Intrinsically disordered proteins of the European corn borer Ostrinia nubilalis (Hbn, 1796)

UNIVERSITY OF NOVI SAD FACULTY OF SCIENCES DEPARTMENT OF BIOLOGY AND ECOLOGY INTRINSICALLY DISORDERED PROTEINS OF THE EUROPEAN CORN BORER OSTRINIA NUBILALIS (HBN, 1796) Doctoral dissertation Advisor: Candidate: Prof. Željko D. Popović, PhD Miloš Avramov, MSc Novi Sad, 2023 UNIVERSITY OF NOVI SAD FORM – 5a FACULTY OF SCIENCES KEY WORD DOCUMENTATION 1 Document type: Doctoral dissertation Author: Miloš Avramov, MSc Supervisor (title, first name, last name, position, institution) Prof. Željko D. Popović, PhD, Faculty of Sciences Thesis title: Intrinsically disordered proteins of the European corn borer Ostrinia nubilalis (Hbn, 1796) Language of text (script): English language, latin script Physical description: Number of: Pages – 166 Chapters – 9 References – 240 Tables – 28 Illustrations – 6 Graphs – 22 Appendices – 1 Scientific field: Biology Scientific subfield (scientific discipline): Biochemistry Subject, Key words: intrinsically disordered proteins; intrinsically disordered protein regions; Ostrinia nubilalis; cold hardiness; diapause; gene expression Abstract in English language: Intrinsically disordered proteins (IDPs) are a specific and diverse group of proteins that, under physiological environmental conditions, do not possess higher levels of structural organization, but are found in a form resembling denatured proteins. When IDPs are exposed to conditions that are denaturing for ordered proteins, such as high and low temperatures or strongly acidic or basic pH of environment, they acquire transient forms of ordered structure. Also, a large number of typically ordered, globular proteins have intrinsically disordered regions (IDRs) in their structure. Contrary to the established view that the function of proteins depends on their ordered structure, research has shown that IDPs possess various molecular functions regardless of the absence of structure. Due to the lack of higher orders of structure, IDPs have the ability to recognize a large number of different biomolecules and interact with them, and during these interactions they partially fold and acquire a more ordered structure. Due to their flexibility and plasticity in intermolecular interactions, IDPs and proteins with intrinsically disordered regions are often involved in cell signaling processes, regulation of transcription and translation, and also function as molecular chaperones. Based on their diverse roles, as well as resistance to denaturation under unfavorable conditions, IDPs probably have a pronounced biological significance in adaptive processes of organisms to abiotic stress factors. In order to 1 The author of the doctoral dissertation has signed the following Statements: 5б – Statement on the authorship, 5в – Statement that the printed and e-version of the doctoral dissertation are identical and about personal data, 5г – Statement on copyright licenses. The paper and e-versions of the Statements are held at he faculty and are not included into the printed thesis. evaluate their potential importance in the process of adaptation to hypometabolic living conditions during phases of arrested development and developing cold resistance, in this dissertation 5th instar larvae of the European corn borer Ostrinia nubilalis were used. In order to survive the cold winter months this species enters a specific resting state called diapause. During diapause, corn borer larvae undergo biochemical and molecular changes that allow them to survive only on stored energy sources and to develop resistance to extremely low winter temperatures. For this reason, the European corn borer is often used as an in vivo model system for studying these processes. In this dissertation, a method was established and optimized for enriching the content of IDPs, validation of the enrichment procedure with specific 2D polyacrylamide gel electrophoresis and isolation of disordered proteins from whole-body homogenates of larvae that were subjected to different temperature treatments to develop low temperature resistance. Isolated proteins were identified using liquid chromatography coupled with tandem mass spectrometry. Also, the degree of intrinsic disorder of the identified proteins was determined by in silico analyses, quantitative and qualitative parameters of their disorder were evaluated, and the functional characterization of the proteins was performed. The relative expression of selected genes for proteins with a significant degree of intrinsic disorder was also examined in 5th instar larvae that were reared under non-diapausing conditions, as well as diapause-inducing conditions that also lead to the development of cold resistance. The results showed that in this species there is a significant content of IDPs and proteins with disordered regions, and that the proportion of proteins with IDRs is correlated with the acquisition of cold resistance. The expression of most of the analyzed genes is also correlated with this adaptation. The results of this dissertation provide new insights into the molecular basis of processes that enable the European corn borer to adapt to unfavorable living conditions characterized by a reduced metabolic rate, limited energy sources for basal metabolism, as well as the negative effects of exposure to low temperatures on the functional and structural homeostasis of proteins and processes they are involved in. Accepted by the Scientific Board on: 30th March 2023 Defended on: (Filled by the faculty service) Thesis Defense Board: (title, first name, last name, position, institution) President: Prof. Edward Petri, PhD, Faculty of Sciences Member: dr Ágnes Tantos, Senior Research Fellow, Research Centre for Natural Sciences, “Eötvös Loránd” Research Network, Budapest, Hungary Member (advisor): Prof. Željko D. Popović, PhD, Faculty of Sciences Note: УНИВЕРЗИТЕТ У НОВОМ САДУ ОБРАЗАЦ – 5а ПРИРОДНО-МАТЕМАТИЧКИ ФАКУЛТЕТ КЉУЧНA ДОКУМЕНТАЦИЈСКА ИНФОРМАЦИЈА 2 Врста рада: Докторска дисертација Име и презиме аутора: MSc Милош Аврамов Ментор (титула, име, презиме, звање, институција) др Жељко Д. Поповић, ванредни професор, Природно-математички факултет Наслов рада: Структурно-неуређени протеини кукурузног пламенца Ostrinia nubilalis (Hbn, 1796) Језик публикације (писмо): Енглески језик, латиница Физички опис рада: Унети број: Страница – 166 Поглавља – 9 Референци – 240 Табела – 28 Слика – 6 Графикона – 22 Прилога – 1 Научна област: Биологија Ужа научна област (научна дисциплина): Биохемија Кључне речи / предметна одредница: структурно-неуређени протеини; структурно-неуређени региони протеина; Ostrinia nubilalis; отпорност на хладноћу; дијапауза; експресија гена Резиме на српском језику: Структурно-неуређени протеини (енгл. intrinsically disordered proteins, IDP-ови) су специфична и разноврсна група протеина који при физиолошким условима средине не поседују више нивое структурне организације, већ се налазе у облику који наликује денатурисаним протеинима. Када су IDP-ови изложени условима који су денатуришући за уређене протеине, као што су високе и ниске температуре или изразито кисела или базна pH средине, они задобијају пролазне облике уређене структуре. Такође, велик број типично уређених, глобуларних протеина у својој структури поседују регионе који могу бити структурно-неуређени (енгл. intrinsically disordered regions, IDR-ови). У супротности са устаљеним виђењем да функција протеина зависи од њихове уређене структуре, истраживањима је показано да IDPови поседују разноразне молекуларне функције без обзира на одсуство структуре. Услед одсуства виших нивоа структуре, IDP-ови имају способност препознавања великог броја других биомолекула и интераговања са њима, а приликом тих интеракција се делимично савијају и задобијају пролазу уређену структуру. Због своје флексибилности и пластичности у међумолекулским интеракцијама, IDP-ови и протеини са структурно-неуређеним регионима често су укључени у процесе ћелијског сигналинга, регулацију транскрипције и транслације, а поседују и 2 Аутор докторске дисертације потписао је и приложио следеће Обрасце: 5б – Изјава о ауторству; 5в – Изјава o истоветности штампане и електронске верзије и о личним подацима; 5г – Изјава о коришћењу. Ове Изјаве се чувају на факултету у штампаном и електронском облику и не кориче се са тезом. функције молекуларних шаперона. На основу њихових разноликих улога, као и отпорности на денатурацију у неповољним условима, IDP-ови вероватно имају изражен биолошки значај у процесима адаптација организама на абиотске факторе стреса животне средине. Како би се проценио њихов претпостављени значај у процесу адаптације организма на хипометаболичке услове живота током фазе мировања и развијања отпорности на хладноћу, у овој дисертацији коришћене су гусенице 5. инстара врсте кукурузног пламенца Ostrinia nubilalis. За ову врсту је карактеристично да у зимском периоду улази у фазу мировања звану дијапауза, како би преживела хладне зимске месеце. Током дијапаузе, у гусеницама кукурузног пламенца долази до промена на биохемијском и молекуларном нивоу које им омогућавају да преживе само на ускладиштеним изворима енергије као и да развију отпорност на изузетно ниске зимске температуре. Због тога се кукурузни пламенац често користи као in vivo модел-систем у истраживањима ових процеса. У овој дисертацији успостављена је и оптимизована метода за обогаћивање садржаја IDPова, валидацију поступка обогаћивања специфичном 2Д електрофорезом на полиакриламидном гелу и изолацију неуређених протеина из хомогената целих гусеница које су подвргаване различитим температурним третманима за развој отпорности на ниске температуре. Изоловани протеини су идентификовани помоћу течне хроматографије купловане са тандемском масеном спектрометријом. Такође, in silico анализама су утврђени степен структурне неуређености идентификованих протеина, процењени квантитативни и квалитативни параметри њихове неуређености и урађена је функционална карактеризација протеина. Испитана је и релативна експресија одабраних гена за протеине са утврђеним значајним степеном структурне неуређености у гусеницама 5. инстара држаним у условима недијапаузе, као и дијапаузе уз развијање отпорности на хладноћу. Резултати су показали да у овој врсти постоји значајан удео IDP-ова и протеина са неуређеним регионима, као и да је удео протеина са IDR-овима корелисан са стицањем отпорности на хладноћу. Експресија већине анализираних гена је такође корелирана са том адаптацијом. Резултати ове дисертације дају нове увиде у молекуларне основе процеса који омогућавају врсти кукурузног пламенца да се прилагоди неповољним условима живота које карактеришу смањена стопа метаболизма, ограничени извори енергије за потребе базалног метаболизма, као негативне ефекте излагања ниским температурама на структурну и функционалну хомеостазу протеина и процеса у којима су они укључени. Датум прихватања теме од стране надлежног већа: 30. март 2023. године Датум одбране: (Попуњава одговарајућа служба) Чланови комисије: (титула, име, презиме, звање, институција) Председник: др Едвард Петри, ванредни професор, Природно-математички факултет Члан: др Агнеш Тонтош, виши научни сарадник, Истраживачки центар за природне науке, Истраживачка мрежа “Етвеш Лоранд”, Будимпешта, Мађарска Члан (ментор): др Жељко Д. Поповић, ванредни професор, Природно-математички факултет Напомена: “New kids on the block…” In memoriam – Miloš Avramov (1943 – 2021) Acknowledgements Having reached this point, people usually ask themselves, “Where do I begin?” That is not the case here. This dissertation is dedicated to my grandparents, Miloš and Nada, whom I owe the biggest gratitude for becoming the person who I am today. They have been my greatest source of inspiration, as well as my biggest supporters, throughout my entire life. Nothing brought me more joy than seeing them be proud of my accomplishments, as I then knew that their trust and faith in me was repaid. Baba i deda, hvala vam. A special mention goes to my high school biology teacher, Biserka Nenadović, who, in a school dedicated to the study of languages and social sciences, made sure that we were prepared for the world of natural sciences, as well. Thank you, Biso, for setting me on this path. I must also express my gratitude to my advisor, Prof. Željko D. Popović, for guiding me not only throughout my doctoral studies, but also during my undergraduate and master studies, ever since I met him as a 2nd year student in the practical course for „Fundamentals of molecular biology“, more than a decade ago. Through his own example, sage advice and constructive criticism, he always pushed me to better myself. How much I owe my grandparents for the person I am today, that much I owe Željko for where I am as a researcher and man of science. I would also like to thank dr Ágnes Tantos for being an integral part in defining the research scope of this dissertation, as well as providing me with the means to carry out my experimental designs, both material as well as educational. Being hosted in her lab group at the Research Centre for Natural Sciences in Budapest as part of a joint project is one of the highlights of my life and it is a period that I will always fondly reminisce on. If only that year did not fly by as quickly as it did. In the same regard I would like to thank dr Éva Schád for lending me her bioinformatics expertise in matters of in silico analyses performed in the dissertation, and dr Ágnes Révész and dr Lilla Turiák for their assistance with the identification of the incredible number of proteins in this work. To Prof. Edward Petri I owe gratitude first for introducing me to the field of structural biology and teaching me the skills which were indispensable during my PhD studies, and, second, for his assistance with the preparation of this body of work with his helpful comments, critique and advice on how to best improve the manuscript. I would also like to thank all my colleagues from the Laboratory for biochemistry and molecular biology for the many wonderful years we have spent together – from the long days in the lab to our outings when we put the work behind us, talk about anything and everything, and enjoy each other’s company. When I doubted myself the most, and would worry about every single issue and obstacle that came my way, one person was by me to reassure me and help me push forward at all times. Thank you, Teodora, for your love, support and for always finding the right words and making me believe in myself, time after time. Finally, I want to thank my family – my mother Nada, father Srđan and brothers Nestor (Avram) and Mihailo (also Avram) who, along with my closest friends, supported me in countless ways throughout my entire formal education which has come to an end after 24 long years. Again, thank you all for everything. Miloš (Prime Avram) Table 20. Relative expression of hsp70 gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 ................................................ 68 Table 21. Relative expression of hsp20.4 gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 ................................................ 70 Table 22. Relative expression of hsp20.1 gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 ................................................ 72 Table 23. Relative expression of tropmy2 gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 ................................................ 74 Table 24. Relative expression of tnt gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 ................................................................ 76 Table 25. Relative expression of thym gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 ................................................................ 78 Table 26. Relative expression of moe gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 ................................................................ 80 Table 27. Primer pairs used for qPCR analysis. ......................................................................... 140 Table 28. Results of gene expression analysis in O. nubilalis larvae ........................................ 141 Table of contents Abstract .......................................................................................................................................... 1 Извод .............................................................................................................................................. 3 1. Introduction ............................................................................................................................... 5 1.1. Intrinsically disordered proteins – IDPs ............................................................................... 6 1.1.1. The code for protein intrinsic disorder .......................................................................... 7 1.1.2. Localized intrinsic disorder – intrinsically disordered regions ..................................... 8 1.1.3. Environmental hardiness of IDPs .................................................................................. 9 1.1.4. Natural abundance of intrinsic disorder ....................................................................... 10 1.1.5. Intrinsic disorder and protein function ........................................................................ 11 1.2. The European corn borer, Ostrinia nubilalis (Hbn., 1796) ................................................ 12 1.2.1. History, distribution and ecology ................................................................................ 12 1.2.2. Insect morphology ....................................................................................................... 13 1.2.3. Life cycle of Ostrinia nubilalis ................................................................................... 14 1.2.4. Ostrinia nubilalis as a model system for diapause and cold adaptation ...................... 16 1.2.4.1. Diapause as an overwintering strategy ................................................................. 