Proteome Characterization of Caenorhabditis Elegans During

Home , Apyrase, Deoxyribonuclease II, GMP reductase, Major sperm protein, Peroxin, Prefoldin subunit 6

PROTEOME CHARACTERIZATION OF CAENORHABDITIS ELEGANS DURING

DEVELOPMENTAL STAGES

ASIFA KHATOON ZAIDI

Submitted in partial fulfillment of the requirements for the degree

Master of Science

Systems Biology And Bioinformatics

CASE WESTERN RESERVE UNIVERSITY

May 2016 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis of

Asifa Khatoon Zaidi

Candidate for the degree of

Master of Science

Committee Chair

Dr. Masaru Miyagi

Committee Member

Dr. David Lodowski

Committee Member

Dr. Gurkan Bebek

Date of Defense

March 25, 2016

*We also certify that written approval has been obtained for any proprietary material contained therein

2 Table of Contents

Table of Contents 3

List of Tables 6

List of Figures 10

Acknowledgments 12

Abstract 13

A: CHAPTER 1: Proteome characterization of Caenorhabditis elegans at different developmental stages 14

1. Summary 14

2. Introduction 15

3. Methods and material 18

3.1. Materials 18

3.2. Methods 18

3.2.1. C. elegans Strain, Maintenance, and Age Synchronization 18

3.2.1.1. Preparation of Nematode Growth Media (NGM) plates 19

3.2.1.2. Equipment and reagents 19

3.2.1.3. Methods 19

12 13 3.3. Labeling Bacteria with Light-( C6-Lys) and Heavy-( C6-Lys) Lysine 19

3.4. Preparation of labeled C. elegans at different larval stages 20

3.5. Separation of Live and Dead Worms 21

3.6. Sample preparation for proteomic analysis 21

3.7. LC-MS/MS analysis 23

3.8. Identification and Quantification of Peptides and Proteins 25

3 3.9. Data analysis 26

4. Results 27

4.1. Overview of the data set 27

4.2. Clustering analysis 29

4.2.1. Principal component analysis (PCA) 29

4.2.2. Unsupervised clustering analysis 31

4.3. Overall distribution appearance rate of newly synthesized proteins 35

4.4. Gene ontology and pathway association of the proteins for which PAR was rapid

at different development stages 37

4.4.1. Gene ontology: molecular function 37

4.4.2. Gene ontology: biological function 44

4.4.3. Pathways (KEGG) association 51

5. Discussion 59

B. CHAPTER 2: Effect of environmental stress on proteome of Caenorhabditis elegans

during development 62

1. Summary 62

2. Introduction 63

3. Materials and methods 66

3.1. Preparation of unlabeled C. elegans L3 larvae 66

3.2. Preparation of labeled C. elegans dauer larvae 66

13 3.3. Preparation of C6-Lys-labeled reference L3 and dauer larvae 68

3.4. Data analysis 68

4. Results 69

4 4.1. Overall expression of total proteins in dauer and L3 larvae 69

4.2. Clustering analysis 71

4.2.1. Principal component analysis (PCA) 71

4.2.2. Unsupervised clustering analysis 74

4.3. Differentially expressed proteins between L3 and dauer larva 76

4.4. Gene ontology and pathway association 79

4.5.1. Gene Ontology and Pathway Association of up regulated proteins in dauer

larvae 79

4.5.2. Gene Ontology and Pathway Association of down regulated proteins in dauer

larvae 94

4.5.3. Gene Ontology and Pathway Association of unique proteins present in dauer

larvae 118

4.5.4. Gene Ontology and Pathway Association of unique proteins in present in L3

larvae 128

5. Discussion 152

C. Appendix 155

D. Bibliography 181

5 List of Tables

CHAPTER 1

Table-1.1: Gene ontology (GO)-molecular function (MF) for proteins with rapid PAR at different developmental stages. 40

Table-1.2: The statistical overrepresentation test (PANTHER GO-Molecular Function) of proteins with rapid PAR at different development stages. 43

Table-1.3: Gene ontology (GO): Biological Process (BP) for proteins with rapid PAR at different developmental stages. 46

Table-1.4: The statistical overrepresentation test (PANTHER GO-Biological Process) of proteins with rapid PAR at different development stages. 50

Table-1.5: The pathway (KEGG) association for proteins with rapid PAR at L1→L2 stages. 55

Table-1.6: The pathway (KEGG) association for the proteins with rapid PAR at L2→L3 stages. 56

Table-1.7: The pathway (KEGG) association for the proteins with rapid PAR at L4→YA stages. 57

Table-1.8: The pathway (KEGG) association for the proteins with rapid PAR at L4→YA stages. 58

CHAPTER 2

Table-2.1: Proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm 83

6 Table -2.2: GO-MF terms for the proteins for which the level of expression is higher than

1.5-fold in dauer larvae compared to L3 worm 84

Table-2.3: GO-BP terms for the proteins for which the level of expression is higher than

1.5-fold in dauer larvae compared to L3 worm. 85

Table-2.4: Statistical overrepresentation test (PANTHER) for GO-MF and GO-BP terms for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm. 86

Table-2.5: KEGG pathways for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm. 88

Table-2.6: REACTOME pathways for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm. 89

Table-2.7: Proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm 98

Table-2.8: GO-MF terms for the proteins for which the level of expression is lower than

1.5-fold in dauer larvae compared to L3 worm 102

Table-2.9: GO-BP terms for the proteins for which the level of expression is lower than

1.5-fold in dauer larvae compared to L3 worm. 106

Table-2.10: KEGG pathway for the proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm. 114

Table-2.11: REACTOME overrepresentation pathway for the proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm. 117

Table-2.12: Unique proteins present in dauer. 120

Table-2.13: GO-MF terms for the unique proteins in dauer. 121

7 Table-2.14: GO-BP terms for the unique proteins in dauer. 122

Table-2.15: KEGG pathway for the unique proteins in dauer. 126

Table-2.16: REACTOME overrepresentation pathway for the unique proteins in dauer.

127

Table-2.17: Unique proteins present in L3. 130

Table-2.18: GO-MF terms for the unique proteins in L3. 135

Table-2.19: GO-BP terms for the unique proteins in L3. 140

Table-2.20: KEGG pathway for the unique proteins in L3. 145

Table-2.21: REACTOME overrepresentation pathway for the unique proteins in L3. 151

APPENDIX

Supplementary Table-1.1: Proteins for which PAR is rapid at L1→L2 stage 155

Supplementary Table-1.2: Proteins for which PAR is rapid at L2→L3 stage 156

Supplementary Table-1.3: Proteins for which PAR is rapid at L3→L4 stage 158

Supplementary Table-1.4: Proteins for which PAR is rapid at L4 →YA stage 161

Supplementary Table-1.5: Proteins for which PAR is slow at L1→L2 stage 164

Supplementary Table-1.6: Proteins for which PAR is slow at L2→L3 stage 166

Supplementary Table-1.7: Proteins for which PAR is slow at L3→L4 stage 168

Supplementary Table-1.8: Proteins for which PAR is slow at L4 →YA stage 169

Supplementary Table-1.9: Gene ontology (GO): molecular function (MF) for proteins with slow PAR at different developmental stages. 170

Supplementary Table-1.10: The Statistical overrepresentation test (PANTHER GO-

Molecular Function) of proteins with slow PAR at different development stages. 172

8 Supplementary Table-1.11: Gene ontology (GO): Biological Process (BP) for proteins with slow PAR at different developmental stages. 173

Supplementary Table-1.12: Statistical overrepresentation test (PANTHER GO-Biological

Process) of proteins with slow PAR at different development stages. 176

Supplementary Table-1.13: KEGG pathway for proteins with slow PAR at L1→L2 stages. 177

Supplementary Table-1.14: KEGG pathway for proteins with slow PAR at L2→L3 stages. 178

Supplementary Table-1.15: KEGG pathway for proteins with slow PAR at L4→YA stages. 179

Supplementary Table-1.16: KEGG pathway for proteins with slow PAR at L4→YA stages. 180

9 List of Figures

CHAPTER 1

Figure 1.1: Experimental Workflow 22

Figure 1.2: Proteins identified during different developmental stages 28

Figure 1.3: Principle component analysis (PCA) of the proteins identified during different development stage. 30

Figure 1.4: Scree plot depicting variance of the all variance of the principle component axis’s present in the different development stage. 33

Figure 1.5: Heat map of the proteins identified during different development stage. 34

Figure 1.6: Overall distribution of the PAR measured for identified proteins during different larval development stages 36

Figure 1.7: Gene Ontology (GO)-Molecular function (MF) terms for proteins with rapid

PAR. 39

Figure 1.8: Gene Ontology-Biological Process terms for proteins with rapid PAR. 45

Figure 1.9: Pathway (KEGG) association for proteins with rapid PAR mapped to metabolic process. 54

CHAPTER 2

Figure 2.1: Overview of workflow 67

Figure 2.2: Overall distribution of newly synthesized proteins at L3 and dauer larval stages 70

Figure 2.3: Principal component analysis (PCA) 72

10 Figure 2.4: Scree plot depicting variance of the all principle component axis’s present in the L3 and dauer larvae samples in triplicates. 73

Figure 2.5: Heat map 75

Figure 2.6: Volcano plot 77

Figure 2.7: Fold change of proteins in dauer larvae to L3 Larvae 78

Figure 2.8: Gene Ontology-Molecular Function terms for 1.5-fold up regulated dauer proteins. 80

Figure 2.9: Pathway association for 1.5-fold up regulated proteins in dauer. 93

Figure 2.10 Gene ontology (GO)-molecular function (MF) and biological process (BP) terms for 1.5-fold down regulated dauer proteins. 97

Figure 2.11: Pathway association for 1.5-fold down regulated proteins in dauer. 113

Figure 2.12: Pathway association for unique proteins of dauer. 125

Figure 2.13: Pathway association for unique proteins of L3. 150

11 Acknowledgements

I enjoyed working with my advisor, Dr. Masaru Miyagi, on my master thesis project. I am very grateful for his invaluable guidance, constructive discussion, and continuous support throughout this research project. I would like to thank him and acknowledge for sharing his wealth of knowledge and wisdom in the field of mass spectrometry and proteomics. I would also like to thank and express my gratitude to Drs.

Gurkan Bebek and David Lodowski for their continuous support, guidance and their availability throughout this project work. I would like to acknowledge the help and guidance of Dr. Sheng Quanhu, Vanderbilt University, Nashville, TN for his constant support and guidance for analysis of data using his software “ProteomicsTools” and providing need-based update on the software. I would also like to thank Dr. Vukoti

Krishna for the help for generating proteomic data for the analysis. I would like to acknowledge that it was a great experience for me to get acquainted to the system biology and bioinformatics field and I am extremely thankful to all my mentors including Drs.

Masaru Miyagi, Dr. David Lodowski, Dr. Gurkan Bebek for their help and support.

12 Proteome Characterization Of Caenorhabditis Elegans During Developmental

Stages

Abstract

ASIFA KHATOON ZAIDI

The goal of the present proteomic study on Caenorhabditis elegans is to determine the appearance rates of newly synthesized proteins at the individual protein level during different developmental stages and to identify differentially expressed proteins during development under nutritional stress. Our SILAC-based proteomic approaches revealed that proteins with rapid appearance rates belong primarily to metabolic processes in all developmental stages. We also found that the expressions of proteins involved in normal metabolic processes such as translation, fatty acid synthesis and glycolysis/gluconeogenesis decrease during development under nutritional stress, while proteins involved in calorie regulation and structural molecular activity including proteins involved in fatty acid degradation and highly active fermentative pathways increase. These results provide new insights into the physiological changes of

Caenorhabditis elegans proteome during development under normal and nutritional stress.

13 A. CHAPTER 1:

Proteome characterization of Caenorhabditis elegans (C. elegans) at different developmental stages

Summary:

The goal of this chapter is to determine the appearance rates of newly synthesized protein (PAR) at individual protein level during normal larval development of

Caenorhabditis elegans (C. elegans). The PAR was monitored during normal larval development consists of four larval (L1, L2, L3, and L4) stages using a “stable isotope labeling by amino acids in cell culture” (SILAC)-based label-chase proteomic approach.

The average PAR for all the proteins quantified was highest at the L1→L2 larval stage, slowed down gradually in the subsequent larval stages, and was slowest at L4→young adult (YA) stage. The analysis of the data suggested that proteins with rapid PAR are mainly involved in metabolic, cellular and biological regulation process gene ontology terms. The proteins exhibiting rapid PAR that belong to overrepresented gene ontology terms are peptidase activities at early development stages (L1→L2 and L2→L3) and translation and translation-regulated processes at L3→L4 and L4→YA stage. Our results provide a new insight into how regulation of protein expression during larval development shapes the adult life of this organism.

14 Introduction:

Caenorhabditis elegans (C. elegans) is a small (about 1 mm in length) and free- living (non-pathogenic and non-parasitic) organism. C. elegans lives in the soil especially decomposing vegetation and feeds primarily on microbes such as bacteria. It is a powerful tool for studying development, genomics, cell biology, neuroscience and aging

(http://www.wormbook.org/) due to evolutionary conservation of gene function between worm and mammal [1-3]. In addition, there are many advantages of C. elegans as a model system that includes its short life cycle, compact genome, conventional development, small size, ease of propagation and produces a large number of progeny per adult. Visualization of the worm and expressed fluorescent proteins in C. elegans is easy because they are transparent throughout its life cycle. Under the optimum environmental condition, it reproduces with a life cycle of about 3 days. It can be readily raised in the laboratory on agar plates or liquid cultures on a diet of Escherichia coli (E. coli).

Furthermore, a wealth of public and proprietary information, databases and knowledge are available that includes RNAi library and several mutants [4, 5]. Despite its simple anatomy, the animal displays a large repertoire of behavior including locomotion, foraging, feeding, defecation, egg laying, dauer larva formation, sensory responses to touch, smell, taste and temperature as well as some complex behaviors like male mating, social behavior and learning and memory [6, 7].

Several studies have demonstrated that alteration in major signaling pathway during development leads to significant modulations in Caenorhabditis elegans (C. elegans) physiology, including extended life span, increased innate immunity and stress- resistance, and extensively altered metabolism [8-21]. Examples of these pathways are

15 insulin/IGF-1 signaling that include daf-2 and daf-16 pathways [9, 10, 13, 16, 18], canonical RTK-Ras-ERK signaling [11, 22], Eph receptor signaling [23], heterotrimeric

G protein signaling [24], Notch signaling [25], TGF-β signaling [26-28], small GTPase signaling [24, 29, 30], Wnt signaling [31, 32], nuclear receptor signal transduction pathways [33, 34] and signaling in the immune response including innate immune response [35-39]. These pathways are mostly related to major normal physiological processes and regulate dauer formation, stress resistance, longevity, and metabolism in C. elegans. Previous high-throughput “omics” studies of C. elegans were mostly carried out at the genomic or transcriptomic level because of the availability of thousands of mutant worms, RNA interference libraries, and whole-genome transcript profiling techniques

[40-43]. These studies have provided a plethora of information on the array of genes expressed during the development of C. elegans [40-43], However, a few proteomic studies have measured the expressions of individual proteins during the larval stages [44-

46], and to the best of our knowledge no study has monitored protein flux (synthesis and/or degradation of proteins).

The proteome of a biological system is highly dynamic and adaptive. A proteome- wide static measurement of protein concentration does not reflect the underlying dynamic nature of these physiological processes. The static levels of proteins are the net results of their synthesis and clearance that are determined by the activities of associated proteins involved in protein synthesis, degradation, and transportation. To capture the dynamic nature of proteome, it is necessary to measure the flux of individual proteins. We have established a label-pulse-chase approach to monitoring the synthesis of proteins in C. elegans [47] using a protein-labeling technique known as “stable isotope labeling by

16 amino acids in cell culture” (SILAC) [48-50], in which heavy carbon-13 labeled lysine

13 ( C6-Lys) was used as a precursor. The SILAC-worm has an identical life span to the

13 unlabeled worm, demonstrating that incorporation of C6-Lys (heavy lysine) residues in proteins does not affect the physiological processes in C. elegans [47]. Using this

SILAC-label-chase technique, Vukoti et al have successfully monitored the newly synthesized proteins over the adult life span of C. elegans [47, 51].

In this study, we monitored the synthesis of individual proteins in C. elegans at different larval development stages in normal growth condition using a SILAC-based label-chase approach. The average rate of appearance of newly synthesized protein

(PAR) for all the proteins quantified was highest at the L1→L2 larval stage, slowed down gradually in the subsequent larval stages, and was slowest at L4→young adult

(YA) stage. The bioinformatics analysis on the data suggested that proteins with rapid

PAR belong mainly to metabolic, cellular and biological regulation processes. The proteins exhibiting rapid PAR that belong to overrepresented gene ontology terms are peptidase activities at early development stages (L1→L2 and L2→L3) and translation and translation-regulated processes at L3→L4 and L4→YA stage. Our results provide a new insight into how regulation of protein expression during larval development under normal environmental condition shapes the adult life of this organism.

17 Materials and methods

Materials

12 13 C6- and C6-lysine (Lys) was purchased from Sigma-Aldrich (St. Louis, MO) and Cambridge Isotope Laboratories (Tewksbury, MA), respectively. Lys-C was purchased from Wako, USA (Richmond, VA). All other chemicals were either reagent grade or were of highest quality that was commercially available.

Methods:

C. elegans: Strain, Maintenance, and Age Synchronization

WT Bristol N2 strain nematodes were used in this study, as they are the strain that

12 13 has its genome sequenced [2, 52]. For the incorporation of C6-Lys (light-Lys) and C6-

Lys (heavy-Lys), nematodes were maintained by standard methods that included culture on peptone-free nematode growth media (NGM) plates (51 mM NaCl, 25 mm K3PO4, 5

µg/mL cholesterol, 1 mM CaCl2, 1 mM MgSO4) seeded with Escherichia coli strain lysine auxotroph, AT713 raised on the appropriately isotopically labeled media. To synchronize their age, gravid nematodes were treated with bleach (5% sodium hypochlorite solution) for 3 to 9 minutes. The bleaching resulted in death of worms while the eggshell protected the embryos. After bleaching, the surviving eggs (embryos) were incubated in liquid media without food and were allowed to hatch as age-synchronized nematodes [47, 53]. The post-hatched period of the egg was counted as the L1 larva.

Each larval stage was identified (or harvested) as described: L2 larva (15 h after L1), L3 larva (9 h after L2), L4 larva (10 h after L13), and young adult (YA; 12 h after L4). The composition of the media, solutions and protocols are reported below [54].

18 Preparation of nematode growth media (NGM) plates

Equipment and Reagents:

NaCl; Agar; Peptone (5 mg/mL); cholesterol in ethanol; 1 M KPO4 buffer pH 6.0

[KH2PO4 (108.3 g); K2HPO4 (35.6 g); H2O to 1 liter]; 1 M MgSO4; Petri plates;

Peristaltic pump.

Methods:

Sodium Chloride (NaCl, 3 g), agar (17 g), and peptone (2.5 g) was mixed in a 2-

liter Erlenmeyer flask. Volume was made up to 975 mL with water (H2O). The

mouth of the flask was covered with aluminum foil and was autoclaved for 50 min.

The flask was cooled in a 55 °C water bath for 15 min. After cooling, 1 mL of 1 M

CaCl2, 1 ml of 5 mg/mL cholesterol in ethanol, 1 mL of 1 M MgSO4 and 25 mL of

1 M KPO4 buffer was added to the flask. The flask was swirled to mix the solution

well. NGM solution was dispensed into the petri plates aseptically. The petri plates

were filled up to 2/3 of their volume. The plates were then incubated at room

temperature for 2-3 days to detect any contamination, and to allow excess moisture

to evaporate. Plates were stored in an airtight container at 4 °C and were used

within several weeks.

12 13 Labeling Bacteria with Light ( C6)-Lys and Heavy ( C6)-Lys

Arginine and lysine auxotrophic Escherichia coli strain AT713 were obtained from the E. coli Genetic Stock Center at Yale University. To label AT713 bacteria with light- or heavy-Lys, bacteria were first streaked on a lysogeny broth (LB) plate and cultured overnight at 37 °C. A single bacterial colony was then inoculated into 10 mL of

19 LB and incubated overnight in an incubator shaker (37 °C, 180 rpm). 100 µL of bacterial culture was next inoculated into 50 mL of M9 minimal medium (50 mM Na2HPO4, 20 mM KH2PO4, 10 mM NaCl, 20 mM NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, and 0.2% glucose) supplemented with arginine (100 µg/mL), cysteine (100 µg/mL), and lysine (100

12 13 µg/mL, either light ( C6)- or heavy ( C6)-Lys) and continuously cultured in an incubator shaker (37 °C, 200 rpm) until the absorbance of the culture at 600 nm (A600) reached

1.0. Then, 10 mL of the resulting labeled bacteria were inoculated into 1000 mL of M9 basal medium, with amino acid supplementation, and cultured in an incubator shaker (37

°C, 200 rpm) until A600 reached 2.0. Bacteria were then harvested by pelleting by centrifugation (8,000 × g, 10 min), and resuspended in 15 mL of sterile water. Bacteria then were spread onto a peptone-free NGM plate (500 µL for each 100-mm plate and 200

µL for each 60-mm plate) and exposed to 1000 mJ/cm2 of ultraviolet light (SpectroLinker

12 XL-1500, Spectronics Corp, Westbury, NY). The plates with the light ( C6)- and heavy

13 ( C6)-Lys containing bacteria were stored at 4 °C until they were used.

Preparation of labeled C. elegans at different larval stages

Wild type (WT) Bristol N2 strain nematodes were transferred onto a peptone-free NGM

12 plate that had been seeded with light ( C6)-Lys labeled E. coli. Gravid animals from the next generation of these nematodes were bleached to collect their live eggs (embryos), according to a protocol described in detail in previous section “ C. elegans: Strain,

Maintenance, and Age Synchronization” [47]. Eggs were transferred onto peptone-free

12 NGM plates seeded with light ( C6)-Lys E. coli. This step of bleaching, synchronization, and plating on NGM plates seeded with unlabeled bacteria is repeated for one more time.

