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Journal of Integrative Agriculture  2023, Vol. 22 Issue (12): 3720-3730    DOI: 10.1016/j.jia.2023.05.019
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Serine protease inhibitors LmSPN2 and LmSPN3 co-regulate embryonic diapause in Locusta migratoria manilensis (Meyen) via the Toll pathway
FENG Shi-qian*, ZHANG Neng*, CHEN Jun*, ZHANG Dao-gang, ZHU Kai-hui, CAI Ni, TU Xiong-bing#, ZHANG Ze-hua

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

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摘要  

飞蝗雌成虫可以感知季节性光周期的变化,从而诱导胚胎滞育,并以此作为越冬的关键策略。丝氨酸蛋白酶抑制剂基因(SPNs)在滞育诱导中发挥重要作用,但只有少数SPNs基因得到了功能验证。LmSPN2在滞育和非滞育卵之间差异表达,然而其生物学功能仍需进一步探究。因此,我们首先对LmSPN2进行了RNAi敲降,结果表明滞育率显著降低了29.7%。使用酵母双杂交、免疫共沉淀和pull-down等技术,我们验证了LmSPN2与LmSPN3之间存在相互作用,且作用位点为LmSPN2的331位谷氨酸。LmSPN3RNAi敲降会使滞育率增加14.6%,表明LmSPN2LmSPN3在滞育调控上具有相反的功能。两个SPN基因的双重敲降导致滞育率降低了26.4%,表明LmSPN2是主导的调节信号。此外,我们发现敲降LmSPN2后,四个Toll通路基因(easterspätzlepelledorsal)显著上调,而敲降LmSPN3后则下调,因此我们推测这两个SPN基因通过Toll通路调节滞育过程。综上LmSPN2对蝗虫卵滞育具有正调控作用,而LmSPN3作用相反,二者通过LmSPN2的331位谷氨酸进行互作,并通过Toll通路影响飞蝗滞育。这种滞育调控机制扩展了我们对昆虫发育调控的理解,并为研发新的蝗虫防控策略的提供了新方向。



Abstract  

Female adults of the migratory locust, Locusta migratoria manilensis (Meyen), can sense seasonal photoperiod changes, which induces embryonic diapause as a key strategy to overwinter.  Serine protease inhibitor genes (SPNs) were thought to play key roles during diapause, while few SPNs were functionally characterized.  LmSPN2 was one of those genes differentially expressed between diapause and non-diapause eggs; however, its biological function remained to be explored.  So, we conducted RNAi knockdown of LmSPN2, resulting in a significant decrease of the egg diapause rate by 29.7%.  Using yeast two-hybrid assays, co-immunoprecipitation, and pull-down methods, we found an interaction between LmSPN2 and LmSPN3, which was proved to be mediated by a glutamate (E331) binding site of LmSPN2.  RNAi knockdown of LmSPN3 resulted in a significant increase in diapause rate by 14.6%, indicating an inverse function of LmSPN2 and LmSPN3 on diapause regulation.  Double knockdown of two SPN genes resulted in a 26.4% reduction in diapause rate, indicating that LmSPN2 was the dominant regulatory signal.  Moreover, we found four Toll pathway genes (easter, spätzle, pelle, and dorsal) upregulated significantly after the knockdown of LmSPN2 while downregulated after the knockdown of LmSPN3.  Therefore, we speculate that two SPNs regulate diapause through the Toll pathway.  Our results indicated that LmSPN2 positively regulates locust egg entry into diapause, while LmSPN3 is a negative regulator of embryonic commitment to diapause.  Their interaction is mediated by the binding site of E331 and influences egg diapause through the Toll pathway.  This mechanistic understanding of diapause regulation expands our understanding of insect developmental regulation and provides functional targets for developing locust management strategies.

Keywords:  Locusta migratoria        insect diapause regulation        Toll pathway        protein interaction        serine protease inhibitor  
Received: 19 January 2023   Accepted: 12 April 2023
Fund: 

This work was supported by the National Key R&D Program of China (2022YFD1400500), the China Agriculture Research System of MOF and MARA (CARS-34-07), the Public-interest Scientific Institution Basal Research Fund, China (Y2022GH12) and the Central Public-interest Scientific Institution Basal Research Fund, China (S2021XM22 and S2022XM21).