16 1.2.4.2. Effects of low and subzero temperatures on biological systems .......................... 17 1.2.4.3. Molecular adaptations in O. nubilalis during diapause and cold hardening ......... 19 2. Thesis hypothesis and aims .................................................................................................... 21 3. Materials and methods ........................................................................................................... 23 3.1. Experimental design of the study ....................................................................................... 24 3.1.1. Pilot sampling and experimental design ...................................................................... 24 3.1.2. Main experiment sampling and design ........................................................................ 25 3.2. Proteomic and bioinformatics analyses .............................................................................. 27 3.2.1. Sample preparation for proteomic analyses ................................................................. 27 3.2.2. 2D PAGE enrichment validation ................................................................................. 27 3.2.3. LC-MS/MS protein identification ............................................................................... 29 3.2.4. Bioinformatic analyses ................................................................................................ 31 3.3. Gene expression analysis of selected IDPs ........................................................................ 33 3.3.1. Isolation of total RNA ................................................................................................. 33 3.3.2. RNA concentration and quality assessment ................................................................ 34 3.3.3. RT-PCR cDNA synthesis ............................................................................................ 35 3.3.4. Primer design ............................................................................................................... 35 3.3.5. Primer specificity analysis ........................................................................................... 37 3.3.6. Primer efficiency determination .................................................................................. 38 3.3.7. Quantitative PCR of genes of interest ......................................................................... 39 3.3.8. Statistical analysis of qPCR results ............................................................................. 40 4. Results ...................................................................................................................................... 41 4.1. Proteomic analyses ............................................................................................................. 42 4.1.1. 2D PAGE confirmation of IDP enrichment procedure ............................................... 42 4.1.2. LC-MS/MS identification of proteins.......................................................................... 43 4.1.3. Effect of sample heating on protein identification ...................................................... 45 4.2. Bioinformatic analyses ....................................................................................................... 47 4.2.1. Determination of protein disorder ............................................................................... 47 4.2.2. Effect of sample heating on protein disorder distribution ........................................... 50 4.2.2.1. Pilot experimental setup ....................................................................................... 50 4.2.2.2. Main experimental setup ....................................................................................... 52 4.2.3. Determination of long intrinsically disordered regions (long IDRs) ........................... 54 4.2.3.1. Pilot experimental setup ....................................................................................... 54 4.2.3.2. Main experimental setup ....................................................................................... 56 4.2.4. Amino acid compositional analyses ............................................................................ 58 4.2.4.1. Total amino acid content ...................................................................................... 58 4.2.4.2. Individual amino acid content .............................................................................. 59 4.2.5. Functional analysis of disordered proteins .................................................................. 60 4.3. Relative gene expression analyses ..................................................................................... 62 4.3.1. Ct values of reference genes ........................................................................................ 62 4.3.2. Relative expression of hsp90 gene .............................................................................. 63 4.3.3. Relative expression of hsc70 gene ............................................................................... 65 4.3.4. Relative expression of hsp70 gene .............................................................................. 67 4.3.5. Relative expression of hsp20.4 gene ........................................................................... 69 4.3.6. Relative expression of hsp20.1 gene ........................................................................... 71 4.3.7. Relative expression of tropmy2 gene ........................................................................... 73 4.3.8. Relative expression of tnt gene .................................................................................... 75 4.3.9. Relative expression of thym gene ................................................................................ 77 4.3.10. Relative expression of moe gene ............................................................................... 79 5. Discussion................................................................................................................................. 81 5.1. Proteomic analyses ............................................................................................................. 82 5.1.1. Enrichment of IDP content and enrichment validation ............................................... 82 5.1.2. Identification of proteins by LC-MS/MS .................................................................... 84 5.1.3. Effect of sample heating on protein identification ...................................................... 85 5.2. Bioinformatical analyses .................................................................................................... 86 5.2.1. Determination of intrinsic disorder.............................................................................. 86 5.2.2. Effect of sample heating on protein disorder distribution ........................................... 87 5.2.3. Long intrinsically disordered regions (long IDRs) ...................................................... 87 5.2.4. Amino acid compositional analyses ............................................................................ 89 5.2.4.1. Glutamic acid, lysine, glutamine and proline ....................................................... 90 5.2.4.2. Compositional bias ............................................................................................... 92 5.2.5. Functional analysis of disordered proteins .................................................................. 92 5.3. Ecophysiological aspects of proteins with intrinsic disorder in cold hardiness ................. 94 5.3.1. IDP content in O. nubilalis cold hardiness .................................................................. 94 5.3.2. Gene expression analyses of disorder-containing proteins .......................................... 97 5.2.4.1. Heat shock protein genes ...................................................................................... 97 5.2.4.2. Structural protein genes ...................................................................................... 102 5.2.4.3. Thymosin beta and moesin genes ........................................................................ 105 5.3.3. IDPs as proposed mediators of O. nubilalis cold adaptation ..................................... 107 6. Conclusions ............................................................................................................................ 109 7. Literature ............................................................................................................................... 112 8. Supplementary materials ..................................................................................................... 139 9. Extended abstract in Serbian ............................................................................................... 142 Author biography...................................................................................................................... 165 Abstract 1 Abstract Intrinsically disordered proteins (IDPs) are a specific and diverse group of proteins that, under physiological environmental conditions, do not possess higher levels of structural organization, but are found in a form resembling denatured proteins. When IDPs are exposed to conditions that are denaturing for ordered proteins, such as high and low temperatures or strongly acidic or basic pH of environment, they acquire transient forms of ordered structure. Also, a large number of typically ordered, globular proteins have intrinsically disordered regions (IDRs) in their structure. Contrary to the established view that the function of proteins depends on their ordered structure, research has shown that IDPs possess various molecular functions regardless of the absence of structure. Due to the lack of higher orders of structure, IDPs have the ability to recognize a large number of different biomolecules and interact with them, and during these interactions they partially fold and acquire a more ordered structure. Due to their flexibility and plasticity in intermolecular interactions, IDPs and proteins with intrinsically disordered regions are often involved in cell signaling processes, regulation of transcription and translation, and also function as molecular chaperones. Based on their diverse roles, as well as resistance to denaturation under unfavorable conditions, IDPs probably have a pronounced biological significance in adaptive processes of organisms to abiotic stress factors. In order to evaluate their potential importance in the process of adaptation to hypometabolic living conditions during phases of arrested development and developing cold resistance, in this dissertation 5th instar larvae of the European corn borer Ostrinia nubilalis were used. In order to survive the cold winter months this species enters a specific resting state called diapause. During diapause, corn borer larvae undergo biochemical and molecular changes that allow them to survive only on stored energy sources and to develop resistance to extremely low winter temperatures. For this reason, the European corn borer is often used as an in vivo model system for studying these processes. In this dissertation, a method was established and optimized for enriching the content of IDPs, validation of the enrichment procedure with specific 2D polyacrylamide gel electrophoresis and isolation of disordered proteins from whole-body homogenates of larvae that were subjected to different temperature treatments to develop low temperature resistance. Isolated proteins were identified using liquid chromatography coupled with tandem mass spectrometry. Also, the degree of intrinsic disorder of the identified proteins was determined by in silico analyses, quantitative and qualitative parameters of their disorder were evaluated, and the functional characterization of the proteins was Abstract 2 performed. The relative expression of selected genes for proteins with a significant degree of intrinsic disorder was also examined in 5th instar larvae that were reared under non-diapausing conditions, as well as diapause-inducing conditions that also lead to the development of cold resistance. The results showed that in this species there is a significant content of IDPs and proteins with disordered regions, and that the proportion of proteins with IDRs is correlated with the acquisition of cold resistance. The expression of most of the analyzed genes is also correlated with this adaptation. The results of this dissertation provide new insights into the molecular basis of processes that enable the European corn borer to adapt to unfavorable living conditions characterized by a reduced metabolic rate, limited energy sources for basal metabolism, as well as the negative effects of exposure to low temperatures on the functional and structural homeostasis of proteins and processes they are involved in. key words: intrinsically disordered proteins; intrinsically disordered protein regions; Ostrinia nubilalis; cold hardiness; diapause; gene expression Извод 3 Извод Структурно-неуређени протеини (енгл. intrinsically disordered proteins, IDP-ови) су специфична и разноврсна група протеина који при физиолошким условима средине не поседују више нивое структурне организације, већ се налазе у облику који наликује денатурисаним протеинима. Када су IDP-ови изложени условима који су денатуришући за уређене протеине, као што су високе и ниске температуре или изразито кисела или базна pH средине, они задобијају пролазне облике уређене структуре. Такође, велик број типично уређених, глобуларних протеина у својој структури поседују регионе који могу бити структурно-неуређени (енгл. intrinsically disordered regions, IDR-ови). У супротности са устаљеним виђењем да функција протеина зависи од њихове уређене структуре, истраживањима је показано да IDP-ови поседују разноразне молекуларне функције без обзира на одсуство структуре. Услед одсуства виших нивоа структуре, IDP-ови имају способност препознавања великог броја других биомолекула и интераговања са њима, а приликом тих интеракција се делимично савијају и задобијају пролазу уређену структуру. Због своје флексибилности и пластичности у међумолекулским интеракцијама, IDP-ови и протеини са структурно-неуређеним регионима често су укључени у процесе ћелијског сигналинга, регулацију транскрипције и транслације, а поседују и функције молекуларних шаперона. На основу њихових разноликих улога, као и отпорности на денатурацију у неповољним условима, IDP-ови вероватно имају изражен биолошки значај у процесима адаптација организама на абиотске факторе стреса животне средине. Како би се проценио њихов претпостављени значај у процесу адаптације организма на хипометаболичке услове живота током фазе мировања и развијања отпорности на хладноћу, у овој дисертацији коришћене су гусенице 5. инстара врсте кукурузног пламенца Ostrinia nubilalis. За ову врсту је карактеристично да у зимском периоду улази у фазу мировања звану дијапауза, како би преживела хладне зимске месеце. Током дијапаузе, у гусеницама кукурузног пламенца долази до промена на биохемијском и молекуларном нивоу које им омогућавају да преживе само на ускладиштеним изворима енергије као и да развију отпорност на изузетно ниске зимске температуре. Због тога се кукурузни пламенац често користи као in vivo моделсистем у истраживањима ових процеса. У овој дисертацији успостављена је и оптимизована метода за обогаћивање садржаја IDP-ова, валидацију поступка обогаћивања специфичном 2Д електрофорезом на полиакриламидном гелу и изолацију неуређених протеина из Извод 4 хомогената целих гусеница које су подвргаване различитим температурним третманима за развој отпорности на ниске температуре. Изоловани протеини су идентификовани помоћу течне хроматографије купловане са тандемском масеном спектрометријом. Такође, in silico анализама су утврђени степен структурне неуређености идентификованих протеина, процењени квантитативни и квалитативни параметри њихове неуређености и урађена је функционална карактеризација протеина. Испитана је и релативна експресија одабраних гена за протеине са утврђеним значајним степеном структурне неуређености у гусеницама 5. инстара држаним у условима недијапаузе, као и дијапаузе уз развијање отпорности на хладноћу. Резултати су показали да у овој врсти постоји значајан удео IDP-ова и протеина са неуређеним регионима, као и да је удео протеина са IDR-овима корелисан са стицањем отпорности на хладноћу. Експресија већине анализираних гена је такође корелирана са том адаптацијом. Резултати ове дисертације дају нове увиде у молекуларне основе процеса који омогућавају врсти кукурузног пламенца да се прилагоди неповољним условима живота које карактеришу смањена стопа метаболизма, ограничени извори енергије за потребе базалног метаболизма, као негативне ефекте излагања ниским температурама на структурну и функционалну хомеостазу протеина и процеса у којима су они укључени. кључне речи: структурно-неуређени протеини; структурно-неуређени региони протеина; Ostrinia nubilalis; отпорност на хладноћу; дијапауза; експресија гена 1. Introduction Introduction 12 1.2. The European corn borer, Ostrinia nubilalis (Hbn., 1796) The European corn borer (ECB), Ostrinia nubilalis (Hbn., 1796) is an insect species belonging to the order Lepidoptera which includes moths and butterflies. It is a member of the Pyraloidea superfamily which is comprised of two families – Crambidae and Pyralidae. The Ostrinia genus in general belongs to the subfamily Pyraustinae, which has a contentious classification. For a long time the subfamily was placed under the Pyralidae family, while more recently phylogenomic studies have firmly placed the Pyraustinae subfamily as members of the Crambidae family (Table 2) (Mutuura and Munroe, 1970; Solis, 2007; Léger et al., 2020; Yang et al., 2021). Even the species name was not without contention, and had undergone several changes (e.g. Pyralis