Possibilities of Systemic RNAi by Feeding Experiments in Colorado Potato Beetle

Faculty of Bioscience Engineering

Academic year 2010-2011

Possibilities of Systemic RNAi by FeedingExperiments in Colorado Potato Beetle

by

Koen Rombouts

[email protected]

Promotor: Prof. Dr. Ir. G. Smagghe

Tutor: Dr. H. Huvenne

Master dissertation submitted in partial fulfilment of the requirements for the degree ofBio-Engineer in cell and gene biotechnology

I, Koen Rombouts, declare that this thesis titled, ”Possibilities of systemic RNAi byfeeding experiments in Colorado Potato Beetle” and the work presented in it are my own.I confirm that:

� This work was done wholly or mainly while in candidature for a research degree atthis University.

� Where any part of this thesis has previously been submitted for a degree or anyother qualification at this University or any other institution, this has been clearlystated.

� Where I have consulted the published work of others, this is always clearly attributed.

� Where I have quoted from the work of others, the source is always given. With theexception of such quotations, this thesis is entirely my own work.

� I have acknowledged all main sources of help.

� Where the thesis is based on work done by myself jointly with others, I have madeclear exactly what was done by others and what I have contributed myself.

Signed: Signed:

Date: Date:

Prof. Dr. Ir. Guy Smagghe Koen Rombouts

i

PREFACE

When looking back at the months that preceded the final version of this master thesis, Iwant to compare it, as a middle distance runner, to a hard race with a lot of changingweather conditions. The start was fast with a tailwind pushing me forward. During theyear the direction of the wind changed numerous times, from tail to cross or even a fullheadwind, but the important thing, as well in running as in science, is to keep progressingand give it the best you have. This is maybe the most important lesson that I learnedduring this thesis: when there is an obstacle in your path, take a moment to analyse itand find a way to circumvent it. Therefore, I would like to thank my promotor, Prof. dr.ir. G. Smagghe, for the opportunities he gave me during the course of this thesis, to takesteps to, eventually one day, become a good scientist. He provided me with a challengingresearch question, asked the right questions when we met and made the decisions, whenobstacles faced the surface.A very big ”thank you” is also addressed to dr. H. Huvenne, for her daily advice andfor stimulating me, to take my scientific attitude and my writing skills to a higher level.Her work ethics are an example that I will take with me in my future career. I wouldalso like to thank everyone else in the Laboratory of Agrozoology (Departement of CropProtection) for their help.On a more personal note, I want to thank my parents for giving me the opportunitiesto develop myself as a person over the last years, their support and advice were alwaysappreciated. Last but not least, I would like to thank my girlfriend, Birgit. She was mymental chearleader and that special person that was able to clear my head, when troubledand I bless myself daily, for having such an unique person like her, at my side.

Koen RomboutsGhent, June 2011

ii

ABSTRACT

The discovery of RNA interference (RNAi) and its mechanism has led to advances in avariety of research domains. Crop protection is one of the domains that is interested inthe development of the application of RNAi, additional to chemical pest control. The useof RNAi to control insect pests, has been studied in different organisms, e.g., Diabroticavirgifera virgifera, but most of the time only the effectivity of target genes was studied,without interest in the location of expression. In this work, the possibility of the systemicmechanism of RNAi in Leptinotarsa decemlineata (Colorado Potato Beetle) is studied onmorphological and gene expression level.

A bio-assay was optimized to designate and rule out effects, not related to RNAi, thatinfluence development of larvae of L. decemlineata. Next to a good experimental setup,potentially interesting target genes, specific to other tissues than the midgut, were se-lected. Three targets, a putative neural protein: Dopamine Transporter Protein (DAT),a putative cuticular protein: Chitin Synthase 1 (ChS1) and another putative cuticularprotein: Glycine-Rich Protein 1 (GRP1), were selected. Only one, GRP1, satisfied allrequirements to test for systemic RNAi during this research. To determine the level ofexpression of GRP1, a semi-quantitative PCR was optimized. Finally, two in vivo ex-periments were used to monitor the development of L. decemlineata from first instar tofourth instar larva, after treatment with dsGRP1. In parallel, a duplicate experiment wasperformed, where the expression of GRP1 was compared between the dsGRP1 treatmentand the controls.

The optimization of the bio-assay showed two important factors that need to be consid-ered when doing in vivo experiments with L. decemlineata: the moment the experimentis performed and the quality of the potato plants, used as a food source. In winter andwhen the food quality is low, the larvae in the experiment were weaker and the lab popu-lation collapsed. The best time to perform a in vivo experiments would be from April toSeptember.The tissue specificity of three targets was assessed in silico and with PCR on differenttissues. For DAT, it was the first time this was investigated in an insect. The experimentalevidence suggested that DAT is not expressed specific in neural tissue. Chitin Synthase1, was not picked up in a specific way with the first primer pair. GRP1 is not expressedin the midgut and was used for the bio-assay.Indications of the presence of systemic RNAi were observed in two instances. In themolecular experiment performed in October 2010, a lowering of expression of GRP1 wasseen after six days, in the dsGRP1 treated larvae. The mortality, on the other hand, washigher in the dsGRP1 treated larvae from the May 2011 experiment. Both results were notconfirmed by the respective parallel experiments. Further repetitions and a more sensitivetechnique (quantitative PCR) are needed to confirm these findings.

iii

SAMENVATTING

De ontdekking van RNA interference (RNAi), en het achterliggende mechanisme, heeft inverschillende onderzoeksdomeinen geleid tot nieuwe inzichten. Gewasbescherming is eenvan de domeinen waar een sterke interesse heerst naar de toepassing van deze techniekals additionele methode , naast de klassieke, chemische methoden van gewasbescherming.Interessante resultaten zijn reeds geboekt met verschillende organismen, vb. Diabroticavirgifera virgifera. Hierbij werd vooral gekeken naar de efficientie van de gebruikte target-genen, maar de locatie van de expressie was van ondergeschikt belang. In dit werk wordtgekeken naar genen die zich uitsluitend buiten de darm bevinden, waardoor de mogelijk-heid van systemische RNAi in Leptinotarsa decemlineata (coloradokever) onderzocht kanworden, op het niveau van de insect morfologie en van de genexpressie.

Het ontwerp en de optimalisatie van een biotoets was noodzakelijk om effecten op de larvenvan L. decemlineata, die niet worden veroorzaakt door de toediening van dsRNA, te de-tecteren. Een geschikte biotoets is een zaak, maar dit moet gepaard gaan met zorgvuldiggeselecteerde targetgenen, die zich in weefsels buiten de darm bevinden. Drie targets wer-den geselecteerd: een neuraal proteıne Dopamine Transport Proteıne (DAT) en cuticulaireproteınen Chitine Synthase 1 (ChS1) en Glycine-Rich Proteıne 1 (GRP1). GRP1 was hetenige van de drie dat voldeed aan de eisen, die hierboven werden vermeld. De genexpressiewerd met een semi-quantitatieve PCR opgevolgd. Deze voorbereidende stappen kwamenvervolgens samen in twee in vivo experimenten, waarin de ontwikkeling van L1 larve totL4 larve werd gevolgd, na behandeling met dsGRP1. Naast de insect morfologische exper-imenten werd, op hetzelfde moment, dezelfde proef herhaald waarin de genexpressie vanGRP1 in de dsGRP1 behandeling werd vergeleken met die van de controles.

Uit de optimalisatie van de biotoets konden twee belangrijke factoren worden geextra-heerd: het tijdstip van de experimenten en de kwaliteit van de aardappelplanten die di-enst doen als voeding van de larven. In de wintermaanden en wanneer de kwaliteit van deaardappelplanten zeer laag was, werd een verzwakking van de labpopulatie waargenomendie culmineerde in een ineenstorting van de populatie in januari. Als conclusie, kunnen wedus stellen dat de proeven best uitgevoerd worden in de periode tussen april en september.De weefselspecificiteit van de drie targets werd door in silico analyse en analyse met PCR,op zes verschillende weefsels, bepaald. Bij DAT, was dit de eerste maal dat dit werd on-derzocht voor een insect en DAT bleek niet specifiek te zijn voor neurale weefsels. Chitinesynthase 1 kon niet specifiek genoeg opgepikt worden door het eerst ontwikkelde primer-paar. GRP1 daarentegen werd niet geexpresseerd in de darm en kon gebruikt worden inde biotoetsen.Aanwijzingen dat systemische RNAi aanwezig is in L. decemlineata werden in de tweeexperimenten gevonden. De expressie van GRP1 was lager voor de dsGRP1 behandeldelarven op dag zes en de mortaliteit was hoger voor de dsGRP1 behandelde larven voorrespectievelijk het experiment van oktober 2010 en het experiment van mei 2011. Beide

iv

Samenvatting

resultaten werden echter niet bevestigd door de respectievelijke experimenten die gelijk-tijdig werden uitgevoerd. Herhalingen en een gevoeligere techniek voor het moleculaireexperiment (quantitatieve PCR) lijken dus nodig om uitsluitsel te geven.

v

Contents

Preface ii

Abstract iii

Samenvatting iv

Contents vi

List of Figures x

List of Tables xi

List of Abbreviations xii

Part I Literature Study 1

1. RNA Interference 21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Cell Autonomous RNAi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.2.1 Processing of Precursors . . . . . . . . . . . . . . . . . . . . . . . . . 51.2.2 RNA-Induced Silencing Complex Activation . . . . . . . . . . . . . . 61.2.3 Post-Transcriptional Gene Silencing . . . . . . . . . . . . . . . . . . 8

1.3 Non-Cell Autonomous RNAi . . . . . . . . . . . . . . . . . . . . . . . . . . 81.3.1 Uptake of dsRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.3.2 Transport of dsRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.3.3 Gene Silencing via the Cell Autonomous RNAi Machinery . . . . . . 9

2. Applications 102.1 Gene Knockdown and Functional Genomics . . . . . . . . . . . . . . . . . . 102.2 Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102.3 RNAi in Agriculture - Pest Control . . . . . . . . . . . . . . . . . . . . . . . 11

3. Leptinotarsa decemlineata 143.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143.2 Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143.3 Life Stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

3.3.1 Egg Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153.3.2 Larval Stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153.3.3 Pupal Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.3.4 Adult Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4. Targets 19

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Contents

4.1 DAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.2 ChS1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194.3 GRP1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Part II Objectives 21

Part III Material and Methods 24

1. Breeding of L. decemlineata 25

2. Bio-assay 262.1 Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262.2 Experimental Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

2.2.1 Insect Physiological Experiment . . . . . . . . . . . . . . . . . . . . 272.2.2 Molecular Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . 27

2.3 Statistic Analysis of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3. Dissection of L. decemlineata 293.1 Dissection of the Heads of L4 larvae . . . . . . . . . . . . . . . . . . . . . . 293.2 Dissection of the Midgut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.3 Dissection of Six Different Tissues . . . . . . . . . . . . . . . . . . . . . . . 293.4 Dissection of L2 Larvae in Ecdysis . . . . . . . . . . . . . . . . . . . . . . . 29

4. Buffers 304.1 Insect Physiological Buffer (IPS) pH 7.0 . . . . . . . . . . . . . . . . . . . . 304.2 TRIS-EDTA (TE) Buffer pH 8.0 . . . . . . . . . . . . . . . . . . . . . . . . 304.3 TRIS-Acetic Acid-EDTA (TAE) Buffer pH 8.0 . . . . . . . . . . . . . . . . 30

5. Agarose Gel Electroforese 315.1 1.5% Agarose Gel Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 315.2 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315.3 Photography of the Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

6. cDNA Synthesis 326.1 Total RNA Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326.2 DNase Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326.3 RNA Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326.4 First Strand cDNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . 33

7. Polymerase Chain Reaction 347.1 Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

7.1.1 Standard Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . 347.1.2 Degenerated Primer Design . . . . . . . . . . . . . . . . . . . . . . . 34

7.2 PCR Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347.3 PCR Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357.4 Semi-Quantitative PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357.5 Long Distance PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357.6 Colony PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357.7 Amplifying Linearized Plasmids DNA . . . . . . . . . . . . . . . . . . . . . 36

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Contents

7.8 PCR Product Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367.9 Overview of Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367.10 PCR programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

8. Cloning 428.1 Ligation of PCR Product in a Cloning Vector . . . . . . . . . . . . . . . . . 428.2 Heat-Shock Transformation of Escherichia coli . . . . . . . . . . . . . . . . . 428.3 Selection of the Colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428.4 Growing of the Selected Colonies . . . . . . . . . . . . . . . . . . . . . . . . 428.5 Plasmid Recovery and Purification . . . . . . . . . . . . . . . . . . . . . . . 438.6 Linearisation of the Plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

9. Sequencing 45

10.Double-stranded RNA Synthesis 4610.1 MegaScript RNAi Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

10.1.1 Transcription Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . 4610.1.2 Annealing the RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 4610.1.3 Nuclease Digestion to remove DNA and ssRNA . . . . . . . . . . . . 4610.1.4 Purification of dsRNA . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Part IV Results 48

1. Introduction 49

2. Selection of Targets 502.1 Target Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502.2 Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502.3 Tissue Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

2.3.1 In silico Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512.3.2 Experimental Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 52

3. Optimization of the bio-assay 543.1 Neonates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

3.1.1 Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543.1.2 Duration of Life Stages . . . . . . . . . . . . . . . . . . . . . . . . . 563.1.3 No Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

3.2 Neonates after 24 Hours of Starvation . . . . . . . . . . . . . . . . . . . . . 573.2.1 Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573.2.2 Duration of Life Stages . . . . . . . . . . . . . . . . . . . . . . . . . 603.2.3 No Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

3.3 Result of the Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

4. In vivo Effects of dsRNA 634.1 October 2010 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

4.1.1 Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634.1.2 Duration of Life Stages . . . . . . . . . . . . . . . . . . . . . . . . . 644.1.3 Molecular Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . 65

4.2 March 2011 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664.2.1 Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

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Contents

4.2.2 Duration of Life Stages . . . . . . . . . . . . . . . . . . . . . . . . . 674.2.3 Molecular Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . 68

4.3 May 2011 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694.3.1 Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694.3.2 Duration of Life Stages . . . . . . . . . . . . . . . . . . . . . . . . . 704.3.3 Molecular Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . 71

5. Chitin Synthase 735.1 In silico Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735.2 Determination of ChS1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Part V Discussion & Conclusion 74

Bibliography 79

ix

List of Figures

I.1.1 Overview of the different types of RNAi . . . . . . . . . . . . . . . . . 3I.1.2 General overview of the siRNA pathway . . . . . . . . . . . . . . . . . 4I.1.3 Small RNA biogenesis in animals . . . . . . . . . . . . . . . . . . . . . 6I.1.4 The RISC assembly pathway for siRNA in D. melanogaster . . . . . . 7

I.2.1 F1 plants expressing a V-ATPase A dsRNA are protected from D.virgifera feeding damage . . . . . . . . . . . . . . . . . . . . . . . . . . 12

I.3.1 The eggs of L. decemlineata ovipositioned on the underside of a leaf . 15I.3.2 Identification key to distinguish the different developmental stages of

L. decemlineata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15I.3.3 Larval stages of L. decemlineata . . . . . . . . . . . . . . . . . . . . . 16I.3.4 Pupa of L. decemlineata . . . . . . . . . . . . . . . . . . . . . . . . . . 17I.3.5 Adult of L. decemlineata . . . . . . . . . . . . . . . . . . . . . . . . . 18

II.1 Schematic drawing of the location of the targets of systemic RNAiused in this work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

III.2.1 Schematic drawing of a compartement of a box for the bio-assay . . . 27

III.8.1 Map of the pGEM-T Vector . . . . . . . . . . . . . . . . . . . . . . . . 44