About author:  #Correspondence TU Xiong-bing, Tel: +86-10-82109569, E-mail: tuxiongbing@caas.cn * These authors contributed equally to this study.

Cite this article: 

FENG Shi-qian, ZHANG Neng, CHEN Jun, ZHANG Dao-gang, ZHU Kai-hui, CAI Ni, TU Xiong-bing, ZHANG Ze-hua. 2023. Serine protease inhibitors LmSPN2 and LmSPN3 co-regulate embryonic diapause in Locusta migratoria manilensis (Meyen) via the Toll pathway. Journal of Integrative Agriculture, 22(12): 3720-3730.

An C, Kanost M R. 2010. Manduca sexta serpin-5 regulates prophenoloxidase activation and the Toll signaling pathway by inhibiting hemolymph proteinase HP6. Insect Biochemistry and Molecular Biology40, 683–689.

Baglin T P, Carrell R W, Church F C, Esmon C T, Huntington J A. 2002. Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism. Proceedings of the National Academy of Sciences of the United States of America99, 11079–11084.

Belvin M P, Anderson K V. 1996. A conserved signaling pathway: The Drosophila Toll-Dorsal pathway. Annual Review of Cell and Developmental Biology12, 393–416.

Bian G, Shin S W, Cheon H M, Kokoza V, Raikhel A S. 2005. Transgenic alteration of Toll immune pathway in the female mosquito Aedes aegyptiProceedings of the National Academy of Sciences of the United States of America102, 13568–13573.

Chen J, Cui D, Ullah H, Hao K, Tu X, Zhang Z. 2020a. Serpin7 controls egg diapause of migratory locust (Locusta migratoria) by regulating polyphenol oxidase. FEBS Open Bio10, 707–717.

Chen J, Cui D, Ullah H, Li S, Pan F, Xu C, Tu X, Zhang Z. 2020b. The function of LmPrx6 in diapause regulation in Locusta migratoria through the insulin signaling pathway. Insects11, 763.

Chu Y, Zhou F, Liu Y, Hong F, Wang G, An C. 2015. Ostrinia furnacalis serpin-3 regulates melanization cascade by inhibiting a prophenoloxidase-activating protease. Insect Biochemistry and Molecular Biology61, 53–61.

Cui D, Tu X, Hao K, Raza A, Chen J, McNeill M, Zhang Z. 2019. Identification of diapause-associated proteins in migratory locust, Locusta migratoria L. (Orthoptera: Acridoidea) by label-free quantification analysis. Journal of Integrative Agriculture18, 2579–2588.

Das D, Arur S. 2017. Conserved insulin signaling in the regulation of oocyte growth, development, and maturation. Molecular Reproduction and Development84, 444–459.

Denlinger D L. 2002. Regulation of diapause. Annual Review of Entomology47, 93–122.

Denlinger D L, Armbruster P A. 2016. Molecular physiology of mosquito diapause. In: Advances in Insect Physiology. Elsevier, Netherlands. pp. 329–361.

Eappen A G, Smith R C, Jacobs-Lorena M. 2013. Enterobacter-activated mosquito immune responses to Plasmodium involve activation of SRPN6 in Anopheles stephensiPLoS ONE8, e62937.

El-Zayat S R, Sibaii H, Mannaa F A. 2019. Toll-like receptors activation, signaling, and targeting: An overview. Bulletin of the National Research Centre43, 1–12.

Etebari K, Asgari S. 2013. Conserved microRNA miR-8 blocks activation of the Toll pathway by upregulating Serpin 27 transcripts. RNA Biology, 10, 1356–1364.

Gangloff M, Murali A, Xiong J, Arnot C J, Weber A N, Sandercock A M, Robinson C V, Sarisky R, Holzenburg A, Kao C, Gay N J. 2008. Structural insight into the mechanism of activation of the Toll receptor by the dimeric ligand Spätzle. The Journal of Biological Chemistry283, 14629–14635.

Garrett M, Fullaondo A, Troxler L, Micklem G, Gubb D. 2009. Identification and analysis of serpin-family genes by homology and synteny across the 12 sequenced Drosophilid genomes. BMC Genomics10, 489.

Gregg P. 1985. Reversal of embryonic diapause in the Australian plague locust, Chortoicetes terminifera (Walker), by temperatures above the development threshold. Journal of Insect Physiology31, 959–962.