nubilalis, P. silacealis, Pyrausta nubilalis) before being settled on the present Ostrinia nubilalis (Caffrey and Worthley, 1927; Mutuura and Munroe, 1970). Table 2. Scientific classification of the European corn borer, O. nubilalis. Taxonomic category Kingdom Animalia Phylum Arthropoda Class Insecta Order Lepidoptera Superfamily Pyraloidea Family Crambidae Subfamily Pyraustinae Genus Ostrinia Species Ostrinia nubilalis 1.2.1. History, distribution and ecology Ostrinia nubilalis is a polyphagous pest moth species that is widespread in temperate regions of Europe, as well as those of northern Africa and western Asia. It is believed that the moth originated in Europe, as the first records of the insect’s presence on this continent are dated to around the 1500s (Bethenod et al., 2005), while its earliest economic impact on European crops was recorded in 1835 (Caffrey and Worthley, 1927). Apart from the Old World, the ECB was introduced to the North American continent most likely by way of broom corn shipments (Sorghum bicolor) from Introduction 13 Austria-Hungary and Italy between 1909 and 1914, with the first specimens being discovered near Boston, Massachussets in 1917 (Smith, 1920; Caffrey and Worthley, 1927). From there, the insect spread westward to the Rocky Mountains in both the United States and Canada (Willet and Harrison, 1999). Corn (Zea mays) is the primary host plant that larvae of the ECB attack and feed on. Young larvae largely feed on the corn tassel, moving into the stalk as they grow and undergo several molting phases. Once inside, the larvae cause significant structural damage to the corn stalk while feeding, which can often lead to the plant snapping and lodging if it is infested with multiple larvae. O. nubalis infestation of corn stalks can be recognized by the characteristic tunnels that they bore throughout the plant. Apart from corn, the ECB has been confirmed as a pest of over 200 different crops, such as hop (Humulus lupulus), mugwort (Artemisia vulgaris), broom corn (Sorghum vulgare), potato (Solanum tuberosum), sweet pepper (Capsicum anuum) and tomato (Solanum licopersicum) (Capinera, 2000; Kuhar et al., 2004; Sole et al., 2010). Common for all these plants is that their stems provide enough space for ECB larvae to bore and settle into. 1.2.2. Insect morphology The European corn borer is a holometabolous insect and undergoes a complete metamorphosis during its development, passing through four distinct stages: embryo or egg, larva, pupa and imago or adult. ECB eggs are oval shaped and flattened, and deposited in clusters of 15 to 20 at a time. Freshly laid eggs are creamy or greenish white in color and opaque in the middle (Fig. 1A). As they mature, the eggs take on a yellowish hue and the black head capsule of the enclosed larva can be seen due to rapid chitinization (Caffrey and Worthley, 1927; Capinera, 2000). Larvae hatch around 24 hours after the head capsule has visibly formed. Newly hatched larvae are less than 2 mm in length, their body pale yellow in color with a pinkish dorsum, and with a dark brown head capsule. The body is divided into 14 segments – 3 thoracal with a pair of true legs on each, 10 abdominal with pairs of prolegs on the 3rd, 4th and 5th abdominal segments, and one terminal segment, also with a pair of prolegs. A dark streak is present on the dorsal side, as well as small dark marks on every segment. As larvae feed and grow, they molt and in field conditions usually go through a total of five developmental stages or instars (Fig. 1B). However, in some cases they can go through six or even seven (Caffrey and Worthley, 1927; Capinera, 2000). Final instar larvae pupate inside the bored tunnels. Early pupae are light brown in color and darken with age Introduction 14 (Fig. 1C). Sexual dimorphism is pronounced at this stage, as male pupae are smaller than the female ones, and the genital openings are differently positioned between the sexes (Caffrey and Worthley, 1927; Capinera, 2000). Adult moths that emerge from pupae are small, again with pronounced sexual dimorphism. Males have a wingspan of 20 to 26 mm and grayish brown in color with dark zigzag lines on their wings (Fig. 1D). Females have a larger wingspan of 25 to 34 mm and are of lighter color than the males. Their wings also contain dark zigzag lines (Fig. 1E). Despite the difference in wingspan, male and female moths are of similar body length – 13 to 14 mm (Caffrey and Worthley, 1927; Capinera, 2000). Figure 1. Life stages of O. nubilalis: (A) egg cluster; (B) 5th instar larva; (C) pupae – male (above), female (below); (D) male adult; (E) female adult. Image credits: (A) and (B) United States Department of Agriculture; (C) Fabrizio Santi (CC BY-NC-ND 4.0); (D) and (E) ©entomart (entomart.be). 1.2.3. Life cycle of Ostrinia nubilalis The four aforementioned life stages constitute the life cycle of one ECB generation. In field conditions, one life cycle takes between 4 and 6 weeks to complete. Up until the 1980s, O. nubilalis was predominantly a univoltine species in Europe, producing one generation per year (Bača et al., 2007). However, in the last 30 years, voltinism of the ECB has been affected by climate changes, primarily temperature fluctuations and amounts of precipitation. The ECB has progressed to being a multivoltine species, most often producing two generations per year – one summer and one winter. That said, there have been years when three generations were detected, with an additional summer generation being produced due to adult moth flights happening in late spring and early Introduction 15 summer (Vajgand, 2010), and the number of yearly generations is likely to increase following the rise of global temperatures (Kocmánková et al., 2008). In the upper parts of North America the ECB usually produces one or two generations per year, while in southern locations three generations are more common with some regions experiencing four per year. In such locations adult moth flights were recorded from as early as April and as late as September (Capinera, 2000). The first generation of the year spawns from 5th instar larvae that had overwintered in a state of arrested development called diapause (explained in detail in 1.2.4.1. Diapause as an overwintering strategy). In late spring, overwintering larvae terminate their diapause program and pupate. This developmental stage lasts around 12 days in field conditions, with pupae requiring an ambient temperature of at least 13°C to develop correctly. First generation adult moths emerge from the cocoons and are mostly active during the nighttime. Imagos feed on plant nectar and live between 18 and 24 days. Female moths begin to lay eggs from 3 to 4 days old. Eggs, 15 to 20 at a time, are usually deposited on the underside of leaves, close to the midrib. The period of oviposition lasts around two weeks. With this, the life cycle of the winter generation is completed (Capinera, 2000; Boyd and Bailey, 2001; Sekulić et al., 2008). Depending on environmental conditions, egg development can last from 4 to 9 days, and generally requires an ambient temperature of at least 15°C. Larvae that hatch from these eggs represent the first generation of the year, which is also considered the summer generation (Fig. 2). Figure 2. Life cycle of bivoltine O. nubilalis specimens. Image by Vanja Tatić (created with BioRender.com). Introduction 16 As has been said, during larval development individuals go through at least 5 instar stages. On average it takes around 50 days for larvae to fully develop, depending on weather conditions, after which they pupate and metamorphose into adult moths (Capinera, 2000; Boy and Bailey, 2001). Regardless of whether there is one or more summer generations of moths during a year, the latest one will lay eggs from which the winter generation will hatch (Capinera, 2000). As summer comes to an end and autumn begins, the days shorten and ambient temperatures drop, all of which serves as an environmental signal that will trigger the larvae prepare for the coming winter. 1.2.4. Ostrinia nubilalis as a model system for diapause and cold adaptation Apart from being an economically important and widespread pest insect species, the ECB is also known for being able to seasonally arrest its development by entering diapause and waiting out the cold winter months, after which it resumes its regular ontogeny. The life cycle of this species has been extensively described, allowing for experiments to be set up with great consistency. Because of this, the ECB has for several decades been used as a reliable model system for studying the mechanisms that govern diapause and adaptations for enduring cold environmental temperatures. In the ECB, these two processes are tightly linked, as exposure to cold temperatures during diapause is one of the necessary triggers for adequate metabolic changes to occur which ensure the insect’s survival during the winter months (Popović et al., 2021). 1.2.4.1. Diapause as an overwintering strategy One of the most significant seasonal changes in the environment that affects insects inhabiting temperate and polar zones is the gradual onset of cold temperatures which can have lethal consequences unless countered. This period is also accompanied by ever increasing food scarcity, which forces insects to rely on energy reserves that were accumulated in the preceding months. An additional challenge that is posed to insects during this time is how to stretch out the limited reserves and endure until conditions improve and food becomes available again. To that end, insects have, over the long course of evolution, acquired adaptations that help them face and survive such harsh living conditions. In order to conserve their limited energy stores, insects enter a hypometabolic resting state called diapause. During diapause growth and development are arrested, the insect’s metabolic rate is significantly reduced (Danks, 1987; Denlinger, 2009) and tolerance to abiotic stressors is increased Introduction 17 (Rinehart et al., 2000). Despite the reduction in the intensity of metabolic processes, diapause is complex and dynamic, and not just a passive stage in an insect’s life cycle. Diapause is also important for insect survival as it is induced ahead of the seasonal changes to which the insect needs to adapt. The most common environmental cue for insects that is time to begin preparations for diapause is the shortening of the photoperiod (Nylin, 2013). Insects can enter diapause at any life stage, and the exact life stage when that occurs is species-specific (Nation, 2008). Diapause is divided into three distinct ecophases, some with their own subphases (Koštál, 2006; Koštál et al., 2017):  Pre-diapause – induction and preparation;  Diapause – initiation and maintenance;  Post-diapause or quiescence. Once diapause is induced, an insect will undergo numerous changes on behavioral, morphological, physiological, biochemical and molecular level in order to ensure its survival during the harsh period. These include cessation of feeding, reduction of mobility, alterations in the structure of cuticle, suppression of less important physiological processes, the aforementioned lowering of general metabolic rate, expression of specific stress-related genes and others (Denlinger, 2002; Storey and Storey, 2004; MacRae, 2010). Concurrently with these metabolic changes diapausing organisms also develop increased resistance to abiotic stressors such as dehydration, UV radiation and low/high temperatures. In insects of polar and temperate regions, such as O. nubilalis, increased hardiness to cold weather conditions is the most common adaptation that occurs during diapause. 1.2.4.2. Effects of low and subzero temperatures on biological systems Environmental temperatures greatly affect different components of biological systems, which rely on specific temperatures ranges in order to remain functional and ensure the survival of an individual. Cold and sub-zero ambient temperatures present living beings with unique and dangerous challenges which are fatal if they cannot be overcome by adaptations on behavioral, physiological, biochemical and molecular levels. In cold environments, organisms need to cope with issues such as freezing of intraand extracellular water, denaturation of proteins induced by cold temperatures, decreased fluidity of membranes, desiccation, lowered transcription and translation rates, just to name a few. Introduction 18 In living organisms water can constitute 90% or more of their total body weight, depending on environmental and developmental factors (Hadley, 1994). Water is also the most abundant molecule in living systems, accounting for 95–99% of all molecules in a particular organism (Edney, 1977). Because of this overwhelming presence, water is the biomolecule that is most susceptible to be adversely affected by low temperatures. In turn, freezing can cause the most devastating consequences for living beings. As temperatures drop below the freezing point of water, ice crystals begin to form in the extracellular spaces. The growing ice crystals can disrupt and pierce membranes, leading to cell death. As more water turns to ice, less of it is readily available to dissolve biomolecules, minerals and other compounds. Moreover, this establishes a concentration gradient between the extraand intracellular environments and water begins to flow outside of the cell in order to reestablish the concentration equilibrium across the cell membrane. However, this process will ultimately end with the cell dying, as the extracellular freezing will cause the cell to lose a critical amount of water (Mazur, 1970, 1984; Zacchariassen and Kristiansen, 2000; Chian and Quinn, 2010). Less dramatic, but no less dangerous are the effects cold temperatures can have on biological processes in living systems. These effects are mediated by water, as it is the major component of body fluids where biological processes take place. As the temperature of fluids decreases, so does the available kinetic energy needed for enabling biochemical reactions to occur. This slows down biological processes and leads to a general depression of metabolism. Additionally, cold temperatures increase the stability of DNA and RNA secondary structures, making it difficult for transcriptional and translational machinery to access them and carry out protein synthesis (D’Amico et al., 2006). Enzymatic activity is also reduced, or completely absent, at low ambient temperatures. Enzymes undergo reversible denaturation, as the cold temperatures reduce the interactions keeping protein subunits together and they subsequently dissociate from each other. Similar effects occur in non-enzymatic proteins as well, with the reversible disassembly of supramolecular structures of microtubules and collagen fibrils under cold conditions (Gaskin et al., 1974; Gelman et al., 1979; Privalov, 1990). To overcome these obstacles and ensure their survival in harsh conditions, organisms have adopted different strategies. These strategies differ based on the environment they inhabit, whether the Introduction 19 changes in temperature are seasonal or year-round and are depended on the life cycle phase the organism is currently in. 1.2.4.3. Molecular adaptations in O. nubilalis during diapause and cold hardening In O. nubilalis, diapause and acclimation to cold ambient temperatures occur during the 5th instar of the larval stage. Diapause itself is photoperiodically induced by the shortening of days in autumn (Beck, 1962). This signals the larvae to begin preparing for the coming winter by accumulating energy reserves. During the course of diapause, larvae will not feed and will need to rely on these stores as sources of fuel for their metabolism (Hahn and Denlinger, 2007, 2011). At the same time ambient temperatures begin to gradually drop, which induces a cascade of changes on physiological and biochemical levels. One of the major changes is in how glycogen, the major reserve polysaccharide, is utilized. As glycogen is being broken down, the resulting glucose is directed towards the synthesis of glycerol. Glycerol is an important cryoprotective molecule that is synthesized in the larval fat body and exported into the haemolymph. As diapause progresses, the level of glycerol in the haemolymph increases, which lowers the supercooling point of the larva to below –20°C and enables it to become cold hardy (Nordin et al., 1984; Grubor-Lajšić et al., 1991; Andreadis et al., 2008). Apart from glycerol, other cryoprotective compounds are produced and accumulated, all of them making the insect increasingly cold hardy. These include sorbitol, trehalose and free amino acids such as alanine, proline and arginine (Koštál et al., 2007; Clark and Worland, 2008; Kojić, 2009; Popović, 2014; Purać et al., 2015). Due to the redirection of glycogen and lack of feeding during diapause, the general metabolic rate needs to decrease in order to conserve energy and ensure survival. Diapausing larvae experience a depression of metabolism by cutting down on energy intensive processes, such as tissue differentiation, cell division and general organism growth and development (Popović et al., 2021). In addition, the cold temperatures slow down metabolic processes, further lowering an organism’s metabolic rate (Storey and Storey, 2004). The energy profile of cold-acclimated diapausing larvae is also distinct from non-acclimated diapausing ones, indicating that exposure to low temperatures during diapause is likely an important component for triggering the necessary metabolic changes for survival (Storey and Storey, 2012). The levels of energy metabolism-related molecules, such as adenine, uridine and guanosine nucleotides, as well as NAD+/NADP+ coenzymes, are depleted in non-acclimated larvae compared to cold-acclimated larvae. In addition to this, the relative Introduction 20 expression of genes encoding different components of the electron transport chain in the nonacclimated diapausing larvae is indicative of increased metabolic rate. Taken together, these findings show that diapausing larvae which have not been exposed to cold temperatures are metabolically very active, which leads to the untimely depletion of their already restricted energy reserves and high mortality (Popović et al., 2021). Other changes that have been reported to occur in regards to diapause and cold hardiness include shifting from aerobic to anaerobic metabolism (Uzelac et al., 2020), increase in the number of aquaporins in cell membranes (Izumi et al., 2007), alterations in the lipid composition of membranes by increasing the content of unsaturated fatty acids (Vukašinović et al., 2013, 2015, 2018) and expression of specific sets of stress protection genes and genes regulating the cell cycle and apoptosis (Denlinger, 2002; Rinehart et al., 2007; MacRae, 2010; Popović et al., 2015; Koštál et al., 2017). Despite this extensive research, the molecular mechanisms governing insect diapause and development of stress-tolerance, in this case to cold ambient temperatures, have not been completely elucidated. While the biochemical and physiological aspects of these processes in the model system O. nubilalis have been broadly covered, one aspect that remains insufficiently studied is the effect of these metabolic and physiological changes on the proteomic level. 