IV.2.1 Gradient PCRs for GRP1, DAT and ChS1 . . . . . . . . . . . . . . . 51IV.2.2 Gel of tissue specifity for GRP1, DAT and ’ChS1’ . . . . . . . . . . . 53

IV.3.1 Accumulated percentage of mortality for neonates . . . . . . . . . . . 55IV.3.2 Accumulated percentage of mortality for the starvation experiment

with 10 neonates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57IV.3.3 Accumulated percentage of mortality for neonates after 24h starva-

tion: November 2010 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58IV.3.4 Accumulated percentage of mortality for neonates after 24h starva-

tion: March 2011 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59IV.3.5 Accumulated percentage of mortality for neonates after 24h starva-

tion: April 2011 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60IV.3.6 Accumulated percentage of mortality of the starvation experiment

with 24h starved neonates . . . . . . . . . . . . . . . . . . . . . . . . . 61

IV.4.1 Accumulated percentage of mortality: October 2010 . . . . . . . . . . 64IV.4.2 Gels of the molecular experiment: October 2010 . . . . . . . . . . . . 65IV.4.3 Accumulated percentage of mortality: March 2011 . . . . . . . . . . . 67IV.4.4 Gels of the molecular experiment: March 2011 . . . . . . . . . . . . . 68IV.4.5 Accumulated percentage of mortality: May 2011 . . . . . . . . . . . . 70IV.4.6 Gels of the molecular experiment: May 2011 . . . . . . . . . . . . . . 71

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List of Tables

I.3.1 Taxonomy of L. decemlineata . . . . . . . . . . . . . . . . . . . . . . . 14

III.2.1 Samples collected in the molecular experiment . . . . . . . . . . . . . 28

III.4.1 Composition IPS pH 7.0 . . . . . . . . . . . . . . . . . . . . . . . . . . 30III.4.2 Composition 1X TE stock solution pH 8.0 . . . . . . . . . . . . . . . . 30III.4.3 Composition 50X TAE stock solution pH 8.0 . . . . . . . . . . . . . . 30

III.6.1 Composition of the TURBO DNA-free kit reaction . . . . . . . . . . . 32III.6.2 Composition SuperScript II Reverse Transciptase reactions . . . . . . 33

III.7.1 Composition of a PCR reaction . . . . . . . . . . . . . . . . . . . . . . 34III.7.2 Composition of a Long Distance PCR reaction (for 0.5 - 9kb) . . . . . 35III.7.3 Primers used during the course of this work . . . . . . . . . . . . . . . 37III.7.4 PCR programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

III.8.1 Composition of the pGEM-T Vector System ligation mix . . . . . . . 42III.8.2 Composition of the restriction reaction for SalI . . . . . . . . . . . . . 43

III.10.1 Composition of the transcription reaction for dsRNA synthesis . . . . 46III.10.2 Composition of the nuclease digestion reaction for dsRNA synthesis . 46III.10.3 Composition of the dsRNA binding mix . . . . . . . . . . . . . . . . . 47

IV.3.1 Average duration of life stages of neonates . . . . . . . . . . . . . . . . 56IV.3.2 Average duration of life stages of neonates after 24h starvation . . . . 60

IV.4.1 Average duration of life stages with SEM (in days): October 2010 . . 65IV.4.2 Molecular results: October 2010 . . . . . . . . . . . . . . . . . . . . . 66IV.4.3 Average duration of life stages with SEM (in days): March 2011 . . . 68IV.4.4 Molecular results: March 2011 . . . . . . . . . . . . . . . . . . . . . . 69IV.4.5 Average duration of life stages with SEM (in days): May 2011 . . . . 71IV.4.6 Molecular results: May 2011 . . . . . . . . . . . . . . . . . . . . . . . 72

xi

LIST OF ABBREVIATIONS

AA Amino acidBt Bacillus thuringiensisChS Chitin synthaseCNS Central nervous systemDAT Dopamine transporter proteindsRBP Double-stranded RNA binding proteindsRNA Double-stranded RNAEtBr Ethidium bromideGlcNAc N-acetyl-glucosamineGRP1 Glycine-rich protein 1IPS Insect physiological bufferLB Luria BertanimiRNA micro RNAMQ Ultrapure waterNC Negative controlNCBI National center of biotechnology informationNoRTC No reverse transcriptase controlNTC No template controlPC Positive controlPCR Polymerase chain reactionpiRNA Piwi-interacting RNAPM Peritrophic membranepri-miRNA Primary miRNARISC RNA-induced silencing complexRLC RISC-loading complexRNAi RNA interferenceRNase RibonucleaseRT Room temperatureSEM Standard error of the meanSID Systemic RNAi defectivesiRNA Short interfering RNAssRNA Single-stranded RNATA Annealing temperatureTM Melting temperatureTAE Tris-acetate-EDTATE Tris-EDTATLRs Toll like receptorsV-ATPase Vacuolar H+ ATPase

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Part I

LITERATURE STUDY

1

Chapter 1

RNA Interference

1.1 Introduction

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that occurs ina variety of eukaryotic cells. In 1998, Fire et al. (1998) documented that double-strandedRNA (dsRNA) has a gene silencing effect in Caenorhabditis elegans. This groundbreakingdiscovery let to a whole new field of research in genomics. In only a couple of years, thegeneral mechanism and its machinery were discovered (Jinek & Doudna, 2008).

RNAi can be defined as a sequence-specific process of mRNA degradation or translationalrepression, that is induced by dsRNA.

What is the significance of RNAi and what are the consequences? (Daneholt, 2011)

• RNAi protects, plants, worms and flies against viral infections. It is still an openquestion if vertebrates share this mechanism

• RNAi secures genome stability by keeping mobile elements (transposons) silent

• RNAi-like mechanisms repress protein synthesis and regulate the development of or-ganisms. Messenger RNA (mRNA) molecules (see Subsection 1.2.1) are the mediatormolecules that are found in almost every species

• RNAi-like mechanisms keep chromatin condensed and suppress transcription. Thisis an important feature for the proper functioning of the genome and for maintenanceof genome integrity

• RNAi offers a new experimental tool to repress genes in a specific way. This resultsin advances in studies concerning gene functions

• RNAi might be a useful approach in future gene therapy

Three types of RNAi are distinguished (see Figure I.1.1). First, there is cell autonomousRNAi, which describes the gene silencing reaction that is provoked, by introduction ofdsRNA in a cell. When RNAi is not restricted to a single cell, it is called non-cellautonomous RNAi. In environmental RNAi, dsRNA molecules are taken up from theenvironment of the cell and this can result in gene silencing in those cells, through cellautonomous RNAi. The last type is systemic RNAi. A dsRNA molecule is introduced inone cell and a signal will be transported to other cells. This results in a gene silencingreaction, in each individual cell, through cell autonomous RNAi.

2

Part I: Literature Study

Figure. I.1.1: Overview of the different types of RNAi (Huvenne & Smagghe, 2010).

1.2 Cell Autonomous RNAi

In cell autonomous RNAi, the silencing proces is limited to the cell in which the dsRNAmolecule is introduced (see Figure I.1.1) (Huvenne & Smagghe, 2010). This mechanismis well conserved in a wide range of eukaryotic organisms throughout the kingdoms (Fire,2007; Meister & Tuschl, 2004; Whangbo & Hunter, 2008; Huvenne & Smagghe, 2010).After the discovery of dsRNA, as the most effective startpoint of the RNAi mechanism inC. elegans by Fire et al. (1998), the mechanism of dsRNA mediated mRNA degradationor repression had to be exposed.

3


Figure. I.1.2: General overview of the siRNA pathway from Gaynor et al. (2010)

In this figure, the general mechanism of the cell autonomous RNAi reaction, starting froma long exogenous dsRNA, is described:

1. The dsRNA molecule is processed by a protein with ribonuclease activity, that iscalled Dicer in C. elegans. This protein cleaves the dsRNA molecule into smallRNA duplexes, called micro RNAs (miRNA), small interfering RNAs (siRNAs) orpiwi-interacting RNAs (piRNA). In this example, siRNAs are used (see further).

2. This molecule is loaded onto the RNA-induced silencing complex (RISC) LoadingComplex (RLC), a protein complex formed by Dicer (in C. elegans) and the dsRNABinding Protein (dsRBP)

3. From the RLC, the RNA duplex is loaded onto the RISC

4. The RISC is activated by cleaving one of the two strands of the double-strandedsmall RNA molecule

5. The active RISC has siRNA-mediated recognition of complementary target mRNAs

6. The target mRNA is cleaved by the RISC

7. The activated RISC can be recycled, leading to a new cycle of mRNA recognitionand degradation

Now, the sequence of steps that leads to an interference reaction will be described in moredetail.

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1.2.1 Processing of Precursors

Small RNA molecules are needed for cell autonomous RNAi. Until now, three types ofthese small RNA molecules are known: miRNAs, siRNAs and piRNAs. miRNAs are regu-lators of indigenous genes. By now, it is embedded in the general vision, that siRNAs arethe defenders of the genomic integrity in response to foreign and invasive nucleic acids. Forexample, experimental delivered dsRNA molecules, viruses, transposons and transgenes(Carthew & Sontheimer, 2009). piRNAs are different in that they are primarily found inanimals, exert their functions most clearly in the germline and derive from precursors thatare poorly understood, but appear to be single-stranded. The clearest difference howeveris the affinity for a different subset of effector proteins (Carthew & Sontheimer, 2009).Since the piRNAs are not as well understood yet, the focus in this work will be on themiRNAs and siRNAs.

Although there is no real functional difference, a distinction has to be made betweenthe biogenesis of miRNAs and siRNAs (see Figure I.1.3).miRNAs are formed from a precursor molecule, that is formed by duplex formation of non-coding genomic sequences and is called primary miRNA (pri-miRNA). This pri-miRNAis formed by RNA polymerase II and is cleaved in the nucleus by a ribonuclease type III(RNase III), often called Drosha in Drosophila melanogaster, forming a pre-miRNA. Thismolecule is then exported to the cytoplasm (Tomari & Zamore, 2005), where it is cleavedby a second enzyme called Dicer in D. melanogaster and C. elegans. It is a RNase III typeprotein that cleaves the pre-miRNA into miRNA molecules (see Figure I.1.3A).Two cleavages of exogenous dsRNA molecules, by only one Dicer-like molecule, are nec-essary to produce a siRNA (see Figure I.1.3B).

The resulting miRNAs and siRNAs are 21-25 nucleotide duplexes with a phosphate groupat both 5’ ends, and hydroxyl groups and two-nucleotide overhangs at both 3’ ends (Siomi& Siomi, 2009).

5


Figure. I.1.3: Small RNA biogenesis in animals. (A) miRNA biosynthesis (B) siRNA biosynthesis(Tomari & Zamore, 2005).

1.2.2 RNA-Induced Silencing Complex Activation

RISC is a multi-protein complex that interacts with small RNA molecules and facilitatesmRNA degradation (See Figure I.1.2). The most important protein involved, is a proteinof the Argonaute family. The multi-protein RISC complex that results from the bindingof Argonaute proteins consist of a variable amino-terminal domain and three conserveddomains (the PAZ, middle and PIWI domains).Phylogenetically, there are three subfamilies or clades of Argonaute proteins: the AGO,the PIWI and the WAGO subfamily (Siomi & Siomi, 2009). The AGO subfamily is themost important subfamily with respect to RNAi. It contains a number of proteins thatall seem to have their own role in the different RNAi pathways.

6


Figure. I.1.4: The RISC assembly pathway for siRNA in D. melanogaster (Tomari & Zamore, 2005)

Figure I.1.4 illustrates the RISC loading process. The Dicer - dsRNA Binding Proteincomplex, the RLC, transfers a dsRNA molecule to an AGO protein. The complex thatis formed with the AGO protein, the dsRNA and the RLC is now called the holo-RISCand forms the core of the RISC (Tomari & Zamore, 2005). The dsRNA molecule willbe reduced to a single-stranded RNA (ssRNA) molecule. The strand that is bound tothe holo-RISC is called the guide strand and the strand that is destroyed is called thepassenger strand. The selection of which strand is guide and which is passenger strand, isbased on the relative thermodynamic stability of the two duplex ends. There is an assym-metry between the two strands and the one with the less stable 5’ terminus will be favoredas the guide strand (Carthew & Sontheimer, 2009). It is believed that the dsRBP playsan important role in the selection of the passenger/guide strand (Tomari & Zamore, 2005).

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1.2.3 Post-Transcriptional Gene Silencing

The final step of the RNAi pathway is the gene silencing. This process can be divided intotwo parts: the siRNA mediated and the miRNA mediated mRNA degradation.First, the siRNA mediated post-transcriptional silencing will be discussed. The siRNAguide strand is responsible for the binding of the holo-RISC to a perfect complementarymRNA target, which is then degraded. The degradation of RNA is induced by the PIWIdomain of the Ago protein. This domain exerts a slicer function that is very accurate. Thefollowing steps lead to the total degradation of the target mRNA (Carthew & Sontheimer,2009):

• Cleavage of the phosphodiester bond from the target strand between the number 10and 11 base pair counting from the 5’ end of the siRNA. This cleavage leaves 5’-monophosphate and 3’-hydroxyl termini on the mRNAs (Tomari & Zamore, 2005)

• After the target is cleaved, it dissociates from the siRNA. This leaves the RISC freeto cleave other mRNA targets. This is called multiple turnover and it is mentionedthat this process might be driven by ATP hydrolysis (Tomari & Zamore, 2005)

• Exonucleases from the cellular environment target the sliced mRNA fragments tocomplete the degradation process (Orban & Izaurralde, 2005)

Secondly, the miRNA mediated post-transcriptional silencing is discussed.Like the siRNAs, miRNAs are loaded into the RISC complex forming the miRISC. Theguide strand is the template for the binding of the miRISC to mRNA targets. Thishappens direct, through base-pairing. In animals, this base-pair complementary is, mostof the time, imperfect. Two outcomes are possible: translational repression or mRNAdegradation. Translational repression is called non-cleavage degradation, but there is noconsensus on what the mechanism is. However, it is most common when there is animperfect match between miRNA and mRNA (Gu & Kay, 2010). When perfect base-pairing is present, mRNA degradation is more frequent (see above) (Jaubert et al., 2007).

1.3 Non-Cell Autonomous RNAi

RNAi is exclusively cell autonomous in many organisms. It is however fascinating, thatthere is also non-cell autonomous RNAi in plants and in a couple of animals. This meansthat locally administered dsRNA can initiate gene silencing in other parts of the organism.Presumably, this happens through the movement of dsRNA, or an induced component,from cell to cell (systemic RNAi) or through the uptake of dsRNA from the environment(environmental RNAi). (Whangbo & Hunter, 2008; Huvenne & Smagghe, 2010).In C. elegans, it was first observed that an interference effect can happen after exposure todsRNA in the environment. For example, by soaking the organisms in a dsRNA solutionor feeding them dsRNA producing bacteria (Fire et al., 1998; Timmons & Fire, 1998). Inunicellular organisms, environmental RNAi consists of the uptake of dsRNA and the sub-sequent cell autonomous silencing pathway (See Section 1.2). In multicellular organisms,a combination of environmental and systemic RNAi can be present when dsRNA is takenup from the environment by a set of cells and there is a spread to other cells. When a genesilencing effect is observed, the non-cell autonomous pathways are always followed by cellautonomous RNAi in each individual cell. The combination of these mechanisms, thatstart with dsRNA in the environment and lead to gene silencing throughout the organism,consists of three steps (Whangbo & Hunter, 2008).