20 After hatching, age-synchronized animals were then cultured up to the L1, L2, L3 or L4

12 larval stage on the peptone-free NGM plates seeded with light ( C6)-Lys E. coli plates containing 25 mg/L 5-fluoro-2-deoxyuridine (Acros Organics, Pittsburgh, PA) to prevent

12 egg production. The worms labeled with light ( C6)-Lys, grown up to L1, L2, L3, or L4 larval stages were then transferred to peptone-free NGM plates seeded with heavy-Lys E. coli, and grown for another 15 h, 9 h, 10 h, and 12 h to allow them to reach L2, L3, L4, and young adult (YA) stages, respectively. After attaining L2, L3, L4 and YA stage in

13 heavy ( C6)-Lys, worms were harvested. The experimental workflow is shown in Figure

1.1. This label-chase experiment was performed in triplicate.

Separation of Live and Dead Worms

Common practice is to separate live worms from dead and use only live worms for the study. However, in this study dead worms were not observed, therefore, this procedure was not necessary.

Sample preparation for proteomic analysis

The worms were suspended in 250 µL of 100 mM ammonium bicarbonate containing 4% perfluorooctanoic acid (PFOA) (w/v) [13], protease inhibitor mixture (Sigma-Aldrich), and phosphatase inhibitor mixture 3 (Sigma-Aldrich), and proteins were then extracted by ultrasonication (4.5 kHz three times for 9 s with a 3-min pause on ice between the strokes) using a Virsonic 100 ultrasonic cell disrupter (SP Scientific, Warminster, PA) as described previously [55, 56]. The resulting protein extract was centrifuged at 15,000 × g

Synchronized eggs Hatching L1

L1 L2 L3 L4 Grown on E. coli 12 Light- C6-Lys 0 h 15 h 9 h 10 h

Grown on E. coli 13 Heavy- C6-Lys 15 h 9 h 10 h 12 h E. coli AT713-Light-12C-Lys L2 L3 L4 YA Harvest E. coli AT713-Heavy-13C-Lys

Protein extraction, Protein extraction, digestionDigestion with with Lys-C Lys-C

LC-MS/MS LC-MS/MS

L L H Protein identification & Fraction of heavy/time t 6 Da Protein quantification Relativeintensity Time: 0 t PAR Protein identification, protein quantification, data analysis

Figure 1.1: Experimental Workflow. After hatching, age-synchronized worms were grown on plates seeded with light-Lys E. coli until they reached to L1, L2, L3 or L4 larval stage. The worms at each larval stage were then transferred to plates seeded with heavy-Lys E. coli, and grown from L1→L2 (15 h), from L2→L3 (9 h), from L3→L4 (10 h) or from L4 →young adult (12 h) respectively, and then harvested. Proteins extracted from these samples were digested by Lys-C and analyzed by LC-MS/MS. This label- chase experiment was performed in triplicate.

22 for 10 min, and the supernatant was collected. The supernatant was reduced with 10 mM dithiothreitol (DTT) at 37 °C for 30 min, and then S-alkylated by 25 mM iodoacetamide at 25 °C for 45 min in the dark. Proteins were then precipitated by mixing with a 9-fold excess volume of ice-cold acetone and left for 2 hr at -20 °C. The precipitated proteins were then centrifuged at 2,400 × g for 10 min at 4 °C, and the pellet was again washed with ice-cold 90 % acetone. Protein pellets were air-dried for 5 min and then redissolved in 50 µL of 100 mM ammonium bicarbonate, containing 8 M urea by sonication in a water bath for 1 min in a Bransonic Ultrasonic 2510R-MT water bath (Danbury, CT).

The resulting solution was diluted with 450 µL of 100 mM ammonium bicarbonate to reduce urea concentration to 0.8 M, and the amount of dissolved protein was determined with a DC protein assay kit (Bio-Rad, Hercules, CA). A total of 25 µg of protein was digested by protease Lys-C protease at a Lys-C: protein ratio of 1:25 (w/w) at 37 °C for

18 hrs. The peptide mixture was desalted using a Vydac C18 UltraMicro Tip Column

(The Nest Group) and resuspended in 0.1 % formic acid.

LC-MS/MS analysis

LC-MS/MS analysis used an UltiMate 3000 LC systems (Dionex Inc.) interfaced to Velos Pro Ion Trap and Orbitrap Elite Hybrid Mass Spectrometer (Thermo Scientific,

Bremen, Germany). The platform was operated in the nano-LC mode, using a standard nano-ESI (Proxeon Biosystems) source. The spray voltage was set to 1.2 kV and the temperature of the heated capillary was set to 200 °C. The solvent flow rate through the column was maintained at 300 nL/min. Lys-C peptide digests (typically 2 µg/2 µL) were injected into a reversed-phase 0.3 × 5 mm C18 PepMap trapping column with a 5-µm

23 particle size (Dionex Inc.) that was equilibrated with 0.1% formic acid, 1% acetonitrile

(v/v). The column was washed for 5 min with the equilibration solution at a flow rate of

25 µL/min, using an isocratic-loading pump operated through an auto sampler. Next, the trapping column was switched in-line with a reversed-phase 0.075 × 150-mm C18

Acclaim PepMap 100 column (Dionex Inc.), and the peptides were chromatographically separated by using a linear gradient of acetonitrile from 2% to 37% over 202 min in aqueous 0.1% formic acid at a flow rate of 300 nL/min.

The eluent was directly introduced to the mass spectrometer. The mass spectrometer was operated in a data-dependent MS to MS/MS switching mode, with the

25 most intense ions in each MS scan subjected to MS/MS analysis. The full MS scan was performed at a resolution of 120,000 (full width at half-maximum) in the Orbitrap detector, and the MS/MS scans were performed in the ion trap detector in collision- induced dissociation mode. The threshold intensity for the MS/MS trigger was always set at 3000. The fragmentation was carried out using the collision-induced dissociation mode with normalized collision energy of 35eV. The data were entirely collected in the profile mode for the full MS scan and the centroid mode for the MS/MS scans. The dynamic exclusion function for previously selected precursor ions applied the following parameters: repeat count of 1, repeat duration of 40 s, exclusion duration of 90 s, and exclusion size list of 500. Xcalibur software (Version 2.2 SP1 build 48, Thermo-Finnigan

Inc., San Jose, CA) was used for instrument control, data acquisition, and data processing.

24 Identification and quantification of peptides and proteins

Proteins were identified by comparing all of the experimental peptide MS/MS spectra against the Wormpep #WS248 (c_elegans.PRJNA13758.WS248.protein.fa.gz data file obtained from ftp://ftp.sanger.ac.uk/pub/wormbase/release; released 7-May-

2015) using Mascot database search software (Version 2.2.0, Matrix Science, London,

UK). To account for the S-alkylation of protein samples, carbamidomethylation of cysteine was set as a fixed modification, whereas variable modifications included oxidation of methionine-to-methionine sulfoxide, acetylation of N-terminal amino groups, and replacement of C-terminal Lys with heavy Lys. The mass tolerance was set at

10 ppm for precursor ions and 0.8 Da for product ions. Strict Lys-C specificity was applied and missed cleavages were not allowed. BuildSummary [57] was used to generate a confident protein list with a peptide false discovery rate ≤ 0.01. Other criteria for significant peptide identifications included the following: peptides must be composed of at least six amino acid residues and have a minimum Mascot score of 20, which corresponds to Mascot absolute probability of 0.01. Only proteins identified with at least two unique peptides were considered to be positive identification. Protein isoforms and proteins that could not be distinguished based on the peptides identified were grouped and reported as a single protein group. The SILAC Quantification Suite in

ProteomicsTools software [57], version 3.9.9, was used to determine the abundance of light- and heavy-labeled proteins, from which the fraction of heavy protein (heavy protein/[light protein + heavy protein]) was calculated. The rate of appearance of newly synthesized protein was obtained by dividing the fraction of heavy protein by the duration of time for the label-chase experiment.

25 Data analysis

Principal component analysis (PCA) by k-means clustering was performed using the PAM algorithm, unsupervised clustering analysis, and heat map and boxplot plotting were done using R (version 3.2.1 (2015-06-18) accessed on 2015-06-24). (The base, stats, cluster, ggplot2, gplots, ggfortify, graphics packages was used. Gene ontology and pathway association were performed using PANTHER (http://www.pantherdb.org/; [58]) and KEGG (http://www.genome.jp/kegg/tool/map_pathway1.html) respectively.

26 RESULTS:

Overview of the data set.

Since this study was a SILAC label-chase experiment, all the identified proteins

13 provided us quantitative values, which was the ratio between the C6-Lys (heavy) labeled and unlabeled peptide species, from which the appearance rate of newly synthesized protein (we abbreviated “appearance rate of newly synthesized protein” as

PAR (protein appearance rate) for ease of pronunciation) during a specified duration of the experiment was calculated. The mean coefficient of variation (CV) values from the

PAR values from all the proteins identified in the triplicate samples at the four different developmental larval stages were as follows: 2.3% (L1→L2), 3.16% (L2→L3), 2.9%

(L3→L4), and 3.2% (L4→YA). Over 96% of proteins have the CV values for PAR

<10% and 83% to 92% of proteins have the CV values < 5%. These results indicate that the variations of the measured PAR values between the replicates for individual proteins are small. Therefore, we included all the proteins identified as either triplicates, duplicates or singles in the triplicate samples of four different developmental larval stages.

The total number of proteins identified in each developmental stage was for

L1→L2, 1200 for L2→L3, 1337 for L3→L4, and 1388 for L4→YA stage, of which 813 proteins were common to all four developmental stages (Figure 1.2). There were 103, 41,

84, and 168 proteins that were identified uniquely in L1→L2, L2→L3, L3→L4, and

L4→YA stage, respectively.

L2→L3 L3→L4 L1→L2 L4→YA

38 28

Figure 1.2: Proteins identified during different developmental stages. The number of proteins that are commonly or uniquely present in each larval stage is labeled within the circles represented by different color for each larval stage. The total number of proteins was 1087 for L1→L2, 1200 for L2→L3, 1337 for L3→L4, and 1388 for L4→YA stage.

28 Clustering analysis:

Principal component analysis (PCA):

PCA analysis (k mean clustering method) on the PAR of the proteins present in triplicate samples (362 out of 813 common proteins) in all developmental stages clearly indicated that triplicate samples were similar to each other (Figure 1.3a). However, each developmental stage was well separated from each other, suggesting profound differences exist in protein expression between different developmental stages. To determine the average dissimilarity of clusters of each developmental stage to one another, we plotted the silhouette width of the PCA (Figure 1.3b). The silhouette width was shown in a silhouette plot comparing the minimum average dissimilarity of each object to other clusters with the average dissimilarity to objects in its own cluster. A value close to one indicates that sample which is much closer to its own cluster than to any other cluster, while a value to zero implies that the given sample lies somewhere between two clusters.

Thus, the average of all objects silhouette widths gives an indication of how well the clusters are defined. The silhouette widths of 0.59 for L1→L2, 0.56 for L2→L3, and 0.61 for L3→L4 indicates that these clusters are of reasonable structure (a measure of how tightly grouped all the data in a cluster; silhouette width >0.51-0.7). While the silhouette width of 0.89 for L4→YA cluster suggests that strong cluster structure (silhouette width

>0.71-1) is present (Figure 1.3b). Overall there is a reasonable separation of clusters present based on average silhouette width of 0.66. The variance of each of the principal components obtained from PCA analysis was shown as scree plot in Figure 1.4 and their associated standard deviation, proportion of variance, and cumulative proportion are listed as table on x-axis. The scree plot indicates that the maximum variance in sample is

Figure 1.3: Principal component analysis (PCA) on proteins identified during different development stages: (a) PCA plot showing biological triplicates samples (total 362 samples in each development stage) from L1→L2, L2→L3, L3→L4, and L4→YA stages labeled with a different color. (b) The silhouette plot, where each silhouette represents a cluster (composed of a horizontal line representing each observation), and their width close to 1 represents the strength of each observation’s membership in a cluster.

30 due to PC1 (88.03%) and PC2 contributes to 6.15% variance. The variance of 3% exists up to PC3 in this dataset.

Unsupervised clustering analysis:

To determine whether there are distinct clusters of proteins present in different development stages, we further analyzed the data set by unsupervised clustering analysis and represented it with a heat map (a graphical visualization tool with unsupervised hierarchal clustering for proteins and samples). As shown in Figure 1.5, the column clustering clearly indicates that triplicate samples are similar and four developmental stages are distinct from each other as shown by PCA. Similar to PCA, the most distinct developmental stage was L4→YA stage. The dendrogram of the row clustering (based on identified proteins in each developmental stage) was cut (using cutree function of stat package in R) at specific level to obtain different distinct clusters of two (clusters of 346 and 16 proteins), three (clusters of 3, 343, and 16 proteins), four (clusters of 1, 146, 197, and 16 proteins) or six (clusters of 3, 128, 197, 18, 12, and 4 proteins). We noticed that two clusters consist of 3 (c-1) and 16 (c-4) proteins exhibiting approximately similar slow and rapid PAR respectively at all developmental stages when dendrogram was cut up to six levels. Therefore, we removed those two clusters consist of 3 and 16 proteins and reanalyzed remaining 343 proteins by unsupervised clustering analysis. Reanalysis revealed that there was two distinct clusters of proteins exist (consist of 110 and 129 proteins) when dendrogram was cut to get up to ten clusters. Remaining five clusters consist of small number of proteins exhibiting similar slow and rapid PAR.

The statistical overrepresentation test for gene ontology (GO)-biological process

31 (BP) terms for proteins in these two clusters demonstrated that one cluster consist of translation, protein complex assembly and biogenesis, protein folding and rRNA metabolic process GO-BP terms. While GO-BP terms for proteins in second cluster were gluconeogenesis, TCA cycle, glycolysis, fatty acid biosynthesis, amino acid synthesis and catabolism, protein folding and translation. There were distinct and overlapping GO-BP terms in both clusters and several isoforms of similar proteins were present in different clusters. The unsupervised clustering analysis of proteins demonstrated that proteins based on rapid PAR were not segregated distinctly in a particular GO terms. However, proteins exhibiting rapid PAR were involved in the metabolic process but structural proteins were clustered in slow PAR group.

Figure 1.4: Scree plot depicting variance of the all principle component axis’s present in the different development stage. The scree plot displaying the eigenvalues as variance determined by “prcomp” PCA analysis associated with twelve principal components from the PAR for individual proteins present in four larval stages. The standard deviation, proportion of variance and cumulative proportion associated with each principal component are listed on the x-axis. Variance = (standard deviation)2.

L1→L2 L2→L3 L3→L4 L4→YA

Figure 1.5: Heat map of the proteins identified during different development stage. The heat map showing triplicate samples of four larval development stages (L1→L2, L2→L3, L3→L4, and L4→YA). Only proteins detected in all three replicate samples were subjected to the unsupervised clustering analysis using Euclidean distance and hierarchal clustering of PAR in different development stages. Only row clustering was shown here.

34 Overall distribution of PAR during different larval development stages

To analyze the overall distribution of the PAR for individual proteins identified during each developmental stage (L1→L2, L2→L3, L3→L4, and L4→YA), the mean values of the PAR from proteins identified in triplicate samples (Figure 1.6) were plotted as a boxplot. The analysis showed that the median value for the PAR for L1→L2 stage is the highest (0.05), slow down gradually as worms grow, and the PAR for L4→YA stage is the lowest (0.03), implying that overall protein synthesis activity is high in L1→L2 stage among the developmental stages and it gradually slows down.

It is apparent from the box plot that in each developmental stage there are many proteins for which the PAR are exceptionally rapid or slow (the proteins that are outside whiskers). Interestingly, these rapid or slow PAR proteins were quite different between different developmental stages. There were very few proteins for which the PAR was extremely rapid (only one proteins) at the L1→L2 larval stage. However, there were large numbers of proteins (~100 proteins) with high PAR at other developmental stages.

On the other hand, proteins for which the PAR was exceptionally slow had a reversed trend. There were more proteins with slow PAR at earlier developmental stages (~ 50 proteins at L1→L2 and L2→L3 larval stages) than the later stages (<10 proteins at

L3→L4 and L4→YA stages).

PAR (Frac*on of heavy/hour)

L1→L2 L2→L3 L3→L4 L4→YA

Stages

Figure 1.6: Overall distribution of the PAR for proteins detected during different larval development stages. Boxplot showing the variability in the PAR for all the proteins identified at different developmental stages. The line inside the box represents median value, the bottom line of the box represents the first quartile and the upper line of the box represents the third quartile. Data points outside the whiskers are greater or less than 1.5 times the interquartile range (75th percentile – 25th percentile).

36 Gene Ontology (GO) and Pathway Association of the proteins exhibiting rapid PAR at different development stages:

In order to understand better the molecular function of the proteins with rapid

PAR (>mean PAR + 1.5 SD) and with slow PAR (

KEGG pathways associated with these proteins were obtained and statistical overrepresentation test was performed on these proteins. The proteins exhibiting rapid and slow PAR at different development stages were listed in the Supplementary Table-

1.1, 1.2, 1.3, and 1.4. The results from the analysis on the proteins with rapid PAR are presented in the main text below and on the proteins with slow PAR are provided as supplemental data (Supplementary Table-1.9-1.16).

Gene ontology (GO)-molecular function (MF)

The identified GO-MF terms of the proteins with rapid PAR was shown in Figure

1.7. The proteins with rapid PAR from all developmental stages were categorized in the total of nine GO-MF terms (Table-1.1). Among the categories, catalytic activity (~37%-

50%) and binding (12%-22%) categories were common in all the developmental stages.

Most of the proteins with rapid PAR at L2→L3 stage (~50%) were in catalytic activity category. Table-1.1 shows the proteins with rapid PAR present in each GO-MF terms at each development stages. Proteins in Table-1.1 were color-coded based on presence in only one (blue), two (black), three (pink), and all four (green) of the developmental stages. Cathepsin B-like cysteine proteinase (cpr)-4 and -5 was common at all the

37 development stages, while there were many other proteases such as aspartyl proteases

(asp-1, -2, -3 -5, 6, 13) that were distinct between the larval stages. The binding group consisted of proteins mainly belong to RNA or DNA helicases, and histone proteins.

They were present at different development stages except at the L1→L2 stage.

Interestingly, the protein in the antioxidant category was present only at L3→L4 and

L4→YA. These proteins were from peroxidase class and represented by glutathione peroxidase (gpx-5) and peroxidase (protein F49E12.1) respectively.

The statistical overrepresentation test for GO-MF terms (Table 1.2) showed that proteins with rapid PAR at L1→L2 and L2→L3 are significantly (p<0.05) overrepresented by aspartic type-endopeptidase and peptidase activity. While proteins from extracellular matrix structural activity and helicase activity GO-MF terms were overrepresented at L3→L4. However, proteins with translation initiation factor activity

GO-MF terms were overrepresented at both L3→L4 and L4→YA stages. This result indicates that proteins with rapid PAR in different development stages had distinct overrepresented GO-MF terms that might correlate with the physiological necessity of the early versus late developmental stage.

1, 2% 1, 1% 1, 2% 1, 1% 1, 1% 1, 2% 3, 4% 3, 4% 2, 3% 1, 13% 4, 5% 3, 5% 5, 7% 7, 9% 4, 6%

1, 13% 3, 37% 26, 38% 7, 10% 4, 6% 30, 47% 9, 12% 41, 53% 5, 8% 1, 12% 10, 15%

12, 16% 14, 22% 14, 21% 2, 25% 1%

4% L1→L2 L2→L3 L3→L4 L4→YA 5% 13% (8) (77) (67) (63) 9% cataly-c ac-vity cataly/c ac/vity cataly0c ac0vity cataly0c ac0vity binding binding 13% 37% structural molecule ac-vity binding receptor ac/vity nucleic acid binding structural molecule ac0vity transla0on regulator ac0vity 12% transcrip-on factor ac-vity structural molecule ac/vity 2% 2% 53% 2% receptor ac0vity transporter ac0vity binding 1% transporter ac/vity 3% 1% 4% 5% receptor ac-vity transla/on regulator ac/vity transla0on regulator ac0vity receptor ac0vity 12% enzyme regulator ac/vity transporter ac0vity enzyme regulator ac0vity 7% 6% 16% 6% enzyme regulator ac0vity 38% 47% structural molecule ac0vity 10% 8% nucleic acid binding nucleic acid binding 25% transcrip0on factor ac0vity transcrip0on factor ac0vity an0oxidant ac0vity 15% an0oxidant ac0vity 22% 21% Figure 1.7: Gene Ontology (GO)-Molecular function (MF) terms for proteins with rapid PAR. The pie graph shows GO-MF for proteins with rapid PAR found in different larval stages (L1→L2 = 13, L2→L3 = 99, L3→L4 =121, L4→YA =144). The total number of proteins mapped to GO-MF terms was written in parenthesis under individual development stage out of the total number of input proteins. Those input proteins are listed in Supplementary Table 1.1, 1.2, 1.3 and 1.4. Each GO-MF term (a slice of a pie graph) is marked with gene counts and percent of the count of genes present in individual GO-MF term to the total gene count (written in parenthesis under each development stage) mapped to GO terms for individual development stage.