2. Thesis hypothesis and aims Materials and Methods 28 Table 4. Composition of gels prepared for first dimension PAGE separation. Component 12.5% separating gel 4% stacking gel dH2O 2.0 mL 2.5 mL 1.5M Tris-HCl pH 8.8 1.5 mL / 0.5M Tris-HCl pH 6.8 / 1.0 mL 30% acrylamide-bisacrylamide 2.5 mL 0.54 mL 10% APS 100 µL 75 µL TEMED 10 µL 10 µL Total (mL) ~6.1 mL ~4.9 mL First, the separating gel was cast by pipetting ~4.5 mL of the separating gel mixture between the assembled glass plates and overlaying it with dH2O until it polymerized after ~20 minutes. Next, the water was discarded and the assembly was filled to the top with the stacking gel mixture. A 10-well comb was placed inside the stacking gel and removed after polymerization, which took ~20 minutes. The finished discontinuous gel was assembled together with the electrophoresis module and placed in a buffer tank filled with Tris-glycine pH 8.3 running buffer. Samples were prepared by taking aliquots from the heated and non-heated sample types and pooling them in two mixtures, respectively. The mixtures were loaded onto the gel with 2X native sample buffer (0.5M Tris-HCl pH 6.8, glycerol, 0.5% bromophenol blue). In total, wells were loaded with 20 µL containing 20 µg of protein. All remaining empty wells were filled with a mix of dH2O and sample buffer to ensure even distribution of the current throughout the gel. Electrophoresis was run for 50 min at 180 V. After the separation, individual lanes were cut out as strips and placed in 10 mL of 1.5 M Tris-HCl pH 8.8 containing 8M urea for 45 min to solubilize the proteins. The separating gel (12.5%) for the second dimension was prepared according to the recipe outlined in Table 5 below: Table 5. Composition of second dimension PAGE separating gel. Component 12.5% separating gel dH2O 0.75 mL 1.5M Tris-HCl pH 8.8 1.5 mL 30% acrylamide-bisacrylamide 2.5 mL Urea (final conc. 8M) 2.88 g 10% APS 50 µL TEMED 5 µL Total (mL) ~6.0 mL Materials and Methods 29 The total volume of the mixture ended up being ~6 mL after the urea dissolved completely. Less APS and TEMED were added compared to the previous separating gel as the urea also makes the gel polymerize much quicker. Additionally, a spacer was placed between the assembled glass plates in order to reduce the width of the gel, compared to its default dimensions. The separating gel containing 8M urea was cast by pipetting ~4.5 mL of the separating gel mixture between the assembled glass plates and overlaying it with dH2O until it polymerized after ~20 minutes. After casting the gel, the water was discarded and a strip with solubilized proteins was placed on top of it instead of a stacking gel, making sure not to introduce any bubbles between the separating gel and the strip. The spacer that was placed beforehand ensured that the width of the separating gel corresponded to the length of the strip. A small volume of 4% separating gel mixture was added to fix the strip onto the separating gel. The second dimension was run for 30 min at 400 V with Tris-glycine pH 8.3 as the running buffer. Following the separation, protein spots were visualized by staining the gels with the Pierce™ Silver Stain Kit (Thermo Scientific, Waltham, MA, USA, cat. no. 24612), according to the manufacturer’s instructions. 3.2.3. LC-MS/MS protein identification Protein identification was done using shotgun LC–MS/MS and the Mascot search engine. Up to 20 µg of total protein from whole-body homogenates were double-digested in-solution using Trypsin/Lys-C mixture, followed by Trypsin digestion. Samples were first prepared for digestion in Microcon-10 kDa centrifugal filters according to the following steps: 1. Added 200 µL of LC-MS grade H2O to the filter and centrifuged at 13 500 g, 4°C for 10 min to rinse it, with ~30 µL of water remaining in the filter after the rinse; discarded the eluate from the outer vial; 2. Added a solution containing up to 20 µg of protein to the filter and topped up to 200 µL with 200 mM NH5CO3, centrifuged at 13 500 g, 4°C for 10 min; discarded the eluate from the outer vial; 3. Added 200 µL of 200 mM NH4CO3, centrifuged at 13 500 g, 4°C for 10 min; discarded the eluate from the outer vial; 4. Added 200 µL of 50 mM NH4CO3, then centrifuged at 13 500 g, 4°C for 10 min; discarded the eluate from the outer vial; Materials and Methods 30 5. Placed the filter upside-down in a new outer vial and centrifuged at 1000 g for 2 min to transfer the protein solution from the filter to the outer vial, then pipetted the solution into a 0.5 mL Lo-Bind Eppendorf tube. After the preparation steps, protein samples were digested according to the following protocol: 1. Added 1.5 µL of LC-MS MeOH to the protein sample for a final MeOH concentration of 5%; 2. Added 5 µL of 0.5% Rapigest and 2 µL of 200 mM DTT to the protein sample and incubated at 60°C for 30 min; 3. Cooled the sample to room temperature, then added 5 µL of 200 mM NH5CO3 and 2.5 µL of 200 mM iodoacetamide; 4. Incubated the sample in the dark for 30 min at room temperature; 5. Added 1 µL of stock Trypsin/Lys-C Mix (20 µg of the mixture in 80 µL of LC–MS grade H2O) to the sample and incubated at 37°C for 1 h; 6. Added trypsin in a 1:25 trypsin:protein ratio at 37°C for 1 h; 7. Terminated the digestion by adding 1.5 µL of formic acid for a final concentration of at least 2% v/v; 8. Completely dried the sample in a vacuum dryer at 50°C. After the digestion, samples were desalted and cleaned up using Pierce C 18 Spin Columns placed in 2 mL Lo-Bind Eppendorf tubes according to the following steps: 1. Added 200 µL of 50% MeOH to the column and centrifuged at 1500 rpm for 1 min. This step was repeated once more and the eluate discarded after; 2. Added 200 µL of 0.5% TFA, 5% ACN solution to the column and centrifuged at 1500 rpm for 1 min. This step was repeated once more and the eluate discarded after; 3. Added 200 µL of 0.1% TFA to the column and centrifuged at 1500 rpm for 1 min. This step was repeated once more and the eluate discarded after; 4. Dissolved the dried sample in 50 µL of 0.1% TFA and applied to the column, then centrifuged at 1500 rpm for 1 min; 5. Collected the eluate and reapplied to the column, then centrifuged at 1500 rpm for 1 min; 6. Added 100 µL of 0.1% TFA to the column and centrifuged at 1500 rpm for 1 min. This step was repeated once more; Materials and Methods 31 7. Placed the column was in a new 2 mL Lo-Bind Eppendorf tube; 8. Added 50 µL of 0.1% TFA, 70% ACN solution to the column and centrifuged at 1500 rpm for 1 min to elute the sample. This step was repeated once more; 9. Completely dried the sample in a vacuum dryer at 50°C and stored in at –20°C until analysis. Tryptic digests were subjected to nano-LC–MS/MS analysis using a Dionex Ultimate 3000 RSLC nanoLC system (Dionex, Sunnyvale, CA, USA) coupled to Bruker Maxis II ETD Q-TOF instrument (Bremen, Germany) via a CaptiveSpray nanoBooster ionization source. Peptides were separated online using Acquity M-Class BEH130 C18 analytical column (1.7 μm, 130 Å, 75 μm × 250 mm Waters, Milford, MA, USA) following trapping on an Acclaim PepMap 100 C-18 trap column (5 μm, 100 Å, 100 μm × 20 mm, Thermo Fisher Scientific, Waltham, MA, USA). The temperature was set at 48°C, and a flow rate of 300 nl/min was applied. The gradient method was from 4% B to 50% B in 90 min; solvent A was 0.1% formic acid in water, whilst solvent B was 0.1% formic acid in acetonitrile. Sample ionization was achieved in the positive electrospray ionization mode. Data-dependent analysis was performed using a fixed cycle time of 2.5 s. MS spectra were acquired over a mass range of 150–2200 m/z at 3 Hz, while CID was performed at 16 Hz for abundant precursors and at 4 Hz for ones of low abundance. Data were evaluated with ProteinScape 3.0 software (Bruker Daltonic GmbH, Bremen, Germany) using the Mascot search engine version 2.5.1 (Matrix Science, London, UK). Considering the limited availability of O. nubilalis protein sequences in published databases, MS/MS spectra were also searched against O. nubilalis, O. furnacalis (a species closely related to the ECB), as well as all lepidopteran protein sequences available in the NCBI database. The following parameters were applied: trypsin as enzyme, 7 ppm peptide mass tolerance, 0.05 Da fragment mass tolerance, and 2 missed cleavages. Carbamidomethylation on cysteines was set as a fixed modification, while deamidation (NQ) and oxidation (M) were set as variable modifications. 3.2.4. Bioinformatic analyses After protein identification, FASTA sequences for all identified proteins were downloaded from the NCBI database and used for the prediction of structural disorder. The structural disorder of proteins was determined with the IUPred long disorder predictor (https://iupred3.elte.hu/). The predictor is based on estimating the total pair-wise inter-residue interaction energy gained when a Materials and Methods 32 polypeptide chain folds. An amino acid is considered to be disordered if its IUPred score is at least 0.5. Mean disorder was computed as the average of residue scores, which range from 0.0 to 1.0. Overall disorder rate (percental disorder, ranging from 0 to 100%) represents the fraction of disordered amino acids in a polypeptide chain. Proteins are considered globular if their overall disorder rate is below 10%; nearly ordered if the rate is between 10% and 30%; partially disordered if the rate is between 30% and 70%; (mostly) disordered if the rate is above 70%. All proteins were further analyzed for the presence of long intrinsically disordered regions (long IDRs) — sequences of at least 20 consecutive disordered amino acids. Additionally, the amino acid composition of the proteins was analyzed to determine the absolute number of individual amino acids that make up each polypeptide, as well as their ratios. Lastly, functional characterization was performed on the identified sequences. Functional information on the identified proteins was collected from various databases such as UniProt (www.uniprot.org/, Consortium, 2021), Pfam (http://pfam.xfam.org/, Mistry et al., 2021), Interpro (https://www.ebi.ac.uk/interpro/, Blum et al., 2021), and GeneOntology (http://geneontology.org/, Ashburner et al., 2000). Data on their molecular functions, cellular localization, the biological processes they are involved in, and the do-mains they contained was collected. All of the analyses were performed using homemade PERL scripts run locally. Materials and Methods 33 3.3. Gene expression analysis of selected IDPs 3.3.1. Isolation of total RNA Total RNA was isolated from whole bodies of larvae from the Main experiment. Each of the five experimental groups was comprised of three biological pools and each pool contained three larvae. RNA isolation was performed using the TRIzol Reagent (Invitrogen, Waltham, MA, USA, cat. no. 15596026) and mortar and pestle. Microtubes (2 mL) containing 1.5 mL of TRIzol Reagent were prepared in advance. The mortar and pestle were placed at -20°C for chilling before sample homogenization. Larvae were placed into the mortar and liquid nitrogen was poured over to freeze them and turn them brittle. After the samples were pulverized into fine dust, they were transferred into the prepared microtubes for the next RNA isolation steps. The mortar and pestle were wiped with 70% EtOH between each sample. Next, the steps outlined in the manufacturer’s protocol for TRIzol Reagent were followed: 1. Centrifuged for 5 minutes at 12 000 g and 4°C, transfered the supernatants to new 2 mL microtubes due to the high fat content of the larvae; 2. Incubated for 5 minutes and added 0.5 mL of chloroform; 3. Gently inverted the microtubes for 3 minutes in order to mix the contents; 4. Centrifuged for 15 minutes at 12 000 g and 4°C to separate the phases; 5. Transferred the top, aqueous phase containing total RNA to new 1,5 mL microtubes; 6. Added 1 mL of ice cold isopropanol to precipitate the RNA; 7. Incubated for 10 minutes, then centrifuged for another 10 minutes at 16 000 g and 4°C; 8. Discarded the supernatant and resuspended the RNA pellet in 1.5 mL of 75% ethanol for salt cleanup; 9. Centrifuged for 10 minutes at 16 000 g and 4°C to precipitate the RNA again; 10. Discarded the supernatant and repeated steps 8 through 9 two more times; 11. Incubated the microtubes with purified RNA pellets for 10 minutes at 60°C in a TSC ThermoShaker (Biometra, Jena, Germany) to evaporate residual ethanol; 12. Added 40 µL of DEPC-treated H2O to the RNA pellets and incubated on the thermoshaker for 10 minutes at 60°C to improve the dissolving of RNA. Materials and Methods 34 3.3.2. RNA concentration and quality assessment Total RNA concentration and purity were assessed using the BioSpec-nano spectrophotometer (Shimadzu, Kyoto, Japan). Absorbance was measured at 230, 260 and 280 nm. The 260 nm wavelength absorbance corresponded to the concentration of the RNA, and the instrument calculated the results according to the following formula: RNA[µg µL]=A260 x R x F where: A260 – the absorbance measured at the 260 nm wavelength, R – sample dilution and F – 40, indicating 40 µg/µL of purified RNA when OD (optical density) equals 1. The 260/230 nm and 260/280 nm absorbance ratios indicated how pure the samples were from salts and proteins/phenol, respectively. To determine the integrity of the isolated RNA, samples were run on 1.5% agarose gel. The gel was prepared as follows:  Dissolved 1.5 g of agarose (VWR Peqlab, Erlangen, Germany, cat. no. 732-2788P) in 100 mL of tris-acetate-EDTA (TAE) buffer and heated until the solution cleared up;  Cooled the solution and added 3 µL of GelRed Nucleic Acid Stain (Biotium, Fremont, CA, USA, cat. no. #41003), swirling to mix thoroughly;  Poured the gel in the cast, placed the well comb and let the gel harden;  Removed the well comb and rinsed the gel with TAE buffer to remove excess stain;  Placed the gel in the electrophoresis tank and equilibrated in running buffer for 30 minutes. The samples were mixed with 10X Blue Juice Gel Loading Buffer (Thermo Fisher Scientific, Waltham, MA, USA, cat. no. 10816015) and loaded onto the gel (1 µL RNA, 1 µL loading buffer, 8 µL DEPC-treated H2O). Electrophoresis was run on a Biometra Compact M system (Biometra, Jena, Germany) at 90V for 45 minutes until the dye reached the halfway point on the gel. After the run, the gel was placed in the ChemiDoc XRS+ System (Bio-Rad, Hercules, CA, USA) to image 28S and 18S RNA bands and measure their intensities. The 28S/18S intensity ratio was used to Materials and Methods 35 calculate the RNA integrity number (RIN) which shows whether the isolated RNA was intact or degraded. 3.3.3. RT-PCR cDNA synthesis Complementary DNA for qPCR analysis was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA, cat. no. 4368814). The standard reaction mixture of 20 µL was up-scaled to 40 µL to produce a higher amount of cDNA. Complementary DNA was synthesized from 4 µg of total RNA from each sample. The reverse transcription process was carried out on an Eppendorf Mastercycler EP Gradient S (Eppendorf, Germany). Reaction mixtures were prepared as follows: Table 6. Composition of individual reaction mixtures for RT-PCR cDNA synthesis. Component Volume 10X RT Buffer 4.0 µL 25X dNTP Mix (100 mM) 1.6 µL 10X RT Random Primers 4.0 µL Multiscribe™ Reverse Transcriptase 2.0 µL DEPC-treated H2O 8.4 µL RNA Template (200 ng/µL) 20.0 µL Total per reaction 40.0 µL Next, the mixtures were pipetted into 200 µL PCR microtubes, vortexed briefly and centrifuged to spin down the contents. The prepared microtubes were placed in the thermal cycler and reverse transcription was carried out according to the following amplification steps: Table 7. Thermal cycler settings for RT-PCR cDNA synthesis. Step Settings Temperature (°C) Time (min) 1 25 10 2 37 120 3 85 5 4 4 Hold After reverse transcription, the synthesized cDNA was diluted 8 times with nuclease-free H2O, bringing the concentration to 12.5 ng/µL, and stored at –20°C until further analysis. 