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1.3.1 Uptake of dsRNA from the Environment

So far, two mechanisms for dsRNA uptake from the environment are known: a transmem-brane channel protein-mediated uptake and an endocytosis-mediated uptake mechanism.In the first mechanism, two transmembrane channel proteins play a role in the uptake. Thismechanism was discovered in C. elegans. In this organism, the multispan transmembraneproteins are Systemic RNAi Defective (SID)-1 and SID-2. There are three hypotheses onhow those two proteins facilitate dsRNA uptake (Huvenne & Smagghe, 2010; Whangbo &Hunter, 2008):

• SID-2 modifies SID-1 to activate transport of dsRNA

• SID-2 binds dsRNA from the environment and delivers it to SID-1

• The endocytosis pathway of dsRNA is induced by SID-2. SID-1 delivers the dsRNAto the cytoplasm

The SID proteins are found in C. elegans and orthologs are present in insects, but in silicoanalysis suggests that they are not essential for dsRNA uptake in insects. Therefore, an-other mechanism is probably responsible for uptake of dsRNA in insects (Tomoyasu et al.,2008; Huvenne & Smagghe, 2010).In insects where no SID protein orthologs are present (e.g., D. melanogaster), a robustsystemic RNAi respons was detected. This means, there is an ’alternative’ uptake mech-anism. Vacuolar H+ ATPase (V-ATPase) and a combination of scavenger receptors playan important role in this uptake. Furthermore, labeled dsRNA was seen to be associatedwith vesicles, which probably means that the ’alternative’ mechanism is an endocytosismechanism that is receptor-mediated. The scavenger receptors are responsible for therecognition in this ’alternative’ mechanism and are known for their involvement in theancestral innate immune response in D. melanogaster. This illustrates that antiviral im-munity, in some multicellular organisms, is closely linked to the mechanisms involved inenvironmental and systemic RNAi (Saleh et al., 2009; Huvenne & Smagghe, 2010).

1.3.2 Transport of dsRNA, or a dsRNA-derived Signal, to other Cells

The cell-to-cell transport is called systemic RNAi. It is only possible in multicellularorganisms, because it involves at least two cells. In C. elegans, SID-1 plays a role in thesystemic spread of dsRNA. In worms that are defective in this protein, no silencing wasobserved in other cells than the cells who were in contact with the dsRNA (Winston et al.,2002). In certain insects however, SID-1 does not seem to be essential for systemic RNAi(Tomoyasu et al., 2008).

1.3.3 Gene Silencing via the Cell Autonomous RNAi Machinery

See Section 1.2.

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Chapter 2

Applications

2.1 Gene Knockdown and Functional Genomics

Starting with the first eukaryotic genome sequence in 1995 (Fleischmann et al., 1995),more and more genome and gene sequences became available. A new domain called func-tional genomics, tried to decipher the functions of these genes. A lot of the knowledgecurrently available, about those genes and their function, is acquired by techniques basedon RNAi. Prior to the use of RNAi, the two methods that were used for investigating agene function in vivo, were gain-of-function and loss-of-function studies, using transgenicand knockout animal technology. These studies required a vast amount of time and moneyand sometimes, it was hard to unambiguously link the result to the mutated gene (Caplenet al., 2001). When RNAi was discovered in C. elegans, the specific gene knockdown,which results in a reduction of the expression of the gene that is targeted, appeared to bean alternative method for the studie of gene functions (Caplen et al., 2001; Vanhecke &Janitz, 2005). The advantages are:

• Knockdown organisms, when compared to knockout organisms, are more likely tosurvive or even produce offspring, because the proteins are still expressed, althoughat a lower level (Dann et al., 2006)

• Very efficient downregulation of the gene expression

• Cheaper and less time consuming than knockout models

• Different degrees of knockdown are possible (Dann et al., 2006)

• Possibility for use in genome-wide, high-throughput studies (Lettre et al., 2004;Boutros et al., 2004)

2.2 Medicine

Immediately after its discovery, scientists saw the potential of RNAi as a therapeutic toolfor human medicine. Because it uses cellular machinery, it allows an efficient targetingwhich often results in a potent down-regulation of gene expression. This is an advantageof RNAi when compared to other antisense based approaches (Aagaard & Rossi, 2007).In theory, this is true, but in reality there are drawbacks that have to be overcome beforetherapies can be developed for a broad range of diseases. The three most important con-cerns when working with mammalian cells are off-target effects, the interferon responses(see further) and effective in vivo delivery (Aagaard & Rossi, 2007).It has been shown that large numbers of non-target genes can be silenced after treatmentwith siRNA (Jackson et al., 2003). These off-target effects can be toxic to cells, whichleads to unwanted cell death (Fedorov et al., 2006).Secondly, the innate immune system can interact in the RNAi pathway. These interactionsare called the interferon responses and occurs when long dsRNA strands are introducedinto the cellular environment. Evolutionary conserved anti-viral mechanisms, including thedouble-stranded-RNA-activated protein kinase receptor, 2’,5’-oligoadenylate synthetase-RNaseL system and the Toll Like Receptors (TLRs), are all capable of inducing two types

10


of interferon responses (Aagaard & Rossi, 2007). These responses inhibit cellular proteinsynthesis and result in cell death. However, it was found that dsRNAs shorter than 30 ntare capable of escaping these systems. The exception to this rule are sequences containingso-called ”danger motifs”. These stimulate the immune system, because they are recog-nized by TLRs (Aagaard & Rossi, 2007; Hornung et al., 2005).Another bottleneck is the delivery of the therapeutics in vivo. Several barriers lie betweenthe extracellular environment, where the products are delivered, and the cytosol, wherethe target mRNAs are localized.

Despite the bottlenecks in the therapeutical use of RNAi, a couple of clinical trails werestarted. They targeted a variety of diseases (Lares et al., 2010):

• Genetic diseases like pachyonychia congenita, Alzheimer’s disease, amyotrophic lat-eral sclerosis, familial adenomatous polyposis (Lares et al., 2010)

• Several different cancers like chronic lymphocytic leukemia, metastatic lymphomaand solid tumors (breast cancer, prostate cancer, lung cancer, sarcomas and lym-phomas) (Lares et al., 2010; Tiemann & Rossi, 2009)

• Viral diseases like hepatitis C virus, human immunodefficiency virus, respiratorysyncytial virus (Lares et al., 2010)

• Others like rheumatic diseases (de Franca et al., 2010), acute renal failure (Tiemann& Rossi, 2009), age-related macular degeneration

2.3 RNAi in Agriculture - Pest Control

Worldwide, the agrochemical market was estimated to be worth 134,000 million US dol-lars in 2010 (marketsandmarkets.com, 2010). Crop protection is a fast evolving business,where innovation is necessary to keep a concurential position and, more practically, toavoid resistance to the products used. RNAi has the potential to gain ground on tra-ditional pesticides against nematodes and insects, due to its unique properties. First,a short overview of the progress of RNAi in crop protection, with regard to insects, willbe given. To conclude, the future prospects of RNAi in pest management will be described.

An insecticide needs to be effective and easy to apply (considering all human safety re-quirements fulfilled). Effectiveness of gene knockdown by RNAi was shown through variousinjection and cell line experiments (Dzitoyeva S & H, 2001; Jarosch & Moritz, 2011). Forcrop protection, autonomous uptake of dsRNA is a condition that has to be met. Priorto 2007, it was therefore believed that protection of crops against insects using RNAi wasnot feasible. The breakthrough paper by Baum et al. (2007) provided evidence for thepotential use of RNAi for pest control. Silencing of target genes was achieved by feedingplant material that expressed hairpin dsRNA constructs for well chosen target genes.Selection of target genes is a critical point with respect to the efficacy of a treatment.Targets would be preferable expressed in cells exposed to the dsRNA, because the effectsare expected to be most prominent there. These cells would be those of the midgut andassociated structures, because these are the only regions of the insect not covered by achitin exoskeleton (Price & Gatehouse, 2008). In some insects, a robust systemic RNAieffect (see Section 1.3) is found, thus opening the door to selection of target genes through-out the body (Tomoyasu et al., 2008).A couple of targets were used succesful for gene silencing in bioassays with coleoptera (e.g.,D. virgifera, L. decemlineata and others): V-ATPase subunits A, D and E, α-tubulin,

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rpL19, . . . (Baum et al., 2007; Zhu et al., 2011)V-ATPase A dsRNA expressing plants have been used to repress the root damage doneby D. virgifera. A result is shown in Figure I.2.1.

Figure. I.2.1: F1 plants expressing a V-ATPase A dsRNA are protected from D. virgifera feedingdamage. On the left a non-transgenic control with average root damage. The planton the right shows the average root protection seen when the transgene is expressed(Baum et al., 2007).

In the future, RNAi can play a role in integrated pest management. Integrated pestmanagement is an approach to pest management, in which the most effective and environ-mentally sensitive practice is selected, based on all the knowledge available on the pests.These methods are used to manage pest damage by the most economical means, andwith the least possible hazard to people, property, and the environment (United StatesEnvironmental Protection Agency (EPA), 2011). The main strategy would be to designtransgenic dsRNA expressing plants. This technique has been used with success to makeplants more resistant against plant viruses, bacteria, nematodes and insects (Baum et al.,2007; Price & Gatehouse, 2008). The assets of RNAi-mediated pest management are thefollowing (Price & Gatehouse, 2008):

• A wide range of potential targets for gene knockdown can be identified per targetspecies by cDNA library screening

• Species specific. Little non-target species will be affected

• Little to no resistance of the insect against RNAi is possible. The nature of theRNAi mechanism makes it hard for insects to circumvent it

• Supporting natural defensive mechanisms of plants. For example, Helicoverpa armigera(the cotton bollworm) has evolved to be able to (partly) detoxify gossypol, whichis a secondary metabolite of cotton. When the protein that is responsible for the

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detoxification, is silenced by artificial food, a high mortality is observed for larvae ofH. armigera (Mao et al., 2007)

• Complementing other pest control techniques to counter broad-range resistance topesticides. RNAi could knockdown the resistance mechanisms developed by insects,thus making the insects susceptible to the pesticide again. For example, Bacillusthuringiensis (Bt) toxins are implemented in transgene plants for 16 years and re-sistance, by upregulation of specific proteinase genes, is already observed in somepopulations (Karumbaiah et al., 2007)

In conclusion, the question can be asked if RNAi-mediated pest control will have animmediate effect on pest management. For coleopteran and lepidopteran plant pests,where Bt-based strategies are more powerful and offer a higher protection, so this seemsunlikely on a short term but with the first instances of resistance to Bt-crops found,RNAi based strategies are becoming a sustainable alternative for the future (Tabashniket al., 2008). For dipterans and phloem-sucking homopteran pests, Bt approaches can beinsufficient. RNAi expressed in the plant can be a possible pest control technique, if it isfigured out how the effector RNA (dsRNA or siRNA) molecules can be transported in thephloem (Price & Gatehouse, 2008).

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Chapter 3

Leptinotarsa decemlineata

3.1 Introduction

L. decemlineata (Colorado Potato Beetle) is a phytophagous beetle that feeds predomi-nantly on Solanum tuberosum, better known as the potato plant. The yellow-orange beetlewith ten black stripes on the elytron occurs generally in all potato-growing areas (Euro-pean and Mediterranean Plant Protection Organisation, 2004). L. decemlineata is usedin this thesis, because it is an important crop pest throughout the world and it is wellknown for the high level of resistance to many insecticide classes (Casagrande, 1987; We-ber, 2003; Alyokhin, 2009). Although the English name indicates the insect feeds mainlyon potato plants, they can sustain their population on other Solanacea, weeds and otherplants (Duchesne & Parent, 1991). In the following sections, the taxonomy and the stagesof development will be discussed.

3.2 Taxonomy

L. decemlineata is a leaf beetle, see Table I.3.1.

Table. I.3.1: Taxonomy of L. decemlineata

Kingdom: AnimaliaPhylum: ArthropodaSubphylum: HexapodaClass: InsectaSubclass: PterygotaInfraclass: NeopteraOrder: Coleoptera (Linnaeus, 1758)Suborder: Polyphaga (Emery, 1886)Infraorder: Cucujiformia (Lameere, 1938)Superfamily: Chrysomeloidea (Latreille, 1802)Family: Chrysomelidae (Latreille, 1802)Subfamily: Chrysomelinae (Latreille, 1802)Tribe: Chrysomelini (Latreille, 1802)Genus: Leptinotarsa (Chevrolat in Dejean, 1836)Species: Leptinotarsa decemlineata (Say, 1824)

3.3 Life Stages

The description of the life stages is based on the beetle in its natural environment, but it isclear that the insects that live in a controlled lab culture undergo the same stages (Boiteau& Blanc, 1992). The complete life cycle of L. decemlineata consists of seven stages: anegg stage, four larval stages, one pupal stage and the adult stage.

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3.3.1 Egg Stage

Following the copulation, the yellow-orange eggs are deposited on the underside of plantleaves in batches of 20-30. This is mainly on potato or other crop plants, nearby weeds orsometimes directly on the soil. The eggs have a typical elongate-oval shape with a lengthof ± 1.5 mm and a width of ± 0.7 mm (see Figure I.3.1).

Figure. I.3.1: The eggs of L. decemlineata ovipositioned on the underside of a leaf (Bergen, 2008).

3.3.2 Larval Stages

The larvae of L. decemlineata have a large red arched abdomen with black spots onboth flanks. The abdomen consists of nine segments. The last segment (posterior) hasa tubelike structure that has an adhesive function. The colour and the size of the headand the pronotum, a sclerotized structure that is situated above the first pair of legs, arethe main characteristics to distinguish between the different developemental stages (seeFigure I.3.2). In Figure I.3.2, an identification key is given to recognize a specific larvalstage.

Figure. I.3.2: Identification key to distinguish the different developmental stages of L. decemlineata.Drawings from Bounhiol (1927)

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The time it takes an organism to complete a larval stage is dependent on the temperatureand photoperiod (optimal temperature: 30°C and optimal photoperiod; European andMediterranean Plant Protection Organisation (2004)). It varies from 2.5 days to 4 daysfor the first three stages and the last stage can take up to 9 days. In total, a duration of13.5 to 21 days (Boiteau & Blanc, 1992; European and Mediterranean Plant ProtectionOrganisation, 2004).

Figure. I.3.3: Larval stages of L. decemlineata. (A) first-instar, (B) second-instar, (C) third-instar,(D) fourth-instar

L1 Stage

When the larvae emerge from their eggs, they are called neonates. They are av. 1.5 mmin length with a black head and pronotum. In this stage, they stay mostly around the eggmass and cause little damage to the plant.

L2 Stage

In this stage, the larvae are av. 3 mm in length with a small head and black pronotum.They are dispersed on the plant, but the damage done is still small.

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L3 Stage

In this stage, the larvae are av. 5 mm long. The head is clearly bigger and the pronotumis not full black (see Figure I.3.2 and I.3.3). The larvae eat non-stop and the damage issevere.

L4 Stage

The fourth-instar larvae are av. 8 mm in length. This stage is clearly recognized, becausethe pronotum is more orange than black (see Figure I.3.3). These larvae, together withthe L3 larvae, do significant damage to the leaves of plants.

3.3.3 Pupal Stage

At the end of the fourth-instar stage, the larvae burrow themselves into the soil. Thepre-pupal stage can take as long as the L4 stage. The larvae are called pupae when amoult encapsulates them. This stage is called the metamorphosis and takes 5 to 7 daysbefore the transformation into adults takes place. In this stage, the sex of the beetle canbe determined (Boiteau & Blanc, 1992).

Figure. I.3.4: Pupa of L. decemlineata

3.3.4 Adult Stage

After the metamorphosis, adult beetles emerge from the soil. Their body is av. 10 mmin length. The elytron shows a characteristic pattern with 10 black stripes on a yellowbackground. In the adult stage, the beetles cause severe damage to plants. Copulation andsubsequent deposition of eggs is their most important occupation all year round, except

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for the winter. The adult beetles hibernate in the soil down to 10 cm under the surface.When temperature conditions get better (68 days a temperature above 10.5°C (Europeanand Mediterranean Plant Protection Organisation, 2004)), they emerge from the soil, feedand deposit their eggs on the plants.

