated posttranscriptionally during the metamorphosis of Drosophila imaginal discs. Developmental Biology 177, 114.
Bayer, C.A., von Kalm, L. & Fristrom, J.W. (1997) Relationships between protein isoforms and genetic functions demonstrate functional redundancy at the Broad-Complex during Drosophila metamorphosis. Developmental Biology 187, 267282.
Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J. & Wittwer, C.T. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55, 611622.
Cingel, A., Savić, J., Lazarević, J., Ćosić, T., Raspor, M., Smigocki, A. & Ninković, S. (2016) Extraordinary adaptive plasticity of Colorado potato beetle: ‘Ten-striped spearman’ in the era of biotechnological warfare. International Journal of Molecular Sciences 17, 1538.
Clements, J., Schoville, S., Clements, N., Chapman, S. & Groves, R.L. (2017) Temporal patterns of imidacloprid resistance throughout a growing season in Leptinotarsa decemlineata populations. Pest Management Science 73, 641650.
Cornette, R., Gotoh, H., Koshikawa, S. & Miura, T. (2008) Juvenile hormone titers and caste differentiation in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Termopsidae). Journal of Insect Physiology 54, 922930.
Crossley, M.S., Chen, Y.H., Groves, R.L. & Schoville, S.D. (2017) Landscape genomics of Colorado potato beetle provides evidence of polygenic adaptation to insecticides. Molecular Ecology 26, 62846300.
Daimon, T., Uchibori, M., Nakao, H., Sezutsu, H. & Shinoda, T. (2015) Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle. Proceedings of the National Academy of Sciences of the United States of America 112, E4226E4235.
DiBello, P.R., Withers, D.A., Bayer, C.A., Fristrom, J.W. & Guild, G.M. (1991) The Drosophila broad-complex encodes a family of related proteins containing zinc fingers. Genetics 129, 385397.
Emery, I.F., Bedian, V. & Guild, G.M. (1994) Differential expression of Broad-Complex transcription factors may forecast tissue-specific developmental fates during Drosophila metamorphosis. Development 120, 32753287.
Erezyilmaz, D.F., Riddiford, L.M. & Truman, J.W. (2006) The pupal specifier broad directs progressive morphogenesis in a direct-developing insect. Proceedings of the National Academy of Sciences of the United States of America 103, 69256930.
Fu, K.-Y., Guo, W.-C., Ahmat, T. & Li, G.-Q. (2015) Knockdown of a nutrient amino acid transporter gene LdNAT1 reduces free neutral amino acid contents and impairs Leptinotarsa decemlineata pupation. Scientific Reports 5, 18124.
Fu, K.-Y., Li, Q., Zhou, L.-T., Meng, Q.-W., , F.-G., Guo, W.-C. & Li, G.-Q. (2016) Knockdown of juvenile hormone acid methyl transferase severely affects the performance of Leptinotarsa decemlineata (Say) larvae and adults. Pest Management Science 72, 12311241.
Guo, W.-C., Tan, W.-Z. & Zhang, Q.-W. (2013) Biology and Integrated Management of the Invasive Alien Pest: Colorado Potato Beetle. Beijing, Science Press.
Guo, W.-C., Fu, K.-Y., Yang, S., Li, X.-X. & Li, G.-Q. (2015) Instar-dependent systemic RNA interference response in Leptinotarsa decemlineata larvae. Pesticide Biochemistry and Physiology 123, 6473.
Hameed, A., Tahir, M.N., Asad, S., Bilal, R., Van Eck, J., Jander, G. & Mansoor, S. (2017) RNAi-mediated simultaneous resistance against three RNA viruses in potato. Molecular Biotechnology 59, 7383.
Huang, J.-H., Lozano, J. & Belles, X. (2013) Broad-complex functions in postembryonic development of the cockroach Blattella germanica shed new light on the evolution of insect metamorphosis. Biochimica et Biophysica Acta 1830, 21782187.
Karim, F.D., Guild, G.M. & Thummel, C.S. (1993) The Drosophila Broad-complex plays a key role in controlling ecdysone-regulated gene expression at the onset of metamorphosis. Development 118, 977988.
Kiss, I., Bencze, G., Fodor, G., Szabad, J. & Fristrom, J.W. (1976) Prepupal larval mosaics in Drosophila melanogaster. Nature 262, 136138.
Kiss, I., Beaton, A.H., Tardiff, J., Fristrom, D. & Fristrom, J.W. (1988) Interactions and developmental effects of mutations in the broad-complex of Drosophila melanogaster. Genetics 118, 247259.
