Lethal effects of recombinant CYP307A1 RNAi Chlorella by feeding on Aedes albopictus

doi:10.11853/j.issn.1003.8280.2023.03.010

Abstract

Abstract: Objective To investigate the effects of silencing the ecdysone synthesis pathway enzyme gene CYP307A1 of Aedes albopictus on the growth and development of Aedes larvae, and to provide new ideas for the prevention and control of mosquito-borne infectious diseases.Methods An RNAi expression vector targeting CYP307A1 was constructed using RNAi technology. Chlorella was used as the recipient for electrotransformation. The selected recombinant Chlorella strains were fed to Ae. albopictus larvae. WPS 2020 software was used to pool data on the mortality, pupation rate, emergence rate, body length, and quantitative real-time PCR results of the expression of CYP307A1 gene in larvae. One-way analysis of variance was performed on the mean values of all groups using SPSS 25. Origin 2019 and WPS 2020 were used to make charts.Results The 300-mosquito experiment showed that the mortality rates of larvae in the control groups fed with filtered water, feed, wild Chlorella, and Chlorella transferred with pMaa7/IR vector were 1.50%, 0.17%, 0.83%, and 8.00%, respectively; the mortality rate of larvae fed with recombinant CYP307A1 RNAi Chlorella was 80.33%, which was statistically higher than that of the control groups (P<0.05). The pupation rate and emergence rate of Aedes larvae fed with feed were 99.83% and 98.50%, respectively, while those of the group fed with recombinant CYP307A1 RNAi Chlorella were 18.50% and 98.50%, respectively. The pupation rate and emergence rate with recombinant CYP307A1 RNAi Chlorella were statistically lower than those of the control group (P<0.05). The expression of the CYP307A1 gene in Aedes larvae fed with the recombinant Chlorella was decreased by 79.00% compared with the control group (P<0.01).Conclusion Recombinant CYP307A1 RNAi Chlorella has anti-mosquito function, which shows promise in the field of biological mosquito control.

Key words: Aedes albopictus, CYP307A1, RNA interference, Chlorella

摘要： 目的了解沉默白纹伊蚊蜕皮激素合成途径酶基因CYP307A1对伊蚊幼蚊生长发育的影响,为蚊媒传染病的防控提供新的思路。方法利用RNA干扰（RNAi）技术,构建CYP307A1的RNAi表达载体,以小球藻为受体进行电击转化,筛选得到的重组藻株喂食白纹伊蚊幼蚊,使用WPS 2020软件统计并汇总幼蚊的死亡率、化蛹率、羽化率、体长和CYP307A1基因表达量实时荧光定量PCR数据；SPSS 25软件进行各组平均值的单因素ANOVA检验,使用Origin 2019和WPS 2020软件进行图表的制作。结果 300只规模实验结果显示,对照组中饲喂过滤水、饲料、野生小球藻和转pMaa7/IR载体小球藻的白纹伊蚊幼蚊死亡率分别为1.50%、0.17%、0.83%和8.00%,而饲喂CYP307A1 RNAi重组小球藻幼蚊死亡率为80.33%,死亡率显著高于各对照组,且差异有统计学意义（P<0.05）。饲喂饲料的伊蚊幼蚊化蛹率为99.83%,羽化率为98.50%,而饲喂CYP307A1 RNAi重组小球藻幼蚊化蛹率为18.50%,羽化率为98.50%,与对照组相比饲喂CYP307A1 RNAi重组小球藻幼蚊的化蛹率和羽化率显著下降,差异有统计学意义（P<0.05）。喂食重组藻株伊蚊幼蚊体内CYP307A1基因表达量比对照组下降79.00%（P<0.01）。结论 CYP307A1 RNAi重组小球藻具有抑蚊功能,有望在生物杀蚊领域发挥作用。

关键词: 白纹伊蚊, CYP307A1, RNA干扰, 小球藻

CLC Number:

R384.1

HUANG Xiao-dan, XIAO Sa, HE Chang-hao, LI Zhi-jie, ZHANG Xiu-xia, LI Ya-jun, FEI Xiao-wen, DENG Xiao-dong. Lethal effects of recombinant CYP307A1 RNAi Chlorella by feeding on Aedes albopictus[J]. Chinese Journal of Vector Biology and Control, 2023, 34(3): 336-343.