Figure. I.3.5: Adult of L. decemlineata

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Chapter 4

Targets of Systemic RNAi in L.decemlineata

To determine whether systemic RNAi is present in L. decemlineata or not, targets needto be selected. The requirements for a good target are: not expressed in the midgut,expressed in a sufficient amount to pick up with Polymerase Chain Reaction (PCR) andpreferably already sequenced and recorded in a nucleotide database (e.g., National Centerfor Biotechnology Institute (2010)). The three targets that are selected in this thesis are:dopamine transporter protein (DAT), chitin synthase 1 (ChS1) and glycine-rich protein 1(GRP1).

4.1 Dopamine Transporter Protein (DAT)

Dopamine is a catecholamine present in high levels throughout the insect central nervoussystem (CNS). DA-ergic neurons play a role in following processes (Caveney & Donly,2002; Donly & Caveney, 2005):

• Modulation of visceral and skeletal muscle contraction

• Salivary gland secretion in many insects

• Operation of the optic tract, protocerebrum and deutocerebrum in the brain

The DAT protein is a Na+/Cl− dependent, membrane-spanning, monoamine neurotrans-mitter transporter. Neurotransmitter transporters recycle the mono-amines that are re-leased at neural synapses during neural signaling (Donly & Caveney, 2005). They arepresent in neurons all over the CNS.

4.2 Chitin Synthase 1 (ChS1)

Chitin is an extracellular biopolymer or β-1,4-linked polymer of N-acetyl-glucosamine(GlcNAc) that is abundant in the biosphere. Fungi and arthropods are the principalproducers. Chitin is synthesized by chitin synthases (ChS), which are located in theplasma membranes. ChS catalyzes the β-1,4 linkage between GlcNAc molecules suppliedby the cytoplasm, thereby forming chitin fibers. The localization of the ChS ensures thatthe formed homopolymer is deposited in the extracellular matrix (Arakane et al., 2004;Zeigerman et al., 2007). In the extracellular environment of insects, chitin is essential forthe structural integrity of the exoskeletal cuticle and midgut peritrophic membrane (PM).These two locations are covered by two different genes that code for proteins with thesame enzyme function, in Tribolium castaneum (Arakane et al., 2004, 2008):

• ChS1 or ChSA is responsible for the synthesis and deposition of chitin in the cuticularmatrices

• ChS2 or ChSB is responsible for the production of the chitinous component of PMs

Oscillating transcription patterns of ChS are observed. Two or three days before larvaemoult, a maximum presence of ChSs is seen in the integumental tissues. At the formation

19


of a new cuticle, a decline in transcription takes place. These oscillations coincide withchitin formation and deposition in the exoskeletal structures (Zeigerman et al., 2007).

4.3 Glycine-Rich Protein 1

Glycine-rich proteins are a large group of proteins that are characterized by a high contentof glycine residues. This type of protein is abundant in a wide variety of organisms, butwith exception of the glycine-rich domains, they are not well-conserved. Therefore, puttingthem in one family is debatable (Sachetto-Martins et al., 2000).Different types of GRPs are found in insects in characterizing regions:

• Structural cuticle GRPs can be recognized by the signal peptide and glycine-richregions that are usually located at the termini (N- and/or C-terminus) (Andersenet al., 1995)

• Cuticular GRPs contain short conserved GXGX, GGXG or GGGX glycine-rich re-peats. These can form flexible coiling structures (Zhang et al., 2008) and often aconserved AAPA/V motif is present (Nøhr & Andersen, 1993; Andersen et al., 1995)

• Other GRPs are intracellular proteins related to RNA binding. The difference fromthe structural GRPs is the ribonucleoprotein consensus sequence (Mousavi & Hotta,2005; Zhang et al., 2008)

GRP1 in L. decemlineata is a 104 amino acid (AA) long cuticular protein. It is expressedmainly in epidermal tissues and occurs at different growth stages, but mostly it relates tothe moulting process (Kim BY, 2005; Zhong et al., 2006; Zhang et al., 2008). In situationswith induced environmental stress (pesticide and drought), an upregulation of the GRPexpression is noticed. This results in changes in the cuticle that have an impact on stressresistance (Zhang et al., 2008).

20

Part II

OBJECTIVES

21

Part II: Objectives

Since the discovery of RNAi, the mechanism has rapidly established itself as a tool inresearch and work is done to bring applications to daily life (e.g., Baum et al. (2007);Tiemann & Rossi (2009); de Franca et al. (2010)). One of these applications is the use ofRNAi in crop protection. When comparing RNAi with the chemical crop protection tech-niques that are in use at the moment, it is seen that pesticides are often efficient, but theyalso have several disadvantages. Toxic residues in food products, toxicity to non-targetspecies and pesticide resistance in the target species are only a couple of examples (Price& Gatehouse, 2008). RNAi on the other hand, would have the advantage of specificity,which eliminates non-target toxicity and toxic residues, and robustness. Resistance tothe RNAi mechanism is harder to accomplish, because all the siRNAs together, producedfrom one dsRNA, recognize a target sequence over the full length. When one geneticallymodifies a plant to express dsRNA, beetles or other insects that feed on this plant, ingestthe dsRNA, which is taken up from the midgut lumen by the cells lining the midgut.When the dsRNA is present in the cells, the RNAi machinery will induce silencing of thegene, targeted by the dsRNA. If the target is chosen properly, this leads to the deathof the beetle or insect. Since the target organism of this thesis, L. decemlineata, is thelargest insect pest on potato plants with high levels of insecticide resistance, RNAi couldprovide a more sustainable alternative (European and Mediterranean Plant ProtectionOrganisation, 2004; Casagrande, 1987; Weber, 2003; Alyokhin, 2009).

The use of cell autonomous RNAi in the midgut for pest control, was already studiedon a variety of insects, including L. decemlineata (e.g., Baum et al. (2007); Mao et al.(2007); Zhu et al. (2011). Knowledge on the presence of a systemic mechanism of RNAiin L. decemlineata on the other hand, is not available and would open the door to a wholenew collection of potential target genes. The general aim of this master thesis is to eval-uate the possibility of systemic RNAi in L. decemlineata, using an in vivo assay.Following steps had to be taken:

• Since working with living organisms is never 100% controllable, it is important thata good bio-assay is developed

• It is important that target genes, that are not expressed in cells of the midgut, areselected. This is important to distinguish between cell autonomous and non-cellautonomous RNAi. The targets that are selected, are presented, schematically, inFigure II.1 with their location.

• To observe differences in expression of these targets, optimization of the detectionmethod using PCR is necessary. Sensitive primers need to be designed and PCRconditions need to be determined to pick up the target genes in the best possibleway.

• When the steps described above are brought together, the actual in vivo assay, usingdsRNA, is performed. Since we want to see differences on a morphological level andon the level of gene expression, we will use two parallel experiments.

These experiments will help to answer the main research question of this master thesis:”Is there systemic RNAi in L. decemlineata?”.

22

Part II: Objectives

Figure. II.1: Schematic drawing of the location of the targets of systemic RNAi used in this work

23

Part III

MATERIAL AND METHODS

24

Chapter 1

Breeding of L. decemlineata

Performing scientific sound experiments with living insects, is a tedious work that requiresa strong and healthy population. The population of L. decemlineata is maintained at thelaboratory of Agrozoology in breeding cabinets that are kept at a constant temperatureof 25°C, a humidity of 50% and a photoperiod of (16:8, Light:Dark).In the cabinet, at least five different containers were present. The largest one, containedtwo potato plants (Solanum tuberosum, variety: ”Bintje”) and the adult beetles. Everyother day, leafs with deposited eggs were taken from this container and placed in petridishes. These petri dishes were closed, with custom made lids that ensured a sufficientsupply of fresh air. When the neonates emerged from the eggs, the petri dish was placedin a larger container, in which a sufficient amount of fresh folliage was present. The larvaewere kept in this container, until they reached the adult stage and were fed every otherday, with a sufficient amount of fresh foliage.

25

Chapter 2

Bio-assay

In a bio-assay, the effect of a treatment is tested on living organisms. In order to havereliable and repeatable results, several variables have to be optimized. During the opti-mization, it was tested if and how these variables influenced the normal development ofthe L. decemlineata larvae. After the optimization, the actual feeding experiment couldbe performed.

2.1 Optimization

Four different treatments were tested on first instar larvae in two different conditions:neonates, hatched within 24h prior to the treatment and 24h starved neonates. In thelatter case, neonates were removed from the eggs, immediatly after hatching, and placedin a plastic petri dish without food, 24h prior to the start of the experiment.The four treatments were:

• 0.1 mM Na2PO4 buffer, which is an alternative dsRNA elution buffer

• Ultrapure water (MQ)

• No fluid

• Starvation: Only feeding on the first day (no feeding)

A transparant, 18 compartment storage box was cleaned with desinfectol (Chem-Lab N.V.,Belgium, Ref. No. CL00.0112.2500) and used as a growing chamber for the larvae. A 2×2mm fresh piece of potato plant leaf is stuck to the bottom of a compartment with 1 µl ofMQ. On top of the leaf, 1 µl of buffer, MQ or nothing, in case of the other two treatments,was pipetted. A neonate or a 24h starved neonate, was placed on top of the leaf, with thehead in the fluid (see Figure III.2.1). Caution was needed, to prevent injury to the larva.The storage box was covered with Parafilm M (Bemis Flexible Packaging, USA, Ref.No.PM996), to prevent the larvae from escaping.

26

Part III: Material and Methods

Figure. III.2.1: Schematic drawing of a compartement of a box for the bio-assay

Mortality and the growth of the larvae was followed daily, using three parameters: length,width and life stage. Whether a larva was dead or not was determined, using the parame-ters: movement and feeding. When both of these parameters scored negative, a larva wasconsidered to be dead. After each observation, remnants of the feeding of the previousday (not applicable to the no feeding group), were removed and fresh potato plant leafswere given, according to the needs of the larvae. The observations were stopped after alllarvae were dead or after they reached fourth larval stage.

2.2 Experimental Setup

The experimental setup can be seperated in two parts: a insect physiologic experimentand a molecular experiment.

2.2.1 Insect Physiological Experiment

The observational experiment was aimed at finding a correlation between a treatment anda phenotypical effect. Here, dsRNA of the target gene is the product that was tested.Three treatments were compared:

• dsRNA of the target gene, in Na2PO4 buffer

• dsGFP in Na2PO4 buffer, as a negative control (NC) for the toxicity of the dsRNA(a non-species sequence)

• 0.1 mM Na2PO4 buffer, which is an alternative dsRNA elution buffer, as a NC

This setup is performed, as explained in Section 2.1, with the exception that 15 larvae areused, instead of 10 larvae, to increase statistical significance. Neonates after 24h starvationare used for these experiments.

2.2.2 Molecular Experiment

In this experiment, changes in the expression profile of the target gene were examined.In this setup, every box has to be considered as a collection of 3 × 5 larvae. The same

27


treatments, as in Subsection 2.2.1, were used and the start-up is identical (see Section2.1).Ten RNase-free 1.5 ml microcentrifuge tubes, were filled with 100 µl of TRI reagent (SigmaAldrich, U.S.A., Ref. No. T9424) and their mass was measured on an analytical balance(Sartorius, Germany, Ref.No. B 120 S). The samples consisted of five larvae, that werecollected at four points in time:

• D0: the start of the experiment

• D1: after 24h

• D3: after 72h

• D6: after 6 days

Table. III.2.1: Samples collected in the molecular experiment

Point in time Treatment

D0 NAD1 Buffer

dsGFPdsRNA

D3 BufferdsGFPdsRNA

D6 BufferdsGFPdsRNA

The samples were weighted and the mass was compared to the original mass, recordedprior to the experiment. The samples were stored at -20°C, to avoid loss of RNA, asrecommended by Sigma Aldrich (2009).Complementary DNA (cDNA) synthesis was performed on the samples, according to Chap-ter 6. Control of the quality and concentration of cDNA, was performed by PolymeraseChain Reaction (PCR) with primers amplifying the household gene, rpL32 (see Chapter7 for mastermix and Table III.7.3 for the primers). Semi-quantitative PCR with 28, 30,32 and 34 cycli can be performed, to compare the expression profiles in each sample (SeeChapter 7.4).

2.3 Statistic Analysis of Data

Since the conditions for parametric analysis (normal distribution, equal number of samplesizes in the different groups, . . . ) were not fulfilled, a non-parametric Kruskal-Wallis one-way analysis of variance by ranks was performed (Siegel & Castellan, 1988; Weaver, 2002).When the null-hypothesis, no difference, was rejected (p = 0.05) the Dunn test was usedas a post-hoc analysis. The statistics were calculated in R (Version 2.10.0 (R DevelopmentCore Team, 2009)).

28

Chapter 3

Dissection of L. decemlineata

3.1 Dissection of the Heads of L4 larvae

The heads of five L. decemlineata L4 larvae were seperated from the body under sterileconditions. The larvae were anaesthetized with and desinfected in 70% desinfectol prior tothe decapitation. The removed heads were rinsed in IPS (see Subsection 4.1) and storedin 300 µl TRI Reagent at -20°C until cDNA synthesis (see Chapter 6). This temperaturegaranties a minimum of RNA degradation during storage (Sigma Aldrich, 2009).

3.2 Dissection of the Midgut

This dissection was performed on five L4 larvae and five adult specimen. The organismswere anaesthetized and desinfected in 70% desinfectol. A small incision was made in theposterior end of the specimen and the head was cut off, hereby detaching the gut from theepidermis. When applying a small amount of pressure to the abdomen, the gut could bepulled out and hindgut, malphigian tubes, trachea and perithropic matrix were removed,as much as possible. During this process, the tissues were rinsed repeatedly in fresh IPS.The dissected midguts were put in 1 ml TRI Reagent and stored at -20°C until cDNAsynthesis (Chapter 6).

3.3 Dissection of Six Different Tissues

The dissection of carcass, trachea, midgut, heads, neural tissue and fat body was performedon L4 larvae. They were anaesthetized and desinfected in 70% desinfectol. The heads wereseperated from the body and placed in 200 µl of TRI Reagent. A dorsal incision was madein the length of the larve. The midgut was taken out, prepared and stored in 100 µl TRIReagent, as described in Section 3.2. Trachea, fat body and ventral nerve cord were takenout, rinsed in IPS and stored in 100 µl TRI Reagent. The carcass tissue consisted of theempty remnants of the larvae. This was also stored in 200 µl TRI Reagent. All tubes werestored at -20°C until cDNA synthesis (Chapter 6).

3.4 Dissection of L2 Larvae in Ecdysis

Seven larvae were taken during ecdysis. No anaesthesy was applied, because the exoskele-ton is soft at this point. The insects were dissected as described in Section 3.2. Insteadof keeping the gut, the gutless remnants were rinsed in IPS and stored in 200 µL TRIReagent at -20°C until cDNA synthesis (Chapter 6).

29

Chapter 4

Buffers

4.1 Insect Physiological Buffer (IPS) pH 7.0

Table. III.4.1: Composition IPS pH 7.0

Component Concentration (in g l−1)

KCl 2.50MgCl2 · 6H2O 4.10MgSO4 · 7H2O 4.84K2HPO4 0.92Sucrose 25.60CaCl2 · 2H2O 0.1468

The buffer was filter sterilized using a 0.2 µm filter pore size (Nalgene Filtration Products,Nalge Nunc International corp., U.S.A., Ref.No. 514-0027).