3.3.4. Primer design Genes to be analyzed were selected according to the intrinsic disorder content of proteins that are coded by them, as well as their functions. Actin and ribosomal protein s03 genes were selected as Materials and Methods 36 reference genes due to their stable expression levels. The other selected genes of interest cover different function groups, such as regulation of muscle contraction and cytoskeleton formation, immune response, signal transduction and protein metabolism. Due to the limited availability of O. nubilalis nucleotide sequences in online databases, most primers were designed using sequences belonging to the closely related O. furnacalis. Nucleotide sequences were collected from the NCBI database (National Centre for Biotechnology Information) and primers designed using the Primer-BLAST tool (Ye et al., 2012). During the design, in silico analysis was also performed to check whether the primers would recognize non-specific targets. The following sequences were available for O. nubilalis. Their short names and NCBI accession numbers are included: 1. actin (EL928709.1), 2. ribosomal protein s03, rps3 (EL929086.1), 3. heat shock protein 90, hsp90 (EL929806.1), 4. small heat shock protein 20.4, hsp20.4 (AB568467.1) and 5. small heat shock protein 20.1, hsp20.1 (AB568468.1). The following sequences were available for O. furnacalis. Their short names and NCBI accession numbers are included: 1. heat shock cognate 70 kDa protein, hsc70 (JF708084.1) 2. heat shock protein 68-like, hsp70 (XM_028309302.1), 3. troponin T, tnt (XM_028316365), 4. thymosin beta, thym (XM_028305412), 5. tropomyosin-2, tropmy2 (XM_028309660) and 6. moesin/ezrin/radixin homolog 1, moe (XM_028309570). Primer sequences can be found in Table 27 of Supplementary Materials. The primer pairs were ordered from and synthesized by Vivogen LLC (Belgrade, Serbia). Primers were delivered in lyophilized form and centrifuged for 30 seconds at 8000 rpm to spin down the contents. Stock solutions (100 µM) were prepared as follows:  Dissolved lyophilisates in 10 times the synthesized amount of primers (25 nmol) with DEPC-treated H2O;  Vortexed the microtubes to mix the contents; Materials and Methods 37  Spun down microtubes briefly and placed on thermoshaker for 10 minutes at 60°C;  Centrifuged for 30 seconds at 8000 rpm to spin down condensates. Working solutions (10 µM) were prepared by diluting the stock solutions 10 times with DEPCtreated H2O. 3.3.5. Primer specificity analysis The specificity of primer pairs derived from O. furnacalis sequences was additionally confirmed by non-quantitative PCR and running the products on an agarose gel. To minimize potential amplification differences that can exist between the samples and ensure good signal strength, aliquots of synthesized cDNA were taken from every sample, mixed together and used as the template for the amplification reactions. The same instrument that was used for cDNA synthesis, the Eppendorf Mastercycler EP Gradient S, was employed here as well. Single reaction mixtures were prepared with the 2X GoTaq Hot Start Green Master Mix (Promega, Madison, WI, USA, cat. no. M7422) as follows: Table 8. Composition of individual reaction mixtures for primer specificity analysis. Component Volume 2X Master Mix 7.0 µL F primer (10 µM) 0.7 µL R primer (10 µM) 0.7 µL DEPC-treated H2O 0.6 µL cDNA mixture (~12.5 ng/µL) 5.0 µL Total per reaction 14.0 µL Next, the mixtures were pipetted into 200 µL PCR microtubes, vortexed briefly and centrifuged to spin down the contents. The prepared microtubes were placed in the thermal cycler and PCR amplification was carried out according to the following steps: Table 9. Thermal cycler settings for primer specificity analysis. Step Settings Comments Temperature (°C) Time (min:sec) 1 95 10:00 Initial denaturation 2 95 00:30 Repeat steps 2–4 40 times 3 60 00:30 4 72 00:30 5 72 07:00 Final elongation Results 44 the D(–16) experimental group; the remaining 86 proteins were found in all five experimental groups (Fig. 6B). When accounting for duplicate entries between all groups in both experimental setups, there were a total of 892 unique proteins – 292 were identified only in groups from the Pilot setup, 284 were identified only in groups from the Main setup and 316 proteins were identified in groups from both experimental setups. The breakdown of the number of unique proteins present in each experimental group, as well as of common proteins in the two different experimental setups, is presented in Table 11 below: Table 11. Total, unique and shared proteins identified in groups of Pilot and Main experimental setups. Experimental setup Experimental group Total number of proteins Unique proteins Common proteins Pilot Dnca 506 294 212 Dca 314 102 Main ND 304 218 86 D(15) 228 142 D(5) 257 171 D(–3) 244 158 D(–16) 250 164 Dnca – non-cold-acclimated diapausing group; Dca – cold-acclimated diapausing group; ND – non-diapausing group; D(15) – diapausing group acclimated to 15°C for two weeks; D(5) – diapausing group acclimated to 5°C for two weeks; D(–3) – diapausing group acclimated to –3°C for two weeks; D(–16) – diapausing group acclimated to –16°C for two weeks. Results 45 4.1.3. Effect of sample heating on protein identification A comparison of total identified proteins was made between the heat-treated and untreated samples of all experimental groups in both setups. Heating the samples resulted in the identification of additional unique proteins in all groups, when compared with the non-heated samples. In the Pilot setup (Fig. 7A), an additional 180 unique proteins were uncovered in the Dnca group after heating the samples, while 265 heat-sensitive proteins were eliminated. The two sample types had 61 proteins in common. Within the Dca group, 96 proteins were found only in the heated sample, 184 in the non-heated samples, and 34 proteins were shared between the two sample types When it comes to the Main setup, sample heating lead to an unexpected outcome in the ND group (Fig. 7B). Almost the same number of unique proteins were identified in the non-heated and heated ND samples (138 and 129, respectively). The two sample types also had 37 proteins in common. The highest amount of proteins were identified in the non-heated samples of D(5) and D(–3) experimental groups – 165 and 151, respectively. With the exception of the ND group, the heated samples contained significantly less unique proteins, and their amounts distribution between the groups is similar. Figure 7. Effect of sample heating on the number of identified proteins in the (A) Pilot and (B) Main experimental setups. Unique Non-heated — proteins found only in the non-heated samples; Unique Heated — proteins found only in heated samples; Common — proteins that were found in both heated and nonheated samples (Dnca – non-cold-acclimated diapausing group; Dca – cold-acclimated diapausing group; ND – non-diapausing group; D(15) – diapausing group acclimated to 15°C for two weeks; D(5) – diapausing group acclimated to 5°C for two weeks; D(–3) – diapausing group acclimated to –3°C for two weeks; D(–16) – diapausing group acclimated to –16°C for two weeks). Results 46 The breakdown of the number of unique and common proteins per sample type and experimental group is presented in Table 12 below: Table 12. Total numbers of unique and shared proteins identified in different sample types of Pilot and Main experimental setup groups. Experimental setup Experimental group Unique Non-heated Unique Heated Common Pilot Dnca 265 180 61 Dca 184 96 34 Main ND 138 129 37 D(15) 144 61 23 D(5) 165 68 24 D(–3) 151 66 27 D(–16) 143 70 37 Unique Non-heated — proteins found only in the non-heated samples; Unique Heated — proteins found only in heated samples; Common — proteins that were found in both heated and non-heated samples (Dnca – non-cold-acclimated diapausing group; Dca – cold-acclimated diapausing group; ND – non-diapausing group; D(15) – diapausing group acclimated to 15°C for two weeks; D(5) – diapausing group acclimated to 5°C for two weeks; D(–3) – diapausing group acclimated to –3°C for two weeks; D(–16) – diapausing group acclimated to –16°C for two weeks). Results 47 4.2. Bioinformatic analyses 4.2.1. Determination of protein disorder In order to determine the extent of structural disorder present in proteins identified in this study, percental disorder was calculated for the total proteins from every individual experimental group. Proteins with an average percental disorder of 70% or higher were considered as mostly disordered (MDPs), with partially disordered proteins (PDPs) if the average percental disorder was between 30% and 70%, nearly ordered (NOPs) for values between 10% and 30%, and ordered (OPs) if the percental value was no higher than 10%. In the Dnca group of the Pilot setup, MDPs accounted for 31 of all identified proteins; 81 were PDPs, 75 were NOPs, and the remaining 319 were OPs. In the Dca group, 16 proteins were MDPs, 51 were PDPs, and 45 were NOPs; there were 198 OPs (Fig. 8A). Figure 8. Total number of proteins with varying degrees of intrinsic disorder in the Pilot (A) and Main (B) experimental setups. OPs – ordered proteins, 10% at most; NOPs – nearly ordered proteins, 10–30%; PDPs – partially disordered proteins, 30–70%; MDPs – mostly disordered proteins, at least 70% (Dnca – noncold-acclimated diapausing group; Dca – cold-acclimated diapausing group; ND – non-diapausing group; D(15) – diapausing group acclimated to 15°C for two weeks; D(5) – diapausing group acclimated to 5°C for two weeks; D(–3) – diapausing group acclimated to –3°C for two weeks; D(–16) – diapausing group acclimated to –16°C for two weeks). When it comes to the Main setup (Fig. 8B), the ND group had the most number of proteins identified across all four disorder content categories. In this group, OPs accounted for 194 hits, NOPs and PDPs were similar in number (43 and 48, respectively), while 19 proteins were MDPs. The individual diapausing groups generally differ in the amount of OPs that were identified in them. Results 48 Out of all those groups, the highest number of OPs were identified in the D(5) and D(–16) groups – 183 and 175, respectively. In terms of MDPs, PDPs and NOPs, similar numbers of these proteins were identified between the diapausing groups. Percental distribution of proteins with varying degrees of intrinsic disorder was also assessed in every group of the two experimental setups. Between the two groups of the Pilot setup, the percental distributions are almost identical – 63% OPs, 15% NOPs, 16% PDPs and 6% MDPs in the Dnca group versus 64% OPs, 14% NOPs, 16% PDPs and 5% MDPs in the Dca group (Fig. 9A). In the Main setup, the main difference is in the distribution of OPs. It is slightly lower in the ND group compared to the diapausing groups on average – 64% versus 69.8%, respectively, while the other protein groups are of similar distribution (Fig. 9B). Figure 9. Percental distribution of proteins with varying degrees of intrinsic disorder in the Pilot (A) and Main (B) experimental setups. OPs – ordered proteins, 10% at most; NOPs – nearly ordered proteins, 10– 30%; PDPs – partially disordered proteins, 30–70%; MDPs – mostly disordered proteins, at least 70% (Dnca – non-cold-acclimated diapausing group; Dca – cold-acclimated diapausing group; ND – nondiapausing group; D(15) – diapausing group acclimated to 15°C for two weeks; D(5) – diapausing group acclimated to 5°C for two weeks; D(–3) – diapausing group acclimated to –3°C for two weeks; D(–16) – diapausing group acclimated to –16°C for two weeks). Results 49 The breakdown of the number of proteins with varying degrees of intrinsic disorder in different experimental groups is presented in Table 13 below: Table 13. Total numbers of proteins with varying degrees of intrinsic disorder identified in different experimental groups of Pilot and Main setups. Experimental setup Experimental group OPs NOPs PDPs MDPs Pilot Dnca 319 75 81 31 Dca 202 45 51 16 Main ND 194 43 48 19 D(15) 158 29 31 10 D(5) 183 28 32 14 D(–3) 168 33 33 10 D(–16) 175 27 34 14 OPs – ordered proteins, 10% at most; NOPs – nearly ordered proteins, 10–30%; PDPs – partially disordered proteins, 30–70%; MDPs – mostly disordered proteins, at least 70% (Dnca – non-cold-acclimated diapausing group; Dca – cold-acclimated diapausing group; ND – non-diapausing group; D(15) – diapausing group acclimated to 15°C for two weeks; D(5) – diapausing group acclimated to 5°C for two weeks; D(–3) – diapausing group acclimated to –3°C for two weeks; D(–16) – diapausing group acclimated to –16°C for two weeks). Results 50 4.2.2. Effect of sample heating on protein disorder distribution The heat treatment had a profound effect on the distribution of proteins with various degrees of intrinsic disorder in the groups of both Pilot and Main experimental setups. The heat-treated samples contained more partially and mostly disordered proteins compared to the non-heated samples, while still retaining a significant portion of heat-resistant ordered proteins. 4.2.2.1. Pilot experimental setup When it comes to the Pilot setup (Fig. 10), OPs accounted for ~80% of all identified proteins in the non-heated samples, while PDPs and MDPs together made up for only ~7.6%. As for NOPs, on average they made up ~12% of all identified proteins in this sample type. After the heat treatment, OP content was reduced by around half, dropping to around 40% of all identified proteins, compared to ~80% in the non-heated samples. Proteins with higher disorder content (PDPs and MDPs), on the other hand, were significantly enriched by the heat treatment. Going by percental distribution, there were 4.4 times as many PDPs in the heated samples compared to the non-heated samples on average (from ~6.7% to ~30%). Figure 10. Effect of heat treatment on the percental distribution of proteins with varying degrees of intrinsic disorder in the two sample types of Pilot setup experimental groups. OPs – ordered proteins, 10% at most; NOPs – nearly ordered proteins, 10–30%; PDPs – partially disordered proteins, 30–70%; MDPs – mostly disordered proteins, at least 70% (Dnca – non-cold-acclimated diapausing group; Dca – cold-acclimated diapausing group). Results 51 When it comes to MDPs, they accounted for ~12.2% of all identified proteins in the heated samples on average, 13 times more than in the non-heated samples. The content of NOPs was also slightly enriched by heat treatment, going from ~12% in the non-heated samples to ~17% in the heated samples. The breakdown of the absolute values of the data shown in Figure 10 is presented in Table 14 below: Table 14. Total numbers of proteins with varying degrees of intrinsic disorder identified in the two sample types of Pilot setup experimental groups. Experimental group Sample type OPs NOPs PDPs MDPs Dnca Non-heated 263 37 23 3 Heated 97 43 70 31 Dca Non-heated 175 27 14 2 Heated 53 22 40 15 OPs – ordered proteins, 10% at most; NOPs – nearly ordered proteins, 10–30%; PDPs – partially disordered proteins, 30–70%; MDPs – mostly disordered proteins, at least 70% (Dnca – non-cold-acclimated diapausing group; Dca – cold-acclimated diapausing group). Results 52 4.2.2.2. Main experimental setup When it comes to the experimental groups of the Main setup, slightly more OPs were identified compared to the Pilot setup. On average, in the non-heated samples, OPs constituted ~82% of all proteins, compared to ~80% in the same sample type of the Pilot setup. On the other hand, the average content of NOPs, PDPs and MDPs in the Main setup groups is slightly lower in comparison to the Pilot setup. Heat treating these samples (Fig. 11) has led to a similar pattern of enrichment as with the Dnca and Dca groups. Going by percental distribution, the amount of OPs in the heated samples dropped by more than a third (from ~82% to ~49%), while the amounts of MDPs and PDPs went up ~10 and 4 times, respectively. The content of NOPs was also slightly enriched by heat treatment, going from ~11% in the non-heated samples to ~14% in the heated samples. Figure 11. Effect of heat treatment on the percental distribution of proteins with varying degrees of intrinsic disorder in the two sample types of Main setup experimental groups. OPs – ordered proteins, 10% at most; NOPs – nearly ordered proteins, 10–30%; PDPs – partially disordered proteins, 30–70%; MDPs – mostly disordered proteins, at least 70% (ND – non-diapausing group; D(15) – diapausing group acclimated to 15°C for two weeks; D(5) – diapausing group acclimated to 5°C for two weeks; D(–3) – diapausing group acclimated to –3°C for two weeks; D(–16) – diapausing group acclimated to –16°C for two weeks). Results 53 The breakdown of the absolute values of the data shown in Figure 11 is presented in Table 15 below: Table 15. Total numbers of proteins with varying degrees of intrinsic disorder identified in the two sample types of Main setup experimental groups. Experimental group Sample type OPs NOPs PDPs MDPs ND Non-heated 144 19 11 1 Heated 75 31 42 18 D(15) Non-heated 135 19 12 1 Heated 37 14 24 9 D(5) Non-heated 157 21 9 2 Heated 50 10 26 6 D(–3) Non-heated 142 22 12 2 Heated 45 14 25 9 D(–16) Non-heated 148 17 12 3 Heated 55 13 27 12 OPs – ordered proteins, 10% at most; NOPs – nearly ordered proteins, 10–30%; PDPs – partially disordered proteins, 30–70%; MDPs – mostly disordered proteins, at least 70% (ND – non-diapausing group; D(15) – diapausing group acclimated to 15°C for two weeks; D(5) – diapausing group acclimated to 5°C for two weeks; D(–3) – diapausing group acclimated to –3°C for two weeks; D(–16) – diapausing group acclimated to –16°C for two weeks). Results 60 4.2.5. Functional analysis of disordered proteins To gain an insight into the biological importance of IDPs in the cold adaptation process of the ECB, we performed a bioinformatic functional analysis of the identified disordered proteins using the data from online knowledgebases Uniprot, Pfam, Interpro, and Gene Ontology. Our results have revealed that only 458 of the proteins have a Uniprot entry and at least one data point from the other listed knowledgebases. Out of that number, 143 proteins are either mostly or partially disordered or contain at least 1 long IDR (Fig. 16, Total unique). Figure 16. Biological processes and molecular functions of intrinsically disordered proteins and proteins containing long IDRs. The category Other encompasses processes and functions that make up less than 4% of total hits each. Total unique – all uniquely identified proteins; Non-heated – all proteins identified in the non-heated sample types; Heated – all proteins identified in the heated sample types. The largest functional group (40 unique hits) is comprised