39 Table-1.1: Gene ontology (GO)-Molecular Function (MF) for proteins with rapid PAR at different developmental stages. GO Terms Wormbase Gene ID Gene Name L1→L2 L2→L3 L3→L4 L4→YA WBGene00000717 Protein COL-144;col-144;ortholog x WBGene00000784 Cathepsin B-like cysteine proteinase 4;cpr-4;ortholog x x x x WBGene00000785 Cathepsin B-like cysteine proteinase 5;cpr-5;ortholog x x x x WBGene00017881 Protein ASP-13;asp-13;ortholog x x WBGene00000214 Aspartic protease 1;asp-1;ortholog x WBGene00000215 Protein ASP-2, isoform a;asp-2;ortholog x WBGene00000216 Aspartic protease 3;asp-3;ortholog x x x WBGene00000218 Protein ASP-5;asp-5;ortholog x WBGene00000219 Aspartic protease 6;asp-6;ortholog x x x WBGene00000776 Protein CPL-1, isoform a;cpl-1;ortholog x x WBGene00000781 Gut-specific cysteine proteinase;cpr-1;ortholog x x x WBGene00000882 Peptidyl-prolyl cis-trans isomerase 6;cyn-6;ortholog x WBGene00001077 Prolyl 4-hydroxylase subunit alpha-1;dpy-18;ortholog x WBGene00001149 Branched-chain-amino-acid aminotransferase, cytosolic;bcat-1;ortholog x x x WBGene00001598 ATP-dependent RNA helicase glh-1;glh-1;ortholog x WBGene00001599 ATP-dependent RNA helicase glh-2;glh-2;ortholog x WBGene00001856 Histone H1.5;hil-5;ortholog x x WBGene00003093 Protein LYS-4;lys-4;ortholog x WBGene00003096 Protein LYS-7;lys-7;ortholog x WBGene00004259 Protein PYR-1;pyr-1;ortholog x WBGene00004391 Ribonucleoside-diphosphate reductase large subunit;rnr-1;ortholog x x x WBGene00006609 Protein TRE-3, isoform b;tre-3;ortholog x x x WBGene00007605 Protein ASP-10, isoform a;asp-10;ortholog x WBGene00007969 Protein ACS-19, isoform a;acs-19;ortholog x x x WBGene00008741 Uncharacterized serine carboxypeptidase F13S12.6;F13D12.6;ortholog x WBGene00009048 Protein CTH-1, isoform a;cth-1;ortholog x WBGene00009342 Protein FASN-1;fasn-1;ortholog x WBGene00009706 Protein F44G3.2;CELE_F44G3.2;ortholog x WBGene00010260 Protein DDX-17, isoform a;ddx-17;ortholog x WBGene00010988 Probable methionine synthase;metr-1;ortholog x WBGene00011272 Probable thymidylate kinase;R53.2;ortholog x WBGene00012186 Protein MLT-11, isoform b;mlt-11;ortholog x WBGene00012530 Protein Y32F6A.5, isoform a;CELE_Y32F6A.5;ortholog x WBGene00016201 Tryptophan 2,3-dioxygenase;C28H8.11;ortholog x WBGene00016335 Putative glucosylceramidase 1;gba-1;ortholog x WBGene00016422 Protein NOAH-1, isoform c;noah-1;ortholog x WBGene00016957 Protein C55F2.1, isoform b;C55F2.1;ortholog x WBGene00019565 Protein CYP-35A3;cyp-35a3;ortholog x WBGene00019682 Putative serine protease K12H4.7;K12H4.7;ortholog x WBGene00020297 Probable nucleolar GTP-binding protein 1;T07A9.9;ortholog x WBGene00022146 Protein Y71G12B.6, isoform a;CELE_Y71G12B.6;ortholog x WBGene00022781 Protein PMT-1, isoform b;pmt-1;ortholog x x x catalytic activity (GO:0003824) WBGene00022856 Putative cystathionine gamma-lyase 2;cth-2;ortholog x WBGene00044294 Protein C01B10.11;C01B10.11;ortholog x WBGene00000252 Protein BLI-2;bli-2;ortholog x WBGene00000479 ATP-dependent RNA helicase cgh-1;cgh-1;ortholog x WBGene00001386 Fatty-acid and retinol-binding protein 2;far-2;ortholog x WBGene00001601 ATP-dependent RNA helicase glh-4;glh-4;ortholog x WBGene00001694 Protein GRD-5;grd-5;ortholog x WBGene00003448 Major sperm protein 55/57;msp-55;ortholog x WBGene00003776 Protein NMY-1;nmy-1;ortholog x WBGene00003992 P granule abnormality protein 1;pgl-1;ortholog x WBGene00004798 Stress-induced protein 1;sip-1;ortholog x WBGene00006926 Vitellogenin-2;vit-2;ortholog x WBGene00006928 Vitellogenin-4;vit-4;ortholog x WBGene00007479 Protein C09F9.2;C09F9.2;ortholog x WBGene00009213 Protein THN-1;thn-1;ortholog x WBGene00010204 Protein F57F5.1;CELE_F57F5.1;ortholog x WBGene00010651 Protein K08C9.2;CELE_K08C9.2;ortholog x WBGene00011222 Protein R10H10.3;CELE_R10H10.3;ortholog x WBGene00011753 Protein T13F3.6;CELE_T13F3.6;ortholog x WBGene00012351 Protein W09C5.1;CELE_W09C5.1;ortholog x WBGene00017132 Maternal effect lethal protein 47;mel-47;ortholog x WBGene00018342 Protein F42A10.6;CELE_F42A10.6;ortholog x WBGene00020662 Protein T21H3.1, isoform a;CELE_T21H3.1;ortholog x WBGene00022245 Protein ACP-6;acp-6;ortholog x WBGene00043743 Protein Y59E9AL.2;CELE_Y59E9AL.2;ortholog x WBGene00000405 Cyclin-dependent kinase 1;cdk-1;ortholog x WBGene00000838 Cullin-3;cul-3;ortholog x WBGene00003154 Protein MCM-2, isoform a;mcm-2;ortholog x WBGene00003155 Protein MCM-3;mcm-3;ortholog x WBGene00003828 Deoxyribonuclease-2;nuc-1;ortholog x WBGene00003955 Proliferating cell nuclear antigen;pcn-1;ortholog x

40 WBGene00004955 Spindle-defective protein 5;spd-5;ortholog x WBGene00005022 UDP-glucose 6-dehydrogenase;sqv-4;ortholog x WBGene00008546 Protein F07A11.2, isoform a;CELE_F07A11.2;ortholog x WBGene00009035 Protein F22B3.4;CELE_F22B3.4;ortholog x WBGene00009897 Protein F49E12.1;CELE_F49E12.1;ortholog x WBGene00010609 Deoxyuridinetriphosphatase;dut-1;ortholog x WBGene00011038 Protein R05H5.3;CELE_R05H5.3;ortholog x WBGene00012600 Protein Y38E10A.22, isoform a;CELE_Y38E10A.22;ortholog x WBGene00018488 Protein ACS-1, isoform a;acs-1;ortholog x Acidic leucine-rich nuclear phosphoprotein 32-related protein WBGene00020588 x 2;T19H12.2;ortholog WBGene00022610 Protein ZC416.6, isoform a;CELE_ZC416.6;ortholog x WBGene00000717 Protein COL-144;col-144;ortholog x WBGene00006824 Protein UNC-95;unc-95;ortholog x WBGene00017881 Protein ASP-13;asp-13;ortholog x WBGene00001598 ATP-dependent RNA helicase glh-1;glh-1;ortholog x x WBGene00001599 ATP-dependent RNA helicase glh-2;glh-2;ortholog x WBGene00001716 Protein GRL-7, isoform a;grl-7;ortholog x x WBGene00001856 Histone H1.5;hil-5;ortholog x x WBGene00006727 Polyubiquitin-A;ubq-1;ortholog x x WBGene00006728 Ubiquitin-60S ribosomal protein L40;ubq-2;ortholog x WBGene00010260 Protein DDX-17, isoform a;ddx-17;ortholog x WBGene00012186 Protein MLT-11, isoform b;mlt-11;ortholog x WBGene00016201 Tryptophan 2,3-dioxygenase;C28H8.11;ortholog x WBGene00018237 Protein F40F4.6;CELE_F40F4.6;ortholog x WBGene00019957 Protein R08E3.1, isoform a;CELE_R08E3.1;ortholog x x WBGene00020297 Probable nucleolar GTP-binding protein 1;T07A9.9;ortholog x WBGene00000479 ATP-dependent RNA helicase cgh-1;cgh-1;ortholog x x WBGene00001601 ATP-dependent RNA helicase glh-4;glh-4;ortholog x x binding (GO:0005488) WBGene00002064 Eukaryotic translation initiation factor 5A-1;iff-1;ortholog x x WBGene00003159 Protein MCM-7;mcm-7;ortholog x WBGene00003776 Protein NMY-1;nmy-1;ortholog x WBGene00003992 P granule abnormality protein 1;pgl-1;ortholog x WBGene00009444 Enhancer of rudimentary homolog;CELE_F35G12.11;ortholog x x WBGene00012484 Protein CAR-1;car-1;ortholog x x WBGene00077697 Protein F58E6.13, isoform c;CELE_F58E6.13;ortholog x WBGene00002061 Eukaryotic translation initiation factor 4E-3;ife-3;ortholog x WBGene00003154 Protein MCM-2, isoform a;mcm-2;ortholog x WBGene00003155 Protein MCM-3;mcm-3;ortholog x WBGene00004241 Pumilio domain-containing protein 5;puf-5;ortholog x WBGene00004955 Spindle-defective protein 5;spd-5;ortholog x WBGene00010609 Deoxyuridinetriphosphatase;dut-1;ortholog x WBGene00013209 Protein BUB-3;bub-3;ortholog x WBGene00019157 Protein H05C05.1, isoform c;CELE_H05C05.1;ortholog x Acidic leucine-rich nuclear phosphoprotein 32-related protein WBGene00020588 x 2;T19H12.2;ortholog WBGene00000717 Protein COL-144;col-144;ortholog x x x WBGene00006824 Protein UNC-95;unc-95;ortholog x WBGene00000699 Protein COL-125;col-125;ortholog x x WBGene00000730 Protein COL-157;col-157;ortholog x WBGene00005016 Cuticle collagen sqt-1;sqt-1;ortholog x x WBGene00006727 Polyubiquitin-A;ubq-1;ortholog x x WBGene00006728 Ubiquitin-60S ribosomal protein L40;ubq-2;ortholog x structural molecule WBGene00019957 Protein R08E3.1, isoform a;CELE_R08E3.1;ortholog x x activity (GO:0005198) WBGene00003776 Protein NMY-1;nmy-1;ortholog x WBGene00004398 Cuticle collagen 6;rol-8;ortholog x WBGene00006039 Sperm-specific class P protein 10;ssp-10;ortholog x WBGene00009031 Major sperm protein;CELE_F21H7.5;ortholog x WBGene00010091 Protein SSP-35;ssp-35;ortholog x WBGene00000693 Protein COL-119;col-119;ortholog x WBGene00000698 Protein COL-124;col-124;ortholog x WBGene00012186 Protein MLT-11, isoform b;mlt-11;ortholog x WBGene00003776 Protein NMY-1;nmy-1;ortholog x enzyme regulator activity WBGene00003955 Proliferating cell nuclear antigen;pcn-1;ortholog x (GO:0030234) WBGene00012600 Protein Y38E10A.22, isoform a;CELE_Y38E10A.22;ortholog x Acidic leucine-rich nuclear phosphoprotein 32-related protein WBGene00020588 x 2;T19H12.2;ortholog WBGene00006366 Protein SYM-1;sym-1;ortholog x x x WBGene00000699 Protein COL-125;col-125;ortholog x x WBGene00000730 Protein COL-157;col-157;ortholog x WBGene00003497 Transmembrane matrix receptor MUP-4;mup-4;ortholog x x x WBGene00004210 Protein PTC-3, isoform c;ptc-3;ortholog x receptor activity WBGene00012253 Protein CLEC-50;clec-50;ortholog x x x (GO:0004872) WBGene00016422 Protein NOAH-1, isoform c;noah-1;ortholog x x WBGene00018237 Protein F40F4.6;CELE_F40F4.6;ortholog x WBGene00019957 Protein R08E3.1, isoform a;CELE_R08E3.1;ortholog x x

catalytic activity (GO:0003824) receptor activity (GO:0004872)

WBGene00004398 Cutticle collagen;rol-8;ortholog x x WBGene00000693 Protein COL-119;col-119;ortholog x x WBGene00000698 Protein COL-124;col-124;ortholog x x WBGene00001598 ATP-dependent RNA helicase glh-1;glh-1;ortholog x x WBGene00001599 ATP-dependent RNA helicase glh-2;glh-2;ortholog x WBGene00010260 Protein DDX-17, isoform a;ddx-17;ortholog x WBGene00000479 ATP-dependent RNA helicase cgh-1;cgh-1;ortholog x x translation regulator WBGene00001601 ATP-dependent RNA helicase glh-4;glh-4;ortholog x x activity (GO:0045182) WBGene00002064 Eukaryotic translation initiation factor 5A-1;iff-1;ortholog x x WBGene00003992 P granule abnormality protein 1;pgl-1;ortholog x WBGene00002061 Eukaryotic translation initiation factor 4E-3;ife-3;ortholog x WBGene00019157 Protein H05C05.1, isoform c;CELE_H05C05.1;ortholog x WBGene00000699 Protein COL-125;col-125;ortholog x x WBGene00000730 Protein COL-157;col-157;ortholog x WBGene00003497 Transmembrane matrix receptor MUP-4;mup-4;ortholog x x x WBGene00011891 Protein DEL-6, isoform b;del-6;ortholog x antioxidant activity WBGene00004398 Cuticle collagen 6;rol-8;ortholog x (GO:0016209) WBGene00000693 Protein COL-119;col-119;ortholog x WBGene00000698 Protein COL-124;col-124;ortholog x WBGene00006933 Vacuolar protein sorting-associated protein 35;vps-35;ortholog x WBGene00007516 Glutathione peroxidase;gpx-5;ortholog x WBGene00009897 Protein F49E12.1;CELE_F49E12.1;ortholog x nucleic acid binding WBGene00006824 Protein UNC-95;unc-95;ortholog x transcription factor activity (GO:0001071) WBGene00009444 Enhancer of rudimentary homolog;CELE_F35G12.11;ortholog x x

Text in blue font = proteins present in one of the development stages; Text in black font = proteins present in a two of the development stage; Text in pink font = proteins present in three of the development stages; Text in green font = proteins present in all four of the development stages; x indicates proteins is that particular development satge present.

42 Table-1.2: The statistical overrepresentation test (PANTHER GO-Molecular Function) of proteins with rapid PAR at different development stages. C. elegans - Input Input Input Input (fold Input PANTHER GO-Molecular Function REFLIST (n) (expected) (over/under) Enrichment) (p value) (20490) L1→L2* ( n=13) aspartic-type endopeptidase activity (GO:0004190) 22 1 0.01 + > 5 0.01390 cysteine-type peptidase activity (GO:0008234) 70 2 0.04 + > 5 0.00089 peptidase activity (GO:0008233) 370 3 0.23 + > 5 0.00147 L2→L3 ( n=99) aspartic-type endopeptidase activity (GO:0004190) 22 7 0.11 + > 5 3.39E-09 extracellular matrix structural constituent (GO:0005201) 85 5 0.41 + > 5 9.60E-03 peptidase activity (GO:0008233) 370 17 1.79 + > 5 4.71E-10 hydrolase activity (GO:0016787) 1415 22 6.84 + 3.22 1.34E-04 catalytic activity (GO:0003824) 3619 41 17.49 + 2.34 4.33E-06 L3→L4 ( n=121) extracellular matrix structural constituent (GO:0005201) 85 5 0.5 + > 5 2.47E-02 translation initiation factor activity (GO:0003743) 86 5 0.51 + > 5 2.61E-02 helicase activity (GO:0004386) 93 5 0.55 + > 5 3.74E-02 L4→YA ( n=112) transaminase activity (GO:0008483) 18 3 0.1 + > 5 2.17E-02 translation initiation factor activity (GO:0003743) 86 5 0.47 + > 5 1.82E-02 translation factor activity, nucleic acid binding (GO:0008135) 123 6 0.67 + > 5 9.82E-03 translation regulator activity (GO:0045182) 107 5 0.58 + > 5 4.95E-02 *GO-MF terms with p value <0.05 without Bonferroni correction.

43 Gene ontology (GO)-biological process (BP):

All proteins exhibiting rapid PAR were categorized in the twelve GO-BP terms

(Table-1.3). We found that the majority of the proteins with rapid PAR belong to the metabolic processes GO-BP term (38%-48%) at all four developmental stages (Figure

1.7). The cathepsin proteinase and aspartyl proteases were the major proteins that represented the metabolic process GO-BP term. Other proteins were chaperonins, reductases, phosphatases, dehydrogenases, methyltransferases, isomerase/calcium binding proteins, transaminase in the metabolic process GO-BP terms. Many GO-BP terms (such as metabolic process, cellular process, biological regulation, biological adhesion, cellular component organization or biogenesis, developmental process, localization, response to stimulus, multicellular organismal process, immune system process) were common to all four developmental stages, however, the numbers and types of proteins that represented each GO term differ significantly across the four development stages. Interestingly, only L4→YA stage had a protein, nuc-1 ortholog, endodeoxyribonuclease 2, from the apoptotic GO-BP term. Similarly, only the L2→L3 stage had the SCL-2 protein, an extracellular sperm coating protein from the reproduction

GO-BP term. The GO-BP terms such as cellular process, localization, development process, response to stimulus, cellular component, and biological adhesion were mostly represented by different collagen (COL) member family proteins at different developmental stages.

44 1, 1% 3, 3% 3, 3% 1, 1% 1, 1% 3, 3% 2, 2% 3, 3% 2, 2% 1, 6% 4, 4% 1, 6% 4, 4% 3, 4% 4, 4% 3, 4% 1, 6% 5, 5% 6, 33% 5, 5% 1, 5% 5, 5% 35, 38% 7, 8% 6, 5% 48, 44% 41, 48% 6, 7% 2, 11% 7, 6% 11, 13% 6, 7% 8, 7% 2, 11% 2, 11% 10, 11% 14, 17% 16, 17% 2, 11% 19, 18% L1→L2 L2→L3 L3→L4 L4→YA (18) (108) (93) (85)

metabolic process metabolic process metabolic process metabolic process

cellular process cellular process cellular process cellular process biological regula8on biological regula7on biological regula7on biological regula5on localiza8on 3% localiza7on localiza7on 6% developmental process 6% 1% biological adhesion 3% 2% biological adhesion response to s7mulus 4% 1% 6% biological adhesion 3% 1% 4% developmental process 4% 2% 33% 3% cellular component immune system process 4% 4% 5% 5% cellular component mul8cellular organismal organiza7on or biogenesis 5% process developmental process cellular component 5% organiza5on or biogenesis 44% immune system process 38% response to s5mulus 5% 8% organiza7on or biogenesis 11% 6% response to s7mulus developmental process 7% cellular component 48% mul5cellular organismal organiza8on or biogenesis 7% 13% mul7cellular organismal apopto7c process 7% response to s8mulus process process 11% 11% 18% immune system process biological adhesion 11% reproduc8on immune system process 11% 17% 17%

Figure 1.8: Gene Ontology (GO)-Biological Process (BP) terms for proteins with rapid PAR. The pie graph shows GO-BP terms for proteins with rapid PAR found in different larval stages (L1→L2 = 13, L2→L3 = 99, L3→L4 =121, L4→YA =144). The total number of proteins mapped to GO-BP terms was written in parenthesis under individual development stage out of the total number of input proteins. Those input proteins are listed in Supplementary Table 1.1, 1.2, 1.3 and 1.4. Each GO-BP term (a slice of a pie graph) is marked with gene counts and percent of the count of genes present in individual GO-BP term to the total gene count mapped to GO terms (written in parenthesis under each stage) for individual development stage.