Guo P, Dong Z, Zhao P, Zhang Y, He H, Tan X, Zhang W, Xia Q. 2015. Structural insights into the unique inhibitory mechanism of the silkworm protease inhibitor serpin18. Scientific Reports5, 11863.

Hao K, Jarwar A R, Ullah H, Tu X, Nong X, Zhang Z. 2019. Transcriptome sequencing reveals potential mechanisms of the maternal effect on egg diapause induction of Locusta migratoriaInternational Journal of Molecular Sciences20, 1974.

Hao K, Wang J, Tu X, Whitman D, Zhang Z. 2017. Transcriptomic and proteomic analysis of Locusta migratoria eggs at different embryonic stages: Comparison for diapause and non-diapause regimes. Journal of Integrative Agriculture16, 1777–1788.

Huntington J A. 2011. Serpin structure, function and dysfunction. Journal of Thrombosis and Haemostasis9, 26–34.

Irving J A, Pike R N, Lesk A M, Whisstock J C. 2000. Phylogeny of the serpin superfamily: Implications of patterns of amino acid conservation for structure and function. Genome Research10, 1845–1864.

Ito S, Nagata K. 2017. Biology of Hsp47 (Serpin H1), a collagen-specific molecular chaperone. Seminars in Cell & Developmental Biology62, 142–151.

Jiang H, Wang Y, Yu X, Zhu Y, Kanost M. 2003. Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: A clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs. Insect Biochemistry and Molecular Biology33, 1049–1060.

Kanost M R, Gorman M J. 2008. Phenoloxidases in insect immunity. Insect Immunology1, 69–96.

Kanost M R, Jiang H. 2015. Clip-domain serine proteases as immune factors in insect hemolymph. Current Opinion in Insect Science11, 47–55.

Katsukawa M, Ohsawa S, Zhang L, Yan Y, Igaki T. 2018. Serpin facilitates tumor-suppressive cell competition by blocking toll-mediated Yki activation in DrosophilaCurrent Biology28, 1756–1767.

Kubrak O I, Kučerová L, Theopold U, Nässel D R. 2014. The sleeping beauty: How reproductive diapause affects hormone signaling, metabolism, immune response and somatic maintenance in Drosophila melanogasterPLoS ONE9, e113051.

Krysan J L, Jackson J J, Lew A C. 1984. Field termination of egg diapause in Diabrotica with new evidence of extended diapause in Dbarberi (Coleoptera: Chrysomelidae). Environmental Entomology13, 1237–1240.

Law R H P, Zhang Q, McGowan S, Buckle A M, Silverman G A, Wong W, Rosado C J, Langendorf C G, Pike R N, Bird P I, Whisstock J C. 2006. An overview of the serpin superfamily. Genome Biology7, 216.

Lemaitre B. 2004. The road to Toll. Nature Reviews Immunology4, 521–527.

Ligoxygakis P, Roth S, Reichhart J. 2003. A serpin regulates dorsal-ventral axis formation in the Drosophila embryo. Current Biology13, 2097–2102.

Long T, Liu M, Zhu A J. 2019. Culturing Drosophila schneider 2 cells. Bio-101, doi: 10.21769/BioProtoc.1010259.

Louche A, Salcedo S P, Bigot S. 2017. Protein–protein interactions: Pull-down assays. Bacterial Protein Secretion Systems (Methods and Protocols), 1615, 247–255.

MacRae T H. 2010. Gene expression, metabolic regulation and stress tolerance during diapause. Cellular and Molecular Life Sciences67, 2405–2424.

Medzhitov R. 2001. Toll-like receptors and innate immunity. Nature Reviews Immunology1, 135–145.

Meekins D A, Kanost M R, Michel K. 2017. Serpins in arthropod biology. Seminars in Cell & Developmental Biology62, 105–119.

Qin T, Zhao X, Luan H, Ba H, Yang L, Li Z, Hou L, Zou X. 2015. Identification, expression pattern and functional characterization of As-MyD88 in bacteria challenge and during different developmental stages of Artemia sinicaDevelopmental and Comparative Immunology50, 9–18.

Ragland G J, Armbruster P A, Meuti M E. 2019. Evolutionary and functional genetics of insect diapause: A call for greater integration. Current Opinion in Insect Science36, 74–81.

Roberts T H, Marttila S, Rasmussen S K, Hejgaard J. 2003. Differential gene expression for suicide-substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley. Journal of Experimental Botany54, 2251–2263.