Koch, A., Biedenkopf, D., Furch, A., Weber, L., Rossbach, O., Abdellatef, E., Linicus, L., Johannsmeier, J., Jelonek, L., Goesmann, A., Cardoza, V., McMillan, J., Mentzel, T. & Kogel, K.H. (2016) An RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs involves a plant passage and is controlled by the fungal silencing machinery. PLoS Pathogens 12, e1005901.
Konopova, B. & Jindra, M. (2008) Broad-Complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis. Development 135, 559568.
Meng, Q.-W., Xu, Q.-Y., Deng, P., Fu, K.-Y., Guo, W.-C. & Li, G.-Q. (2018a) Transcriptional response of Methoprene-tolerant (Met) gene to three insect growth disruptors in Leptinotarsa decemlineata (Say). Journal of Asia-Pacific Entomology 214, 466473.
Meng, Q.-W., Xu, Q.-Y., Deng, P., Fu, K.-Y., Guo, W.-C. & Li, G.-Q. (2018b) Involvement of methoprene-tolerant (Met) in the determination of the final body size in Leptinotarsa decemlineata (Say) larvae. Insect Biochemistry and Molecular Biology 97, 19.
Minakuchi, C., Tanaka, M., Miura, K. & Tanaka, T. (2011) Developmental profile and hormonal regulation of the transcription factors broad and Krüppel homolog 1 in hemimetabolous thrips. Insect Biochemistry and Molecular Biology 41, 125134.
Muramatsu, D., Kinjoh, T., Shinoda, T. & Hiruma, K. (2008) The role of 20-hydroxyecdysone and juvenile hormone in pupal commitment of the epidermis of the silkworm, Bombyx mori. Mechanisms of Development 125, 411420.
Nagamine, K., Kayukawa, T., Hoshizaki, S., Matsuo, T., Shinoda, T. & Ishikawa, Y. (2014) Cloning, phylogeny, and expression analysis of the Broad-Complex gene in the longicorn beetle Psacothea hilaris. Springerplus 3, 539.
Nishita, Y. (2014) Ecdysone response elements in the distal promoter of the Bombyx Broad-Complex gene, BmBR-C. Insect Molecular Biology 23, 341356.
Nishita, Y. & Takiya, S. (2004) Structure and expression of the gene encoding a Broad-Complex homolog in the silkworm, Bombyx mori. Gene 339, 161172.
Ogura, T., Minakuchi, C., Nakagawa, Y., Smagghe, G. & Miyagawa, H. (2005) Molecular cloning, expression analysis and functional confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata. FEBS Journal 272, 41144128.
Palli, S.R. (2014) RNA interference in Colorado potato beetle: steps toward development of dsRNA as a commercial insecticide. Current Opinion in Insect Science 6, 18.
Parthasarathy, R., Tan, A., Bai, H. & Palli, S.R. (2008) Transcription factor broad suppresses precocious development of adult structures during larval–pupal metamorphosis in the red flour beetle, Tribolium castaneum. Mechanisms of Development 125, 299313.
Piulachs, M.-D., Pagone, V. & Bellés, X. (2010) Key roles of the Broad-Complex gene in insect embryogenesis. Insect Biochemistry and Molecular Biology 40, 468475.
Reza, A.M., Kanamori, Y., Shinoda, T., Shimura, S., Mita, K., Nakahara, Y., Kiuchi, M. & Kamimura, M. (2004) Hormonal control of a metamorphosis-specific transcriptional factor Broad-Complex in silkworm. Comparative Biochemistry and Physiology B: Biochemistry and Molecular Biology 139, 753761.
San Miguel, K. & Scott, J.G. (2016) The next generation of insecticides: dsRNA is stable as a foliar-applied insecticide. Pest Management Science 72, 801809.
Schoville, S.D., Chen, Y.H., Andersson, M.N., Benoit, J.B., Bhandari, A., Bowsher, J.H., Brevik, K., Cappelle, K., Chen, M.M., Childers, A.K., Childers, C., Christiaens, O., Clements, J., Didion, E.M., Elpidina, E.N., Engsontia, P., Friedrich, M., García-Robles, I., Gibbs, R.A., Goswami, C., Grapputo, A., Gruden, K., Grynberg, M., Henrissat, B., Jennings, E.C., Jones, J.W., Kalsi, M., Khan, S.A., Kumar, A., Li, F., Lombard, V., Ma, X., Martynov, A., Miller, N.J., Mitchell, R.F., Munoz-Torres, M., Muszewska, A., Oppert, B., Palli, S.R., Panfilio, K.A., Pauchet, Y., Perkin, L.C., Petek, M., Poelchau, M.F., Record, É, Rinehart, J.P., Robertson, H.M., Rosendale, A.J., Ruiz-Arroyo, V.M., Smagghe, G., Szendrei, Z., Thomas, G.W.C., Torson, A.S., Vargas Jentzsch, I.M., Weirauch, M.T., Yates, A.D., Yocum, G.D., Yoon, J.S. & Richards, S. (2018) A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Scientific Reports 8, 1931.