of cytoskeleton structural components or proteins associated with them, for example regulators of muscle contraction or actin filament organization proteins. The second-largest group encompasses proteins functioning as molecular chaperones (32 unique hits), followed by proteins involved in protein and amino acid metabolism (18 unique hits). The rest of the proteins cover a wide range of biological processes and molecular functions, including translation (15 unique hits), nucleic acid binding (10 unique hits), chitin binding and formation of cuticle (7 unique hits), and others (Fig. 16, Total unique). Additionally, heat treatment of the samples increased the number of proteins that could be identified and functionally analyzed. Proteins that are involved in chitin binding and cuticle formation were found only in the Results 61 heated samples, as were the majority of nucleic acid binding proteins (10 total hits compared to 1 total hit). More proteins belonging to the Cytoskeleton category were also present in the heated samples (23 total hits) compared to the non-heated ones (17 total hits). More proteins that act as molecular chaperones, on the other hand, were present in the non-heated samples (22 total hits) than in the heated samples (14 total hits) (Fig. 16, Non-heated, Heated). When it comes to processes and functions grouped in the Other category (21 unique hit), electron transport chain (ETC) components and proteins involved in lipid metabolism are the most represented with 4 unique hits each (Fig. 17, Total unique). Additionally, ETC components were only identified in the heated samples, as were proteins involved in enzyme regulation, transcription, signaling regulation and TCA cycle. In fact, the majority of proteins from the Other category were identified after sample heating, in comparison to non-heated samples (15 and 8 total hits, respectively). Figure 17. Biological processes and molecular functions of intrinsically disordered proteins and proteins containing long IDRs in the Other category. Total unique – all uniquely identified proteins; Non-heated – all proteins identified in the non-heated sample types; Heated – all proteins identified in the heated sample types; ETC – electron transport chain; TCA – tricarboxylic acid. Due to the volume of data points, a summary of all proteins with intrinsic disorder that have been annotated is presented in a table that is available upon request from the author. The table includes protein names, accession numbers, calculated percental disorder, number of long IDRs and functions that were assigned to the proteins. Results 62 4.3. Relative gene expression analyses All relative gene expression analyses were performed on samples prepared as part of the Main experimental setup, with ND (non-diapause) considered as the control group. 4.3.1. Ct values of reference genes There were no statistically significant differences in the variances for the measured Ct values of the reference genes (Fig. 18). The Ct values of actin were higher than the Ct values of rps3, except in the ND experimental group where the values for rps3 were slightly higher than the values for actin. Figure 18. Ct values of reference genes – rps3 and actin. The results are presented as mean Ct values ± standard error of mean of 6 qPCR reactions per experimental group – 3 biological replicates per group, performed in 2 technical replicates each. One-way ANOVA and post hoc Tuckey test (significance level p<0.05) were used to statistically analyze the results. Results 63 4.3.2. Relative expression of hsp90 gene The results of relative gene expression for hsp90, a major molecular chaperone, not only differ between the experimental groups, but the differences in the diapausing groups are also conspicuously contrasted (Fig. 19). Figure 19. Relative expression of hsp90 gene. The results are expressed in ΔCt and presented as univariate scatterplots. Each dot represents one biological pool comprised of 3 larvae. The black bars represent the mean of ΔCt values for each group. Statistically significant differences in relative expression between groups are labeled with letters above the scatterplots. Compared to the non-diapause control and other diapausing groups which have been cold acclimated, the gene expression of hsp90 is severely down-regulated in the D(15) group which was in early diapause. As diapause and cold acclimation progressed in the D(5), D(–3) and D(–16) groups, the expression of the hsp90 gene increased to values higher than in the ND control. Statistically significant differences in relative expression are also presented in Table 18, while the means of calculated ΔCt values and standard errors of means are presented in Table 28 in the Supplementary Material. Results 64 Table 18. Relative expression of hsp90 gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 (1 – statistically significant; 0 – not statistically significant). ND D(15) D(5) D(–3) D(–16) ND 1 1 1 1 D(15) 1 1 1 D(5) 0 0 D(–3) 0 D(–16) Results 65 4.3.3. Relative expression of hsc70 gene The results of relative gene expression for hsc70, which is considered a constitutively expressed molecular chaperone, are contrasted between the non-diapause control and the diapausing, cold acclimated groups. (Fig. 20). Figure 20. Relative expression of hsc70 gene. The results are expressed in ΔCt and presented as univariate scatterplots. Each dot represents one biological pool comprised of 3 larvae. The black bars represent the mean of ΔCt values for each group. Statistically significant differences in relative expression between groups are labeled with letters above the scatterplots. As with the previous two heat shock protein genes, the relative gene expression of hsc70 is also upregulated in the diapausing groups when compared to the ND control. While the differences in expression between the individual diapausing groups were not statistically significant, the absolute calculated values of ΔCt were higher in the two diapausing groups that were acclimated to temperatures below 0°C (D(–3) and D(–16)), in comparison to the other two diapausing groups. Statistically significant differences in relative expression are also presented in Table 19, while the means of calculated ΔCt values and standard errors of means are presented in Table 28 in the Supplementary Material. Results 66 Table 19. Relative expression of hsc70 gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 (1 – statistically significant; 0 – not statistically significant). ND D(15) D(5) D(–3) D(–16) ND 1 1 1 1 D(15) 0 0 0 D(5) 0 0 D(–3) 0 D(–16) Results 67 4.3.4. Relative expression of hsp70 gene The results of relative gene expression for the inducible molecular chaperone hsp70 differ between the experimental groups (Fig. 21). When compared with the ND group, hsp70 expression steadily increases with the cold acclimation treatment and progression of diapause. Figure 21. Relative expression of hsp70 gene. The results are expressed in ΔCt and presented as univariate scatterplots. Each dot represents one biological pool comprised of 3 larvae. The black bars represent the mean of ΔCt values for each group. Statistically significant differences in relative expression between groups are labeled with letters above the scatterplots. Relative expression of the hsp70 gene begins significantly increasing already in the D(15) group, when compared with the ND control, and reaches the highest recorded values in the D(–16) group. Although differences in expression between groups D(–3) and D(–16) were not statistically significant, the absolute values were higher in the latter group. Statistically significant differences in relative expression are also presented in Table 20, while the means of calculated ΔCt values and standard errors of means are presented in Table 28 in the Supplementary Material. Results 68 Table 20. Relative expression of hsp70 gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 (1 – statistically significant; 0 – not statistically significant) ND D(15) D(5) D(–3) D(–16) ND 1 1 1 1 D(15) 1 1 1 D(5) 1 1 D(–3) 0 D(–16) Results 69 4.3.5. Relative expression of hsp20.4 gene The results of relative gene expression for the small, ATP-independent chaperone hsp20.4 are similar to the pattern of hsp70 expression, and they differ between the experimental groups (Fig. 22). Figure 22. Relative expression of hsp20.4 gene. The results are expressed in ΔCt and presented as univariate scatterplots. Each dot represents one biological pool comprised of 3 larvae. The black bars represent the mean of ΔCt values for each group. Statistically significant differences in relative expression between groups are labeled with letters above the scatterplots. The lowest relative expression was recorded in the ND control, and it steadily increased as diapause and cold acclimation progressed. Expression levels peaked in the D(–3) group, before dropping in the D(–16) group which was acclimated at the lowest temperature and spent the longest time in diapause of all experimental groups. Statistically significant differences in relative expression are also presented in Table 21, while the means of calculated ΔCt values and standard errors of means are presented in Table 28 in the Supplementary Material. Results 76 Table 24. Relative expression of tnt gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 (1 – statistically significant; 0 – not statistically significant). ND D(15) D(5) D(–3) D(–16) ND 0 1 1 1 D(15) 1 1 1 D(5) 0 1 D(–3) 1 D(–16) Results 77 4.3.9. Relative expression of thym gene Thymosin beta is a polypeptide involved insect humoral response to microbial infections. The results of relative gene expression for thym differ between the ND control on one side and the diapausing experimental groups on the other (Fig. 26). Figure 26. Relative expression of thym gene. The results are expressed in ΔCt and presented as univariate scatterplots. Each dot represents one biological pool comprised of 3 larvae. The black bars represent the mean of ΔCt values for each group. Statistically significant differences in relative expression between groups are labeled with letters above the scatterplots. Expression of thym was significantly down-regulated in all diapausing and cold acclimated groups in comparison to the non-diapausing control. When it comes to the individual diapausing groups, expression levels were mostly similar with the exception of the D(5) group, where the lowest expression of thym gene was recorded. Statistically significant differences in relative expression are also presented in Table 25, while the means of calculated ΔCt values and standard errors of means are presented in Table 28 in the Supplementary Material. Results 78 Table 25. Relative expression of thym gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 (1 – statistically significant; 0 – not statistically significant). ND D(15) D(5) D(–3) D(–16) ND 1 1 1 1 D(15) 1 0 0 D(5) 0 1 D(–3) 0 D(–16) Results 79 4.3.10. Relative expression of moe gene Moesin is a cytoskeletal protein involved in many regulatory processes. The results of relative gene expression for moe differ between the experimental groups (Fig. 27). When compared with the ND control, expression is down-regulated during most of diapause when temperatures are above or around 0°C. Figure 27. Relative expression of moe gene. The results are expressed in ΔCt and presented as univariate scatterplots. Each dot represents one biological pool comprised of 3 larvae. The black bars represent the mean of ΔCt values for each group. Statistically significant differences in relative expression between groups are labeled with letters above the scatterplots. Looking at the diapausing groups, expression of moe is lowest in early diapause (D(15) group) and steadily increases as diapause and acclimation to temperatures around and below 0°C progress. By late diapause and at lowest temperatures (D(–16) group) expression returns to levels recorded in the non-diapause control. Statistically significant differences in relative expression are also presented in Table 26, while the means of calculated ΔCt values and standard errors of means are presented in Table 28 in the Supplementary Material. Results 80 Table 26. Relative expression of moe gene in whole-body O. nubilalis larvae – one-way ANOVA and post hoc Tuckey test for significance level p<0.05 (1 – statistically significant; 0 – not statistically significant). ND D(15) D(5) D(–3) D(–16) ND 1 1 0 0 D(15) 0 1 1 D(5) 0 1 D(–3) 1 D(–16) 5. Discussion Discussion 82 5.1. Proteomic analyses Proteomic analyses of intrinsically disordered proteins/protein regions and ID-containing proteins, based on in vitro methodologies and techniques, are fraught with obstacles. The difficulties stem from the fundamental structural properties of these biomolecules. Being intrinsically disordered, these proteins are usually not able to crystallize. Consequently, they are not amenable for structural analyses using the typical methods for resolving protein structure, such as X-ray crystallography (Piovesan et al., 2017). High content of protein disorder is also prohibitive when it comes to structure determination employing cryo-EM. In addition, cryo-EM is quite restrictive when it comes to the size of analytes, which limits its application in determining the structure of individual proteins (Bari and Prakashchand, 2021; Avramov et al., 2022). As such, proteins containing intrinsic disorder are far less studied than proteins that are globular and possess a well-ordered structure (Siemer, 2020). 5.1.1. Enrichment of IDP content and enrichment validation However, the same fundamental physicochemical properties, which present obstacles in the study of IDP structures, can also provide unique advantages in other aspects of proteomic research. As has been stipulated, IDPs can behave vastly different than ordered, globular proteins in environments where conditions are not at physiological levels. Instead of denaturing or, at the very least, losing some level of biological activity, IDPs obtain transient ordered structures under these conditions. Additionally, under such circumstances, that is what allows them to perform their various functions (Uversky, 2009).These unique properties of IDPs can be employed in several ways in order to enrich samples to be analyzed with proteins containing various degrees of intrinsic disorder. One such approach entails treating samples with acidic precipitating agents, e.g. perchloric or trichloracetic acid (PCA and TCA, respectively). Applying either of these chemicals leads to the denaturing and precipitation of globular proteins in the samples, which are then subsequently removed by centrifugation (Cortese et al., 2005; Romero-Pérez et al., 2023). The remaining content is in this way enriched in intrinsically disordered proteins, although some globular proteins are quite stable even at critically low pH and therefore are still present in the sample after acid treatment. Next, the enriching effects of this treatment can be explored and validated by 2D PAGE methods, which offer considerable resolving power. Discussion 83 Apart from treating the samples with precipitating agents, the content of intrinsically disordered proteins can be enriched by exposing the samples to boiling temperatures (Galea et al., 2006, 2009; Zhang et al., 2018). Again owing to their unique physicochemical properties, in particular low mean hydrophobicity and high net charge, IDPs exhibit resistance to heat denaturation and low thermal aggregation. Instead of denaturing, proteins containing intrinsic disorder can adopt transient, higher orders of structure while the high temperature conditions persist (Uversky, 2009). As with the acid treatment, globular proteins will denature and can be removed from the samples with subsequent centrifugation. What will remain in the samples are heat-stable intrinsically disordered proteins. For the purposes of the studies performed in this dissertation, heat sampling was chosen as the preferred method for enriching the samples with intrinsically disordered proteins. The method is simple, straightforward and provides adequate reduction of the content of globular proteins, which in turn uncovers proteins that would have been masked by their ordered counterparts (Galea et al., 2006; Zhang et al., 2018; Avramov et al., 2022). Acid treatment, on the other hand, requires taking into consideration that different precipitating agents and their concentrations lead to different proteomic profiles that can be detected after the treatment (Cortese et al., 2005). Following enrichment, its effects were validated using a particular, modified 2D-PAGE method. This in-house developed method (Csizmók et al., 2006; Tantos and Tompa, 2012) relies on the use of 8M urea to further filter out globular proteins from the samples that are being analyzed. Urea is used to solubilize the proteins after separation in the first dimension, and also as a component of the resolving gel for the second dimension separation. Intrinsically disordered proteins are unaffected by the urea during electrophoresis and align in a distinct diagonal pattern in the second dimension. The movement of globular proteins is impeded by the urea, and they typically end up located above the aforementioned diagonal. Looking at Figures 5A and 5B, it becomes sufficiently apparent why enrichment is the necessary first step in proteomic analyses of intrinsically disordered proteins. Non-heated sample types are abundant in globular, ordered proteins which impeded the separation in the first dimension, as well as transfer into the second dimension gel (Fig. 5A). The likely culprits are high molecular weight arylphorins – storage proteins that are present in large amounts in the haemolymph of insects such as the ECB (Taški et al., 2004). Once the offending proteins were removed by heating the samples up, separation in both the first and second dimension was more successful (Fig. 5B). In the second dimension, it can be seen that Discussion 84 several groups of proteins are much more visible and are aligned along the diagonal. Their positioning, as well as presence after the enrichment procedure, indicate that they are either partially or mostly disordered (Csizmók et al., 2006; Tantos and Tompa, 2012). Some globular proteins can still be noticed, as they appear in several spots above the diagonal. As it can be the case with acid treatment, where some globular proteins exhibit structural stability even at acidic pH levels, so can some globular proteins show they are thermostable and remain in the samples after heat treatment. 