黄小丹, 肖洒, 贺长皓, 李智杰, 张秀霞, 李亚军, 费小雯, 邓晓东. CYP307A1 RNAi重组小球藻口服喂饲对白纹伊蚊的致死作用[J]. 中国媒介生物学及控制杂志, 2023, 34(3): 336-343.

References

[1] Gubler DJ. The global threat of emergent/re-emergent vector-borne diseases[M]//Atkinson PW. Vector biology,ecology and control. Dordrecht:Springer,2010:39-62. DOI:10.1007/978-90-481-2458-9_4.
[2] Yu X,Zhu YB,Xiao XP,et al. Progress towards understanding the mosquito-borne virus life cycle[J]. Trends Parasitol,2019,35(12):1009-1017. DOI:10.1016/j.pt.2019.09.006.
[3] Bhatt S,Gething PW,Brady OJ,et al. The global distribution and burden of dengue[J]. Nature,2013,496(7446):504-507. DOI:10.1038/nature12060.
[4] Brady OJ,Gething PW,Bhatt S,et al. Refining the global spatial limits of dengue virus transmission by evidence-based consensus[J]. PLoS Negl Trop Dis,2012,6(8):e1760. DOI:10.1371/journal.pntd.0001760.
[5] Yue YJ,Liu XB,Ren DS,et al. Spatial dynamics of dengue fever in mainland China,2019[J]. Int J Environ Res Public Health,2021,18(6):2855. DOI:10.3390/ijerph18062855.
[6] Simpson DIH. Zika virus infection in man[J]. Trans R Soc Trop Med Hyg,1964,58(4):335-338. DOI:10.1016/0035-9203(64)90200-7.
[7] Fauci AS,Morens DM. Zika virus in the Americas:Yet another arbovirus threat[J]. N Engl J Med,2016,374(7):601-604. DOI:10.1056/NEJMp1600297.
[8] Beard JR,Officer A,de Carvalho IA,et al. The world report on ageing and health:A policy framework for healthy ageing[J]. Lancet,2016,387(10033):2145-2154. DOI:10.1016/S0140-6736(15)00516-4.
[9] 国家卫生和计划生育委员会,国家质检总局. 黄热病防控方案(2016年版)[J]. 国际流行病学传染病学杂志,2016,43(3):150-152. DOI:10.3760/cma.j.issn.1673-4149.2016.03.002.National Health and Family Planning Commission,General Administration of Quality Supervision,Inspection and Quarantine. Protocol for prevention and control for yellow fever[J]. Inter J Epidemiol Infect Dis,2016,43(3):150-152. DOI:10.3760/cma.j.issn.1673-4149.2016.03.002.(in Chinese)
[10] Njenga MK,Nderitu L,Ledermann JP,et al. Tracking epidemic Chikungunya virus into the Indian Ocean from east Africa[J]. J Gen Virol,2008,89(Pt 11):2754-2760. DOI:10.1099/vir.0. 2008/005413-0.
[11] Shragai T,Tesla B,Murdock C,et al. Zika and chikungunya:Mosquito-borne viruses in a changing world[J]. Ann N Y Acad Sci,2017,1399(1):61-77. DOI:10.1111/nyas.13306.
[12] 杨天赐,傅桂明. 浙江省登革热传播媒介白纹伊蚊调查研究[J]. 中华卫生杀虫药械,2006,12(3):189-191. DOI:10.3969/j.issn.1671-2781.2006.03.012.Yang TC,Fu GM. Investigation on the distribution of dengue vector Aedes albopictus in Zhejiang province[J]. Chin J Hyg Insect Equip,2006,12(3):189-191. DOI:10.3969/j.issn.1671-2781.2006.03.012.(in Chinese)
[13] Wilson AL,Courtenay O,Kelly-Hope LA,et al. The importance of vector control for the control and elimination of vector-borne diseases[J]. PLoS Negl Trop Dis,2020,14(1):e0007831. DOI:10.1371/journal.pntd.0007831.
[14] 王伟伟,刘妮,陆沁,等. RNAi技术的最新研究进展[J]. 生物技术通报,2017,33(11):35-40. DOI:10.13560/j.cnki.biotech.bull.1985.2017-0455.Wang WW,Liu N,Lu Q,et al. Latest research progress on RNA interference technology[J]. Biotechnol Bull,2017,33(11):35-40. DOI:10.13560/j.cnki.biotech.bull.1985.2017-0455.(in Chinese)