Sahoo A, Dutta A, Dandapat J, Samanta L. 2018. Low H2O2 and enhanced oxidative resistance in the diapause-destined pupa of silkworm, Antheraea mylitta (Lepidoptera:Saturniidae) suggest their possible involvement in dormancy and lifespan extension. BMC Zoology3, 1–9.

Sgolastra F, Bosch J, Molowny-Horas R, Maini S, Kemp W P. 2010. Effect of temperature regime on diapause intensity in an adult-wintering Hymenopteran with obligate diapause. Journal of Insect Physiology56, 185–194.

Shin S W, Park S S, Park D S, Kim M G, Kim S C, Brey P T, Park H. 1998. Isolation and characterization of immune-related genes from the fall webworm, Hyphantria cunea, using PCR-based differential display and subtractive cloning. Insect Biochemistry and Molecular Biology28, 827–837.

Silverman G A, Bird P I, Carrell R W, Church F C, Coughlin P B, Gettins P G W, Irving J A, Lomas D A, Luke C J, Moyer R W, Pemberton P A, Remold-O’Donnell E, Salvesen G S, Travis J, Whisstock J C. 2001. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. The Journal of Biological Chemistry276, 33293–33296.

Sugden C, Ross S, Bloomfield G, Ivens A, Skelton J, Mueller-Taubenberger A, Williams J G. 2010. Two novel Src homology 2 domain proteins interact to regulate dictyostelium gene expression during growth and early development. The Journal of Biological Chemistry285, 22927–22935.

Tanaka H. 1994. Embryonic diapause and life cycle in the migratory locust, Locusta migratoria L. (Orthoptera: Acrididae), in Kyoto. Applied Entomology and Zoology29, 179–191.

Tanaka S. 1992. The significance of embryonic diapause in a Japanese strain of the migratory locust, Locusta migratoria (Orthoptera: Acrididae). Japanese Journal of Entomology60, 503–520.

Tsuchiya R, Kaneshima A, Kobayashi M, Yamazaki M, Takasu Y, Sezutsu H, Tanaka Y, Mizoguchi A, Shiomi K. 2021. Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkworm Bombyx moriProceedings of the National Academy of Sciences of the United States of America118, e2020028118.

Tu X, Wang J, Hao K, Whitman D W, Fan Y, Cao G, Zhang Z. 2015. Transcriptomic and proteomic analysis of pre-diapause and non-diapause eggs of migratory locust, Locusta migratoria L. (Orthoptera: Acridoidea). Scientific Reports5, 11402.

Valanne S, Wang J, Rämet M. 2011. The Drosophila Toll signaling pathway. Journal of Immunology , 186, 649–656.

Veillard F, Troxler L, Reichhart J. 2016. Drosophila melanogaster clip-domain serine proteases: Structure, function and regulation. Biochimie122, 255–269.

Viswanathan K, Bot I, Liu L, Dai E, Turner P C, Togonu-Bickersteth B, Richardson J, Davids J A, Williams J M, Bartee M Y, Chen H, van Berkel T J C, Biessen E A L, Moyer R W, Lucas A R. 2012. Viral cross-class serpin inhibits vascular inflammation and T lymphocyte fratricide; a study in rodent models in vivo and human cell lines
in vitroPLoS ONE7, e44694.

Wang X, Fan J, Zhou M, Gao G, Wei L, Kang L. 2021. Interactive effect of photoperiod and temperature on the induction and termination of embryonic diapause in the migratory locust. Pest Management Science77, 2854–2862.

Wartenberg H, Ihmer A, Schwarz S, Miething A, Viebahn C. 2001. Mitotic arrest of female germ cells during prenatal oogenesis. A colcemid-like, non-apoptotic cell death. Anatomy and Embryology204, 421–435.

Ye S, Cech A L, Belmares R, Bergstrom R C, Tong Y, Corey D R, Kanost M R, Goldsmith E J. 2001. The structure of a Michaelis serpin–protease complex. Nature Structural Biology8, 979–983.

Yocum G D. 2001. Differential expression of two HSP70 transcripts in response to cold shock, thermoperiod, and adult diapause in the Colorado potato beetle. Journal of Insect Physiology47, 1139–1145.

Zhang L, Lecoq M, Latchininsky A, Hunter D. 2019. Locust and grasshopper management. Annual Review of Entomology64, 15–34.

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