45 Table-1.3: Gene ontology (GO)-Biological Process (BP) for proteins with rapid PAR at different developmental stages. GO Term Wormbase Gene ID Gene name L1→L2 L2→L3 L3→L4 L4→YA WBGene00000784 Cathepsin B-like cysteine proteinase 4;cpr-4;ortholog x x x x WBGene00000785 Cathepsin B-like cysteine proteinase 5;cpr-5;ortholog x x x x WBGene00004415 60S ribosomal protein L4;rpl-4;ortholog x WBGene00006366 Protein SYM-1;sym-1;ortholog x x x WBGene00006824 Protein UNC-95;unc-95;ortholog x WBGene00017881 Protein ASP-13;asp-13;ortholog x x WBGene00000214 Aspartic protease 1;asp-1;ortholog x WBGene00000215 Protein ASP-2, isoform a;asp-2;ortholog x WBGene00000216 Aspartic protease 3;asp-3;ortholog x x x WBGene00000218 Protein ASP-5;asp-5;ortholog x WBGene00000219 Aspartic protease 6;asp-6;ortholog x x x WBGene00000776 Protein CPL-1, isoform a;cpl-1;ortholog x x WBGene00000781 Gut-specific cysteine proteinase;cpr-1;ortholog x x x WBGene00000882 Peptidyl-prolyl cis-trans isomerase 6;cyn-6;ortholog x x WBGene00001077 Prolyl 4-hydroxylase subunit alpha-1;dpy-18;ortholog x Branched-chain-amino-acid aminotransferase, cytosolic;bcat- WBGene00001149 x x x 1;ortholog WBGene00001598 ATP-dependent RNA helicase glh-1;glh-1;ortholog x x WBGene00001599 ATP-dependent RNA helicase glh-2;glh-2;ortholog x WBGene00001856 Histone H1.5;hil-5;ortholog x x WBGene00003093 Protein LYS-4;lys-4;ortholog x WBGene00003096 Protein LYS-7;lys-7;ortholog x WBGene00003497 Transmembrane matrix receptor MUP-4;mup-4;ortholog x x x WBGene00004210 Protein PTC-3, isoform c;ptc-3;ortholog x WBGene00004259 Protein PYR-1;pyr-1;ortholog x x Ribonucleoside-diphosphate reductase large subunit;rnr- WBGene00004391 x x x 1;ortholog WBGene00006609 Protein TRE-3, isoform b;tre-3;ortholog x x x WBGene00006727 Polyubiquitin-A;ubq-1;ortholog x x WBGene00006728 Ubiquitin-60S ribosomal protein L40;ubq-2;ortholog x WBGene00007605 Protein ASP-10, isoform a;asp-10;ortholog x WBGene00007969 Protein ACS-19, isoform a;acs-19;ortholog x x Uncharacterized serine carboxypeptidase WBGene00008741 x x F13S12.6;F13D12.6;ortholog WBGene00009048 Protein CTH-1, isoform a;cth-1;ortholog x WBGene00009342 Protein FASN-1;fasn-1;ortholog x x WBGene00009706 Protein F44G3.2;CELE_F44G3.2;ortholog x WBGene00010260 Protein DDX-17, isoform a;ddx-17;ortholog x WBGene00010988 Probable methionine synthase;metr-1;ortholog x metabolic process WBGene00011272 Probable thymidylate kinase;R53.2;ortholog x (GO:0008152) WBGene00012186 Protein MLT-11, isoform b;mlt-11;ortholog x WBGene00012530 Protein Y32F6A.5, isoform a;CELE_Y32F6A.5;ortholog x WBGene00016201 Tryptophan 2,3-dioxygenase;C28H8.11;ortholog x WBGene00016335 Putative glucosylceramidase 1;gba-1;ortholog x WBGene00016957 Protein C55F2.1, isoform b;C55F2.1;ortholog x WBGene00019565 Protein CYP-35A3;cyp-35a3;ortholog x x WBGene00019682 Putative serine protease K12H4.7;K12H4.7;ortholog x x WBGene00020297 Probable nucleolar GTP-binding protein 1;T07A9.9;ortholog x WBGene00022146 Protein Y71G12B.6, isoform a;CELE_Y71G12B.6;ortholog x WBGene00022245 Protein ACP-6;acp-6;ortholog x x WBGene00022781 Protein PMT-1, isoform b;pmt-1;ortholog x x WBGene00022856 Putative cystathionine gamma-lyase 2;cth-2;ortholog x WBGene00044294 Protein C01B10.11;C01B10.11;ortholog x WBGene00000479 ATP-dependent RNA helicase cgh-1;cgh-1;ortholog x x WBGene00001601 ATP-dependent RNA helicase glh-4;glh-4;ortholog x x WBGene00002064 Eukaryotic translation initiation factor 5A-1;iff-1;ortholog x x WBGene00003159 Protein MCM-7;mcm-7;ortholog x WBGene00003776 Protein NMY-1;nmy-1;ortholog x WBGene00003992 P granule abnormality protein 1;pgl-1;ortholog x WBGene00004798 Stress-induced protein 1;sip-1;ortholog x WBGene00007516 Glutathione peroxidase;gpx-5;ortholog x WBGene00009444 Enhancer of rudimentary homolog;CELE_F35G12.11;ortholog x x WBGene00010204 Protein F57F5.1;CELE_F57F5.1;ortholog x WBGene00012484 Protein CAR-1;car-1;ortholog x x WBGene00013894 Probable arginine kinase ZC434.8;ZC434.8;ortholog x WBGene00022610 Protein ZC416.6, isoform a;CELE_ZC416.6;ortholog x x WBGene00000405 Cyclin-dependent kinase 1;cdk-1;ortholog x WBGene00000838 Cullin-3;cul-3;ortholog x WBGene00003155 Protein MCM-3;mcm-3;ortholog x WBGene00003828 Deoxyribonuclease-2;nuc-1;ortholog x WBGene00003955 Proliferating cell nuclear antigen;pcn-1;ortholog x WBGene00004241 Pumilio domain-containing protein 5;puf-5;ortholog x WBGene00008546 Protein F07A11.2, isoform a;CELE_F07A11.2;ortholog x WBGene00009035 Protein F22B3.4;CELE_F22B3.4;ortholog x

46 WBGene00009897 Protein F49E12.1;CELE_F49E12.1;ortholog x WBGene00010609 Deoxyuridinetriphosphatase;dut-1;ortholog x WBGene00011038 Protein R05H5.3;CELE_R05H5.3;ortholog x WBGene00013209 Protein BUB-3;bub-3;ortholog x WBGene00018488 Protein ACS-1, isoform a;acs-1;ortholog x WBGene00019157 Protein H05C05.1, isoform c;CELE_H05C05.1;ortholog x WBGene00000717 Protein COL-144;col-144;ortholog x x x WBGene00006366 Protein SYM-1;sym-1;ortholog x x x WBGene00000699 Protein COL-125;col-125;ortholog x x WBGene00000730 Protein COL-157;col-157;ortholog x WBGene00000882 Peptidyl-prolyl cis-trans isomerase 6;cyn-6;ortholog x x WBGene00001716 Protein GRL-7, isoform a;grl-7;ortholog x x WBGene00001856 Histone H1.5;hil-5;ortholog x x WBGene00003497 Transmembrane matrix receptor MUP-4;mup-4;ortholog x x x WBGene00004210 Protein PTC-3, isoform c;ptc-3;ortholog x WBGene00004259 Protein PYR-1;pyr-1;ortholog x x WBGene00005016 Cuticle collagen sqt-1;sqt-1;ortholog x x Uncharacterized serine carboxypeptidase WBGene00008741 x x F13S12.6;F13D12.6;ortholog WBGene00010988 Probable methionine synthase;metr-1;ortholog x WBGene00011272 Probable thymidylate kinase;R53.2;ortholog x WBGene00012530 Protein Y32F6A.5, isoform a;CELE_Y32F6A.5;ortholog x WBGene00016201 Tryptophan 2,3-dioxygenase;C28H8.11;ortholog x WBGene00016422 Protein NOAH-1, isoform c;noah-1;ortholog x x cellular process WBGene00018237 Protein F40F4.6;CELE_F40F4.6;ortholog x (GO:0009987) WBGene00019957 Protein R08E3.1, isoform a;CELE_R08E3.1;ortholog x x WBGene00003159 Protein MCM-7;mcm-7;ortholog x WBGene00003776 Protein NMY-1;nmy-1;ortholog x WBGene00004398 Cuticle collagen 6;rol-8;ortholog x WBGene00077697 Protein F58E6.13, isoform c;CELE_F58E6.13;ortholog x WBGene00000405 Cyclin-dependent kinase 1;cdk-1;ortholog x WBGene00000693 Protein COL-119;col-119;ortholog x WBGene00000698 Protein COL-124;col-124;ortholog x WBGene00000838 Cullin-3;cul-3;ortholog x WBGene00003154 Protein MCM-2, isoform a;mcm-2;ortholog x WBGene00003155 Protein MCM-3;mcm-3;ortholog x WBGene00003828 Deoxyribonuclease-2;nuc-1;ortholog x WBGene00003955 Proliferating cell nuclear antigen;pcn-1;ortholog x WBGene00004042 Serine/threonine-protein kinase plk-1;plk-1;ortholog x WBGene00004955 Spindle-defective protein 5;spd-5;ortholog x WBGene00006933 Vacuolar protein sorting-associated protein 35;vps-35;ortholog x WBGene00010609 Deoxyuridinetriphosphatase;dut-1;ortholog x WBGene00013209 Protein BUB-3;bub-3;ortholog x WBGene00006366 Protein SYM-1;sym-1;ortholog x x x WBGene00006824 Protein UNC-95;unc-95;ortholog x WBGene00000699 Protein COL-125;col-125;ortholog x x WBGene00000730 Protein COL-157;col-157;ortholog x WBGene00001598 ATP-dependent RNA helicase glh-1;glh-1;ortholog x x WBGene00001599 ATP-dependent RNA helicase glh-2;glh-2;ortholog x WBGene00010260 Protein DDX-17, isoform a;ddx-17;ortholog x WBGene00011891 Protein DEL-6, isoform b;del-6;ortholog x WBGene00012186 Protein MLT-11, isoform b;mlt-11;ortholog x WBGene00000479 ATP-dependent RNA helicase cgh-1;cgh-1;ortholog x x WBGene00001601 ATP-dependent RNA helicase glh-4;glh-4;ortholog x x biological regulation WBGene00002064 Eukaryotic translation initiation factor 5A-1;iff-1;ortholog x x (GO:0065007) WBGene00003776 Protein NMY-1;nmy-1;ortholog x WBGene00003992 P granule abnormality protein 1;pgl-1;ortholog x WBGene00004398 Cuticle collagen 6;rol-8;ortholog x WBGene00009444 Enhancer of rudimentary homolog;CELE_F35G12.11;ortholog x x WBGene00000693 Protein COL-119;col-119;ortholog x WBGene00000698 Protein COL-124;col-124;ortholog x WBGene00002061 Eukaryotic translation initiation factor 4E-3;ife-3;ortholog x WBGene00003955 Proliferating cell nuclear antigen;pcn-1;ortholog x WBGene00012600 Protein Y38E10A.22, isoform a;CELE_Y38E10A.22;ortholog x WBGene00019157 Protein H05C05.1, isoform c;CELE_H05C05.1;ortholog x Acidic leucine-rich nuclear phosphoprotein 32-related protein WBGene00020588 x 2;T19H12.2;ortholog WBGene00000699 Protein COL-125;col-125;ortholog x x WBGene00000730 Protein COL-157;col-157;ortholog x WBGene00003497 Transmembrane matrix receptor MUP-4;mup-4;ortholog x x x WBGene00004210 Protein PTC-3, isoform c;ptc-3;ortholog x Uncharacterized serine carboxypeptidase WBGene00008741 x x F13S12.6;F13D12.6;ortholog WBGene00011891 Protein DEL-6, isoform b;del-6;ortholog x localization WBGene00012253 Protein CLEC-50;clec-50;ortholog x x x (GO:0051179) WBGene00003776 Protein NMY-1;nmy-1;ortholog x WBGene00004398 Cuticle collagen 6;rol-8;ortholog x

metabolic process (GO:0008152) localization (GO:0051179)

WBGene00000693 Protein COL-119;col-119;ortholog x WBGene00000698 Protein COL-124;col-124;ortholog x WBGene00001501 Ferritin;ftn-2;ortholog x WBGene00006933 Vacuolar protein sorting-associated protein 35;vps-35;ortholog x WBGene00013209 Protein BUB-3;bub-3;ortholog x WBGene00000717 Protein COL-144;col-144;ortholog x x x WBGene00006366 Protein SYM-1;sym-1;ortholog x x x WBGene00005016 Cuticle collagen sqt-1;sqt-1;ortholog x x developmental WBGene00018237 Protein F40F4.6;CELE_F40F4.6;ortholog x process WBGene00019957 Protein R08E3.1, isoform a;CELE_R08E3.1;ortholog x x (GO:0032502) WBGene00004398 Cuticle collagen 6;rol-8;ortholog x WBGene00003828 Deoxyribonuclease-2;nuc-1;ortholog x WBGene00004042 Serine/threonine-protein kinase plk-1;plk-1;ortholog x WBGene00006824 Protein UNC-95;unc-95;ortholog x WBGene00000699 Protein COL-125;col-125;ortholog x x WBGene00000730 Protein COL-157;col-157;ortholog x WBGene00012186 Protein MLT-11, isoform b;mlt-11;ortholog x response to stimulus WBGene00004398 Cuticle collagen 6;rol-8;ortholog x (GO:0050896) WBGene00004798 Stress-induced protein 1;sip-1;ortholog x x WBGene00007516 Glutathione peroxidase;gpx-5;ortholog x WBGene00000693 Protein COL-119;col-119;ortholog x WBGene00000698 Protein COL-124;col-124;ortholog x reproduction WBGene00009895 Protein SCL-2, isoform a;scl-2;ortholog x (GO:0000003) WBGene00001856 Histone H1.5;hil-5;ortholog x x cellular component WBGene00005016 Cuticle collagen sqt-1;sqt-1;ortholog x x organization or WBGene00003776 Protein NMY-1;nmy-1;ortholog x biogenesis WBGene00004398 Cuticle collagen 6;rol-8;ortholog x (GO:0071840) WBGene00003828 Deoxyribonuclease-2;nuc-1;ortholog x WBGene00004955 Spindle-defective protein 5;spd-5;ortholog x WBGene00006366 Protein SYM-1;sym-1;ortholog x x x multicellular WBGene00011891 Protein DEL-6, isoform b;del-6;ortholog x organismal process WBGene00018237 Protein F40F4.6;CELE_F40F4.6;ortholog x (GO:0032501) WBGene00019957 Protein R08E3.1, isoform a;CELE_R08E3.1;ortholog x x WBGene00004398 Cuticle collagen 6;rol-8;ortholog x WBGene00000717 Protein COL-144;col-144;ortholog x x x WBGene00006366 Protein SYM-1;sym-1;ortholog x x x WBGene00003497 Transmembrane matrix receptor MUP-4;mup-4;ortholog x x x biological adhesion WBGene00005016 Cuticle collagen sqt-1;sqt-1;ortholog x x (GO:0022610) WBGene00016422 Protein NOAH-1, isoform c;noah-1;ortholog x x WBGene00018237 Protein F40F4.6;CELE_F40F4.6;ortholog x WBGene00004398 Cuticle collagen 6;rol-8;ortholog x apoptotic process WBGene00003828 Deoxyribonuclease-2;nuc-1;ortholog x (GO:0006915) WBGene00006366 Protein SYM-1;sym-1;ortholog x x x WBGene00009895 Protein SCL-2, isoform a;scl-2;ortholog x immune system WBGene00012186 Protein MLT-11, isoform b;mlt-11;ortholog x process WBGene00012253 Protein CLEC-50;clec-50;ortholog x x x (GO:0002376) WBGene00004398 Cuticle collagen 6;rol-8;ortholog x WBGene00009897 Protein F49E12.1;CELE_F49E12.1;ortholog x WBGene00018488 Protein ACS-1, isoform a;acs-1;ortholog x Text in blue font = proteins present in one of the development stages; Text in black font = proteins present in a two of the development stage; Text in pink font = proteins present in three of the development stages; Text in green font = proteins present in all four of the development stages; x indicates presence of proteins in particular development stage.

48 The overrepresentation test using PANTHER GO-BP (Table 1.4) shows that proteins with rapid PAR at all development stages were significantly (p<0.05) represented by protein metabolic processes and proteolysis GO-BP terms. However, disaccharide metabolic process and translation regulation were the GO-BP terms that were overrepresented at L3→L4 and L4→YA stage. The DNA metabolic process including DNA transcription and replication, DNA catabolism, chromosome segregation

GO-BP terms were represented by proteins with rapid PAR at L4→YA stage. This indicates that worms at the later stage of development require more gene expression, hence, proteins related to DNA replication/transcription are rapidly synthesized.

49 Table-1.4: The statistical overrepresentation test (PANTHER GO-Biological Process) of proteins with rapid PAR at different development stages. C. elegans - Input Input Input Input (fold Input PANTHER GO-Biological Process REFLIST (n) (expected) (over/under) Enrichment) (p value) (20490) L1→L2 ( n=18) cellular defense response (GO:0006968) 35 1 0.02 + > 5 2.20E-02 cell adhesion (GO:0007155) 193 2 0.12 + > 5 6.46E-03 biological adhesion (GO:0022610) 193 2 0.12 + > 5 6.46E-03 proteolysis (GO:0006508) 474 3 0.3 + > 5 2.97E-03 protein metabolic process (GO:0019538) 1879 5 1.19 + 4.19 4.44E-03 primary metabolic process (GO:0044238) 4498 6 2.85 + 2.1 4.58E-02 L2→L3 ( n=108) disaccharide metabolic process (GO:0005984) 5 1 0.02 + 41.39 2.39E-02 anterior/posterior axis specification (GO:0009948) 5 1 0.02 + 41.39 2.39E-02 cellular amino acid biosynthetic process (GO:0008652) 67 3 0.32 + 9.27 4.34E-03 proteolysis (GO:0006508) 474 19 2.29 + 8.3 1.52E-12 cellular amino acid metabolic process (GO:0006520) 215 8 1.04 + 7.7 1.08E-05 purine nucleobase metabolic process (GO:0006144) 58 2 0.28 + 7.14 3.24E-02 regulation of liquid surface tension (GO:0050828) 64 2 0.31 + 6.47 3.88E-02 cell adhesion (GO:0007155) 193 6 0.93 + 6.43 3.70E-04 biological adhesion (GO:0022610) 193 6 0.93 + 6.43 3.70E-04 regulation of translation (GO:0006417) 117 3 0.57 + 5.31 1.95E-02 receptor-mediated endocytosis (GO:0006898) 132 3 0.64 + 4.7 2.65E-02 mesoderm development (GO:0007498) 184 4 0.89 + 4.5 1.25E-02 protein metabolic process (GO:0019538) 1879 26 9.08 + 2.86 6.64E-07 primary metabolic process (GO:0044238) 4498 44 21.73 + 2.02 5.31E-07 metabolic process (GO:0008152) 5383 48 26.01 + 1.85 1.84E-06 Unclassified (UNCLASSIFIED) 12877 40 62.22 - 0.64 4.89E-06 RNA metabolic process (GO:0016070) 1258 1 6.08 - < 0.2 1.41E-02 L3→L4 ( n=93) disaccharide metabolic process (GO:0005984) 5 1 0.03 + > 5 2.91E-02 regulation of translation (GO:0006417) 117 5 0.69 + > 5 6.97E-04 cell adhesion (GO:0007155) 193 6 1.14 + > 5 1.07E-03 biological adhesion (GO:0022610) 193 6 1.14 + > 5 1.07E-03 receptor-mediated endocytosis (GO:0006898) 132 3 0.78 + 3.85 4.40E-02 mesoderm development (GO:0007498) 184 4 1.09 + 3.68 2.41E-02 proteolysis (GO:0006508) 474 10 2.8 + 3.57 5.39E-04 cellular component movement (GO:0006928) 208 4 1.23 + 3.26 3.55E-02 cellular amino acid metabolic process (GO:0006520) 215 4 1.27 + 3.15 3.93E-02 protein metabolic process (GO:0019538) 1879 18 11.1 + 1.62 2.78E-02 cellular process (GO:0009987) 4064 16 24 - 0.67 3.89E-02 L4→YA ( n=85) DNA catabolic process (GO:0006308) 4 1 0.02 + > 5 2.16E-02 disaccharide metabolic process (GO:0005984) 5 1 0.03 + > 5 2.70E-02 DNA replication (GO:0006260) 87 4 0.48 + > 5 1.40E-03 regulation of translation (GO:0006417) 117 5 0.64 + > 5 4.91E-04 chromosome segregation (GO:0007059) 63 2 0.34 + > 5 4.70E-02 regulation of liquid surface tension (GO:0050828) 64 2 0.35 + > 5 4.84E-02 DNA metabolic process (GO:0006259) 211 5 1.15 + 4.34 6.26E-03 receptor-mediated endocytosis (GO:0006898) 132 3 0.72 + 4.16 3.63E-02 proteolysis (GO:0006508) 474 8 2.59 + 3.09 4.65E-03 translation (GO:0006412) 362 6 1.98 + 3.03 1.49E-02 protein metabolic process (GO:0019538) 1879 17 10.27 + 1.66 2.67E-02 primary metabolic process (GO:0044238) 4498 36 24.59 + 1.46 8.17E-03 metabolic process (GO:0008152) 5383 41 29.42 + 1.39 1.03E-02 cellular process (GO:0009987) 4064 14 22.21 - 0.63 2.89E-02 transcription from RNA polymerase II promoter (GO:0006366) 893 1 4.88 - 0.2 4.15E-02 cell communication (GO:0007154) 1827 2 9.99 - 0.2 2.05E-03 transcription, DNA-dependent (GO:0006351) 928 1 5.07 - < 0.2 3.51E-02 GO-BP terms with p value <0.05 without Bonferroni correction.

50 Pathway (KEGG) association:

The KEGG pathway association (Table-1.5, 1.6, 1.7, and 1.8) revealed that the proteins with rapid PAR at L1→L2 stage mainly belong to lysosomal, ribosomal and fatty acid metabolism. The KEGG pathway is a basic pathway mapping tool. Only five proteins out of thirteen proteins at L1→L2 were mapped to KEGG pathways. The cathepsin B-like cysteine proteinase represented the lysosomal group. While ribosomal structural proteins, RPL-14 and -17 were present in the ribosomal group. The FAT-6 protein mapped to multiple groups including fatty acid metabolism, longevity regulating pathway and unsaturated fatty acid biosynthesis pathway. fat-6 encodes an Acyl-CoA desaturase (similarity to mammalian Acyl-CoA desaturases and also known as delta-9 fatty acid desaturase). FAT-6 readily desaturates long chain but not medium chain fatty acid. In C. elegans, FAT-6 participates in longevity regulating pathway downstream of mTOR signaling denoted in KEGG database.

The number of proteins with rapid PAR at L2→L3, L3→L4, and L4→YA were large and mapped to a large list of pathways. We analyzed top five KEGG pathways based on protein counts in each pathway category and they are shown in Table-1.5, 1.6,

1.7, and 1.8. Most of the proteins with rapid PAR are mapped to metabolic pathways in

KEGG database except L1→L2 stage. There was a clear distinction between proteins with rapid PAR in L2→L3, L3→L4 and L4→YA that represented metabolic pathways.

Proteins of the L3→L4 stage (ACS-9, BCAT-1, RNR-1, TRE-1, FASN-1) that were mapped to a metabolic process were common to both L2→L3 and L4→YA stages.

However, some proteins were only present in either L2→L3 or L4→YA stage. As shown

51 in Figure 1.8, proteins with rapid PAR were involved in quite distinct fatty acid metabolism at L4→YA stages compared to L2→L3 and L3→L4. The proteins at L2→L3 and L3→L4 stage were involved in fatty acid biosynthesis/elongation pathways.

Similarly, at the L4→YA stage proteins were also involved in fatty acid biosynthesis/elongation but in mitochondria.

The lysosomal pathway was common to all developmental stages that include cathepsin B-like cysteine proteinases. At the L2→L3 stage, proteins with rapid PAR are from amino acid metabolism group. While proteins involved in DNA replication (MCM2

(human ortholog of minichromosome maintenance complex component 2 and had helicase activity) and MCM-3 (human ortholog of DNA replication licensing factor), rpa-

1 (replication protein A) that are involved in DNA replication, repair and recombination) were specifically present at L4→YA stage. This suggests that there is a substantial shift in metabolism during a different developmental stage as reported earlier and that is also recognized at the level of PAR.

52 a

Figure 1.9: Pathway (KEGG) association for proteins with rapid PAR mapped to metabolic process. The proteins with rapid PAR from (a) L2→L3 (99); (b) L3→L4 (121); (c) L4→YA (144) stages were mapped to KEGG metabolic pathway highlighted in red on the background of different metabolic pathways denoted in different colors. The color coding for different metabolic pathways in each figure (a, b, and c) are as follows: fatty acid metabolism (green); Amino acid metabolism (orange); Purine and pyrimidine metabolism (red); oxidative phosphorylation (violet); TCA cycle and glyoxylate dicarboxylate metabolism (blue). The proteins from each development stage mapped to metabolic pathways in KEGG are shown in this figure and are listed in Table 1.5, 1.6, 1.7 and 1.8. These tables have mean PAR value from triplicate samples.