4.2 TRIS-EDTA (TE) Buffer pH 8.0

Table. III.4.2: Composition 1X TE stock solution pH 8.0

Component Amount

TRIS 20 mlEDTA (0.5 M) 2 mlDemineralized water 988 mlpH set with HCl

4.3 TRIS-Acetic Acid-EDTA (TAE) Buffer pH 8.0

Table. III.4.3: Composition 50X TAE stock solution pH 8.0

Component Amount

TRIS 60.5 gAcetic Acid 14.3 mlEDTA (0.5 M, pH 8.0) 25 mlDemineralized water 250 mlpH set with NaOH

30

Chapter 5

Agarose Gel Electroforese

5.1 1.5% Agarose Gel Preparation

3.75 g of Electrophoresis Grade Agarose (Invitrogen, U.K., Ref.No. 15510-027) is dissolvedin 250 ml of 0.5X TAE buffer (Composition see Section 4.3).

5.2 Sample Preparation

For each well 5 µl of sample was mixed with 2 µl of 6X Mass Ruler loading Dye (FermentasLife Sciences, Germany, Ref.No. R0621). As a reference 6 µl of Mass Ruler DNA ladderMix (Fermentas Life Sciences, Germany, Ref.No. SM0403) was used. The gel was thensubmersed in 0.5X TAE buffer and ran at 100 V in a Mupid-One electrophoresis system(Advance, Japan, Ref.No. 070648 and 071044). The gel was stained for at least 20’ inEthidium Bromide (EtBr) (40 µl EtBr in 400 ml 0.5X TAE).

5.3 Photography of the Gel

A photograph was taken with a digital camera (Bio-Rad laboratories, U.S.A., Gel DocXR+) and the quantity One Software (version 4.5.2, Bio-Rad laboratories, U.S.A.)

31

Chapter 6

cDNA Synthesis

6.1 Total RNA Isolation

Total RNA, of the whole insect or dissected tissues (see Chapter 3), was extracted usingthe TRI Reagent method, according to the technical bulletin (version June 2009) (SigmaAldrich, 2009). First, the tissue was homogenized in TRI Reagent using a sterile pestle.Then incubated at room temperature (RT) for 5’ and centrifuged (12,000×g for 15’ at4°C). The supernatant was transferred to a fresh tube and 200 µl of chloroform (minimum99%, Sigma Aldrich, Germany, Ref.No. 018K0137) was added per ml TRI Reagent used.The mixture was shaken vigorously and incubated (2-15’ at RT). After centrifugation(12,000×g for 15’ at 4°C), the aqueous layer was transferred to a fresh tube. Per ml ofTRI Reagent, 500 µl of isopropanol (Fluka BioChemica, The Netherlands, Ref.No. 59304)was added. The mixture was shaken and centrifuged (12,000×g for 10’ at 4°C). The pelletwas washed with 1 ml of 75% ethanol (Chem-Lab N.V., Belgium, Ref.No. CL00.0505.2500)and after centrifugation (12,000×g for 5’ at 4°C), the pellet was air-dried (5-10’ at RT or 5’at 37°C). When the pellet was completely dry, 25-50 µl of MQ was added and incubated (5’at 55°C). Using the NanoDrop ND-1000 spectrophotometer (Thermo Scientific, U.S.A.),the RNA concentration, based on 260/280 nm and 260/230 nm ratios, was determined.

6.2 DNase Treatment

The reaction was set up, as described in the TURBO DNA-free kit (Ambion, U.S.A.,Ref.No., AM1907) protocol (version April 2009) (Ambion, 2009).

Table. III.6.1: Composition of the TURBO DNA-free kit reaction

Component Amount (in µl)

TURBO DNase (2 Units µl−1) 110X TURBO DNase Buffer 2.5RNA equivalent 1 µgNuclease-free water to 20

This reaction was incubated for 30’ at 37°C. 2.5 µl DNase Inactivation Reagent was addedto stop the reaction. The mixture was incubated (5’ at RT) with occasional mixing. Aftercentrifugation (10,000×g for 2’ at 4°C), the supernatant was transferred and the RNAconcentration was measured using the NanoDrop ND-1000 spectrophotometer.

6.3 RNA Quality Control

The quality of the extracted RNA was tested by loading 1 µg subsamples, taken beforeand after the DNase treatment, on 1.5% agarose gel. The RNA quality was good whenthe two bands of 18S (± 2kb) and the 28S (± 5kb) ribosomal RNA were visible (Ambion,2001).

32


6.4 First Strand cDNA Synthesis

The SuperScript II Reverse Transcriptase kit (Invitrogen, U.S.A., Ref.No. 100004925) wasused and the protocol (version November 2003) was followed (Invitrogen, 2003).

Table. III.6.2: Composition SuperScript II Reverse Transciptase reactions


Sample No TemplateControl(NTC)

No ReverseTranscriptaseControl(NoRTC)

Oligo (dT)12(500 µg ml−1)

1 1 1

dNTP mix (10mM)

1 1 1

RNA equivalent 1 µg 0 equivalent 1 µgNuclease-freewater

to 12 10 to 12

The mix was heated in the Labcycler for 5’ to 65°C and chilled on ice. 4 µl of 5X First-Strand Buffer and 1 µl of 0.1 mM DDT were added. The samples were incubated for 2’ at42°C. 1 µl of SuperScript II Reverse Transcriptase was added to all tubes, except for theNoRTC, and the mix was incubated for 50’ at 42°C. Inactivation was obtained by heatingthe mixture for 2’ to 70°C. The obtained cDNA can be diluted and used for PCR reactions(see Chapter 7).

33

Chapter 7

Polymerase Chain Reaction

7.1 Primer Design

7.1.1 Standard Primer Design

In standard primer design, the process starts with a sequence obtained from the NationalCenter of Biotechnology Information (NCBI) database (National Center for BiotechnologyInstitute, 2010). The online primer design tools: Primer3 (Rozen & Skaletsky, 1998) andFinnzymes multiple primer analyzer (Finnzymes, 2010), were used. The primers wereordered at Invitrogen and kept as stock solution of 100 µM in TE buffer (Section 4.2) andas a working solution of 10 µM in MQ.

7.1.2 Degenerated Primer Design

In degenerated primer design, the sequence of the target gene, is not yet sequenced forthe species of interest. Several different protein sequences were analysed for conservedregions. In these regions, primers were designed, based on the translated or available DNAsequences. The DNA sequences had codons that coded for the same amino acids, but oftenthey varied in the second or third nucleotide of the codon. Primers were designed withdegenerated nucleotides, resulting in a mixture of primers, which maximizes the chancethat one of these primers is a perfect match to the target sequence (Stanley, 2005). Afteranalysis using the Finnzymes analyzer, primers were ordered at Invitrogen, stored as stocksolutions of 100 µM in TE buffer and used as working solutions of 10 µM in MQ.

7.2 PCR Mix

20 µl PCR reaction volumes were used. Each reaction contained the following components(Invitrogen, U.S.A., Ref.No. L0342-020):

Table. III.7.1: Composition of a PCR reaction


H2O 12.910X PCR buffer 2MgCl2 (50 µM) 0.6dNTP mix (10 µM) 0.4Forward Primer (10 µM) 1Reverse Primer (10 µM) 1Taq DNA Polymerase (5 U µl−1) 0.115 cDNA/Negative Control/Positive Control 2

The NC is MQ. In some cases also a positive control (PC) is used. The PCR reactionsare performed using a labcycler (SensoQuest, Germany, Ref.No. 011-101). The PCRprograms used, are listed in Table III.7.4.

34


7.3 PCR Optimization

The first step in the optimization of the PCR reaction was a gradient PCR, to obtain theoptimal annealing temperature for the given primers. The temperature range that wasprogrammed was based on the primer’s melting temperature (TM ), given by the primerdesign tool, minus 5 degrees (see Table III.7.4). For the gradient PCR, a TM − 5± 3 wasset.When a low efficiency was observed, different primer concentrations were tried (0.5x, 2x).

7.4 Semi-Quantitative PCR

In a semi-quantitative PCR, a PCR reaction is performed and stopped at 3 or 4 differentnumbers of cycli. This was performed by pausing the labcycler after the number of cyclithat were wanted. The PCR products were loaded on gel to compare the amounts of DNAamplified after each cycle number.A visual score was given, based on the intensity of the bands on the photograph of thegel:

• -: no visible amplification

• ±: weak band

• +: clear band

• ++: strong band

7.5 Long Distance PCR

Long DNA fragments (≥ 0.5 kb), that are hard to amplify with normal PCR, can be ampli-fied using the Expand Long Template PCR System (Roche Applied Science, Switzerland,Ref.No. 11 681 834 001). The reaction was performed, as described in the protocol(Version August 2009). One reaction consists of:

Table. III.7.2: Composition of a Long Distance PCR reaction (for 0.5 - 9kb)


H2O 29.5Expand Long Template Buffer 1 (37°C) 5dNTP mix (10 µM) 1.75Forward Primer (10 µM) 1.5Reverse Primer (10 µM) 1.5Expand Long Template Enzyme mix (5 U µl−1) 0.75Template 1

5 cDNA 10

The program used, is listed in Table III.7.4.

7.6 Colony PCR

Colonies were picked up, with a sterile tooth pick, and rinsed in 10 µl MQ. A PCR reactionwas set up, using a standard PCR mix (see Table III.7.1) with SP6 and T7 primers. ThePCR program used, is listed in Table III.7.4. Fragment size was checked on 1.5% agarose

35


gel. Fragments should have been 155bp longer than the insert (65bp of T7 + 90bp ofSP6).

7.7 Amplifying Linearized Plasmids DNA

Starting from linearized plasmids, 1/50 up to 1/2000 dilutions were made for amplificationusing PCR. The mastermix was the standard mix, as described in Table III.7.1, andprimers, designed to contain a T7 fragment (5’-TAA TAC GAC TCA CTA TAG GG-3’)at the 5’-end, were used (see Table III.7.3).

7.8 PCR Product Purification

PCR products were purified using the E.Z.N.A. Cycle Pure Kit (Omega Bio-Tech Inc.,U.S.A., Ref.No. D6492-02). The Cycle-Pure spin protocol (version May 2007) was fol-lowed. Five volumes of CP Buffer were added to the total PCR product and the mixturewas vortexed thoroughly and spinned down. The mixture was applied to the HiBind MiniColumn. Subsequently, the column was centrifuged (13,000×g for 1’ at RT). The flow-through was discarded and the column was washed twice with, respectively, 700 µl and500 µl DNA Wash Buffer and centrifuged (13,000×g for 1’ at RT). The column was com-pletely dried by centrifugation (maximum speed for 2’ at RT). DNA was eluted with 30-50µl of MQ to the center of the column and subsequently centrifuged (13,000×g for 1’, atRT). The concentration of the purified PCR product was determined with the NanoDropspectrophotometer and the product was checked on 1.5 % agarose gel.

7.9 Overview of Primers

36

Tab

le.

III.

7.3:

Pri

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sed

du

rin

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this

work

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eg.:

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NA

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CC

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CC

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40


7.10 PCR programs

Table. III.7.4: PCR programs

PCR name Program Usage

Colony PCR 3’ 94°C; 25 × (30s 94°C; 30s55°C; 30s 72°C); 7’ 72°C; ∞4°C

Colony PCR: Section 7.6

Long Distance PCR 2’ 92°C; 10 × (20s 92°C; 30sTA; 1.5kb min−1 58°C); 30 ×(25s 92°C; 30s TA; 4’+20s percyclus 58°C); 7’ 58°C; ∞ 4°C

Amplification of long frag-ments: Section III.7.2

PCR optimization 3’ 94°C; 35 × (30s 94°C; 30s50 5°C; 30s 72°C); 7’ 72°C; ∞4°C

Primer optimization: Section2.1

Purification 3’ 94°C; 35 × (30s 94°C; 30sTA; 30s 72°C); 7’ 72°C;∞ 4°C

Purification with optimal TA

for DAT and GRP1 primerpairs: See Table III.7.3

Purification T7 3’ 94°C; 5 × (30s 94°C; 30s58°C; 30s 72°C); 30 × (30s94°C; 30s 68°C; 30s 72°C); 7’72°C; ∞ 4°C

Purification of T7 fragments:Section 7.7

RT PCR 5’ 65°C; 2’ 42°C; 50’ 42°C; 15’70°C; ∞ 4°C

Reverse Transcription: Sec-tion 6.4

41

Chapter 8

Cloning

8.1 Ligation of PCR Product in a Cloning Vector

The ligation of the purified PCR product (see Section7.8) was done according to thepGEM-T Vector System (Promega, U.S.A., Ref.No. A3600) protocol (Version December2010), except that half the reaction volume was used. The amount of PCR product needed,was determined by following formula:

ng of vector× kb size of insert

kb size of vector× insert:vector molar ratio = ng of insert

The ratio used in this work was 3:1. This gave following mix:

Table. III.8.1: Composition of the pGEM-T Vector System ligation mix


2X Rapid ligation Buffer. T4 DNA ligase 2.5pGEM-T or pGEM-T Easy Vector (50 ng) 0.5T4 DNA ligase (3 Weiss units µl−1) 0.5PCR product See formulaNuclease-free water To 5 µl

This mixture was incubated for 1 hour at RT.

8.2 Heat-Shock Transformation of Escherichia coli

The reaction volume from the ligation was added to 40 µl of competent cells. This mixturewas incubated for 30’ on ice. The cells were heat shocked for 50s at 42°C, in a heating block(Eppendorf, U.S.A.) and subsequently incubated (2’ on ice). 250 µl of preheated liquidLuria Bertani (LB) medium (Duchefa Biochemie, The Netherlands, Ref.No. L1703.0500)was added and this mixture was incubated (45’ at 37°C, shaking). 100 µl of a 1x and 100µl of a 0.1× dilution, were plated on solid LB plates (1.5% agar)(Duchefa Biochemie, TheNetherlands, Ref.No. L1705.0500), supplemented with 100 µg ml−1 carbenicillin (Sigma-Aldrich, Germany, Ref.No. C1613). These plates were incubated (overnight at 37°C) andthose with the optimal colony density were used.

8.3 Selection of the Colonies

Per plate, 10 single colonies, from small to larger, were picked up with a sterile tooth pickand transferred to a master plate of solid LB (1.5% agar) with 100 µg ml−1 carbenicillin.This plate was incubated overnight at 37°C.

8.4 Growing of the Selected Colonies

From the colonies that showed a band of the appropriate length on gel after colony PCR,three colonies were selected. These colonies were picked from the master plate and trans-

42


ferred to 3 ml liquid LB medium, with 100 µg ml−1 carbenicillin. These solutions wereincubated overnight, at 300 rpm at 37°C, in non-air tight tubes.

8.5 Plasmid Recovery and Purification

The E.Z.N.A. Plasmid Miniprep Kit (Omega Bio-Tek, U.S.A., Ref.No. D6943-02) wasused, according to Protocol I (version July 2008), to obtain purified plasmids from thetransformed E. coli.After centrifugation (10,000×g for 10’ at RT), the bacterial pellet was resuspended com-pletely, by adding 250 µl of Solution I/RNase A solution and vortexing thoroughly. Themixture was transferred to a 1.5 ml microcentrifuge tube and 250 µl of Solution II wasadded, to obtain a clear lysate. 350 µl of Solution III was added and a flocculent whiteprecipitate formed. After centrifugation (maximum speed for 10’ at RT), a compact whitepellet formed. The cleared supernatant was added to a prepared HiBind DNA MiniprepColumn. The preparation consisted of adding 100 µl of Equilibration Buffer and subse-quent centrifugation (maximum speed for 1’) in a 2 ml collection tube. The lysate waspassed through the column by centrifugation (maximum speed for 1’). 500 µl of BufferHB was added, to wash the column. After centrifugation (maximum speed for 1’), thecolumn was washed twice with 700 µl of DNA Wash Buffer. The column was centrifuged(maximum speed for 1’) and dried by centrifugation (maximum speed for 2’). This is acritical step for a good yield. The column was placed in a 1.5 ml microcentrifuge tubeand 75 µl of sterile MQ was added, directly on the column matrix. After centrifugation(maximum speed for 1’), purified plasmids were obtained.The concentration and quality of DNA was determined, using the NanoDrop spectropho-tometer.