5.1.2. Identification of proteins by LC-MS/MS Protein identification in non-heated and heated samples from both experimental setups was performed by matching the MS/MS spectra of detected peptides against the protein sequences that are available in the NCBI database not only for the species in question, O. nubilalis, but also for the Asian corn borer (ACB, Ostrinia. furnacalis) and all other lepidopteran species. The decision to expand the search paramateres to also include these other insects was made due to the limited number of ECB protein sequences available in the aforementioned database (~1600 entries) in comparison to, for example, the ACB (around 28 500 entries). The European corn borer has not been a subject of proteomics-level studies (Purać et al., 2016), and as such its protein entries are relatively few in number when compared with other species. Furthermore, the ACB was selected for protein identification as it is a close relative of the ECB. There is recorded gene flow between the two moth species, and they produce viable hybrid offspring after interspecies mating (Domingue et al., 2008; Li and Yang, 2022). A total of 2103 proteins were identified in the entire study. The majority of the proteins, as was to be expected due to the limited availability of ECB sequences, were identified because the search parameters were expanded to include other lepidopteran species. Despite this, sequence similarity was at such a level that the peptides acquired from protein digestion of ECB homogenates could be matched with existing sequences from these other species with high confidence. The total takes into account that identical proteins could be identified across the different experimental groups and sample types. When the duplicates are addressed, the number of unique proteins that have been identified in this study comes to 892. Of that number, 361 proteins were common for the two experimental setups, while 292 and 284 were unique to the Pilot and Main setups, respectively. Discussion 85 When looking at the breakdown of the proteins identified in every group, it can be noticed that the numbers vary conspicuously. In the Pilot setup, nearly three times as many proteins were uniquely identified in the Dnca group compared to the Dca group – 294 and 102 proteins, respectively. The remaining proteins, 212 of them, were found in both of these groups (Fig. 6A). In the Main setup, on the other hand, more unique proteins were identified in the ND control group (218) than in any of the diapausing, cold-acclimated groups (142, 171, 158 and 164, respectively). These five experimental groups all shared 86 proteins between them (Fig. 6B). These discrepancies in both setups highlight the effects and merits of the experimental designs – cold acclimation and diapause/resting state, on the proteome of the sampled insects (Grubor-Lajšić et al., 1991; Hahn and Denlinger, 2011; Kojić et al., 2018; Uzelac et al., 2020; Popović et al., 2021; Avramov et al., 2022) in increasing the number of unique proteins that can be identified in the study. Lastly, an unintentional finding was made during this process, in regards to the sequences that were recognized in the NCBI database by peptide matching. In both the Pilot and Main setups, nearly half of the identified proteins (47% and 45%, respectively) come from entries that have only been predicted from available nucleotide sequences. It is important to highlight these results, as the findings in this study lend experimental validation for the protein sequence prediction endeavors. 5.1.3. Effect of sample heating on protein identification Protein identification was also largely affected by whether the samples were heat-treated or not. As can be expected, fewer proteins were identified in non-heated samples. On average, non-heated samples contained at least double the amount of unique proteins than their heated counterparts (Fig. 7, Table 12). The only exception to this pattern is the ND control group of the Main experimental setup (Fig. 7B). Surprisingly, the two sample types of this experimental group contained a nearly identical number of unique proteins – 165 in the non-heated samples and 151 in the heated samples. Apart from this exception, the non-heated samples of all other experimental groups had, on average, 30–50% more unique proteins than their heated counterparts. Another way to look at these results is that heating the samples allowed for additional unique proteins to be identified. There are several reasons why these proteins would be detectable only after sample heating. In the non-heated samples, their presence could potentially be masked by the abundant and overrepresented proteins, preventing their signal from being detected by LC-MS/MS identification. Some proteins are embedded as components of multi-subunit complexes, which also Discussion 92 (Theillet et al., 2013). In many scaffold proteins for example, which contain a high amount of intrinsic disorder, PPII helixes are abundant and through them scaffold proteins can recognize and interact with different binding partners (Dosztányi et al., 2006; Cortese et al., 2008). Thus, proline is important in protein intrinsic disorder not only to ensure that proteins remain unstructured and primed for interaction events, but to enable and facilitate those interactions as well. 5.2.4.2. Compositional bias Compositional analyses such as these are usually performed in the context of specific proteins of interest. However, there are indications that the amino acid composition of intrinsically disordered proteins could have species-specific bias. In their study of IDPs in the green algae Chlamydomonas reinhardtii, Zhang and coauthors (2018) have reported that IDPs that were predicted for this species have a biased amino acid composition when compared to “classical” IDPs available in the DisProt database. In particular, the green algae IDPs were depleted in disorder-promoting residues such as glutamic acid, lysine, proline, glutamine and aspartic acid. They have also compared the amino acid composition of experimentally validated IDPs with available green algae proteome data and have found that the IDPs were depleted in serine, threonine, asparagine and leucine residues. Conversely, the content of these amino acids in PDPs and MDPs from our study is generally similar to that of NOPs and OPs (Fig. 15). Taken altogether, these findings could point to the existence of a species-specific compositional bias, however strengthening that claim would require for more studies to take this approach in analyzing intrinsically disordered proteins. 5.2.5. Functional analysis of disordered proteins What immediately stands out, after the functional analysis of proteins identified in this study, is how significant the gap in functional annotation still is, for ordered and disordered proteins alike. Out of the 892 unique proteins identified here, barely 50% of them are listed in Uniprot and annotated with data from at least one other major annotation databases, such as e.g. Pfam. When the focus is narrowed down to just the disorder-containing proteins, the gap further increases as only 143 such proteins could be covered by this analysis. Low annotation efforts are likely further exacerbated by the fact that lepidopteran insects do not have many representative model species, apart from the silkworm Bombyx mori (International Silkworm Genome Consortium, 2008). As such, these findings could serve to motivate and direct future efforts to lessen the gap in functional knowledge of both ordered and disordered proteins in insects. Discussion 93 Here it was shown that the identified proteins perform a wide array of molecular functions and are involved in very diverse biological processes. The vast majority of them (40) are linked with the cytoskeletal network, either as its structural constituents or regulators of muscle fiber contraction. Following are proteins acting as molecular chaperones (32) that either assist with the proper folding of nascent proteins or handle misfolded ones. The last two major groups of proteins are involved in protein and amino acid metabolism (18), regulation of translational processes such as elongation, or they are structural components of ribosomes (15). The rest of the proteins have a plethora of functions and cover various metabolic processes, such as the metabolism of carbohydrates and lipids, insect cuticle formation, binding of nucleic acids, as well as oxidoreductive processes in the electron transport chain (Figs. 16 and 17). Heating the samples also allowed for novel functions and processes to be identified. Proteins that are involved in the formation of insect cuticle were found exclusively in these samples. Similarly the content of proteins that are a part of the cytoskeletal network or are involved in binding nucleic acids was also enriched following sample heating (Fig. 16, Heated). Lastly, sample heating allowed for many of the proteins in the Other category to be uncovered. The number of identified proteins almost doubled after the treatment (Fig. 17). As with the previous proteomic and bioinformatics analyses, these findings again highlight the importance of heat treating the samples in order to more completely identify and functionally analyze intrinsically disordered proteins. Discussion 94 5.3. Ecophysiological aspects of proteins with intrinsic disorder in cold hardiness As has been mentioned in the Introduction, proteins containing intrinsic disorder possess some unique physicochemical properties that are distinct from those found in ordered, globular proteins. Due to these properties, which stem from the protein’s amino acid composition, IDPs and ID-containing proteins are more resilient to adverse environmental factors than ordered proteins. The intrinsic disorder that is present in their structures allows them to either retain their activity and functionality under such conditions, or to undergo disorder-to-order, or vice-versa, transitions which, again, have the same end result of keeping the proteins functional. Because of that, ID-containing proteins are likely majorly involved in cold adaptation processes which allow organisms to survive when outside temperatures fall even well below the freezing point of water. The European corn borer, O. nubilalis, was selected as a model system to explore this aspect of intrinsic disorder in proteins, as this species has been frequently used in studies pertaining to adaptations that allow organisms to survive under hypometabolic and harsh environmental conditions, such as those caused by temperatures close to or below 0°C. This approach aims to further both the functional knowledge on intrinsically disordered proteins, as well as help elucidate the molecular mechanisms that govern stress adaptation processes in a cold-adapted insect species. 5.3.1. IDP content in O. nubilalis cold hardiness Organisms undergo various biochemical, physiological and molecular changes as part of their adaptive processes to perturbations in the environment. These adaptations can often also lead to changes in the content of an organism’s proteome. Considering the diversity of functions that ID-containing proteins possess, it was prudent to determine whether the content of these biomolecules is affected by cold adaptation processes, and to what extent. This issue was approached from two directions, mirroring how disorder content was evaluated for the proteins that were identified in this study. Potential changes in IDP content due to cold acclimation were first assessed from the point of percental intrinsic disorder, i.e. the content of MDPs, PDPs, NOPs and OPs in the different experimental groups. In both the Pilot and Main experimental setups it can be seen that diapause and cold acclimation had a negative effect on the number of proteins that could be detected and identified (Fig. 6). As such, the amount of proteins with considerable Discussion 95 percental disorder (MDPs and PDPs) was also lower in the cold-acclimated groups (Fig. 8). That said, the percental distribution of the four different protein categories – MDPs, PDPs, NOPs and OPs, relative to the total number of proteins, was actually fairly similar between the experimental groups (Fig. 9). Therefore, the content of IDPs, at least according to the percental disorder criteria, did not seem to be affected by cold acclimation. However, protein intrinsic disorder in this study was also assessed by determining how many proteins (regardless of percental disorder) contain long intrinsically disordered regions, which have a higher potential for biological relevance. When looking at the distribution of proteins containing long IDRs in the different experimental groups, the effects of diapause and cold acclimation on IDP content are more evident and pronounced (Figs. 12 and 13). Most notably, the percentage of NOPs that contain at least one long IDR was increased significantly in the diapausing, cold-acclimated groups, at the expense of NOPs without long IDRs. With the exception of OPs, the percentage of proteins containing long IDRs was higher in the aforementioned experimental groups, especially when comparing the groups from the Main setup to the non-diapausing control (Fig. 13A-E). This could be particularly significant for NOPs, which are close to being considered ordered. Intrinsic disorder generally increases the flexibility of polypeptide chains, which is a favorable trait for protein functionality in cold environments (Siddiqui et al., 2006). Possessing at least one long IDR would make NOPs less rigid, allowing them to retain more of their molecular mobility and preserve their functionality at low temperatures. As such, the cold adaptation processes in the ECB seem to affect the IDP content more in a qualitative manner, rather than quantitative. These findings are in line with the notion that environmental pressure can affect proteome composition in regards to intrinsically disordered proteins. In a large study covering 46 fully sequenced prokaryotes that inhabit different mesophilic and extreme habitats, it was shown that there is a correlation between the extremity of the environment and protein intrinsic disorder measured in the percentage of proteins containing long IDRs (Vicedo et al., 2015). Additionally, an interesting observation was made that non-related species sharing the same type of habitat have similar protein disorder content, as opposed to species that are related but inhabit drastically different environments (mesophiles versus extremophiles). Higher than average disorder content was predicted for non-related species of archaea that thrive in habitats saturated with salts (Halobacterium sp. NRC-1 and Haloarcula marismortui ATCC 43049). On the other hand, higher disorder content was predicted for the halophilic Marinobacter aquaeolei VT8 bacterium Discussion 96 compared to its mesophilic taxonomic relative Pseudoalteromonas atlantica T6c. When it comes to hot and cold environments, it was found that thermophiles and psychrophiles actually have reduced protein disorder content compared to mesophilic species, which seems to be an adaptive trait of prokaryotes (Burra et al., 2010; Vicedo et al., 2015). While those findings seem in contrast to the results obtained for IDP content in O. nubilalis in this study, analysis of homologous proteins from two opposing extremophiles – the psychrophilic Colwellia psychrerythraea 34H and the hyperthermophilic Pyrococcus horikoshii OT3, has shown that more homologs from Colwellia contain long IDRs compared to the ones from Pyrococcus (Vicedo et al., 2015). This reinforces the hypothesis that increased protein flexibility, by means of higher content of long IDRs, is an adaptation necessary for them to function in cold environments. Similar studies on the relationship between protein intrinsic disorder and environmental temperatures in eukaryotes, however, are lacking. In the plant model species Arabidopsis thaliana, heat stress lead to the expression of heatinduced proteins that were enriched in electrostatically charged amino acids, while being depleted in hydrophobic and polar residues. Additionally, they were also enriched in long IDRs (AlvarezPonce, 2018). Such amino acid and long IDR compositions are in contrast to proteins found in thermophilic bacteria and archaea, which can point to different strategies for temperature adaptations on the protein level between eukaryotes and prokaryotes. When it comes to animal studies, the influence of different habitat temperature regimes on cold adaptation was explored in two closely related zoarcid fish species. The eurythermal Zoarces viviparus thrives at 15°C, but can experience and survive temperatures as low as 0°C due to seasonal fluctuations (Pörtner and Knust, 2007). The stenothermal Pachycara brachycephalum, on the other hand, is adapted to living at a constant 0°C (Brodte et al., 2006). Amino acid compositional analysis of orthologous sequences from these two species has shown that there is a pattern of distinct amino acid substitutions in the cold-adapted Pachycara brachycephalum. In particular, the frequency of acidic residues, such as glutamic acid, was reduced, while the frequencies of basic amino acids was elevated. A net result of these substitutions is a reduction in the number of salt bridges that can be formed, which would lead to increased flexibility of the polypeptide chain, and better