[15] Timmons L,Fire A. Specific interference by ingested dsRNA[J]. Nature,1998,395(6705):854. DOI:10.1038/27579.
[16] Tabara H,Grishok A,Mello CC. RNAi in C. elegans:Soaking in the genome sequence[J]. Science,1998,282(5388):430-431. DOI:10.1126/science.282.5388.430.
[17] Kumar A,Wang SJ,Ou RG,et al. Development of an RNAi based microalgal larvicide to control mosquitoes[J]. MalariaWorld J,2013,4(6):1-7.
[18] 张职显. 双链RNA介导的甜菜夜蛾CYP314A1基因沉默对甲氧虫酰肼与虱螨脲复配毒力的影响[D]. 北京:中国农业科学院,2021. DOI:10.27630/d.cnki.gznky.2021.000463.Zhang ZX. Effects of double-stranded RNA mediated CYP314A1 gene silencing gene silencing on the toxicity of methoxyfenozide and lufenuron complex on Spodoptera exigua[D]. Beijing:Chinese Academy of Agricultural Sciences,2021. DOI:10.27630/d.cnki.gznky.2021.000463.(in Chinese)
[19] Dorrell RG,Smith AG. Do red and green make brown?:Perspectives on plastid acquisitions within chromalveolates[J]. Eukaryot Cell,2011,10(7):856-868. DOI:10.1128/EC.00326-10.
[20] Jeuken MJW,Zhang NW,McHale LK,et al. Rin4 causes hybrid necrosis and race-specific resistance in an interspecific lettuce hybrid[J]. Plant Cell,2009,21(10):3368-3378. DOI:10.1105/tpc.109.070334.
[21] Altpeter F,Springer NM,Bartley LE,et al. Advancing crop transformation in the era of genome editing[J]. Plant Cell,2016,28(7):1510-1520. DOI:10.1105/tpc.16.00196.
[22] Gilbert LI,Rybczynski R,Warren JT. Control and biochemical nature of the ecdysteroidogenic pathway[J]. Annu Rev Entomol,2002,47:883-916. DOI:10.1146/annurev.ento.47.091201. 145302.
[23] Zhou J,Zhang HL,Li J,et al. Molecular cloning and expression profile of a Halloween gene encoding CYP307A1 from the seabuckthorn carpenterworm,Holcocerus hippophaecolus[J]. J Insect Sci,2013,13(1):56. DOI:10.1673/031.013.5601.
[24] Marchal E,Vandersmissen HP,Badisco L,et al. Control of ecdysteroidogenesis in prothoracic glands of insects:A review[J]. Peptides,2010,31(3):506-519. DOI:10.1016/j.peptides. 2009.08.020.
[25] Abdel-Aal YAI,Hammock BD. Transition state analogs as ligands for affinity purification of juvenile hormone esterase[J]. Science,1986,233(4768):1073-1076. DOI:10.1126/science. 3738525.
[26] Warren JT,Rybczynski R,Gilbert LI. Stereospecific,mechanism-based,suicide inhibition of a cytochrome P450 involved in ecdysteroid biosynthesis in the prothoracic glands of Manduca sexta[J]. Insect Biochem Mol Biol,1995,25(6):679-695. DOI:10.1016/0965-1748(95)00007-I.
[27] Namiki T,Niwa R,Sakudoh T,et al. Cytochrome P450CYP307A1/Spook:A regulator for ecdysone synthesis in insects[J]. Biochem Biophys Res Commun,2005,337(1):367-374. DOI:10.1016/j.bbrc.2005.09.043.
[28] Chavez VM,Marques G,Delbecque JP,et al. The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels[J]. Development,2000,127(19):4115-4126. DOI:10.1242/dev.127.19.4115.
[29] Hentze JL,Moeller ME,Jørgensen AF,et al. Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects[J]. PLoS One,2013,8(2):e55131. DOI:10.1371/journal.pone.0055131.
[30] Ono H,Rewitz KF,Shinoda T,et al. Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera[J]. Dev Biol,2006,298(2):555-570. DOI:10.1016/j.ydbio.2006.07.023.