54 Pathway Search Result

Following object(s) was/were not found cel:CELE_B0222.6 cel:CELE_C44H4.3 cel:CELE_F22A3.6 cel:CELE_F28A12.4 cel:CELE_M03A1.7 cel:CELE_T05C12.10 cel:CELE_T24E12.5 cel:CELE_Y105E8A.6

Sort by the pathway list Table-1.5: The Pathway (KEGG) association for proteins with rapid PAR at L1→L2 Hidestages. all objects

cel04142 Lysosome - Caenorhabditis elegans (nematode) (2)

cel:CELE_F44C4.3 cpr-4; Cathepsin B-like cysteine proteinase 4 cel:CELE_W07B8.5 cpr-5; Cathepsin B-like cysteine proteinase 5

cel03010 Ribosome - Caenorhabditis elegans (nematode) (2)

cel:CELE_B0041.4 rpl-4; 60S ribosomal protein L4 cel:CELE_Y48G8AL.8 rpl-17; 60S ribosomal protein L17

cel01212 Fatty acid metabolism - Caenorhabditis elegans (nematode) (1)

cel:CELE_VZK822L.1 fat-6; Delta(9)-fatty-acid desaturase fat-6

cel04212 Longevity regulating pathway - worm - Caenorhabditis elegans (nematode) (1)

cel:CELE_VZK822L.1 fat-6; Delta(9)-fatty-acid desaturase fat-6

cel01040 Biosynthesis of unsaturated fatty acids - Caenorhabditis elegans (nematode) (1)

cel:CELE_VZK822L.1 fat-6; Delta(9)-fatty-acid desaturase fat-6

55 Pathway Search Result

Following object(s) was/were not found cel:CELE_B0222.6 cel:CELE_B0414.3 cel:CELE_B0491.2 cel:CELE_C01B10.11 cel:CELE_C02A12.4 cel:CELE_C06A8.3 cel:CELE_C09F9.2 cel:CELE_C15C8.3 cel:CELE_C17F4.7 cel:CELE_C23H5.8 cel:CELE_C29F4.1 cel:CELE_C34G6.6 cel:CELE_C38C6.6 cel:CELE_C44H4.3 cel:CELE_C55B7.1 cel:CELE_F01D5.5 cel:CELE_F02A9.3 cel:CELE_F09B9.4 cel:CELE_F15B9.1 cel:CELE_F15E11.1 cel:CELE_F15E11.13 cel:CELE_F15E11.14 cel:CELE_F19C7.1 cel:CELE_F21F8.3 cel:CELE_F21F8.7 cel:CELE_F22A3.6 cel:CELE_F25B5.4 cel:CELE_F28A12.4 cel:CELE_F28D1.3 cel:CELE_F35C5.6 cel:CELE_F40F4.6 cel:CELE_F41E6.2 cel:CELE_F42A10.6 cel:CELE_F42G9.2 cel:CELE_F49E11.10 cel:CELE_F53F4.13 cel:CELE_F55G11.4 cel:CELE_F58B3.1 cel:CELE_K06G5.1 cel:CELE_K07D8.1 cel:CELE_K09D9.2 cel:CELE_K09F5.3 cel:CELE_K12H4.7 cel:CELE_M28.10 cel:CELE_M60.2 cel:CELE_R08E3.1 cel:CELE_R09A8.3 cel:CELE_R10D12.14 cel:CELE_T01B10.2 cel:CELE_T02E9.2 cel:CELE_T08A9.9 cel:CELE_T08G5.10 cel:CELE_T11F9.9 cel:CELE_T18H9.2 cel:CELE_T19B10.2 cel:CELE_T21C9.3 cel:CELE_T21G5.3 cel:CELE_T21H3.1 cel:CELE_T22H6.5 cel:CELE_T28C6.4 cel:CELE_T28C6.6 cel:CELE_W01F3.3 cel:CELE_W02A2.2 cel:CELE_W04E12.8 cel:CELE_W08F4.6 cel:CELE_Y17G7A.1 cel:CELE_Y19D10B.7 cel:CELE_Y32F6A.5 cel:CELE_Y39B6A.20 cel:CELE_Y71G12B.6 cel:CELE_Y73B6BL.24 cel:CELE_ZK6.11 cel:CELE_ZK622.3

Sort by the pathway list Table-1.6: The pathway (KEGG) association for the proteins with rapid PAR at L2→L3 stages. Hide all objects

cel01100 Metabolic pathways - Caenorhabditis elegans (nematode) (15)

cel:CELE_C28H8.11 tdo-2; Tryptophan 2,3-dioxygenase cel:CELE_C33C12.3 gba-1; Putative glucosylceramidase 1 cel:CELE_C36A4.9 acs-19; fatty Acid CoA Synthetase family cel:CELE_C55F2.1 atic-1; 5-Aminoimidazole-4-carboxamide ribonucleotide formylTransferase/IMP Cyclohydrolase homolog cel:CELE_D2085.1 pyr-1; PYRimidine biosynthesis cel:CELE_F22B8.6 cth-1; CystaTHionine gamma lyase cel:CELE_F32H2.5 fasn-1; Fatty Acid SyNthase cel:CELE_K02A4.1 bcat-1; Branched-chain-amino-acid aminotransferase, cytosolic cel:CELE_R03D7.1 metr-1; Probable methionine synthase cel:CELE_R53.2 dtmk-1; Probable thymidylate kinase cel:CELE_T23G5.1 rnr-1; Ribonucleoside-diphosphate reductase large subunit cel:CELE_W05E10.4 tre-3; TREhalase cel:CELE_Y47D3B.10 dpy-18; Prolyl 4-hydroxylase subunit alpha-1 cel:CELE_Y55H10A.1 vha-19; Vacuolar H ATPase cel:CELE_ZK1127.10 cth-2; Putative cystathionine gamma-lyase 2

cel04142 Lysosome - Caenorhabditis elegans (nematode) (8)

cel:CELE_C33C12.3 gba-1; Putative glucosylceramidase 1 cel:CELE_C52E4.1 cpr-1; Gut-specific cysteine proteinase cel:CELE_F13D12.6 F13D12.6; Uncharacterized serine carboxypeptidase F13S12.6 cel:CELE_F44C4.3 cpr-4; Cathepsin B-like cysteine proteinase 4 cel:CELE_H22K11.1 asp-3; Aspartic protease 3 cel:CELE_T03E6.7 cpl-1; CathePsin L family cel:CELE_W07B8.5 cpr-5; Cathepsin B-like cysteine proteinase 5 cel:CELE_Y55H10A.1 vha-19; Vacuolar H ATPase

cel01230 Biosynthesis of amino acids - Caenorhabditis elegans (nematode) (4)

cel:CELE_F22B8.6 cth-1; CystaTHionine gamma lyase cel:CELE_K02A4.1 bcat-1; Branched-chain-amino-acid aminotransferase, cytosolic cel:CELE_R03D7.1 metr-1; Probable methionine synthase cel:CELE_ZK1127.10 cth-2; Putative cystathionine gamma-lyase 2

cel00270 Cysteine and methionine metabolism - Caenorhabditis elegans (nematode) (4)

cel00240 Pyrimidine metabolism - Caenorhabditis elegans (nematode) (3)

cel:CELE_D2085.1 pyr-1; PYRimidine biosynthesis cel:CELE_R53.2 dtmk-1; Probable thymidylate kinase cel:CELE_T23G5.1 rnr-1; Ribonucleoside-diphosphate reductase large subunit

cel00450 Selenocompound metabolism - Caenorhabditis elegans (nematode) (3)

cel:CELE_F22B8.6 cth-1; CystaTHionine gamma lyase cel:CELE_R03D7.1 metr-1; Probable methionine synthase 56 cel:CELE_ZK1127.10 cth-2; Putative cystathionine gamma-lyase 2

cel01212 Fatty acid metabolism - Caenorhabditis elegans (nematode) (2)

cel:CELE_F32H2.5 fasn-1; Fatty Acid SyNthase cel:CELE_VZK822L.1 fat-6; Delta(9)-fatty-acid desaturase fat-6 Pathway Search Result

Following object(s) was/were not found cel:CELE_B0222.6 cel:CELE_B0365.6 cel:CELE_B0414.3 cel:CELE_B0491.2 cel:CELE_B0491.3 cel:CELE_C04F6.1 cel:CELE_C04G2.9 cel:CELE_C09B9.6 cel:CELE_C09F9.2 cel:CELE_C14C6.5 cel:CELE_C14F11.4 cel:CELE_C17F4.7 cel:CELE_C18G1.4 cel:CELE_C23H5.8 cel:CELE_C29F4.1 cel:CELE_C33F10.9 cel:CELE_C34F11.4 cel:CELE_C34G6.6 cel:CELE_C35D10.11 cel:CELE_C38C6.6 cel:CELE_C42D8.2 cel:CELE_C44B12.5 cel:CELE_C44H4.3 cel:CELE_C56G7.1 cel:CELE_E01G4.6 cel:CELE_E03H12.10 cel:CELE_EEED8.1 cel:CELE_F02A9.3 cel:CELE_F20D12.1 cel:CELE_F21F8.7 cel:CELE_F21H7.5 cel:CELE_F25B5.4 cel:CELE_F28D1.3 cel:CELE_F35C5.6 cel:CELE_F35G12.11 cel:CELE_F36H12.7 cel:CELE_F38A5.13 cel:CELE_F41E6.2 cel:CELE_F42A10.6 cel:CELE_F42G9.2 cel:CELE_F44D12.3 cel:CELE_F44D12.5 cel:CELE_F44D12.7 cel:CELE_F44E2.8 cel:CELE_F46F11.2 cel:CELE_F52B10.1 cel:CELE_F53F4.13 cel:CELE_F53H4.2 cel:CELE_F55B11.2 cel:CELE_F55C5.1 cel:CELE_F57F5.1 cel:CELE_F57H12.6 cel:CELE_F58A6.8 cel:CELE_F58E6.13 cel:CELE_F58G1.1 cel:CELE_F59D8.1 cel:CELE_F59D8.2 cel:CELE_F59E12.12 cel:CELE_H23N18.5 cel:CELE_K03H1.4 cel:CELE_K05F1.2 cel:CELE_K06G5.1 cel:CELE_K07D8.1 cel:CELE_K07F5.1 cel:CELE_K07F5.9 cel:CELE_K07H8.6 cel:CELE_K08C9.2 cel:CELE_K09D9.2 cel:CELE_K09F5.2 cel:CELE_K09F5.3 cel:CELE_K09H9.3 cel:CELE_K12H4.7 cel:CELE_M01E11.5 cel:CELE_M03A1.7 cel:CELE_M28.10 cel:CELE_R05F9.13 cel:CELE_R06C7.1 cel:CELE_R08E3.1 cel:CELE_R10D12.14 cel:CELE_R10H10.3 cel:CELE_R13H9.2 cel:CELE_R13H9.4 cel:CELE_T02E9.2 cel:CELE_T05C12.10 cel:CELE_T05G5.10 cel:CELE_T08A9.9 cel:CELE_T12F5.3 cel:CELE_T13F2.1 cel:CELE_T13F3.6 cel:CELE_T19B10.2 cel:CELE_T21G5.3 cel:CELE_T21H3.1 cel:CELE_T28H11.6 cel:CELE_W02A2.2 cel:CELE_W04E12.8 cel:CELE_W09C5.1 cel:CELE_Y17G7A.1 cel:CELE_Y18D10A.11 cel:CELE_Y18D10A.17 cel:CELE_Y41E3.2 cel:CELE_Y45F10C.4 cel:CELE_Y45F10D.4 cel:CELE_Y47G6A.15 cel:CELE_Y57A10A.11 cel:CELE_Y59E9AL.2 cel:CELE_Y59E9AL.3 cel:CELE_Y73B6BL.24 cel:CELE_ZK1025.3 cel:CELE_ZK1248.6 cel:CELE_ZK1290.3 cel:CELE_ZK1320.3 cel:CELE_ZK354.1 cel:CELE_ZK354.11 cel:CELE_ZK354.4 cel:CELE_ZK354.5 cel:CELE_ZK381.4 cel:CELE_ZK622.3

Sort by the pathway list Table-1.7: The Pathway (KEGG) association for proteins with rapid PAR at L3→L4 Hidestages. all objects

cel01100 Metabolic pathways - Caenorhabditis elegans (nematode) (7)

cel:CELE_C36A4.9 acs-19; fatty Acid CoA Synthetase family cel:CELE_D2085.1 pyr-1; PYRimidine biosynthesis cel:CELE_F32H2.5 fasn-1; Fatty Acid SyNthase cel:CELE_K02A4.1 bcat-1; Branched-chain-amino-acid aminotransferase, cytosolic cel:CELE_T23G5.1 rnr-1; Ribonucleoside-diphosphate reductase large subunit cel:CELE_W05E10.4 tre-3; TREhalase cel:CELE_ZC416.6 ZC416.6; Uncharacterized protein

cel04142 Lysosome - Caenorhabditis elegans (nematode) (5)

cel:CELE_C52E4.1 cpr-1; Gut-specific cysteine proteinase cel:CELE_F13D12.6 F13D12.6; Uncharacterized serine carboxypeptidase F13S12.6 cel:CELE_F44C4.3 cpr-4; Cathepsin B-like cysteine proteinase 4 cel:CELE_H22K11.1 asp-3; Aspartic protease 3 cel:CELE_W07B8.5 cpr-5; Cathepsin B-like cysteine proteinase 5

cel01212 Fatty acid metabolism - Caenorhabditis elegans (nematode) (3)

cel:CELE_F32H2.5 fasn-1; Fatty Acid SyNthase cel:CELE_VZK822L.1 fat-6; Delta(9)-fatty-acid desaturase fat-6 cel:CELE_Y67H2A.8 fat-1; Omega-3 fatty acid desaturase fat-1

cel00480 Glutathione metabolism - Caenorhabditis elegans (nematode) (2)

cel:CELE_C11E4.1 gpx-5; Glutathione peroxidase cel:CELE_T23G5.1 rnr-1; Ribonucleoside-diphosphate reductase large subunit

cel01040 Biosynthesis of unsaturated fatty acids - Caenorhabditis elegans (nematode) (2)

cel:CELE_VZK822L.1 fat-6; Delta(9)-fatty-acid desaturase fat-6 cel:CELE_Y67H2A.8 fat-1; Omega-3 fatty acid desaturase fat-1

cel00590 Arachidonic acid metabolism - Caenorhabditis elegans (nematode) (2)

57 Pathway Search Result

Following object(s) was/were not found cel:CELE_B0244.8 cel:CELE_C04F6.1 cel:CELE_C08F11.11 cel:CELE_C18G1.4 cel:CELE_C23H5.8 cel:CELE_C24F3.6 cel:CELE_C34F6.2 cel:CELE_C35D10.13 cel:CELE_C39D10.7 cel:CELE_C42D8.2 cel:CELE_C50B6.2 cel:CELE_C53B4.5 cel:CELE_D1037.3 cel:CELE_D1054.10 cel:CELE_D1086.10 cel:CELE_D1086.11 cel:CELE_EEED8.1 cel:CELE_F13G11.3 cel:CELE_F13H10.8 cel:CELE_F17E9.4 cel:CELE_F20D12.1 cel:CELE_F21F8.7 cel:CELE_F25E2.2 cel:CELE_F35C5.6 cel:CELE_F35G12.11 cel:CELE_F41E6.12 cel:CELE_F42A10.6 cel:CELE_F44E2.8 cel:CELE_F46E10.1 cel:CELE_F46F11.2 cel:CELE_F49E12.1 cel:CELE_F53F4.13 cel:CELE_F54C9.8 cel:CELE_F55B11.2 cel:CELE_F56A3.4 cel:CELE_F56F3.1 cel:CELE_F59D8.1 cel:CELE_F59D8.2 cel:CELE_H05C05.1 cel:CELE_K03H1.4 cel:CELE_K07D8.1 cel:CELE_K07H8.6 cel:CELE_K09F5.2 cel:CELE_K09F5.3 cel:CELE_M01E11.5 cel:CELE_R05H5.3 cel:CELE_R06C7.1 cel:CELE_R10D12.14 cel:CELE_T05G5.10 cel:CELE_T05G5.3 cel:CELE_T05G5.7 cel:CELE_T08A9.9 cel:CELE_T12F5.3 cel:CELE_T13F2.1 cel:CELE_T13F3.6 cel:CELE_T19H12.2 cel:CELE_T21H3.1 cel:CELE_W01A8.1 cel:CELE_W03D2.4 cel:CELE_W03F11.1 cel:CELE_W04E12.8 cel:CELE_W05E7.1 cel:CELE_W05F2.3 cel:CELE_W09C5.1 cel:CELE_Y105C5B.5 cel:CELE_Y18D10A.11 cel:CELE_Y18D10A.17 cel:CELE_Y38E10A.22 cel:CELE_Y45F10C.4 cel:CELE_Y45F10D.4 cel:CELE_Y48A6B.13 cel:CELE_Y54G9A.6 cel:CELE_Y62H9A.4 cel:CELE_Y62H9A.6 cel:CELE_Y69A2AR.28 cel:CELE_Y69H2.3 cel:CELE_Y71H2B.4 cel:CELE_Y73B6BL.24 cel:CELE_Y76A2B.1 cel:CELE_ZK105.1 cel:CELE_ZK1248.7 cel:CELE_ZK622.3

Sort by the pathway list Table-1.8: The Pathway (KEGG) association for proteins with rapid PAR at L4→YA Hidestages. all objects

cel01100 Metabolic pathways - Caenorhabditis elegans (nematode) (12)

cel:CELE_C36A4.4 C36A4.4; Probable UDP-N-acetylglucosamine pyrophosphorylase cel:CELE_C36A4.9 acs-19; fatty Acid CoA Synthetase family cel:CELE_F07A11.2 gfat-1; Glutamine-Fructose 6-phosphate AminoTransferase homolog cel:CELE_F21D5.1 F21D5.1; Uncharacterized protein cel:CELE_F22B3.4 gfat-2; Glutamine-Fructose 6-phosphate AminoTransferase homolog cel:CELE_F29F11.1 sqv-4; UDP-glucose 6-dehydrogenase cel:CELE_K02A4.1 bcat-1; Branched-chain-amino-acid aminotransferase, cytosolic cel:CELE_K07A1.2 dut-1; DeoxyUTPase cel:CELE_T23G5.1 rnr-1; Ribonucleoside-diphosphate reductase large subunit cel:CELE_W05E10.4 tre-3; TREhalase cel:CELE_Y65B4BL.5 acs-13; fatty Acid CoA Synthetase family cel:CELE_ZC416.6 ZC416.6; Uncharacterized protein

cel04142 Lysosome - Caenorhabditis elegans (nematode) (6)

cel:CELE_C07B5.5 nuc-1; Deoxyribonuclease-2 cel:CELE_C52E4.1 cpr-1; Gut-specific cysteine proteinase cel:CELE_F44C4.3 cpr-4; Cathepsin B-like cysteine proteinase 4 cel:CELE_H22K11.1 asp-3; Aspartic protease 3 cel:CELE_T03E6.7 cpl-1; CathePsin L family cel:CELE_W07B8.5 cpr-5; Cathepsin B-like cysteine proteinase 5

cel00520 Amino sugar and nucleotide sugar metabolism - Caenorhabditis elegans (nematode) (5)

cel:CELE_C36A4.4 C36A4.4; Probable UDP-N-acetylglucosamine pyrophosphorylase cel:CELE_F07A11.2 gfat-1; Glutamine-Fructose 6-phosphate AminoTransferase homolog cel:CELE_F21D5.1 F21D5.1; Uncharacterized protein cel:CELE_F22B3.4 gfat-2; Glutamine-Fructose 6-phosphate AminoTransferase homolog cel:CELE_F29F11.1 sqv-4; UDP-glucose 6-dehydrogenase

cel01212 Fatty acid metabolism - Caenorhabditis elegans (nematode) (3)

cel:CELE_VZK822L.1 fat-6; Delta(9)-fatty-acid desaturase fat-6 cel:CELE_Y65B4BL.5 acs-13; fatty Acid CoA Synthetase family cel:CELE_Y67H2A.8 fat-1; Omega-3 fatty acid desaturase fat-1

cel03030 DNA replication - Caenorhabditis elegans (nematode) (3)

cel:CELE_C25D7.6 mcm-3; yeast MCM (licensing factor) related cel:CELE_F18A1.5 rpa-1; Probable replication factor A 73 kDa subunit cel:CELE_Y17G7B.5 mcm-2; yeast MCM (licensing factor) related

cel04931 Insulin resistance - Caenorhabditis elegans (nematode) (2)

cel:CELE_F07A11.2 gfat-1; Glutamine-Fructose 6-phosphate AminoTransferase homolog cel:CELE_F22B3.4 gfat-2; Glutamine-Fructose 6-phosphate AminoTransferase homolog 58

cel00250 Alanine, aspartate and glutamate metabolism - Caenorhabditis elegans (nematode) (2)

cel:CELE_F07A11.2 gfat-1; Glutamine-Fructose 6-phosphate AminoTransferase homolog cel:CELE_F22B3.4 gfat-2; Glutamine-Fructose 6-phosphate AminoTransferase homolog

cel00500 Starch and sucrose metabolism - Caenorhabditis elegans (nematode) (2)

cel:CELE_F29F11.1 sqv-4; UDP-glucose 6-dehydrogenase cel:CELE_W05E10.4 tre-3; TREhalase

cel04120 Ubiquitin mediated proteolysis - Caenorhabditis elegans (nematode) (2)

cel:CELE_F46A9.4 skr-2; SKp1 Related (ubiquitin ligase complex component) cel:CELE_Y108G3AL.1 cul-3; Cullin-3

cel01040 Biosynthesis of unsaturated fatty acids - Caenorhabditis elegans (nematode) (2)

cel:CELE_VZK822L.1 fat-6; Delta(9)-fatty-acid desaturase fat-6 cel:CELE_Y67H2A.8 fat-1; Omega-3 fatty acid desaturase fat-1

cel04212 Longevity regulating pathway - worm - Caenorhabditis elegans (nematode) (2)

cel:CELE_F43D9.4 sip-1; Stress-induced protein 1 cel:CELE_VZK822L.1 fat-6; Delta(9)-fatty-acid desaturase fat-6

cel00240 Pyrimidine metabolism - Caenorhabditis elegans (nematode) (2)

cel:CELE_K07A1.2 dut-1; DeoxyUTPase cel:CELE_T23G5.1 rnr-1; Ribonucleoside-diphosphate reductase large subunit

cel04141 Protein processing in endoplasmic reticulum - Caenorhabditis elegans (nematode) (1)

cel:CELE_F46A9.4 skr-2; SKp1 Related (ubiquitin ligase complex component)

cel04146 Peroxisome - Caenorhabditis elegans (nematode) (1)

cel:CELE_Y65B4BL.5 acs-13; fatty Acid CoA Synthetase family

cel00071 Fatty acid degradation - Caenorhabditis elegans (nematode) (1)

cel:CELE_Y65B4BL.5 acs-13; fatty Acid CoA Synthetase family

cel03013 RNA transport - Caenorhabditis elegans (nematode) (1)

cel:CELE_B0348.6 ife-3; Eukaryotic translation initiation factor 4E-3

cel04310 Wnt signaling pathway - Caenorhabditis elegans (nematode) (1)

cel:CELE_F46A9.4 skr-2; SKp1 Related (ubiquitin ligase complex component)

cel00040 Pentose and glucuronate interconversions - Caenorhabditis elegans (nematode) (1)

cel:CELE_F29F11.1 sqv-4; UDP-glucose 6-dehydrogenase

cel03440 Homologous recombination - Caenorhabditis elegans (nematode) (1)

cel:CELE_F18A1.5 rpa-1; Probable replication factor A 73 kDa subunit

cel01200 Carbon metabolism - Caenorhabditis elegans (nematode) (1) Discussion:

Using a SILAC-based pulse-chase approach, we have monitored newly synthesized proteins in C. elegans at a global level during normal physiological development. This is the first study of this kind that measures the appearance rate of newly synthesized proteins (aka PAR here) during a normal larval development of C. elegans. The present study revealed that the median PAR of all proteins identified during different developmental stages are highest in L1→L2 stage and gradually decreases in the subsequent larval stages, which implies that global protein synthesis activity is highest in the L1→L2 stage that gradually reduces in the subsequent larval stages. This implies high translation activity at the L1→L2 stage and suggests that the first larval stage requires more protein synthesis to grow in size. This might also be associated with the high metabolic requirement of the first post-embryonic developmental stage.