Scott, J.G., Michel, K., Bartholomay, L.C., Siegfried, B.D., Hunter, W.B., Smagghe, G., Zhu, K.Y. & Douglas, A.E. (2013) Towards the elements of successful insect RNAi. Journal of Insect Physiology 59, 12121221.
Shi, X.-Q., Guo, W.-C., Wan, P.-J., Zhou, L.-T., Ren, X.-L., Tursun, A., Fu, K.-Y. & Li, G.-Q. (2013) Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata (Say). BMC Research Notes 6, 93.
Shi, J.-F., Mu, L.-L., Chen, X., Guo, W.-C. & Li, G.-Q. (2016) RNA interference of chitin synthase genes inhibits chitin biosynthesis and affects larval performance in Leptinotarsa decemlineata (Say). International Journal of Biological Sciences 12, 13191331.
Spokony, R.F. & Restifo, L.L. (2007) Anciently duplicated Broad Complex exons have distinct temporal functions during tissue morphogenesis. Development Genes and Evolution 217, 499513.
Suzuki, Y., Truman, J.W. & Riddiford, L.M. (2008) The role of Broad in the development of Tribolium castaneum: implications for the evolution of the holometabolous insect pupa. Development 135, 569577.
Uhlirova, M., Foy, B.D., Beaty, B.J., Olson, K.E., Riddiford, L.M. & Jindra, M. (2003) Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis. Proceedings of the National Academy of Sciences of the United States of America 100, 1560715612.
Ureña, E., Chafino, S., Manjón, C., Franch-Marro, X. & Martín, D. (2016) The occurrence of the holometabolous pupal stage requires the interaction between E93, krüppel-homolog 1 and broad-complex. PLoS Genetics 12, e1006020.
Wang, M. & Jin, H. (2017) Spray-induced gene silencing: a powerful innovative strategy for crop protection. Trends in Microbiology 25, 46.
Whyard, S., Singh, A.D. & Wong, S. (2009) Ingested double-stranded RNAs can act as species-specific insecticides. Insect Biochemistry and Molecular Biology 39, 824832.
Xu, Q.-Y., Meng, Q.-W., Shi, J.-F., Deng, P., Guo, W.-C. & Li, G.-Q. (2017) Novaluron ingestion causes larval lethality and inhibits chitin content in Leptinotarsa decemlineata fourth-instar larvae. Pesticide Biochemistry and Physiology 143, 173180.
Xu, Q.-Y., Meng, Q.-W., Deng, P., Guo, W.-C. & Li, G.-Q. (2018) Leptinotarsa hormone receptor 4 (HR4) tunes ecdysteroidogenesis and mediates 20-hydroxyecdysone signaling during larval-pupal metamorphosis. Insect Biochemistry and Molecular Biology 94, 5060.
Zhou, B. & Riddiford, L.M. (2001) Hormonal regulation and patterning of the Broad Complex in the epidermis and wing discs of the tobacco hornworm, Manduca sexta. Developmental Biology 231, 125137.
Zhou, X. & Riddiford, L.M. (2002) Broad specifies pupal development and mediates the ‘status quo’ of juvenile hormone on the pupal-adult transformation in Drosophila and Manduca. Development 129, 22592269.
Zhou, B., Hiruma, K., Shinoda, T. & Riddiford, L.M. (1998) Juvenile hormone prevents ecdysteroid-induced expression of broad complex RNAs in the epidermis of the tobacco hornworm, Manduca sexta. Developmental Biology 203, 233244.
Zhou, X., Zhou, B., Truman, J.W. & Riddiford, L.M. (2004) Overexpression of broad: a new insight into its role in the Drosophila prothoracic gland cells. Journal of Experimental Biology 207, 11511161.
Zhou, L.T., Jia, S., Wan, P.J., Kong, Y., Guo, W.C., Ahmat, T. & Li, G.Q. (2013) RNA interference of a putative S-adenosyl-L-homocysteine hydrolase gene affects larval performance in Leptinotarsa decemlineata (Say). Journal of Insect Physiology 59, 10491056.


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Impairment of pupation by RNA interference-aided knockdown of Broad-Complex gene in Leptinotarsa decemlineata (Say)

  • Q.-Y. Xu (a1), Q.-W. Meng (a1), P. Deng (a1), K.-Y. Fu (a2), W.-C. Guo (a2) and G.-Q. Li (a1)...


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