8.6 Linearisation of the Plasmid

The pGEM-T Vector contains multiple restriction sites (see Figure III.8.1). The restrictionenzym chosen, should have no restriction sites inside the target sequence. The digestionwas performed, as described in the protocol accompanying the restricion enzymes (Fer-mentas Life Sciences, Germany). In this research, SalI (Fermentas Life Sciences, Germany,Ref.No. ER0641) was used and the reaction was set up, following the Fermentas protocol(version July 2010) (see Table III.8.2).

Table. III.8.2: Composition of the restriction reaction for SalI


10X buffer O 2SalI (10 U µl−1) 1DNA equivalent 1 µgNuclease Free water To 20 µl

This mixture was incubated for 30’ at 37°C. Inactivation occured by heating for 20’ at65°C. The linearised fragment was purified, according to Section 7.8, and could be usedas a template in cDNA synthesis.

43


Figure. III.8.1: Map of the pGEM-T Vector (Promega, 2008)

44

Chapter 9

Sequencing

To confirm the sequence of a PCR product, direct sequencing was performed. The PCRproduct was purified using the method described in Section 7.8. Two tubes were preparedwith 10 µl of PCR product, at a DNA concentration of 10 ng µl−1 (for fragments ≤500bp)or 20 ng µl−1 (for fragments ≥500bp). 4 µl of 5 mM forward primer was added to onetube and 4 µl of 5 mM reverse primer to the other.To confirm the insert in a plasmid, two times, a 1.5 ml microcentrifugation tube was filledwith 10 µl of DNA at 100 ng µl−1. No primers should be added, the vector’s SP6 and/orT7 sequences could be used.

The sequencing was performed by AGOWA-LGC Genomics and consensus sequences werederived with BioEdit software (version 7.0.5.3). With BLAST analysis, the consensussequence was checked against the NCBI nucleotide database.

45

Chapter 10

Double-stranded RNA Synthesis

10.1 MegaScript RNAi Kit

The dsRNA synthesis was performed using the MEGAscript RNAi Kit (Ambion Inc.,U.S.A., Ref.No. 1626) protocol (version 0502).

10.1.1 Transcription Reaction

The transcription reaction consisted of:

Table. III.10.1: Composition of the transcription reaction for dsRNA synthesis


10X T7 Reaction Buffer 2ATP solution (75 mM) 2CTP solution (75 mM) 2GTP solution (75 mM) 2UTP solution (75 mM) 2T7 Enzyme Mix 2linear template DNA Eq. 1 µgNuclease Free water To 20 µl

This reaction was incubated for 4h at 37°C. After 3h and 4h, a 0.5 µl subsample, in 1.5 µlTE Buffer, was taken to see if there is additional yield between 3h and 4h and for qualitycontrol on 1.5% agarose gel.

10.1.2 Annealing the RNA

The transcription reaction was incubated (5’ at 75°C) and left to cool (on the bench, noton ice), until it reached RT. After this step, a 0.5 µl subsample was taken and dilutedwith 49,5 µl of TE buffer for quality control on 1.5% agarose gel.

10.1.3 Nuclease Digestion to remove DNA and ssRNA

The nuclease digestion was performed, to remove the template DNA and ssRNA that didnot anneal.

Table. III.10.2: Composition of the nuclease digestion reaction for dsRNA synthesis


10X Digestion Buffer 5DNase I (2 U/µl) 2RNase I (2 U/µl) 2dsRNA 20Nuclease Free water 21

46


This reaction was incubated for 1h at 37°C. A 0.5 µl subsample, in 1.5 µl TE Buffer, wastaken for quality control on 1.5% agarose gel.

10.1.4 Purification of dsRNA

A binding mix was assembled with the following components:

Table. III.10.3: Composition of the dsRNA binding mix


10X Binding Buffer 50dsRNA 50Nuclease Free water 150100% Ethanol 250

This mixture was transferred onto the filter of the Filter Cartridge and centrifuged (max-imum speed for 2’). The cartridge was washed twice with 500 µl of Wash Solution andcentrifuged (maximum speed for 1’). The filter was dried by centrifugation (maximumspeed for 2’) and the dsRNA was eluted twice with 35 µl Elution Solution, preheated to95°C, in a fresh collection tube and centrifuged (maximum speed for 2’). 0.5 µl of samplewas diluted, with 4.5 µl of TE buffer, as a subsample for quality control on 1.5% agarosegel.The concentration of dsRNA, was determined using the NanoDrop spectrophotometer.The solution was stored at -20°C until further use.

47

Part IV

RESULTS

48

Chapter 1

Introduction

In the search for the answer to the initial research question: ”Is there systemic RNAi inL. decemlineata?”, several steps had to be taken.First, target genes had to be selected and amplification techniques needed to be optimized.These steps ensured a good synthesis of dsRNA and a specific method to identify the ef-fects of a treatment on transcription scale (Chapter 2).

Secondly, an experimental setup had to be developed. A feeding experiment was designedand optimized at two levels: an insect morphological and a molecular level (Chapter 3).When the optimal conditions were determined, the results of the final in vivo experimentswere presented.

The results, of each of the three in vivo assays of the effect of dsRNA on survival anddevelopment of larvae of L. decemlineata, can be divided into two blocks. An insect mor-phological part, in which mortality and developmental time are central, and a molecularpart, in which transcription levels of the target gene are compared (Chapter 4).

49

Chapter 2

Selection of Targets

2.1 Target Selection

The research asked for well-chosen targets. These targets had to be outside the midgut ofthe insect, to be able to link a possible effect to the systemic mechanism of RNAi.The first step, was to see which sequences were available for L. decemlineata on the NCBIwebsite (National Center for Biotechnology Institute, 2010). Two targets were selectedfrom the list, because literature described them to be tissue specific: the DAT gene (seeSection I.4.1) (Caveney & Donly, 2002) and the GRP1 gene (see Section I.4.3)(Zhanget al., 2008). A third target was picked up, because it was discussed extensively in twoarticles on T. castaneum, which gave a point of comparison (Arakane et al., 2004, 2008).This target is the ChS1 gene (see Section I.4.2). It must be noted that the sequence ofthis last gene is not known for L. decemlineata.This selection resulted in three possible target genes: DAT, ChS1 and GRP1.

2.2 Primer Design and PCR Optimization

To amplify the target genes with PCR reactions, good primers needed to be designed.This was essential to synthesize dsRNA and to evaluate gene expression, as a result in thefeeding experiments.

Primers were designed, based on the sequences obtained from NCBI and according toSection III.7.1. For ChS1 however, primers were designed for the T. castaneum sequence.All primer sequences can be seen in Table III.7.3.A 55±3°C gradient PCR was performed with a 1

5 L4 cDNA dilution, to obtain the optimalannealing temperature for primers for GRP1 and DAT. ChS1 needed a different PCRprogram that had a longer extension step (1’ instead of 30s).

• DAT: the amplification with P79 (5’-AAT GGT GGA GGT GCA TTT TT-3’) andP80 (5’-AAA CAG CTA GAA GGC ACA AAG C-3’), was insufficient. Differentprimer concentrations (0.5×, 2×, 5×) were tried, with no result.cDNA was made from the heads of L4 larvae (Subsection III.3.1), because the per-centage of CNS cells should be larger due to the presence of the brain. This gave aclear result for the DAT primers.The DAT primers were further optimized with a gradient PCR to obtain a optimalTA: 52°C

• ChS1: this target proved to be more challenging. First, primers were designed basedon the T. castaneum sequence. Different temperatures, primer concentrations andtemplate concentrations were tried. These attempts proved to be fruitless, so threedegenerated primer pairs were designed based on different known insect sequences(see Subsection III.7.1.2). These primers were tested extensively with different PCRproperties. This was not successful, so two degenerated primer pairs from the above-mentioned paper (Arakane et al., 2004), designed for T. castaneum, were ordered.The combination of P95 (5’-TGY GCN ACN ATG TGG CAY G-3’) and P96 (5’-

50

Part IV: Results

CCA RTG NCC DAT NGC RTA YTC RAA-3’) (see Table III.7.3) delivered a clearsignal (see Figure IV.2.1).With direct sequencing the sequence was obtained and gene specific primers, P101(5’-CGA TGT GGC ACG AGA CTA AA-3’) and P102 (5’-CAG CTC CGA TAGGAT GAT T-3’), were designed and tested with gradient PCR for an optimal TA:58°C

• GRP1: amplification with P77 (5’-ATG AAC ACT TTG GCC GTA GC-3’) andP78 (5’-ACA GAA CAA TTG GCA CAA TCA G-3’) gave a good result. The TA

was determined to be 52°C

In Figure IV.2.1 the results of the gradient PCRs are given for the three targets.

Figure. IV.2.1: Combined photograph of the gels of the gradient PCRs for GRP1, DAT, ChS1:P95-96 and P101-102. (52: TA 52°C; 55: TA 55°C; 58: TA 58°C).

2.3 Tissue Specificity of the Target Genes

Systemic RNAi can only be determined, when it was certain that the target genes involved,were not expressed in cells that had direct contact with the dsRNA food. In this case, thetarget genes should not be expressed in the cells of the midgut.

Tissue specificity was checked for the three targets. Two different approaches were used: anin silico approach, in which the L. decemlineata midgut transcriptome database (Huvenne,in prep.) was used, and an experimental method in which cDNA of six different tissues(carcass, trachea, midgut, heads, ventral nerve chord and fat body) was synthesized andused as a template.

2.3.1 In silico Analysis

In silico analysis was a relatively simple control of the abundancy of the expression ofthe target gene in the midgut. The aim of this analysis was to find if the literature wascorrect, regarding the tissue specificity.

BLAST analysis against the L. decemlineata midgut database made clear that:

• The DAT fragment contained a 90 bp fragment that matched significant (E ≤ 0.001)with the database and a new forward primer, P97 (5’-AAC ATG CTG GGG AAGATT GT-3’), was designed to exclude this fragment (see Table III.7.3).

• The ChS1 fragment gave several matches with the database and with ChS2. As de-scribed in Section I.4.2, ChS2 (or ChSB in this paper) is essential for the PM, while

51

Part IV: Results

ChS1 (or ChSA in this paper) has a role in cuticula formation for T. castaneum(Arakane et al., 2008). The primers used to pick up this fragment could not dis-tinguish between ChS1 of ChS2 and are of no use for testing the research question.Further, see Chapter 5

• GRP1 had no significant match and could be regarded as a target that is not ex-pressed in the midgut.

2.3.2 Experimental Analysis

To confirm the in silico analysis, experimental evidence was acquired by PCR on sixdifferent tissues with cDNA of whole bodies of fourth-instar larvae of L. decemlineata asa positive control (see Figure IV.2.2).

• DAT showed weak bands for all tissues.

• ’ChS1’ confirmed the in silico analysis. Bands were visible at midgut cDNA andcDNA of heads.

• GRP1 had a band at the carcass (weak), trachea (weak), heads (strong) and fatbody (strong) cDNA. Midgut cDNA gave no amplification, which confirmed the insilico analysis.

52

Part IV: Results

Figure. IV.2.2: Combined photograph of the gels of the PCRs with cDNA of six different tissuesfor GRP1, DAT and ’ChS1’. (C: carcass; T: crachea; M: midgut; H: heads; N:ventral nerve chord; FB: fat body; NC: negative Control; PC: positive Control)

In conclusion, it was observed that DAT was present, ’ChS1’ was clearly visible on geland GRP1 was not present in midgut tissue. This means that GRP1 is the only targetgene that can be used for further experiments, to determine the presence of a systemicmechanism for RNAi in L. decemlineata.

53

Chapter 3

Optimization of the bio-assay

In the optimization, it was tried to specify and eliminate the factors that could influencethe outcome of an observational bio-assay. This results in a representative experimentalsetup for doing the feeding experiments with dsRNA.As described in Chapter III.2, the buffer of the dsRNAs, ultrapure water and No Fluidtreatment of the food, was tested. Next to these three parameters, it was tested how longa neonate survived without feeding. This is useful to compare mortality due to starvationwith mortality, induced by treatments.

First, neonates were selected, from the egg mass, at the start of the experiment. Af-ter the first optimization experiment, the observation was made that the neonates do noteat the whole piece of leaf with the treatment. A second experimental setup was designed,where neonates were 24 hours starved before the start of the experiment, because theyare more likely to eat all the food with treatment, supplied at the start of the experiment.The aim of the experiment remained the same as for the neonates without starvation.

3.1 Neonates

As described in Section III.2.1, the experiment was set up with three treatments and oneno feeding box. 10 neonates were picked randomly per box. Two biological replicationswere conducted: the first experiment took place in October 2010 and the second in April2011.

Three parameters were checked daily (± 24 hour interval), until the larvae reached L4stage: body length, body width and remarks (life stage, death, . . .). Two types of datawere used for analysis. The first type contains the mortality and the second group focuseson the duration of life stages, which will be called sublethal effects.

3.1.1 Mortality

The mortality of 10 neonates was followed and is graphically presented in Figure IV.3.1.

• October 2010: A mortality of 10% for the buffer treatment was seen. The MQtreatment showed a mortality of 40% with one dead larva at day 6, 9, 12 and 17.The NF larvae showed one death at day 6 leading to a total of 10% mortality.

• April 2011: the buffer treatment showed one death at day 9. The MQ treatedneonates, all survived the experiment. One NF treated larva died at day 5 and oneat day 7. This resulted in mortalities of 10%, 0% and 20%, respectively.

54

Part IV: Results

Figure. IV.3.1: Accumulated percentage of mortality for neonates. Experiment of October 2010and April 2011. 4: Buffer, �: MQ, ◦: No Fluid

55

Part IV: Results

3.1.2 Duration of Life Stages

An important sublethal parameter is the duration of the life stages. In Table IV.3.1, theaverage duration of each life stage is given with the Standard Error of the Mean (SEM) foreach treatment. Statistical analysis is performed with a Kruskal-Wallis test and post-hocanalysis using the Dunn test at p ≤ 0.05 and p ≤ 0.1.

• October 2010: The L1 stage of buffer treatment was, statistically (p ≤ 0.05), shorterthan the NF treatment and the MQ treatment was, statistically (p ≤ 0.1), shorterthan the NF treatment . The duration of the L2, L3 and total to L4 stage was thesame for all treatments.

• April 2011: The duration of the L1 stage and L3 stage was, statistically (p ≤ 0.05and p ≤ 0.1 respectively), shorter for the buffer treatment than for the NF treatment.In the L2 and total to L4 stage, there was no significant difference between the threetreatments.

Table. IV.3.1: Average duration of life stages with SEM of optimization experiments with 10neonates. B: Buffer, MQ: Ultrapure water, NF: No fluid. a: significant differencewith buffer at p ≤ 0.05, b: significant difference with MQ at p ≤ 0.05, c: significantdifference with no fluid at p ≤ 0.05

October 2010 April 2011B MQ NF B MQ NF

L1 stage 3.70±0.30b 5.11±0.11a 4.30±0.26 5.10±0.28c 5.80±0.25 6.63±0.26a

L2 stage 3.78±0.40 3.25±0.31 3.22±0.30 3.44±0.29 3.10±0.18 3.63±0.46L3 stage 4.56±0.60 5.00±0.52 5.44±0.58 4.00±0.24c 3.40±0.16 3.00±0.00a

Total to L4 12.00±0.78 13.50±0.50 13.00±0.62 12.56±0.34 12.30±0.15 13.25±0.45

3.1.3 No Feeding

After an initial feeding, no food was given. This experiment sets a benchmark for mortalitydue to starvation of larvae of L. decemlineata. This can be useful to compare againsttreatments, who work on functional genes of digestion and absorption of nutrients. In thisexperiment, no larvae reached the second instar life stage. 50% of the larvae (LD50) weredead after 4 days and 100% after 5 days.