The gene ontology analysis on proteins with rapid PAR revealed that aspartyl proteases are the proteins that are rapidly synthesized in early development stages while cysteine proteinases (cpr-4 and -5) are rapidly synthesized in all four larval stages. The cpr-1 was also rapidly synthesized in all larval stages except at L1→L4 stage. We found distinct and common aspartyl proteases that showed rapid PAR at each developmental stage. ASP-3, and -6 were rapidly synthesized in all larval stages except L1→L2, while

ASP-13 was rapidly synthesized only in early two larval stages, and ASP-1, -2 and -5 was only at L2→L3 stage. These aspartyl proteases are mostly expressed in the gut and plausibly involved in protein metabolism and digestion of food in worm [59]. They are also reported to be involved in the necrotic death in different tissues [59, 60]. These

59 proteases are also proposed to be involved in aging process [61]. However, specific roles of these proteases during the larval development stages are poorly understood. Our data suggests that specific requirement of these aspartyl and cysteine proteases in the larval development stages. Further studies will be necessary to clarify the specific roles of these proteases in the larval development stages.

The statistical overrepresentation test (PANTHER) on the proteins exhibiting rapid PAR indicated clearly that early stages (up to L3 stage) had more representation of peptidase activity but later larval stages (L3 →YA) had more proteins involved in translation category such as translation initiation factor, RNA helicases, and mRNA processing factor. This suggests that in the later larval development stage proteins that facilitate protein translation become more important in development.

Gene ontology-biological process term mapping for on proteins with rapid PAR suggested that they are mainly in three groups: metabolic, cellular and biological regulation process. Proteins that are a member of collagen group are rapidly synthesized in the later larval stages, suggesting that they are required for the morphological development of larva. Proteins involved in fatty acid elongation process were rapidly synthesized in middle two larval stages but absent in L1→L2 and L4→YA stages, and proteins in the category of cell cycle, stress proteins, and extracellular matrix proteins were rapidly synthesized in L4→YA stage, suggesting that the dynamic physiological changes during the larval development of C. elegans.

60 In conclusion, the results show the dynamic nature of protein metabolism that reflects the physiological changes of this model organism during its larval development.

61 B: CHAPTER 2:

Effect of environmental stress on proteome of Caenorhabditis elegans during development.

Summary:

The goal of this portion of the project was to identify differentially expressed proteins in dauer larvae compared to normal third (L3) stage larvae. We used a SILAC- based approach to achieve accurate quantification. The results revealed that more than twice as many as proteins are down regulated than up regulated in dauer larvae compared to L3 larvae. We found that differentially up regulated proteins in the dauer stage belong to lipid storage, muscle contraction and stress response as would be expected for a stage which is entered into nutritional environmental stress, while the majority of down regulated proteins represent translation, transcription, gene expression, transport processes and TCA cycle which would be required for normal organismal development.

62 Introduction:

Under adequate nutritional and environmental conditions of growth, C. elegans rapidly progresses from an embryonic stage, through the four (L1 to L4) larval stages, to develop into reproductive adult stage within ~3-4 days at 20 °C. After hatching from an egg, if nutrition is not adequate for the growth of larvae, then growth arrest occurs at the

L1 stage. Larvae arrested at L1 do not survive longer without food. But, when the worms encounter unfavorable conditions after progression to L2 (e.g. high temperature, stress, inadequate food, and over-crowding), they undergo growth arrest at L2 larval stage before reproductive maturity [62] and the formation of a morphologically distinct L2d larvae occurs. Depending on the persistence of the environmental parameters, the L2d larva retains the potential to be either a dauer larva or an L3 larva [63, 64]. If the environment continues to be poor for growth, in the presence of the dauer pheromone, daumone [65], L2d molts into a specialized L3 diapause form termed, dauer larvae [66], enabling long-term survival of the organisms [67, 68]. Dauer larvae can survive for several months in this state, also known as ‘non-aging state’ as its duration does not markedly affect the duration of the post-dauer life span. When conditions again become favorable for growth (within an hour of the presence of needed nutrition), the animal exits from the dauer stage, re-entering into the life cycle and after ~10 hours, molts to become an L4 stage larvae [66].

At the dauer stage, locomotion is remarkably reduced and feeding is indefinitely arrested [54]. Morphological changes in dauer larvae suggest adaptation to the adverse conditions and support the organism’s survival under these adverse conditions [69]. The distinct morphological changes were reported as; they have a very thin body (ratio of

63 length to width is about 30:1) and have a thick altered cuticle, the buccal cavity is sealed by a cuticular block, and the pharyngeal and intestinal lumens are shrunken, with hazy microvilli in the intestine [54]. The excretory gland lacks secretory granules, despite their pores remains open. The gonads of dauer larvae are also arrested at the L2 stage [63, 69].

Genomic analysis of dauer entry as well as the dauer phase has revealed that the dauer state is associated with an increased expression of stress resistance genes, decreased metabolic process, and suppressed insulin-like pathway signaling [9-11, 67, 68,

70-74]. Several studies have demonstrated that the insulin/IGF-1 signaling pathway plays an important role in the long-lived growth arrest state at dauer larval stage in C. elegans

[11, 67, 68, 70-73, 75]. Other studies have shown that posttranslational modifications

(PTMs) on certain proteins such as O-GlcNAcylation, influence stress resistance, protein folding, mitochondrial function and epigenetic memory [76]. These PTMs appear to regulate cytoskeletal modifications and protein turnover during dauer larval stage formation. Previous 2D-gel based proteomic studies demonstrated that up regulated proteins in dauer larvae belonged to three functional categories: oxidative stress-defense- related proteins (HSP12.6, GST-1), muscle proteins (levamisol-resistant proteins, troponin C, and ACT-3, etc.), and energy generation (ATP synthase and NADH- ubiquinone oxidoreductase) [44, 67, 77]. Studies reported by Madi et al and Wadsworth et al showed the decreased levels of high-energy phosphates but increased expressions of inorganic pyrophosphatase, phosphatidylethanolamine-binding protein, alcohol dehydrogenase, and aldehyde dehydrogenase in dauer [62, 78]. While splicing factor rsp-

3 and methionyl-tRNA synthase (proteins related to protein synthesis), and pepsin-like

64 aspartyl protease-1 were significantly down regulated in dauer [62, 78]. The authors inferred that these enzymes might be beneficial for dauer for adaptation to adverse growth conditions and might be linked to the longevity of dauer larva [77, 79]. Although these preceding studies implied several underlying mechanisms to cope with environmental stress due to lack of nutrition, still there is a limitation in our knowledge of how the transformation of nematodes to dauer stage occurs at the molecular level. This includes the need of understanding the sensing mechanism of environmental stress

(temperature, crowding, and food), understanding coordinated regulatory mechanism in the modulation of metabolic and signaling pathways as well as morphological changes that occur at both the gene and protein expression levels.

In this study, we compared the expression levels of individual proteins in dauer and L3 larvae in a global scale using a stable-isotope labeling by amino acids in cell culture (SILAC)-based approach [51]. We found that more proteins in general are down regulated in dauer than up regulated when compared to L3 larvae. Bioinformatics analysis on the data revealed that proteins involved in fatty acid metabolism, stress related and muscular proteins were up regulated and proteins involved in chromatin remodeling, translation, amino acid biosynthesis and regular metabolism were down regulated. These results suggest that there is a shift in metabolism to conserve energy and to resist stress and is consistent with the previous morphological features of dauer larvae.

65 Materials and methods:

The materials and most of the methods (such as C. elegans strain, maintenance,

12 13 and age synchronization, labeling bacteria with light-( C6-Lys) and heavy-( C6-Lys) lysine, Proteomic Sample Preparation, LC-MS/MS analysis, and identification and quantification of peptides and proteins) used for this project are described in chapter 1.

Methods that are not described in chapter 1 and specifically used for the chapter 2 are described below.

Preparation of C. elegans L3 larvae

WT Bristol N2 strain nematodes were transferred onto a peptone-free NGM plate

12 that had been seeded with light ( C6)-Lys labeled E. coli. Gravid animals from the next generation of these nematodes were bleached to collect their live eggs, as described in chapter 1. Eggs were transferred onto peptone-free NGM plates seeded with light -Lys E. coli. After hatching of eggs, age-synchronized worms were then cultured to grow into L3 larval stage on peptone-free NGM plates seeded with light-Lys E. coli containing 25 mg/L 5-fluoro-2′-deoxyuridine (Acros Organics, Pittsburgh, PA), and harvested.

Preparation of C. elegans dauer larva

Dauer larvae were prepared as describe previously by Madi et al [77]. Briefly, age-synchronized L2 larvae that have been fed with light-Lys E. coli were transferred onto 2% agar plates and cultured at 20 °C for 48 h in the absence of peptone and E. coli, and then harvested. The experimental workflow is shown in Figure 2.1.

Synchronized eggs

Hatching L1

Grown in standard culture volume Grown in ¼th culture volume 12 12 Light- C6-Lys Light- C6-Lys L2 L2

NO FOOD FOOD L3 Dauer

Protein extraction, digestion with Lys-C

REFERENCE SAMPLE 13 (Heavy- C6-Lys-labeled L3+dauer [1:1])

LC-MS/MS

Protein identification, protein quantification, data analysis

12 Figure 2.1: Overview of workflow: Proteins extracted from light ( C6)-Lys labeled L3 or dauer larvae samples in triplicates were digested by Lys-C. Separately, a reference 13 Lys-C digest was prepared from a mixture of C6-Lys labeled L3 and dauer larvae in a 13 ratio of 1:1. The C6-Lys labeled reference digest was mixed with the L3 and dauer larvae digests in a 1:1 ratio in protein amount in individual triplicate samples, and subjected to LC-MS/MS.

67 13 Preparation of C6-Lys labeled reference L3 and dauer larvae

13 C6-Lys labeled L3 and dauer larvae were prepared from the eggs laid by the

13 13 C6-Lys labeled animals as described above except C6-Lys E. coli was given as a food

12 source instead of C6-Lys E. coli.

Data analysis:

First, quantitative estimates obtained for individual proteins in each group for dauer or L3 larvae as a ratio of light to heavy lysine was normalized by dividing by the mean value of all proteins (ratio of light to heavy) in that group. Student’s t-test was performed to estimate the significant difference between the mean normalized ratio of light to heavy lysine intensities of individual proteins of dauer and L3 larva present in duplicates or triplicates. After normalization, the mean values of duplicate and triplicate biological samples were calculated. The fold-change was calculated for individual protein by dividing dauer mean ratio of heavy to light by L3 larvae ratio of heavy to light measurements. The fold-change was calculated for all proteins present in singles, duplicates or triplicates but differentially expressed proteins with the statistical significance of p < 0.05 were further analyzed.

Clustering analysis and gene ontology and pathway association were performed as described in the data analysis section of chapter 1.

68 Results:

Overall expression of proteins in dauer and L3 larvae:

We successfully identified and quantified 1321 proteins from L3 larvae and 801 proteins from dauer larvae samples, of which 732 proteins were common to both larvae samples, which corresponds to 55% and 91% of proteins identified from L3 and dauer

13 larvae samples, respectively (Figure 2.2a). Since a constant amount of the C6-Lys reference digest was added to all the digests of the experimental samples prior to the LC-

12 13 MS/MS, we were able to obtain a light ( C6)-Lys to heavy ( C6)-Lys peptide intensity ratio for all the peptides identified. The distributions of the light to heavy ratios for all proteins for dauer and L3 larvae for triplicate samples are shown in Figure 2.2b. The box plot shows that the range and interquartile range of the light to heavy ratios of the individual proteins of dauer larvae sample (8.7, 0.67) are greater than that of the L3 larvae (5.9, 0.27). This result indicates that the expression level of proteins in dauer larvae has more variation than in L3 larvae.

69 a b

732

589 Ra*o (Light/Heavy)

L3 Dauer

Figure 2.2: Proteins identified and quantified from L3 and dauer larvae samples. (a) Venn diagram showing the overlap of a total number of proteins identified from L3 and dauer (D) larvae samples in triplicates. (b) Box plot showing the distribution of light/heavy protein ratios of total number of proteins identified from L3 and dauer larvae samples in triplicates..

70 Clustering analysis:

Principal component analysis (PCA):

PCA analysis was performed on the light to heavy ratios of all the proteins identified in triplicate samples of L3 and dauer larvae. The PCA plot clearly indicates that the L3 and dauer larvae data points are well separated, suggesting they are distinct from each other (Figure 2.3a). The plot also shows that the triplicate samples of L3 larvae are in one cluster (close to each other), while the triplicate samples of dauer larvae are more spread within their cluster. This implies that the physiological state of dauer larvae population has wider variation compared to L3 larvae population. To determine the average dissimilarity within each cluster, we plotted silhouette width of the PCA analysis (Figure 2.3b). An average silhouette width of 0.52 suggests that the overall there is a reasonable separation of clusters between L3 and dauer larvae (Figure 2.3b). The variance of each principal components obtained from “prcomp” PCA analysis was shown as scree plot in Figure 2.4 and their associated standard deviation, proportion of variance, and cumulative proportion were listed as table on x-axis. The scree plot indicates that the maximum variance in sample is due to PC1 (75.27%) and then from PC2 (8.37%). The variance of 8% to 4% exists up to PC5.

71 a

Stage Dauer L3 PC2(8.37%)

PC1(75.27%) b

Dauer

Figure 2.3: Principal component analysis (PCA): (a) PCA plot showing biological triplicates samples from L3 and Dauer larva labeled with different color. (b) The scree plot showing variance of ten principle components from data points of L3 and dauer larva samples on the ratios of light-heavy proteins. The standard deviation, proportion of variance and cumulative proportion associated with each principal component are listed on the x-axis.

Variance

PC1 PC2 PC3 PC4 PC5 PC6 Standard Devia,on 17.1 5.7 5.2 4.7 3.6 1.2e-14

Propor,on of 0.75 0.08 0.07 0.057 0.04 0.0e+0 Varia,on Cumula,ve 0.75 0.84 0.91 0.97 1.00 1.00 Propor,on

Figure 2.4: Scree plot depicting variance of the all principle component axis’s present in the L3 and dauer larvae samples in triplicates. The scree plot displaying the variance determined by “prcomp” PCA analysis associated with six principal components from the light to heavy ratios of the individual proteins present in four larval stages. The standard deviation, proportion of variance and cumulative proportion associated with each principal component are listed on the x-axis. Variance = (standard deviation)2.

73 Unsupervised clustering analysis:

To look at the difference in the expression levels of proteins in L3 and dauer larvae triplicate samples (389), we subjected proteins commonly expressed in both the samples to unsupervised clustering analysis. As shown in Figure 2.5, the triplicates of each group, L3 and dauer larvae, were cluster together in heat map based on column hierarchal clustering (column dendrogram was not shown in the figure). Similar to PCA, unsupervised clustering analysis also implied that the protein expression levels were distinct between L3 and dauer larvae samples. The dendrogram of the row clustering

(based on identified proteins in dauer and L3 larvae) was cut (using cutree function of stat package in R) at specific levels to obtain different distinct clusters of two (clusters of

346 and 16 proteins), three (clusters of 384, 2 and 3 proteins), four (clusters of 384, 1, 3, and 1 proteins) or six (clusters of 17, 350, 17, 1, 3, and 1 proteins). We noticed that there was primarily two clusters consist of 185 and 165 proteins when dendrogram was cut up to twenty clusters. The remaining clusters have small number of proteins as shown in parenthesis above. Reanalysis after removing proteins with small and similar variance revealed that unsupervised clustering analysis again provide two distinct clusters of proteins exist (consist of 185 and 165 proteins) when dendrogram was cut to get up to ten clusters. The majority of the proteins from triplicate samples (389) was present in one cluster (384) or two clusters (185 and 165) and did not map distinctly to any of the particular gene ontology (GO) terms. Therefore, these clusters were not analyzed further for GO and pathway association but additional analysis was performed for differential expression of the proteins present in dauer and L3 larvae.

L3 Dauer

Figure 2.5: Heat map: The heat map showing triplicate samples of L3 and dauer larvae. Proteins samples of dauer and L3 larvae with missing data were omitted and heat map was plotted using Euclidean distance and hierarchal clustering of ratio of light to heavy values. Only row clustering was shown.

75 Differentially expressed proteins between L3 and dauer larvae

First, the fold-change of each individual protein (732) present in both L3 and dauer larvae samples were calculated. Then p values for the significance of the difference between light to heavy ratios of L3 and dauer larvae samples were obtained using

Student’s t test, indicating that there are 357 proteins that are differentially expressed significantly (p <0.05). We have analyzed the overall trends of significant fold changes in all proteins detected in both L3 and dauer larvae samples by volcano plot (Figure 2.6).

The volcano plot clearly indicates that there are more proteins (258 proteins) those expression levels are significantly decreased in dauer larvae than increased (99 proteins), suggesting that many proteins’ expressions are reduced in dauer larvae.

To characterize the up and down regulation of proteins needed for molecular function and, biological processes, we selected the proteins that had significantly altered

1.5-fold up and down regulated in dauer stage compared to L3 larval stages for pathway mapping. The bar graph shows up or down regulated proteins (total of 732) in dauer larvae compared to L3 larvae (Figure 2.7). The blue dashed line indicates 2-fold change and red dashed line indicates 1.5-fold change. The total number of proteins and significant proteins (p <0.05) in parenthesis that are 2-fold changed are marked in blue and 1.5-fold changed are marked in red.

76 -log10 p-value

Log2 Fold Change

Figure 2.6: Volcano plot: Fold changes of protein expressions between proteins expressed in dauer to L3 larvae (log2 values of dauer/L3 larva) are plotted against p value (–log10 p values). Data points that are significant based on p value (< 0.05) were marked with dark blue color. The plot includes 732 proteins that were identified in both L3 and dauer larvae samples.

27 up (7) 77 up (28)

214 down (169) 84 down (62)

Figure 2.7: Fold change of proteins in dauer larvae to L3 Larvae: Fold changes of protein expressions between proteins expressed in dauer to L3 larvae (log2 values of dauer/L3 lava) are plotted from the lowest to highest values. The red and blue dashed lines display 1.5-fold and 2-fold up or down regulated proteins in dauer larvae compared to normal L3 larvae. The numbers of proteins that are up or down regulated (1.5-fold (red) or 2-fold (blue)) are labeled for each line. The numbers in the parenthesis denotes the number of proteins that are significantly differentially expressed at p < 0.05.

78 Gene Ontology and Pathway Association:

Gene ontology and pathway association of proteins up regulated in dauer

The gene ontology term and pathway mapping on 28 proteins for which the expression levels were greater than 1.5-fold higher in dauer larvae compared to L3 larvae

(p <0.05) was shown in Figure 2.8 and up regulated proteins and their fold change is listed in Table-2.1.