interactions with solvents on the protein surface (Windisch et al., 2012). While this study did not explore the mechanisms of cold adaptation on the level of intrinsically disordered proteins, it has demonstrated more evidence for the protein flexibility hypothesis under cold environmental conditions. Discussion 97 5.3.2. Gene expression analyses of disorder-containing proteins In order to determine if and how the results of proteomic analyses correlate with changes on the transcriptional level, relative expression of genes encoding different ID-containing proteins that are implicated in diapause and cold adaptation processes was explored. In total, 9 genes were analyzed – five encoding HSPs (hsp90, hsc70, hsp70, hsp20.4 and hsp20.1), two encoding structural proteins (tropmy2 and tnt), one encoding a protein involved in insect immune response (thym) and one encoding a member of the ERM (ezrin, radixin and moesin) family of proteins (moe). All of the proteins encoded by the genes analyzed here were determined to contain at least 10% intrinsic disorder and/or at least one long intrinsically disordered protein region. Structural proteins, small HSPs and thymosin beta were over 30% disordered and considered PDPs (disorder percentage between 30% and 70%). However, HSP20.1 was the only one without long IDRs. It should be noted that, due to its high ID content, it is likely that this small heat shock protein possesses a segment of consecutive disordered residues that is just shy of the 20 amino acid threshold to be regarded as a long IDR. Additionally, HSP20.4 was determined to be 87% disordered, however only a partial protein sequence was available for this analysis. It is likely that this disorder percentage is inflated because of the missing residues and should be closer to that of HSP20.1 (~46%). Lastly, the three larger HSPs (HSP90, HSC70 and HSP70) were determined to be NOPs (disorder percentage between 10% and 30%) that contain at least one long IDR. HSP70 contained the most disorder out of the three, with 21% disorder percentage and two long IDRs. 5.2.4.1. Heat shock protein genes Molecular chaperones known as heat shock proteins play important roles in the homeostasis of biological systems. They are responsible for ensuring that newly synthesized proteins properly fold into their mature conformations, as well as handling abnormal proteins either by correcting their misfolded structures or directing them towards protein degradation and recycling pathways (MacRae, 2010; King and MacRae, 2015; Storey and Storey, 2022). HSPs also transport nascent proteins across organelle membranes to their subcellular destinations, where they spontaneously fold into their correct conformations (Saibil, 2013). Under physiological conditions, HSPs are moderately expressed because of the said role in protein maturation. However, their expression and accumulation increase when an organism is faced with adverse environmental conditions, such as high or low temperatures, exposure to UV radiation, pressure changes, lack of oxygen or water, Discussion 98 and presence of pollutants. Heat shock proteins alleviate the damaging effects of these abiotic stressors by preserving the structure and function of all manner of affected proteins (Hochachka and Somero, 2002; Kregel, 2002; King and MacRae, 2015). All analyzed HSP genes, excluding hsp20.1, have shown a similar pattern of expression in this study. Their relative expression levels were much higher in cold-acclimated diapausing groups when compared to the non-diapausing control group, while for hsp20.1 the opposite was the case. Additionally, it can be seen that the expression of all these genes gradually increased in the diapausing groups as diapause progressed and the larvae were exposed to increasingly colder temperatures. The only exception is the hsc70 gene, which exhibited equally high expression in all diapausing groups regardless of acclimation temperature. hsp90 Relative expression of the hsp90 gene was markedly high in all experimental groups, with the exception of the D(15) group where it was heavily downregulated. Additionally, expression of hsp90 was higher in the cold-acclimated groups in comparison to the non-diapausing control, and it rose as larvae were exposed to gradually colder temperatures and diapause progressed (Fig. 19). These results are in accordance with previous studies that have analyzed the expression of hsp90 in insects that undergo developmental arrest and/or experience prolonged periods of cold environmental temperatures as part of their life cycle. In several leafminers of the Liriomyza genus, exposure to the cold induces the expression of hsp90 (Huang and Kang, 2007; Huang et al., 2009). It can also be induced during the resting state of diapause, as was detected in pupae of the onion fly Delia antiqua (Chen et al., 2005), as well as larvae of the rice borer Chilo suppresalis. Interestingly, the high expression of hsp90 in diapausing rice borer larvae was not further induced by exposure to cold temperatures, as opposed to the non-diapausing larvae (Sonoda et al., 2006). On the other hand, cold shock upregulated hsp90 expression in both diapausing and nondiapausing pupae of the flesh fly Sarcophaga crassipalpis (Rinehart and Denlinger, 2000). Additionally, in the flesh fly pupae, as well as those of Helicoverpa zea, it was detected that hsp90 expression increases towards the end of diapause and during post-diapausal development (Rinehart and Denlinger, 2000; Zhang and Denlinger, 2010). HSP90 activity is modulated and directed by interactions with a great number of co-chaperones. These interactions enable HSP90 to be involved in many molecular processes, from protein folding Discussion 99 and maturation, modification of transcription factors and various kinases, to regulation of cell communication. HSP90 also differs from other HSPs as it tends to bind and interact with proteins that are already partially folded and help them overcome their intrinsic instability in order to fold into an active conformation, even taking over substrates from HSP70 to do so (MacRae, 2010; Röhl et al., 2013). Taking all of this into consideration, increased hsp90 expression during diapause and cold acclimation in the ECB is to be expected in order to ensure that the aforementioned processes continue unimpeded in an unfavorable metabolic landscape caused by hypometabolism and exposure to cold temperatures. High expression towards the end of diapause could also be necessary to prepare the larvae for resumption of development and intense protein synthesis that will follow. hsc70 and hsp70 Similarly to hsp90, expression of both the constitutive hsc70 and the inducible hsp70 genes was higher in the cold-acclimated diapausing larvae when compared to the non-diapausing control. The expression of hsp70 gradually increased as diapause progressed and temperatures dropped, while hsc70 expression remained at a similar level regardless of diapause status and acclimation temperatures (Figs. 20 and 21). These findings on the expression of the constitutive hsc70 are in accordance with previous similar studies done on the ECB (Popović 2014; Popović et al., 2015). Considering that it does not seem to be affected by acclimation temperatures, increased hsc70 expression could be reflective of the metabolic changes that occur in diapausing larvae that differentiate them from their non-diapausing counterparts. Looking at other insect species, there does not seem to be a conclusive pattern to hsc70 expression. During the adult diapause of the cold-hardy northern malt fly Drosophila montana expression of hsc70 is downregulated (Kankare et al., 2010). Conversely, increase in hsc70 expression was detected over the timecourse of diapause in larvae of the corn stalk borer Sesamia nonagrioides (Gkouvitsas et al., 2009), as well as in the second half of larval diapause in the bamboo borer Omphisa fuscidentalis (Tungjitwitayakul et al., 2008). Cold stress amplified the expression of hsc70 in the fat body of non-diapausing silkworm larvae (Fang et al., 2021), while downregulating it in non-diapausing adults of the European firebug Pyrrhocoris apterus (Koštál and Tollarová-Borovanska, 2009). Expression of hsp70, on the other hand, showed more consistent responsiveness to abiotic stressors, befitting its inducible nature. In the aforementioned firebug, cold stress amplified hsp70 Discussion 100 expression (Koštál and Tollarová-Borovanska, 2009), as well as in the Liriomyza leafminers (Huang and Kang, 2007; Huang et al., 2009). Expression of hsp70 is also upregulated during larval diapause of the gall fly Eurosta solidaginis in late fall and winter periods (Zhang et al., 2011). It is also upregulated during larval diapause in the Antarctic midge Belgica antarctica. HSP70 has also been determined to be critical for survival of this species in the extremely cold environment of the Antarctic (Rinehart et al., 2006, 2007). Considering that HSP90, HSC70 and HSP70 belong to the group of ATP-dependent heat shock proteins, this can call into question what their actual role is in metabolic states when energy sources and reserves are limited, such as during the hypometabolic state of diapause. When ATP is not a limiting factor, HSPs take up abnormal proteins and assist in their folding/refolding, or interact with different receptors and kinases to modulate cellular signaling (King and MacRae, 2015). However, under hypometabolic conditions (i.e. diapause) when ATP is limited (Popović et al., 2021), HSPs are more likely to remain in an ADP-bound state which favors substrate binding and protective sequestering until energy metabolism recovers (King and MacRae, 2015). For example, when HSP70-bound ATP is hydrolyzed, HSP70 transitions into an ADP-bound state with strong substrate affinity, while substrate dissociation rates are decreased. In such a state, HSP70 has high affinity for ATP, rebinding of which causes the substrate to release from the chaperone (Mayer et al., 2000; Arakawa et al., 2011; Mayer and Gierasch, 2019; Wu et al., 2020). Increased expression of hsp90, hsp70 and hsc70 in late diapause (D(–16) group, Figs. 19, 20 and 21) could also have positive effects on post-diapausal development of ECB larvae. Once diapause terminates and regular development resumes, the high content of different HSP transcripts would provide the now active larvae with a pool of adequate amounts of HSPs to assist in the folding of newly synthesized proteins (Popović et al., 2015). Additionally, this would also allow the HSPs to regulate their own expression and decrease it by binding to heat shock factors (HSFs). Monomer HSFs are transcription factors that are usually bound to different HSPs, such as HSP90, HSP70 and HSC70, under normometabolic conditions (Zou et al., 1998; Guo et al., 2001; Zheng et al., 2016). When stressors cause proteins to unfold, the damaged proteins compete with HSFs for binding sites on HSPs. This causes HSF monomers to detach and trimerize, after which they are translocated into the nucleus and bind to heat shock response elements (HREs) to upregulate transcription of HSP genes (Hochachka and Somero, 2002). Once stressors subside and Discussion 101 normometabolism is reestablished, HSPs are free to rebind HSFs and inhibit their transcription pathway, freeing up energy to be used for other metabolic processes. hsp20.4 and hsp20.1 Small heat shock proteins (sHSPs) belong to a group of molecular chaperones that share several structural commonalities. Their C-termini contain a conserved α-crystallin domain typical for sHSPs, while the secondary structure features a conserved β-sheet structure. The α-crystallin domain is necessary for the chaperone function of sHSPs, while the β-sheet enables the oligomerization of individual sHSPs. True to their name, these chaperones are smaller than other HSPs, with their molecular weights ranging from 12 to 43 kDa (Haslbeck, et al., 2005; Stetler et al., 2010). The majority of identified sHSPs in insects do not contain introns in their sequences, which is believed to facilitate rapid stress-responses. Lack of introns facilitates the rapidity of the response, as such mRNA do not undergo post-transcriptional processing (Li et al., 2009; King and MacRae, 2015). This type of stress response is reminiscent of prokaryotic organisms, which feature simpler transcriptional and translational machinery which can and must respond quickly to changes in the environment in order to ensure their survival. Small HSPs are incredibly diverse in number, size and molecular processes they are involved in. This diversity of sHSPs allows organisms to finely tune their cellular responses to stressful factors, as can be witnessed in the great adaptability of insects to almost any type of environment (Li et al., 2009). Small HSPs are the first line of defense against protein damage induced by stress factors, preventing aggregation and subsequent irreversible denaturation of substrate proteins. They bind abnormal proteins in an ATP-independent manner until they can be taken over by one of the members of the larger HSP families for refolding (King and MacRae, 2015; Żwirowski et al., 2017). Analyses in this study have revealed that the two small HSPs – hsp20.4 and hsp20.1, have opposite expression patterns in regards to diapause and acclimation to cold temperatures. Expression of hsp20.4 was higher in the diapausing groups when compared to the non-diapausing control. It steadily increased throughout the diapause and as temperatures dropped, with a slight decrease towards the end of the resting phase (Fig. 22). Conversely, hsp20.1 was highly upregulated in the non-diapausing group in comparison to any of the diapausing, cold-acclimated groups (Fig. 23). Such results could indicate that hsp20.4 and hsp20.1 have distinct functional roles in the different development phases and responses to stress in O. nubilalis. Additionally, of the two sHSPs, Discussion 108 Figure 28. Proposed mechanism of proteomic changes induced by the decrease in ambient temperature and progression of a hypometabolic resting state over time. These changes would lead to an increase of proteins with more flexibility in their structure, allowing them to remain mobile and active in thermodynamically unfavorable conditions of life at low environmental temperatures. In turn, they ensure that the functional and structural homeostasis of biological processes and biomolecules is maintained during this hypometabolic and energy-restricted period. Image created with Biorender.com. 6. Conclusions Conclusions 110 The following conclusions can be made according to the proposed hypothesis, defined aims and results of analyses performed on O. nubilalis 5th instar larvae that were subjected to different experimental conditions in order to trigger the development of cord hardiness:  the methods for isolating IDPs and proteins with long IDRs were successfully implemented and optimized in O. nubilalis larvae, while heat treating the crude homogenates enriched the content of IDPs and proteins with long IDRs which was also validated by 2D PAGE and LC-MS/MS;  numerous proteins, that were only predicted from nucleotide sequences available in databases, were detected by LC-MS/MS;  the presence of IDPs and proteins with long IDRs in all experimental groups was detected by bioinformatical analysis, as were the strong effects of IDP enrichment by sample heating;  there is a considerable fraction of heat-stable ordered proteins, as a large number of structured proteins was detected after sample heating;  proteins with varying degrees of structural disorder were identified and confirmed by bioinformatical analyses;  the content of specific disorder-promoting amino acids is more pronounced in proteins with higher degree of determined intrinsic disorder, which is in accordance with the results of previous studies on IDPs;  the ratio of IDPs does not increase during the gradual cold acclimation process, which partially disproves the proposed hypothesis;  the ratio of proteins with long IDRs increases during the gradual cold acclimation process, which partially confirms the proposed hypothesis;  bioinformatical analysis has shown that the identified IDPs and proteins with long IDRs are involved in numerous biological processes, exhibited by the variety of their molecular functions, with most of the belonging to groups of cytoskeletal proteins, molecular chaperones, proteins involved in translation and protein metabolism, as well as regulatory proteins with nucleic acid-binding activity; Conclusions 111  a fraction of proteins remained unidentified and many proteins could not be assigned any functions due to information gaps in available databases;  the expression of most genes encoding IDPs and proteins with long IDRs was upregulated during diapause and gradual development of cold hardiness;  IDPs and proteins with long IDRs are involved in the maintenance of structural and functional homeostasis during a hypometabolic resting phase (diapause) and they contribute to the total potential of cold hardiness. To my knowledge this is the first study on IDPs and proteins with long IDRs in an in vivo model that attains cold hardiness during its resting phase. These findings enrich the knowledge of the ecophysiological connection between diapause and cold hardiness, as well as the molecular mechanisms that allow not only the development but also the maintenance of cold hardiness during the long time course of the resting phase. Additionally, these results have proven, in no uncertain terms, the existence of heat-resistant ordered proteins that should be further investigated. The results of this study have potential applications in various fields – from cryopreservation in human and veterinary medicine to food and pharmaceutical industries. 7. Literature Literature 113 A Alvarez-Ponce, D., Ruiz-González, M. X., Vera-Sirera, F., Feyertag, F., Perez-Amador, M. A., & Fares, M. A. (2018). 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