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Figure. IV.3.2: Accumulated percentage of mortality for the starvation experiment with 10neonates.

3.2 Neonates after 24 Hours of Starvation

As explained, it was noticed that the neonates seemed to have not eaten all of the food,given at the start of the experiment. When neonates were starved for 24h prior to thestart of the experiment, all food should be eaten. Again, three treatments were studied fortheir effect on mortality and duration of life stages. In this case, three biological replicateswere performed: November 2010, March 2011 and April 2011.

3.2.1 Mortality

The mortality of 10 neonates was followed and is graphically presented in Figures IV.3.3,IV.3.4 and IV.3.5.

Mortality for the buffer treatment was lowest at a total mortality of 50%, with one deadlarva at day 2, 9, 10, 12 and 19. The MQ treated larvae showed a mortality of 100% after14 days, with one dead larva on day 4, 5, 6, 7, 10, 11, 12 and two dead larvae on day 9 and14. The NF treatment was intermediate with a total mortality of 70%. One dead larvaon day 3, 7, 12, 14 and 22 and two dead larvae on day 11 were observed.

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Figure. IV.3.3: Accumulated percentage of mortality for neonates after 24h of starvation. Experi-ment of November 2010. 4: Buffer, �: MQ, ◦: No Fluid

The buffer treatment showed a total mortality of 40% with one dead larva on day 2 and7 and two dead larvae on day 6. At day 4, 5 and 7 one larva died for the MQ treatment,to result in a total mortality of 30%. The NF treatment showed a total mortality of 40%with two dead larvae at day 3 and 5.

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Figure. IV.3.4: Accumulated percentage of mortality for neonates after 24h of starvation. Experi-ment of March 2011. 4: Buffer, �: MQ, ◦: No Fluid

The buffer treatment showed one dead larva on day 11. For the MQ treated larvae, therewas one dead larva at day 5. The NF treatment had one dead larva at day 12. Thisresulted in a total mortality of 10% for all treatments.

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Figure. IV.3.5: Accumulated percentage of mortality for neonates after 24h of starvation. Experi-ment of April 2011. 4: Buffer, �: MQ, ◦: No Fluid


The duration of life stages was recorded, in the same way it was done for the neonates with-out starvation. All values are average durations (in days) of a life stage with their SEM, forthe three treatments and replicates. Statistical analysis is performed with Kruskal-Wallistest with post-hoc analysis using the Dunn test at p ≤ 0.05 and p ≤ 0.1.No statistical differences were observed at p ≤ 0.05 for the optimization experiments withneonates after 24h starvation prior to the start of the experiment. At p ≤ 0.1, NF treat-ment took significant longer than MQ treatment for the April 2011 experiment at the L1stage. In all other stages, no significant differences (p ≤ 0.1) were observed.

Table. IV.3.2: Average duration of life stages with SEM of neonates 24h starved prior to theexperiment. B: Buffer, MQ: Ultrapure water, NF: No fluid. a: difference withbuffer at p ≤ 0.05, b: difference with MQ at p ≤ 0.05, c: difference with no fluid atp ≤ 0.05

November 2010 March 2011 April 2011B MQ NF B MQ NF B MQ NF

L1 stage 5.44±0.60 5.67±0.49 5.33±0.21 3.22±0.49 3.50±0.46 4.00±0.52 4.90±0.18 4.80±0.39 5.67±0.29L2 stage 5.00±0.35 5.00±0.00 5.00±0.00 4.33±0.21 5.14±0.34 5.67±0.84 3.50±0.31 3.30±0.26 3.22±0.15L3 stage 5.00±0.84 NA 4.33±0.33 4.17±0.60 4.29±0.61 3.33±0.21 3.33±0.33 3.33±0.29 3.00±0.29

Total toL4

15.00±1.67 NA 14.67±0.67 11.50±0.67 13.00±1.02 13.00±1.06 11.78±0.22 11.56±0.29 11.89±0.20

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3.2.3 No Feeding

As presented for neonates, the mortality of 24h starved neonates is given in Figure IV.3.6.Two biological replicates were conducted (November 2010 and March 2011). In November2010, the LD50 was 2.5 days and all larvae were dead after six days. In March 2011, ittook 4.6 days for 50% of the larvae to die and seven days for all larvae.

Figure. IV.3.6: Accumulated percentage of mortality of the starvation experiment with neonates24h starved prior to the start of the experiment. �: November 2010, ◦: March2011

3.3 Result of the Optimization

The aim of the optimization was to see if there was an effect of the buffer of dsRNAor MQ on the survival and development of neonates of L. decemlineata with a no fluidtreatment as a control, because it has no influence on development. A second aim camealong with the optimization. It was observed that neonates do not eat all of the food, atthe start of the experiment, therefore the neonates were 24h starved prior to the start ofthe experiment. This resulted in two optimization rounds with two biological replicationsfor neonates and three replications for neonates after 24h starvation.The results consisted of two parts and one experiment on its own: the survival of treatedlarvae and the duration of life cycles, and the survival of larvae when no food was sup-plied. First, the mortality was observed. For the neonates, this showed a low mortalityfor both replicates, but in April 2011 the mortality was lowest. For the neonates after24h starvation, the mortality was high for the experiments in November 2011 and March2011. In April 2011 however, the mortality was low for all three treatments.

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Secondly, the duration of life stages was recorded. For neonates, the results were compa-rable for the two optimization rounds and did only show significant differences betweenthe buffer treatment and the control in which the treatment performed better than thecontrol. For the neonates after 24h starvation, no statistical differences (p ≤ 0.05) wereseen between the three treatments in the three experiments.Finally, the experiment in which no food was supplied after the start of the experiment,gave an idea about the time it takes for the larvae to die of starvation. Five to seven dayswas the duration that came back in the no feeding experiment with neonates and withneonates after 24h of starvation prior to the only feeding.

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Chapter 4

In vivo Effects of dsRNA onSurvival and Development ofLarvae of L. decemlineata

In this chapter, the results of the feeding experiments, performed to test the systemic effectof dsGRP1 are presented. Each experiment will be discussed on its own. An experimentcan be divided into two parts. First, the results of the insect morphological data (mortalityand duration of life stages) are presented and secondly, the results of the molecular dataare given.

4.1 October 2010: dsGRP1

4.1.1 Mortality

Mortality was followed every day during the development from L1 up to L4 life stages.The collected data are presented in Figure IV.4.1.The mortality is lowest for the dsGRP1 treatment with 27%. At day 4 and 9 one larvadied and at day 14 two larvae died. The dsGFP treatment has the highest mortality atthe end of the experiment with 47%. At day 3 and 4 one larva died, at day 10 three larvaedied and at day 13 two larvae died. The buffer treated series had two dead larvae at day4 and one at day 6 and 7.It can be seen that day 4 was a day in which four larvae died over the three treatments.This coincides roughly with the moulting from L1 to L2 stage.

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Figure. IV.4.1: Accumulated percentage of mortality in the October 2010 experiment. 4: Buffer,�: dsGFP , ◦: dsGRP1


In the feeding experiments, executed as described in Chapter III.2, the duration of eachlife stage of every treatment (buffer, dsGFP and dsGRP1) was recorded. When a genesilencing effect would be present, it was expected to see a significant difference betweenthe two controls (buffer and dsGFP) and the treatment (dsGRP1).When looking to the listing of average values with the SEM (in days) (Table IV.4.1),a comparison is made of the three treatments for every life stage using Kruskal-wallisanalysis with a post-hoc Dunn test at p ≤ 0.05 and p ≤ 0.1 (which showed the same as p≤ 0.05).

• L1 stage: No statistic difference was found between buffer, dsGFP and dsGRP1.

• L2 stage: All treatments had, statistically (p ≤ 0.05), the same duration of the lifestage.

• L3 stage: Difference (p ≤ 0.05) was found between the treatment with buffer anddsGFP. Buffer treated larvae showed a shorter L3 stage than dsGFP treated larvae.

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• Total to L4: As in the L3 stage, a statistical difference was found between bufferand dsGFP.

Table. IV.4.1: Average duration of life stages with SEM (in days) in October 2010. a: differencewith buffer at p ≤ 0.05, b: difference with dsGFP at p ≤ 0.05, c: difference withdsGRP1 at p ≤ 0.05

Buffer dsGFP dsGRP1

L1 stage 4.00±0.17 3.46±0.14 3.86±0.10L2 stage 3.00±0.27 3.31±0.13 3.14±0.14L3 stage 3.73±0.24b 5.00±0.19a 4.36±0.15

Total to L4 10.73±0.14b 11.88±0.23a 11.36±0.15


The molecular experiment, as described in Subsection III.2.2.2, resulted in cDNA fromthree points in time: D0: Day 0, D1: Day 1, D3: Day 3 and D6: Day 6; and threetreatments. All cDNA was checked for consistent expression using the rpL 32 primers (seeFigure IV.4.2). Three repetitions of a semi-quantitative PCR (see Section III.7.4) wereperformed, the gels compared and scored (see Table IV.4.2).

Figure. IV.4.2: Gels of the molecular experiment of October 2010. (A) Gel of cDNA from themolecular experiment of FE1 amplified with rpL32 primers (25 cycli). (B) part ofa gel of semi-quantitative PCR (D6 and 34 cycli)

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Table. IV.4.2: Molecular results for the October 2010 experiment. Scores based on three repeats.D0: Day 0, D1: Day 1, D3: Day 3 and D6: Day 6, ++: strong band, +: clear band,±: weak band, -: no band

D0 D1 D3 D6

Buffer dsGFP dsGRP1 Buffer dsGFP dsGRP1 Buffer dsGFP dsGRP1

31 cycli + ± ± + + ± + + + -34 cycli + + + ++ ++ + ++ ++ ++ ±

The expression of GRP1, for dsGRP1 treated larvae, is compared with the control treat-ments. At 31 cycli, bands were seen at all time points and treatments except for D6 GRP1.The expression of GRP1 at D1 was higher at GRP1 treated larvae than for the other twotreatments. This same effect was seen for 34 cycli. Expression of D1, D3 GRP1 was asstrong or stronger expressed than their controls, but for D6 GRP1 the expression wasslower than the controls.

4.2 March 2011: dsGRP1

4.2.1 Mortality

Mortality was followed every day, during the development from L1 up to L4 life stages.The collected data are presented in Figure IV.4.3.All treatments reached a mortality of 100% before transition from L2 stage to L3 stage.The dsGRP1 treatment had one dead larva at day 4, 6, 8 and 15. Two larvae died at day10, four at day 7 and five at day 9. For the dsGFP treatment, one larva died at day 3, 10and 16. Two larvae died at day 4 and 8, and four larvae died at day 6 and 7. The buffertreated series had one death at day 3, 8 and 14. Two dead larvae at day 7 and 10, andfour larvae died at day 6 and 9.Day 6, 7 and 9 seem to be crucial for all the treatments. This are approximatly the daysthe moulting from L2 to L3 takes place.

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Figure. IV.4.3: Accumulated percentage of mortality in the March 2011 experiment. 4: Buffer,�: dsGFP , ◦: dsGRP1

All larvae were dead in life stage 2. The larvae treated with dsGFP had the highestmortality in life stage 1 (60%) and dsGRP1 treatment the lowest (7%).


In the feeding experiments, executed as described in Chapter III.2, the duration of eachlife stage of every treatment (buffer, dsGFP and dsGRP1) was recorded. When a genesilencing effect would be present, it was expected to see a significant difference betweenthe two controls (buffer and dsGFP) and the treatment (dsGRP1).When looking to the listing of average values with the SEM (in days) (Table IV.4.1) acomparison is made of the three treatments for every life stage using the Kruskal-Wallistest and a post-hoc Dunn test (p ≤ 0.05 and p ≤ 0.1).

• L1 stage: There was no statistic evidence (p ≤ 0.05 and p ≤ 0.1) for a differencebetween any of the treatments.

• L2, L3 and total to L4 stage: No larvae reached these life stages.

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Table. IV.4.3: Average duration of life stages with SEM (in days) in the March 2011 experiment.

Buffer dsGFP dsGRP1

L1 stage 5.30±0.15 4.83±0.17 5.36±0.23L2 stage NA NA NAL3 stage NA NA NA

Total to L4 NA NA NA


The molecular experiment, as described in Subsection III.2.2.2, resulted in cDNA fromthree points in time: D0: Day 0, D1: Day 1, D3: Day 3 and D6: Day 6; and threetreatments. All cDNA was checked for consistent expression using the rpL 32 primers (seeFigure IV.4.4). Three repetitions of a semi-quantitative PCR were performed, the gelscompared and scored (see Table IV.4.4).

Figure. IV.4.4: Gels of the molecular experiment of March 2011. (A) Gel of cDNA from themolecular experiment of March 2011 amplified with rpL32 primers (25 cycli). (B)part of a gel of semi-quantitative PCR (D1 and 34 cycli)

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Table. IV.4.4: Molecular results for the March 2011 experiment. Scores based on two repeats. D0:Day 0, D1: Day 1, D3: Day 3, D6: Day 6, ++: strong band, +: clear band, ±:weak band, -: no band

D0 D1 D3 D6


30 cycli - - + ++ - ± + + - -34 cycli + ± + ++ + + + + - -

The results for the dsGRP1 treated larvae will be compared to the two control treatments.At 30 cycli, it was seen for D1 that the GRP1 expression is higher for dsGRP1 treatmentthan for the controls. No expression was seen at D6 GFP and D6 GRP1 treatments. At34 cycli, bands are visible for all treatments, except for D6 GFP and D6 GRP1. Theexpression of GRP1 for D1 GRP1 was still higher.

4.3 May 2011: dsGRP1

4.3.1 Mortality

Mortality was followed every day during the development from L1 up to L4 life stages.The collected data are presented in Figure IV.4.5.dsGRP1 has the highest mortality of the three treatments with 87%. At day 2, 3 and 7one larva died. Two larvae died at day 5 and 11, and at day 6 and 14 three larvae died.dsGFP has a lower mortality of 27%, with one dead larva at day 8 and 14 and two deadlarvae at day 7. The buffer treated larvae showed a mortality of 40% and had one deadlarva at day 2 and 9 and two dead larvae at day 6 and 8.

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Figure. IV.4.5: Accumulated percentage of mortality in the May 2011 experiment. 4: Buffer, �:dsGFP, ◦: dsGRP1


In the feeding experiments, executed as described in Chapter III.2, the duration of eachlife stage of every treatment (buffer, dsGFP and dsGRP1) can be compared to each other.When a gene silencing effect would be present, it was expected to see a significant differencebetween the two controls (buffer and dsGFP) and the treatment (dsGRP1).When looking to the listing of average values with the SEM (in days) (Table IV.4.5) acomparison is made of the three treatments for every life stage.

• L1 stage: Statistic analysis showed a significant (p ≤ 0.05) difference between dsGFPand dsGRP1. The dsGFP treated larvae had a shorter L1 stage the dsGRP1 treatedlarvae.

• L2 stage: A significant difference (p ≤ 0.05) between buffer and dsGFP treatmentwas observed with the duration of the L2 stage of the buffer treated larvae shorterthan that of the dsGFP treated larvae.