[31] Rewitz KF,Rybczynski R,Warren JT,et al. Identification,characterization and developmental expression of Halloween genes encoding P450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm,Manduca sexta[J]. Insect Biochem Mol Biol,2006,36(3):188-199. DOI:10.1016/j.ibmb.2005.12.002.
[32] Kraemer MUG,Sinka ME,Duda KA,et al. The global distribution of the arbovirus vectors Aedes aegypti and Ae. albopictus[J]. Elife,2015,4:e08347. DOI:10.7554/eLife.08347.
[33] Silva JVJ,Lopes TRR,de Oliveira-Filho EF,et al. Current status,challenges and perspectives in the development of vaccines against yellow fever,dengue,Zika and Chikungunya viruses[J]. Acta Trop,2018,182:257-263. DOI:10.1016/j.actatropica. 2018.03.009.
[34] LaTourette II PC,Awasthi S,Desmond A,et al. Protection against herpes simplex virus type 2 infection in a neonatal murine model using a trivalent nucleoside-modified mRNA in lipid nanoparticle vaccine[J]. Vaccine,2020,38(47):7409-7413. DOI:10.1016/j.vaccine.2020.09.079.
[35] World Health Organization. A WHO report on global strategy for dengue prevention and control,2012-2020[R]. Geneva:WHO,2012.
[36] Zhou R,Mohr S,Hannon GJ,et al. Inducing RNAi in Drosophila cells by soaking with dsRNA[J]. Cold Spring Harb Protoc,2014,2014(5):pdb.prot080747. DOI:10.1101/pdb.prot080747.
[37] Magalhaes T,Brackney DE,Beier JC,et al. Silencing an Anopheles gambiae catalase and sulfhydryl oxidase increases mosquito mortality after a blood meal[J]. Arch Insect Biochem Physiol,2008,68(3):134-143. DOI:10.1002/arch.20238.
[38] Sim C,Denlinger DL. Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens[J]. Proc Natl Acad Sci USA,2008,105(18):6777-6781. DOI:10.1073/pnas.0802067105.
[39] 张萍,李晨曦,郝晓冉,等. 新型隐球酵母非编码小RNAs的研究进展[J]. 菌物学报,2018,37(10):1357-1363. DOI:10.13346/j.mycosystema.180159.Zhang P,Li CX,Hao XR,et al. Small non-coding RNAs in Cryptococcus neoformans[J]. Mycosystema,2018,37(10):1357-1363. DOI:10.13346/j.mycosystema.180159.(in Chinese)
[40] Duman-Scheel M. Saccharomyces cerevisiae (Baker's yeast) as an interfering RNA expression and delivery system[J]. Curr Drug Targets,2019,20(9):942-952. DOI:10.2174/1389450120666181126123538.
[41] 费小雯,张阳,李亚军,等. 伊蚊3HKT基因RNAi载体构建及口服对伊蚊的致死作用[J]. 热带生物学报,2021,12(3):356-362. DOI:10.15886/j.cnki.rdswxb.2021.03.012.Fei XW,Zhang Y,Li YJ,et al. Construction of RNAi vector of 3HKT gene and its lethal effect on Aedes aegypti[J]. J Trop Biol,2021,12(3):356-362. DOI:10.15886/j.cnki.rdswxb.2021.03. 012.(in Chinese)
[42] 贾爽. 白背飞虱和灰飞虱蜕皮激素合成相关Halloween基因的克隆与功能验证[D]. 南京:南京农业大学,2013.Jia S. Cloning and characterization of Halloween genes involving in ecdysteriodogenesis in Sogatella furcifera and Laodelphgax striatellus[D]. Nanjing:Nanjing Agricultural University,2013. (in Chinese)
[43] Pondeville E,David JP,Guittard E,et al. Microarray and RNAi analysis of P450 s in Anopheles gambiae male and female steroidogenic tissues:CYP307A1 is required for ecdysteroid synthesis[J]. PLoS One,2013,8(12):e79861. DOI:10.1371/journal.pone.0079861.