Only 14 proteins out of 28 proteins mapped to four gene ontology (GO)- molecular function (MF) terms as catalytic activity, binding, structural molecular activity and transport activity (Table-2.2). The majority of the up regulated proteins in the dauer larvae were from the catalytic group (36%). However, these up regulated proteins were not represented by the aspartic proteases as observed in the normal development stages in chapter 1, rather these proteins in catalytic groups were OIG-2 (involved in lipid storage),

HMG-11 (predicted to have DNA binding activity and expressed also in the body wall musculature), MCA-3 (encodes plasma membrane Ca2+ ATPases required for coelomoycte endocytosis), CEC-5 (meiotic chromosome segregation), PPFR-2 protein phosphatase 4, regulatory subunit 2, a human ortholog of PPP4R2), and uncharacterized protein C53C9.2. However, binding proteins are rnp-4 (RNA transport), and H2A.Z histone variant (HTZ-1 regulate gene expression during pharyngeal organogenesis). The binding group consisted of ACT-4 (actin expressed on body wall) and ACT-5

(cytoplasmic actin expressed only in intestinal microvilli), and TNT-2 (ortholog of human troponin T type I, II and III skeletal muscles expressed in muscles of anal, body wall and reproductive system of C. elegans). Most of these up regulated proteins such as

79 a b

1, 7% 1, 5% 1, 5%

1, 5% 5, 26% 5, 36% 1, 5% 4, 29%

2, 11%

3, 16% 2, 11%

4, 28% 3, 16%

(14) (19) cataly.c ac.vity (GO: metabolic process (GO: 0003824) 0008152) binding (GO:0005488) localiza;on (GO:0051179) 7% structural molecule ac.vity developmental process (GO: 36% (GO:0005198) 0032502) 29% transporter ac.vity (GO: 0005215) cellular component organiza;on or biogenesis (GO:0071840) cellular process (GO:0009987) 28% 5% 5% biological regula;on (GO: 5% 0065007) 5% 26% response to s;mulus (GO: 0050896) 11% mul;cellular organismal 16% 11% process (GO:0032501) 16% immune system process (GO: 0002376)

Figure 2.8: Gene ontology (GO)-molecular function (MF) and biological process (BP) terms for 1.5-fold up regulated dauer proteins. The pie graph shows (a) GO-MF and (b) GO-BP terms for the proteins (28) that are significantly (p <0.05) 1.5-fold up regulated in dauer larvae. Those proteins are listed in Table 2.1 Each GO-MF and GO- BP term (a slice of a pie graph) is marked with gene counts and percent of the count of genes present in individual GO-BP term to the total gene count mapped to GO terms (written in parenthesis under each stage) for individual development stage.

80 ACT-4, TNT-2, HTZ-1, HMG-1 are expressed in muscles of different body part of C. elegans and involved in muscle function (www.wormbase.org).

The GO-biological processes (BP) are represented by nine groups and 26% of them were involved in metabolic processes (SODH-1, ALH-1, TNT-2, RNP-4, MCA-3) .

Most of them were described in GO_MF section except SODH-1 (involved in determination of adult lifespan and expressed in the muscle cell, hypodermis, nervous system, and the intestine) and ALH-1 (aldehyde dehydrogenase 2, mitochondria and involved in lipid storage) proteins that were from dehydrogenase and reductase group.

Both SODH-1 and ALH-1 are also involved in fermentative metabolism. There was a chaperone proteins lea-1 that is predicted to be hydrophilic and heat-resistant, and that might participate in anhydrobiosis, ametabolic state for survival in the extreme environmental condition.

The statistical overrepresentation test (PANTHER) with Bonferroni correction had no significant GO terms for MF and BP. However, significant (p<0.05) GO-MF and -

BP terms were found without Bonferroni correction; they were proteins involved in desiccation, muscle processes including contraction, and calcium ion transport, ATPase, dehydrogenase, oxidoreductase activity (Table-2.4). These processes might also beneficial for dauer survival by protecting them against environmental stress including nutritional stress.

81 Next, we mapped these proteins to KEGG pathway and listed only top 5 hits in the Table-2.5. Pathway mapping indicated that SODH-1 and ALH-1 are the two proteins that were mapped to multiple pathways including fatty acid degradation, and energy metabolism including glycolysis, pentose and glucuronate interconversions, and amino acid degradation (Table-2.5 and Figure 2.9b). The results clearly indicated that in dauer larvae metabolism is altered to conserve energy and is consistent with the previous findings of gene expression. KEGG tools do not provide an overrepresentation test, therefore, we used REACTOME.org to estimate the significant overrepresented pathways. This analysis revealed that significant overrepresented pathways were homeostasis, muscle contraction, extracellular matrix organization, signal transduction and small molecule transmembrane transport, and neuronal system. These results complement the PANTHER GO-MF, BP and KEGG pathways (Table-2.6 and Figure

2.9a).

82 Table-2.1: Proteins for which the level of expression is higher than 1.5-fold (p <0.05) in dauer larvae compared to L3 worm. Wormbase Gene ID Wormpep ID Protein names Gene names Ratio (light/heavy) Fold Change p value Dauer L3 WBGene00021048 CE05036 Uncharacterized protein 4.52056 1.34335 3.36515 0.00007 WBGene00009888 CE31529 Uncharacterized protein 2.85282 1.11066 2.56857 0.00001 WBGene00012759 CE20253 Protein SUP-1 sup-1 2.99672 1.22206 2.45220 0.00061 WBGene00007296 CE05210 ARRestin Domain protein arrd-25 2.65564 1.11790 2.37555 0.00000 WBGene00001976 CE04890 HMG (High mobility group protein I alpha) hmg-11 5.73209 2.42194 2.36673 0.00203 Integrin alpha pat-2 (Paralyzed arrest at two- WBGene00003929 CE00194 1.71586 0.80210 2.13922 0.00561 fold protein 2) WBGene00016898 CE27882 Uncharacterized protein C53C9.2 2.85484 1.37067 2.08281 0.00001 Transthyretin-like protein 16 (Unknown spot 4 WBGene00012382 CE19022 ttr-16 2.21370 1.15792 1.91180 0.00013 protein from 2D-page) WBGene00018459 CE23736 Uncharacterized protein 1.41357 0.74977 1.88534 0.00133 WBGene00017064 CE09101 Protein Phosphatase Four Regulatory subunit ppfr-2 2.26748 1.23763 1.83212 0.00295 WBGene00017193 CE39239 Uncharacterized protein 1.84951 1.04495 1.76995 0.00916 WBGene00015328 CE29018 EGF-like domain-containing protein C02B10.3 1.85605 1.06340 1.74539 0.00025 WBGene00003860 CE21614 One IG domain oig-2 1.39958 0.82145 1.70380 0.00273 WBGene00002263 CE47024 Plant Late Embryo Abundant (LEA) related lea-1 2.72968 1.63297 1.67160 0.00088 WBGene00002263 CE46912 Plant Late Embryo Abundant (LEA) related lea-1 2.72909 1.64136 1.66270 0.00129 WBGene00017993 CE04479 C.Elegans Chromodomain protein cec-5 1.87825 1.13016 1.66193 0.00225 WBGene00000475 CE19148 C. Elegans Y-box cey-4 2.08326 1.26512 1.64669 0.00009 WBGene00000067 CE16463 ACTin act-5 1.46586 0.89637 1.63533 0.00001 WBGene00006587 CE34313 TropoNin T 1.94124 1.19424 1.62550 0.00000 WBGene00000107 CE29809 ALdehyde deHydrogenase 2.48165 1.56184 1.58893 0.04478 WBGene00003153 CE28372 Calcium-transporting ATPase (EC 3.6.3.8) mca-3 1.82389 1.15512 1.57897 0.00008 WBGene00019947 CE07426 Histone H2A.V (H2A.F/Z) htz-1 2.08324 1.31953 1.57878 0.00004 WBGene00004387 CE01044 RNA-binding protein 8A rnp-4 1.49106 0.95390 1.56311 0.04833 WBGene00005655 CE30386 Serpentine Receptor, class R srr-4 2.52780 1.62003 1.56034 0.00528 WBGene00000066 CE12358 Actin-4 act-4 1.42958 0.92091 1.55236 0.00000 WBGene00011977 CE35910 Uncharacterized protein 2.00567 1.30285 1.53944 0.02236 WBGene00007966 CE46883 Microtubule-Associated Protein Homolog maph-1.3 1.70634 1.11844 1.52564 0.00498 Alcohol dehydrogenase 1 (EC 1.1.1.1) WBGene00010790 CE12212 sodh-1 8.36660 5.48411 1.52561 0.00007 (Sorbitol dehydrogenase family protein 1)

83 Table-2.2: GO-MF terms for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm GOTERM Wormbase Gene ID Protein name Protein Class Uncharacterized protein non-motor actin binding WBGene00016898 C53C9.2;C53C9.2;ortholog protein(PC00085) catalytic WBGene00003860 Protein OIG-2;oig-2;ortholog activity High mobility group protein I WBGene00001976 (GO:0003824) alpha;hmg-11;ortholog WBGene00017993 Protein CEC-5;cec-5;ortholog WBGene00017064 Protein PPFR-2;ppfr-2;ortholog Uncharacterized protein non-motor actin binding WBGene00016898 C53C9.2;C53C9.2;ortholog protein(PC00085) binding WBGene00004387 Protein RNP-4;rnp-4;ortholog mRNA splicing factor(PC00171) (GO:0005488) EGF-like domain-containing protein WBGene00015328 C02B10.3;C02B10.3;ortholog WBGene00019947 Histone H2A.V;htz-1;ortholog Uncharacterized protein non-motor actin binding WBGene00016898 structural C53C9.2;C53C9.2;ortholog protein(PC00085) molecule WBGene00000066 Actin-4;act-4;ortholog actin and actin related protein(PC00085) activity WBGene00000067 Protein ACT-5;act-5;ortholog actin and actin related protein(PC00085) (GO:0005198) Protein TNT-2, isoform a;tnt- WBGene00006587 actin binding motor protein(PC00085) 2;ortholog transporter Protein MCA-3, isoform b;mca- cation transporter(PC00227);ion activity WBGene00003153 3;ortholog channel(PC00068);hydrolase(PC00133) (GO:0005215)

84 Table-2.3: GO-BP terms for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm GO terms Wormbase Gene ID Protein name Protein class WBGene00004387 Protein RNP-4;rnp-4;ortholog mRNA splicing factor(PC00171) WBGene00006587 Protein TNT-2, isoform a;tnt-2;ortholog actin binding motor protein(PC00085) cation transporter(PC00227);ion metabolic process WBGene00003153 Protein MCA-3, isoform b;mca-3;ortholog channel(PC00068);hydrolase(PC00133) (GO:0008152) dehydrogenase(PC00176);reductase(PC0 WBGene00010790 Alcohol dehydrogenase 1;sodh-1;ortholog 0092) WBGene00000107 Protein ALH-1, isoform a;alh-1;ortholog dehydrogenase(PC00176) WBGene00000066 Actin-4;act-4;ortholog actin and actin related protein(PC00085) localization WBGene00000067 Protein ACT-5;act-5;ortholog actin and actin related protein(PC00085) (GO:0051179) cation transporter(PC00227);ion WBGene00003153 Protein MCA-3, isoform b;mca-3;ortholog channel(PC00068);hydrolase(PC00133) WBGene00000066 Actin-4;act-4;ortholog actin and actin related protein(PC00085) developmental EGF-like domain-containing protein process WBGene00015328 C02B10.3;C02B10.3;ortholog (GO:0032502) WBGene00000067 Protein ACT-5;act-5;ortholog actin and actin related protein(PC00085) cellular WBGene00000066 Actin-4;act-4;ortholog actin and actin related protein(PC00085) component WBGene00000067 Protein ACT-5;act-5;ortholog actin and actin related protein(PC00085) cellular process WBGene00000066 Actin-4;act-4;ortholog actin and actin related protein(PC00085) (GO:0009987) WBGene00000067 Protein ACT-5;act-5;ortholog actin and actin related protein(PC00085) biological cation transporter(PC00227);ion regulation WBGene00003153 Protein MCA-3, isoform b;mca-3;ortholog channel(PC00068);hydrolase(PC00133) (GO:0065007) response to stimulus WBGene00002263 Protein LEA-1, isoform k;lea-1;ortholog chaperone(PC00072) (GO:0050896) multicellular organismal WBGene00006587 Protein TNT-2, isoform a;tnt-2;ortholog actin binding motor protein(PC00085) process (GO:0032501) immune system process WBGene00002263 Protein LEA-1, isoform k;lea-1;ortholog chaperone(PC00072) (GO:0002376)

85 Table-2.4: Statistical Overrepresentation test (PANTHER) for GO-MF and GO-BP terms for the proteins for which the level of expression is higher than 1.5-fold in dauerlarvae compared to L3 worm. C. elegans - Input Input Input Input (fold Input GO Term REFLIST (n) (expected) (over/under) Enrichment) (p value) (20490) GO molecular function (n=27) alcohol dehydrogenase (NAD) activity (GO:0004022) 3 1 0 + > 5 3.95E-03 calcium-transporting ATPase activity (GO:0005388) 5 1 0.01 + > 5 6.57E-03 ATPase activity, coupled to transmembrane movement of ions, 6 1 0.01 + > 5 7.88E-03 phosphorylative mechanism (GO:0015662) ribonucleoprotein complex binding (GO:0043021) 13 1 0.02 + > 5 1.70E-02 oxidoreductase activity, acting on the aldehyde or oxo group of 20 1 0.03 + > 5 2.60E-02 donors, NAD or NADP as acceptor (GO:0016620) structural constituent of cytoskeleton (GO:0005200) 22 1 0.03 + > 5 2.86E-02 oxidoreductase activity, acting on the aldehyde or oxo group of 25 1 0.03 + > 5 3.24E-02 donors (GO:0016903) calcium ion transmembrane transporter activity (GO:0015085) 29 1 0.04 + > 5 3.75E-02 divalent inorganic cation transmembrane transporter activity 32 1 0.04 + > 5 4.13E-02 (GO:0072509) cation-transporting ATPase activity (GO:0019829) 37 1 0.05 + > 5 4.76E-02 ATPase activity, coupled to transmembrane movement of ions 37 1 0.05 + > 5 4.76E-02 (GO:0042625) GO biological process (n=27) microvillus organization (GO:0032528) 1 1 0 + > 5 1.32E-03 response to water (GO:0009415) 1 1 0 + > 5 1.32E-03 response to water deprivation (GO:0009414) 1 1 0 + > 5 1.32E-03 negative regulation of cytosolic calcium ion concentration 1 1 0 + > 5 1.32E-03 (GO:0051481) microvillus assembly (GO:0030033) 1 1 0 + > 5 1.32E-03 response to desiccation (GO:0009269) 1 1 0 + > 5 1.32E-03 tube formation (GO:0035148) 2 1 0 + > 5 2.63E-03 cytosolic calcium ion homeostasis (GO:0051480) 2 1 0 + > 5 2.63E-03 response to acid chemical (GO:0001101) 5 1 0.01 + > 5 6.57E-03 positive regulation of endocytosis (GO:0045807) 11 2 0.01 + > 5 1.00E-04 positive regulation of engulfment of apoptotic cell 6 1 0.01 + > 5 7.88E-03 (GO:1901076) multicellular organismal homeostasis (GO:0048871) 6 1 0.01 + > 5 7.88E-03 positive regulation of apoptotic cell clearance (GO:2000427) 6 1 0.01 + > 5 7.88E-03 integrin-mediated signaling pathway (GO:0007229) 6 1 0.01 + > 5 7.88E-03 cellular divalent inorganic cation homeostasis (GO:0072503) 8 1 0.01 + > 5 1.05E-02 positive regulation of phagocytosis (GO:0050766) 8 1 0.01 + > 5 1.05E-02 cellular calcium ion homeostasis (GO:0006874) 8 1 0.01 + > 5 1.05E-02 positive regulation of phagocytosis, engulfment (GO:0060100) 8 1 0.01 + > 5 1.05E-02 calcium ion homeostasis (GO:0055074) 9 1 0.01 + > 5 1.18E-02 divalent inorganic cation homeostasis (GO:0072507) 10 1 0.01 + > 5 1.31E-02 regulation of endocytosis (GO:0030100) 25 2 0.03 + > 5 5.12E-04 regulation of engulfment of apoptotic cell (GO:1901074) 13 1 0.02 + > 5 1.70E-02 striated muscle contraction (GO:0006941) 13 1 0.02 + > 5 1.70E-02 cellular metal ion homeostasis (GO:0006875) 13 1 0.02 + > 5 1.70E-02 regulation of apoptotic cell clearance (GO:2000425) 13 1 0.02 + > 5 1.70E-02 striated muscle contraction involved in embryonic body 13 1 0.02 + > 5 1.70E-02 morphogenesis (GO:0061199) regulation of vesicle-mediated transport (GO:0060627) 29 2 0.04 + > 5 6.87E-04 positive regulation of transport (GO:0051050) 32 2 0.04 + > 5 8.34E-04 regulation of phagocytosis, engulfment (GO:0060099) 17 1 0.02 + > 5 2.22E-02 regulation of phagocytosis (GO:0050764) 18 1 0.02 + > 5 2.34E-02 positive regulation of cellular component organization 37 2 0.05 + > 5 1.11E-03 (GO:0051130) hyperosmotic response (GO:0006972) 19 1 0.03 + > 5 2.47E-02 cellular ion homeostasis (GO:0006873) 20 1 0.03 + > 5 2.60E-02 cellular cation homeostasis (GO:0030003) 20 1 0.03 + > 5 2.60E-02 metal ion homeostasis (GO:0055065) 20 1 0.03 + > 5 2.60E-02 tube morphogenesis (GO:0035239) 21 1 0.03 + > 5 2.73E-02 positive regulation of locomotion (GO:0040017) 42 2 0.06 + > 5 1.43E-03 regulation of muscle contraction (GO:0006937) 22 1 0.03 + > 5 2.86E-02 regulation of muscle system process (GO:0090257) 22 1 0.03 + > 5 2.86E-02 positive regulation of growth rate (GO:0040010) 22 1 0.03 + > 5 2.86E-02 cellular chemical homeostasis (GO:0055082) 24 1 0.03 + > 5 3.11E-02 muscle system process (GO:0003012) 26 1 0.03 + > 5 3.37E-02 muscle contraction (GO:0006936) 26 1 0.03 + > 5 3.37E-02 calcium ion transmembrane transport (GO:0070588) 26 1 0.03 + > 5 3.37E-02 regulation of system process (GO:0044057) 27 1 0.04 + > 5 3.50E-02

86 regulation of growth rate (GO:0040009) 27 1 0.04 + > 5 3.50E-02 response to osmotic stress (GO:0006970) 29 1 0.04 + > 5 3.75E-02 feminization of hermaphroditic germ-line (GO:0040022) 34 1 0.04 + > 5 4.38E-02 calcium ion transport (GO:0006816) 36 1 0.05 + > 5 4.64E-02 inorganic ion homeostasis (GO:0098771) 36 1 0.05 + > 5 4.64E-02 cation homeostasis (GO:0055080) 37 1 0.05 + > 5 4.76E-02 ion homeostasis (GO:0050801) 38 1 0.05 + > 5 4.89E-02 regulation of transport (GO:0051049) 113 2 0.15 + > 5 9.74E-03 homeostatic process (GO:0042592) 138 2 0.18 + > 5 1.42E-02 secretion by cell (GO:0032940) 140 2 0.18 + > 5 1.46E-02 secretion (GO:0046903) 141 2 0.19 + > 5 1.48E-02 meiotic chromosome segregation (GO:0045132) 142 2 0.19 + > 5 1.50E-02 inductive cell migration (GO:0040039) 146 2 0.19 + > 5 1.58E-02 regulation of locomotion (GO:0040012) 158 2 0.21 + > 5 1.84E-02 nuclear chromosome segregation (GO:0098813) 165 2 0.22 + > 5 1.99E-02 chromosome segregation (GO:0007059) 180 2 0.24 + > 5 2.34E-02 regulation of localization (GO:0032879) 217 2 0.29 + > 5 3.30E-02 meiotic nuclear division (GO:0007126) 231 2 0.3 + > 5 3.70E-02 meiotic cell cycle process (GO:1903046) 236 2 0.31 + > 5 3.85E-02 meiotic cell cycle (GO:0051321) 237 2 0.31 + > 5 3.88E-02 regulation of cellular component organization (GO:0051128) 260 2 0.34 + > 5 4.58E-02 cell cycle process (GO:0022402) 463 3 0.61 + 4.92 2.25E-02 apoptotic process (GO:0006915) 484 3 0.64 + 4.7 2.53E-02 programmed cell death (GO:0012501) 494 3 0.65 + 4.61 2.66E-02 cell cycle (GO:0007049) 498 3 0.66 + 4.57 2.72E-02 death (GO:0016265) 534 3 0.7 + 4.26 3.25E-02 cell death (GO:0008219) 534 3 0.7 + 4.26 3.25E-02 hermaphrodite genitalia development (GO:0040035) 734 4 0.97 + 4.14 1.49E-02 genitalia development (GO:0048806) 740 4 0.98 + 4.1 1.54E-02 lipid storage (GO:0019915) 559 3 0.74 + 4.07 3.65E-02 maintenance of location (GO:0051235) 573 3 0.76 + 3.97 3.88E-02 lipid localization (GO:0010876) 591 3 0.78 + 3.85 4.19E-02 endocytosis (GO:0006897) 823 4 1.08 + 3.69 2.18E-02 development of primary sexual characteristics (GO:0045137) 848 4 1.12 + 3.58 2.40E-02 gonad development (GO:0008406) 848 4 1.12 + 3.58 2.40E-02 regulation of biological quality (GO:0065008) 851 4 1.12 + 3.57 2.43E-02 reproductive system development (GO:0061458) 857 4 1.13 + 3.54 2.49E-02 reproductive structure development (GO:0048608) 857 4 1.13 + 3.54 2.49E-02 sex differentiation (GO:0007548) 924 4 1.22 + 3.29 3.17E-02 vesicle-mediated transport (GO:0016192) 931 4 1.23 + 3.26 3.24E-02 organ development (GO:0048513) 952 4 1.25 + 3.19 3.48E-02 locomotion (GO:0040011) 1577 6 2.08 + 2.89 1.50E-02 nematode larval development (GO:0002119) 2010 7 2.65 + 2.64 1.33E-02 larval development (GO:0002164) 2012 7 2.65 + 2.64 1.33E-02 post-embryonic development (GO:0009791) 2029 7 2.67 + 2.62 1.39E-02 reproductive process (GO:0022414) 1491 5 1.96 + 2.54 4.29E-02 localization (GO:0051179) 2795 8 3.68 + 2.17 2.39E-02 reproduction (GO:0000003) 2536 7 3.34 + 2.09 4.20E-02 single-multicellular organism process (GO:0044707) 4626 11 6.1 + 1.8 2.65E-02 multicellular organismal process (GO:0032501) 4739 11 6.24 + 1.76 3.13E-02 multicellular organismal development (GO:0007275) 4328 10 5.7 + 1.75 4.29E-02 GO terms with p value <0.05 without Bonferroni correction.