• L3 stage: The difference seen in the L2 stage between buffer and dsGFP treatmentwas also observed here.

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• total to L4 stage: No differences could be found by statistical analysis (p ≤ 0.05 andp ≤ 0.1).

Table. IV.4.5: Average duration of life stages with SEM (in days) in the May 2011 experiment.a: difference with dsGRP1 at p ≤ 0.05, b: difference with dsGFP at p ≤ 0.05, c:difference with buffer at p ≤ 0.05

Buffer dsGFP dsGRP1

L1 stage 5.70±0.37 5.40±0.21a 7.13±0.55b

L2 stage 3.11±0.26b 4.67±0.40c 3.80±0.58L3 stage 4.33±0.17 3.55±0.15 5.50±1.50

Total to L4 13.11±0.48 13.45±0.33 15.00±1.00


The molecular experiment, as described in Subsection III.2.2.2, resulted in cDNA fromthree points in time: D0: Day 0, D1: Day 1, D3: Day 3 and D6: Day 6; and threetreatments. All cDNA was checked for consistent expression using the rpL 32 primers (seeFigure IV.4.6). Three repetitions of a semi-quantitative PCR were performed, the gelscompared and scored (see Table IV.4.6).

Figure. IV.4.6: Gels of the molecular experiment of May 2011. (A) Gel of cDNA from the molecularexperiment of May 2011, amplified with rpL32 primers (25 cycli). (B) part of a gelof semi-quantitative PCR (D6 and 34 cycli)

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Table. IV.4.6: Molecular results for the May 2011 experiment. Scores based on three repeats. D0:Day 0, D1: Day 1, D3: Day 3, D6: Day 6, +: clear band, ±: weak band, -: no band

D0 D1 D3 D6


30 cycli - - ± + - - - - + +34 cycli ± ± ± + - ± ± + + +

The results for the dsGRP1 treated larvae will be compared to the two control treatments.At 30 cycli, a stronger signal is seen for the dsGRP1 treatment at D1. D3 had no expressionfor all treatments. At D6 GRP1 and GFP treatment had higher expression of GRP1 thanthe buffer treatment. The same trend was visible at 34 cycli.

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Chapter 5

Chitin Synthase

The sequences of ChS1 and ChS2 are not known in a database. It was tried to determinethese sequences in two ways: a molecular method for ChS1 and an in silico analysis forChS2.

5.1 In silico Analysis

Using the L. decemlineata midgut transcriptome database and the protein sequence forChS2 of T. castaneum, it was tried to find the protein sequence of ChS2 of L. decemlin-eata and the nucleotide sequence. By BLASTing the ChS2 protein sequence against thedatabase (pBLAST), a protein sequence of 1452 AA with alignment score: 4125, identities:0,5414993 and similarities: 0,7048193, was retrieved and it might be possible the ChS2protein (similarity matrix used: BLOSUM62 in BioEdit (Hall, 1999)). Reverse transcrip-tion of this sequence gave an idea of the possible nucleotide sequence. A nucleotide BLASTreturned a number of sequences to reconstruct part of the total sequence. ± 1200bp werebetween 1624 and 4052 bp of the reverse transcribed sequence. PCR products amplifiedwith P101 and P102, matched perfectly with these sequences, reinforcing the idea thesesequences belong to ChS2.

5.2 Determination of ChS1

Steps taken to retrieve the sequence of ChS1:

• Design of six primers for sequences that were different between ChS1 and ChS1 ofT. castaneum

• The test of the primers with cDNA from L2 larvae moulting to L3 stage without themidgut, gave amplification for one primerset: P122-126, but subsequent PCR reac-tions could not amplify enough material for cloning, even after long distance PCR.P101-102 gave clear amplification, but not enough for cloning. After purification,long distance PCR gave a concentration high enough for cloning

• The sequence was cloned according to Chapter III.8

• The inserts of the cloning were send for sequencing to retrieve a partial sequence.The cloned sequences, appeared very similar to ChS2 sequences that already wereretrieved in silico

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Part V

DISCUSSION & CONCLUSION

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Part V: Discussion & Conclusion

The aim of this thesis was to investigate the possibility of systemic RNAi in L. decemlin-eata. The genes for proteins DAT, ChS1 and GRP1, expressed in the neural tissues, thecuticular tissue and the epidermal tissue, respectively, were the targets used in this work.First, the bio-assay to perform the analysis, needed to be optimized.

During the optimization of the bio-assay, the mortality was a parameter that was easy tofollow. For neonates, the differences between October 2010 and April 2011 were small.The MQ treatment showed a higher mortality in October than in April (40% against 0%),but this could probably be accounted for by interindividual variations in the population orin the experiment. For neonates after 24h of starvation on the other hand, it is clear thatthe variation of the mortality, between the three experiments, is bigger. The mortalitieslower from November 2010 (maximum mortality of 100%) to the third experiment of April2011 (maximum mortality of 10%).The higher mortalities in October and November 2010 are probably due to winter and aleaf disease (Phytophtora infestans (Fry, 2008)) on the potato plants, decreasing the freshleaf surface and nutritional value of the food. In the lab population, a negative effect wasalready seen in October and November, in January, the population totally collapsed andonly regained strength by the middle of March.These observations learned that variability in population strength, over the course of theyear, is an important factor. The optimal period to conduct a bio-assay would be fromApril to September, when the quality of potato plants is at its best (Westermann, 1993).Between October and the end of February, the populations are weak, because the qualityof food is low, which could give unwanted results.Secondly, a possible explanation for the higher mortality in MQ treated larvae, in theOctober and November 2010 experiments, is that MQ is depleted from minerals, whichleads to a negative mineral homeostasis in the larva. When the population is weak, maybethis negative effect can be more profound.

Although, there are some statistical diffences found, between two treatments (buffer vs.MQ treatment and buffer vs. no fluid treatment) regarding the life stages, no recurringpatterns were observed. This indicates that these differences should not be regarded assignificant and are probably linked to interindividual variations in the population or inthe experiment. When developing the in vivo dsRNA experiments, the buffer can be usedas a ’no effect’ control.Choosing between neonates and neonates after 24h starvation, seems not necessary. Usingneonates 24h starved prior to the experiment is a practice, that was seen in other feedingexperiments (Huvenne & Smagghe (2010) for an overview of experiments performed priorto 2009). In the bio-assays performed in this work, it was decided to use neonates after24h of starvation, because they eat the whole piece of leaf, with fluid, that is providedat the start of the experiment. This is important, because in the future experiments, wewant to control the quantity of product that is ingested by the larvae.

Next to the optimization of the bio-assay, target selection and tissue specificity were avery important step. DAT is a Na+-dependent high-affinity neurotransmitter transporter.It is stated that DAT is expressed in certain types of neural cells and all information inliterature on insect DATs is related to the CNS. We, hereby, concluded that the expressionis specific for the CNS (Caveney & Donly, 2002; Donly & Caveney, 2005; Boudanova et al.,2008; Bai & Burton, 2009). Later, a new search in literature found instances in mammals,where DAT was localized in other tissues than the CNS, in rats (Mignini et al., 2009) and

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humans (Lemmer et al., 2002; Frankhauser et al., 2006). Unique in this work, is that thetissue specificity of DAT was actually tested in an insect: L. decemlineata. The in silicoanalysis using the BLAST analysis on the L. decemlineata midgut transcriptome database,showed no match with the sequence amplified by our specific primer pair. Surprisingly,the experimental analysis did not confirm this. A weak signal for all tissues indicated thatDAT is not expressed tissue specific or that contamination from CNS tissue is present inall samples. This is a possibility, because dissections can not rule out that small parts ofneurons contaminate the tissues.

Two ChS are known in various insect species (e.g., D. melanogaster (Gagou et al., 2002),T. castaneum (Arakane et al., 2004), Manduca sexta (Hogenkamp et al., 2005), Spodopterafrugiperda (Bolognesi et al., 2005), Spodoptera exigua (Chen et al., 2007), Ostrinia fur-nacalis (Qu et al., 2011)). ChS1 is responsible for biosynthesis of the chitin, found inthe cuticular exoskeleton and other tissues that are ectodermal in origin (foregut, hindgutand trachea)(Arakane et al., 2004; Hogenkamp et al., 2005; Merzendorfer, 2006). ChS2is responsible for biosynthesis of the chitin associated with the PM in epithelial cells ofthe midgut (Merzendorfer, 2006; Qu et al., 2011). The aim was to select ChS1, becausethere should be no expression in the midgut (Arakane et al., 2004; Hogenkamp et al., 2005;Merzendorfer, 2006). In silico analysis with the midgut transcriptome database, learnedthat the fragment, we amplified with our first primer set, was present in the midgut.This led to the assumption that the primers were not specific for ChS1. This idea wassupported by PCR analysis on the different tissues. There was amplification in head andmidgut cDNA. Head cDNA contained several types of tissues, like cuticular, foregut andCNS tissue, due to the dissection method. This probably explains the strong expressionof ChS found here. The expression in midgut insinuated, that we amplified ChS2. Thismeans, we probably are not able to distinguish between ChS1 and ChS2 with the firstprimer set that was designed.A (partial) sequence for ChS2 and ChS1 was sought. For ChS2, a possible protein sequenceand part of a possible nucleotide sequence were found in silico and with the sequencingdata from P101-102 product. To obtain certainty, a 3’-RACE and 5’ RACE with theseprimers could be performed. The ChS1 sequence was not amplified in a sufficient amount,with P122-126 (see Table III.7.3), for sequencing or cloning, after an initial weak signal.To obtain this sequence, fresh cDNA of L2 larvae moulting to L3 stage, or cDNA of larvaetwo or three days before the moult would be even better (Arakane et al., 2004), shouldbe made and a long distance PCR with a higher concentration, than the dilution of 1

5used here, could be performed to obtain a sufficient amount of material for cloning orsequencing. Another possibility is performing a 3’ or 5’ RACE directly with the P122 orP126 primers.

GRP1 is a putative cuticular protein, expressed mainly in epidermis tissue, that is upregu-lated during moulting and during environmental stress situations like drought or insecticidetreatment (Kim BY, 2005; Zhong et al., 2006; Zhang et al., 2008; Togawa et al., 2008).In silico analysis with the midgut transcriptome database, confirmed that GRP1 was notexpressed in the midgut. A PCR reaction on cDNA from different tissues confirmed this.Expression seemed to be the highest in head and in fat body. A possible explanation forthis is, that the cDNA of head contains a lot of different tissues, like epidermis tissue,which explains the strong band. The relatively strong band at fat body cDNA on theother hand is puzzling. Perhaps contamination of other tissues like epidermal tissue, dueto the method of dissection, is responsible for the data, but this remains unclear. Against

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expectations expression in carcass tissue was low, which is probably due to a low expres-sion of GRP1 or the loss of strong GRP1-expressing epidermal tissue in the dissection.

GRP1 was used as the target gene in the bio-assays that were conducted at three ex-perimental data: October 2010, March 2011 and May 2011. A silencing effect of thedsGRP1 treatment was expected to lead to effects like: a higher mortality, longer devel-opmental time, lower expression of GRP1, deformations in the epidermis, . . . (e.g., Baumet al. (2007); Mao et al. (2007); Meyering-Vos & Muller (2007); Li et al. (2011)). Thehigher mortality and the lower expression of GRP1 were seen in two experiments, respec-tively, May 2011 and October 2010.

Mortality was not higher for dsGRP1 treated larvae for October 2010 and March 2011,but March 2011 is a special case, because not one larva reached the third instar larvalstage. This indicated that there was a problem with the whole experiment or even withthe whole population at this moment. Hereby, the results obtained in this experimentseem irrelevant when drawing conclusions, so they are left out.In May 2011 however, a higher mortality in the dsGRP1 treatment was visible when com-pared to the buffer and the dsGFP treatment groups. This observation indicates thatthere was a negative effect of dsGRP1 on larvae of L. decemlineata, this can also be linkedto post-transcriptional gene silencing by RNAi. When looking at the parallel molecularexperiment, no effect of silencing through dsGRP1 was seen. Expression is a highly vari-able parameter, that can change significant in the time span of only a couple of hours.Therefore, it is posible that we ”missed” a silencing effect by sampling too early (high mor-talities at days 4 and 5) or too late. The responsability of dsGRP1 for a higher mortalityin the May 2011 experiment, is strengthened by the observation that most of the larvaethat did die, died at the moments of moulting. In Figure IVIV.4.5, each plateau in thegraph is approximately linked to a stage and the inclined sections approximate the daysthat moulting is observed in all treatments. The moulting from L1 to L2 stage, is situatedafter four to six days. The L2 to L3 moulting takes place around days 10 to 12, and thefinal larval moulting to the L4 stage after 13 to 14 days. These days coincide with thedays of high mortality, suggesting a link between the death, the dsGRP1 treatment andthe moulting. Since only one of the experiments showed that there might be an effect ofdsGRP1 on the mortality of larvae of L. decemlineata. Repetitions of these experiments,in the April-September period, should probably give a confirmation of this result.

In the October 2010 molecular experiment on the other hand, a downregulation of theGRP1 expression in dsGRP1 treated larvae was observed compared to the expression inbuffer and dsGFP treated larvae at day six. This is the effect that is expected when post-transcriptional gene silencing is present (e.g., Baum et al. (2007)). This is an importantindication that systemic RNAi in larvae of L. decemlineata can be present.

The duration of the life stages was also followed and significant differences (p ≤ 0.05)occured, but no consistent pattern was present. This shows a lack of evidence for a dif-ference in total developmental time between the three treatments, at the two points intime. Even when the a clear effect on mortality was seen in the May 2011 experiment,no significant effect was visible on the duration of life stages. Perhaps, this indicates thatGRP1 is not essential for development, but has an impact on particular moments in thelife cycle, like the moulting or environmental stress, that lead to the death of the organism

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at that moment or it leads to survival without significant disturbance of the development.

Next to the observations regarding the RNAi effects of dsGRP1, other data were col-lected, that provoked questions and possible explanations. A remarkable observation wasthat the expression of GRP1 was higher in the dsGRP1 treated larvae, than for the bufferand dsGFP treated larvae after one day for the two experiments. A possible explana-tion could be that the dsGRP1, that is delivered at the start of the experiment, is stillpresent in the midgut or in cells. During RNA extraction, dsRNA would not be removedand literature supports the idea that reverse transcription PCR (Section 6.4) can amplifydsRNA (Aradaib, 2009). This would explain the stronger signal at this point in time. Asecond hypothesis is that the larvae picked, were not all in the same developmental periodfor the three treatments. This could give differences in expression. A third option is thatthe larvae have a high level of environmental stress, in the dsGRP1 treatment during dayone. GRP1 has a stress-induced upregulation in adults of L. decemlineata which wouldexplain the higher expression (Zhang et al., 2008). It is not clear to us, if this effect ispresent in larvae and which stress factor would be responsible. Temperature does notseem to be a likely factor of influence, because all boxes were kept in the same climatecabinet. Drought stress should not be present in the experiment, because fresh potatoplant material is supplied daily. Experiments with induced stress in larvae (as describedin Zhang et al. (2008), would be able to support this hypothesis. Since these results arenowhere described in literature, it is hard to say which hypothesis is most probable.

To conclude, it is clear that evidence for a systemic RNAi effect can be found in theexperiments performed. To clarify and confirm the results, new experiments would needto be performed. In these experiments, the seasonal variability and more accurate meth-ods for analysis should be considered. What would be a huge increase in accuracy, wouldbe the implementation of (reverse transcriptase) quantitative PCR. This would make itpossible to accurately and quantitatively measure and analyze the data with statistics.Next to that a first step was taken, to find the sequence of ChS1 and 2. With the primers,designed for this work, and the steps mentioned above, it would be fairly simple to acquirethese sequences.

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