87 Pathway Search Result

Following object(s) was/were not found cel:CELE_C02B10.3 cel:CELE_C04C11.2 cel:CELE_C36A4.5 cel:CELE_C53C9.2 cel:CELE_D2092.2 cel:CELE_F07C3.2 cel:CELE_F32E10.6 cel:CELE_F45D11.14 cel:CELE_F49E2.5 cel:CELE_F53A9.10 cel:CELE_F54F2.1 cel:CELE_K08H10.1 cel:CELE_K11D12.3 cel:CELE_R08C7.3 cel:CELE_T05A7.4 cel:CELE_T24B8.3 cel:CELE_T25C8.2 cel:CELE_W05H9.1 cel:CELE_Y38F1A.9 cel:CELE_Y39A1C.3 cel:CELE_Y41C4A.13 cel:CELE_Y5F2A.1

Sort by the pathway list Table-2.5: KEGG pathways for the proteins for which the level of expression is higher Hidethan all objects 1.5-fold in dauer larvae compared to L3 worm

cel00010 Glycolysis / Gluconeogenesis - Caenorhabditis elegans (nematode) (2)

cel:CELE_F54D8.3 alh-1; ALdehyde deHydrogenase cel:CELE_K12G11.3 sodh-1; Alcohol dehydrogenase 1

cel01100 Metabolic pathways - Caenorhabditis elegans (nematode) (2)

cel:CELE_F54D8.3 alh-1; ALdehyde deHydrogenase cel:CELE_K12G11.3 sodh-1; Alcohol dehydrogenase 1

cel00071 Fatty acid degradation - Caenorhabditis elegans (nematode) (2)

cel:CELE_F54D8.3 alh-1; ALdehyde deHydrogenase cel:CELE_K12G11.3 sodh-1; Alcohol dehydrogenase 1

cel00340 Histidine metabolism - Caenorhabditis elegans (nematode) (1)

cel:CELE_F54D8.3 alh-1; ALdehyde deHydrogenase

cel00280 Valine, leucine and isoleucine degradation - Caenorhabditis elegans (nematode) (1)

cel:CELE_F54D8.3 alh-1; ALdehyde deHydrogenase

cel00330 Arginine and proline metabolism - Caenorhabditis elegans (nematode) (1)

cel:CELE_F54D8.3 alh-1; ALdehyde deHydrogenase

cel03040 Spliceosome - Caenorhabditis elegans (nematode) (1)

cel:CELE_R07E5.14 rnp-4; RNP (RRM RNA binding domain) containing

cel04020 Calcium signaling pathway - Caenorhabditis elegans (nematode) (1)

cel:CELE_Y67D8C.10 mca-3; Membrane Calcium ATPase

cel03013 RNA transport - Caenorhabditis elegans (nematode) (1)

cel:CELE_R07E5.14 rnp-4; RNP (RRM RNA binding domain) containing

cel00561 Glycerolipid metabolism - Caenorhabditis elegans (nematode) (1)

cel:CELE_F54D8.3 alh-1; ALdehyde deHydrogenase

cel00982 Drug metabolism - cytochrome P450 - Caenorhabditis elegans (nematode) (1)

cel:CELE_K12G11.3 sodh-1; Alcohol dehydrogenase 1 88 cel00310 Lysine degradation - Caenorhabditis elegans (nematode) (1)

cel:CELE_F54D8.3 alh-1; ALdehyde deHydrogenase Table-2.6: REACTOME pathways for the proteins for which the level of expression is higher than 1.5-fold in dauer larvae compared to L3 worm. Wormbase Gene Pathway name pValue FDR ID WBGene00003153 Reduction of cytosolic Ca++ levels 6.44E-08 2.37E-06 WBGene00003153 Platelet calcium homeostasis 8.18E-08 2.37E-06 WBGene00003153 Platelet homeostasis 4.89E-07 9.30E-06 WBGene00003153 Ion homeostasis 2.46E-06 3.44E-05 WBGene00003153 Ion transport by P-type ATPases 3.15E-06 3.47E-05 WBGene00003153 Muscle contraction 1.15E-05 9.16E-05 WBGene00003153 Cardiac conduction 1.15E-05 9.16E-05 WBGene00000107 Metabolism of serotonin 3.30E-04 1.98E-03 WBGene00000107 Serotonin clearance from the synaptic cleft 3.30E-04 1.98E-03 WBGene00003929; Hemostasis 1.55E-03 7.75E-03 WBGene00003153 WBGene00000107 Ethanol oxidation 1.76E-03 8.80E-03 WBGene00000107 Neurotransmitter Clearance In The Synaptic Cleft 3.53E-03 1.41E-02 WBGene00003153 Ion channel transport 9.30E-03 3.72E-02 Cross-presentation of particulate exogenous antigens WBGene00003929 1.74E-02 6.57E-02 (phagosomes) WBGene00000107 RA biosynthesis pathway 2.19E-02 6.57E-02 WBGene00000107 Signaling by Retinoic Acid 3.21E-02 9.63E-02 WBGene00003929 Syndecan interactions 3.45E-02 1.03E-01 WBGene00003929 Non-integrin membrane-ECM interactions 3.45E-02 1.03E-01 WBGene00003929 Integrin cell surface interactions 8.41E-02 2.47E-01 WBGene00003929 p130Cas linkage to MAPK signaling for integrins 1.24E-01 2.47E-01 GRB2:SOS provides linkage to MAPK signaling for WBGene00003929 1.39E-01 2.52E-01 Integrins WBGene00003929 Signal transduction by L1 1.54E-01 2.52E-01 WBGene00004387 Transport of Mature Transcript to Cytoplasm 1.69E-01 2.52E-01 Transport of Mature mRNA derived from an Intron- WBGene00004387 1.69E-01 2.52E-01 Containing Transcript WBGene00000107 Phase 1 - Functionalization of compounds 2.22E-01 2.52E-01 WBGene00003929 Integrin alphaIIb beta3 signaling 2.39E-01 2.52E-01 WBGene00003929 Platelet Aggregation (Plug Formation) 2.52E-01 2.52E-01 WBGene00004387 mRNA 3'-end processing 2.97E-01 2.97E-01 WBGene00004387 RNA Polymerase II Transcription Termination 2.97E-01 2.97E-01 WBGene00004387 Cleavage of Growing Transcript in the Termination Region 2.97E-01 2.97E-01 WBGene00003929 L1CAM interactions 2.97E-01 2.97E-01 WBGene00003153 Transmembrane transport of small molecules 3.00E-01 3.00E-01 WBGene00003929 Extracellular matrix organization 3.16E-01 3.16E-01 WBGene00000107 Transmission across Chemical Synapses 3.26E-01 3.26E-01 WBGene00003929 Antigen processing-Cross presentation 3.28E-01 3.28E-01 WBGene00000475 mRNA Splicing - Minor Pathway 3.45E-01 3.45E-01 WBGene00004387; mRNA Splicing 3.78E-01 3.78E-01 WBGene00000475

WBGene00004387; mRNA Splicing - Major Pathway 3.78E-01 3.78E-01 WBGene00000475

WBGene00004387; Processing of Capped Intron-Containing Pre-mRNA 3.84E-01 3.84E-01 WBGene00000475

89 WBGene00003929 Platelet degranulation 3.96E-01 3.96E-01 WBGene00003929 Response to elevated platelet cytosolic Ca2+ 4.06E-01 4.06E-01 WBGene00000107 Biological oxidations 4.58E-01 4.58E-01 WBGene00003929 Class I MHC mediated antigen processing & presentation 4.71E-01 4.71E-01 WBGene00000107 Neuronal System 5.35E-01 5.35E-01 WBGene00004387 RNA Polymerase II Transcription 6.28E-01 6.28E-01 WBGene00003929 Platelet activation, signaling and aggregation 6.66E-01 6.66E-01 Nonsense Mediated Decay (NMD) enhanced by the Exon WBGene00004387 6.75E-01 6.75E-01 Junction Complex (EJC) WBGene00004387 Nonsense-Mediated Decay (NMD) 6.75E-01 6.75E-01 WBGene00003929 Adaptive Immune System 6.90E-01 6.90E-01 WBGene00003929 Axon guidance 8.05E-01 8.05E-01 WBGene00003929 Developmental Biology 8.35E-01 8.35E-01 WBGene00004387; Gene Expression 9.27E-01 9.27E-01 WBGene00000475

WBGene00003929; Signal Transduction 9.57E-01 9.57E-01 WBGene00000107 WBGene00003929 Immune System 9.59E-01 9.59E-01 WBGene00000107 Metabolism 1.00E+00 1.00E+00

90 a

91 b

Figure 2.9: Pathway association for 1.5-fold up regulated proteins in dauer. Proteins mapped to (a) the REACTOME pathways (b) KEGG pathways. (a) REACTOME pathways graph showing mapped proteins to pathway in light pink and significantly overrepresented pathways are highlighted in dark brown color (see Table-2.6). (b) KEGG pathways mapped are highlighted in red on the background of different metabolic pathways denoted in different colors (see Table-2.5). The color coding for different metabolic pathways are as follows: fatty acid metabolism (green); Amino acid metabolism (orange); Purine and pyrimidine metabolism (red); oxidative phosphorylation (violet); TCA cycle and glyoxylate dicarboxylate metabolism (blue).

93 Gene ontology and pathway sssociation of down regulated proteins in dauer

The gene ontology and pathway mapping for the 169 proteins for which the expression levels were 1.5-fold lower in dauer larvae compared to L3 larvae (p <0.05) was shown in Table-2.7 and Figure 2.10. The gene ontology (GO)-molecular function

(MF) mapping demonstrated that there were 194 GO terms for 169 down regulated proteins. These proteins mapped to the following eight MF groups as catalytic activity, binding, structural molecular activity, translation regulatory activity, transporter activity, enzyme regulatory activity, nucleic acid binding and transcription activity (Table-2.8).

The majority of the down regulated proteins in the dauer larvae was from binding group

(34%) and related to translation including ribosomal proteins. The catalytic activity group proteins were aspartic proteases, cathepsin B cysteine proteases, translation elongation factors, oxidases, transaminases, phosphatases, kinases, and hydrolases. These down regulated proteins represent mostly translation and related proteins and transcription factor activity proteins, which suggest that protein synthesis pathway is mainly down regulated to conserve energy.

The GO-biological processes (BP) of down regulated proteins are represented by eleven groups similar to the up regulated proteins except apoptotic process and biological adhesion groups are included. It is to be noted among all proteins, metabolic process is the major group that is down regulated (53%). The remaining proteins fall into ten other groups that include proteins related to stress (heat shock proteins and chaperone proteins,

GST, PDI), translation (ribosomal proteins, translation initiation factors and elongation

94 factors), and ATP synthase proteins, transcription factor proteins. This analysis revealed that protein synthesis is significantly decreased and other regular metabolic process is down regulated.

The statistical overrepresentation test (PANTHER) with Bonferroni correction had very long list of significant overrepresented GO terms for MF (~50 terms ) and BP

(~100 terms). The top five GO-MF terms based on p values were protein-glutamine gamma-glutamyltransferase activity, rRNA binding, intramolecular oxidoreductase activity, transposing S-S bonds, protein disulfide isomerase activity, and translation elongation factor activity. Similarly top five GO-BP terms were protein deglutathionylation, maturation of SSU-rRNA including from tricistronic rRNA transcript, ribosomal small subunit biogenesis, translational elongation translation.

The KEGG pathway mapping for top five pathways is shown in Table-2.5.

Pathway mapping indicated that the top five pathways down regulated are fatty acid synthase, nucleotide synthesis, oxidative phosphorylation, pentose-phosphaste and amino acid metabolism (Table-2.5 and Figure 2.11b). The results clearly indicate that the regular metabolic process is down regulated. In addition, the REACTOME mapping

(Figure 2.11a) and overrepresentation test (Table-2.9) revealed that GTP hydrolysis

(joining of the 60S ribosomal subunit), SRP-dependent cotranslational protein targeting to membrane, formation of a pool of free 40S subunits, nonsense mediated decay (NMD) dependent/independent of the exon junction complex (EJC), translation (including initiation and elongation), metabolism of proteins, gene expression, peptide chain

95 elongation, NADPH regeneration, HSF1 activation, branched chain amino acid catabolism, secretory pathways (Golgi cisternae reorganization), and integration of energy metabolism pathways are significantly overrepresented pathways among 212 mapped pathways in REACTOME.

96 a b 1, 1% 1, 1% 2, 1% 1, 1% 2, 1% 4, 2% 1, 0% 5, 3% 5, 2%

8, 4% 12, 5% 14, 6%

66, 34% 17, 7%

51, 26% 19, 8% 126, 53%

38, 16%

57, 29%

(194) (236) 1% metabolic process (GO: 2% 1% binding (GO:0005488) 0008152)

3% cataly;c ac;vity (GO: cellular process (GO:0009987) 4% 0003824)

structural molecule ac;vity cellular component organizaAon or biogenesis 34% (GO:0005198) (GO:0071840) 1% biological regulaAon (GO: transla;on regulator ac;vity 0065007) (GO:0045182) 26% 1% localizaAon (GO:0051179) transporter ac;vity (GO: 1% 0005215) 0% 2% response to sAmulus (GO: enzyme regulator ac;vity 0050896) 5% (GO:0030234) 6% developmental process (GO: nucleic acid binding 0032502) 7% transcrip;on factor ac;vity (GO:0001071) mulAcellular organismal 29% 8% protein binding transcrip;on process (GO:0032501) 53% factor ac;vity (GO:0000988) apoptoAc process (GO: 16% 0006915) biological adhesion (GO: 0022610)

immune system process (GO: 0002376) Figure 2.10 Gene ontology (GO)-molecular function (MF) and biological process (BP) terms for 1.5-fold down regulated dauer proteins. The pie graph shows (a) GO- MF and (b) GO-BP for the proteins (169) that are significantly (p <0.05) 1.5-fold down regulated in dauer larvae. Those proteins are listed in Table 2.7. Each GO-MF and GO- BP term (a slice of a pie graph) is marked with gene counts and percent of the count of genes present in individual GO-BP term to the total gene count mapped to GO terms (written in parenthesis under each stage) for individual development stage.

97 Table-2.7: Proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm Wormbase Gene ID Wormpep ID Protein names Gene names Ratio (light/heavy) Fold Change p value Dauer L3 Probable 4-aminobutyrate aminotransferase, mitochondrial (EC 2.6.1.19) ((S)-3-amino-2- methylpropionate transaminase) (EC 2.6.1.22) WBGene00001794 CE06092 gta-1 0.62792 0.94267 0.66610 0.00023 (GABA aminotransferase) (GABA-AT) (Gamma-amino-N-butyrate transaminase) (GABA transaminase) (L-AIBAT) Transitional endoplasmic reticulum ATPase WBGene00008053 CE05402 homolog 2 (Cell division cycle-related protein cdc-48.2 0.62380 0.93725 0.66557 0.00031 48.2) (p97/CDC48 homolog 2) Isocitrate dehydrogenase [NADP] (EC WBGene00010317 CE03436 idh-1 0.61679 0.92780 0.66478 0.00028 1.1.1.42) WBGene00003964 CE11570 Protein disulfide-isomerase (EC 5.3.4.1) pdi-3 0.51837 0.78240 0.66255 0.00022 WBGene00004408 CE09655 60S acidic ribosomal protein P0 rpa-0 0.48932 0.73919 0.66197 0.00000 Transitional endoplasmic reticulum ATPase WBGene00007352 CE02114 homolog 1 (Cell division cycle-related protein cdc-48.1 0.62894 0.95070 0.66156 0.00034 48.1) (p97/CDC48 homolog 1) Protein disulfide-isomerase 1 (PDI 1) (EC WBGene00003962 CE00897 pdi-1 0.54868 0.82974 0.66126 0.00263 5.3.4.1) (Prolyl 4-hydroxylase subunit beta-1) GDI (RabGDP Dissociation Inhibitor) family WBGene00001558 CE14944 gdi-1 0.68447 1.03991 0.65820 0.04703 (RabGDI) WBGene00011629 CE13443 Uncharacterized protein 0.73443 1.11654 0.65777 0.00310 Peptidyl-prolyl cis-trans isomerase 5 (PPIase WBGene00000881 CE17730 cyn-5 0.53695 0.81905 0.65558 0.00390 5) (EC 5.2.1.8) (Cyclophilin-5) (Rotamase 5) WBGene00004438 CE02777 60S ribosomal protein L23a 1 rpl-25.1 0.44673 0.68175 0.65527 0.00012 Isoleucine--tRNA ligase, cytoplasmic (EC WBGene00002152 CE06304 irs-1 0.49710 0.76424 0.65045 0.00065 6.1.1.5) (Isoleucyl-tRNA synthetase) (IleRS) Eukaryotic translation initiation factor 3 WBGene00001225 CE47851 subunit B (eIF3b) (Eukaryotic translation eif-3.B 0.52099 0.80146 0.65005 0.00039 initiation factor 3 subunit 9) WBGene00003829 CE16096 Aspergillus NUclear Division related (NUD-1) nud-1 0.50633 0.78018 0.64899 0.00417 WBGene00001744 CE29472 Glycyl Amino-acyl tRNA Synthetase gars-1 0.50603 0.78119 0.64777 0.00189 WBGene00016250 CE00103 Uncharacterized protein C30C11.4 0.50289 0.77735 0.64692 0.00464 Ubiquinol-Cytochrome c oxidoReductase WBGene00012158 CE20123 ucr-2.1 0.68406 1.05782 0.64667 0.00014 complex WBGene00006924 CE11254 VIG (Drosophila Vasa Intronic Gene) ortholog vig-1 0.74363 1.15192 0.64555 0.00252 WBGene00000542 CE37743 Calpain clp-1 (EC 3.4.22.-) clp-1 0.91102 1.41214 0.64514 0.01321 WBGene00022170 CE22939 Uncharacterized protein 0.60756 0.94248 0.64464 0.00896 WBGene00019727 CE04757 2 (Zwei) IG domain protein zig-12 0.55832 0.86624 0.64453 0.00191 WBGene00004266 CE16905 RAB family rab-1 0.63434 0.98748 0.64238 0.00003 26S proteasome non-ATPase regulatory WBGene00004463 CE03368 subunit 6 (26S proteasome regulatory subunit rpn-7 0.51694 0.80820 0.63961 0.00008 rpn-7) mitochondrial genome encoded NADH- WBGene00010959 CE34065 (NaDh) Ubiquinone Oxidoreductase chain nduo-1 0.66360 1.04049 0.63777 0.00162 homolog WBGene00002008 CE07244 Heat shock 70 kDa protein D hsp-4 0.49479 0.77866 0.63544 0.00008 WBGene00016493 CE30433 60S acidic ribosomal protein P2 rpa-2 0.47332 0.74572 0.63471 0.00043 Eukaryotic translation initiation factor 3 WBGene00001227 CE00291 subunit D (eIF3d) (Eukaryotic translation eif-3.D 0.48905 0.77097 0.63432 0.00602 initiation factor 3 subunit 7) WBGene00002258 CE14426 Fatty acid-binding protein homolog 6 lbp-6 0.54783 0.86457 0.63364 0.00019 Alanine--tRNA ligase, cytoplasmic (EC WBGene00000197 CE09768 aars-2 0.53491 0.84904 0.63002 0.01470 6.1.1.7) (AlaRS A) (Alanyl-tRNA synthetase) WBGene00004477 CE04561 40S ribosomal protein S8, Small subunit rps-8 0.47975 0.76174 0.62980 0.00080 Phosphate carrier protein, mitochondrial WBGene00008505 CE09162 0.56942 0.90472 0.62939 0.00021 (PTP), Uncharacterized protein Acidic leucine-rich nuclear phosphoprotein 32- WBGene00009367 CE17766 related protein 1 (ANP32/acidic nuclear 0.90176 1.44182 0.62543 0.00012 phosphoprotein-like protein 1) WBGene00007836 CE08497 Uncharacterized protein 0.52661 0.84503 0.62319 0.00003 WBGene00006439 CE46432 Adenine Nucleotide Translocator ant-1.1 0.54773 0.87965 0.62267 0.00039 WBGene00006626 CE02626 Tudor Staphylococcal Nuclease homolog tsn-1 0.46458 0.74747 0.62154 0.00012 Heat shock protein 90 (Abnormal dauer WBGene00000915 CE05441 daf-21 0.50517 0.81384 0.62073 0.00000 formation protein 21) DAF-16/FOXO Controlled, germline Tumor WBGene00012615 CE19095 dct-16 0.45299 0.72993 0.62059 0.00018 affecting

98 Probable succinyl-CoA ligase [ADP-forming] subunit beta, mitochondrial (EC 6.2.1.5) (ATP- WBGene00009812 CE03351 specific succinyl-CoA synthetase subunit beta) 0.64262 1.03963 0.61812 0.00130 (Succinyl-CoA synthetase beta-A chain) (SCS- betaA) WBGene00004488 CE13265 40S ribosomal protein S19 rps-19 0.43588 0.70575 0.61761 0.00005 WBGene00020915 CE18307 NucleOLar protein nol-5 0.50007 0.80995 0.61741 0.00042 WBGene00012344 CE14708 OLA (Obg-Like ATPase) homolog ola-1 0.46601 0.76125 0.61217 0.04027 WBGene00006889 CE13349 Probable prefoldin subunit 3 pfd-3 0.50505 0.83118 0.60764 0.00427 WBGene00002005 CE09682 Heat shock 70 kDa protein A hsp-1 0.47907 0.78870 0.60742 0.00004 WBGene00004428 CE01030 60S ribosomal protein L13a rpl-16 0.48732 0.80358 0.60644 0.00275 WBGene00000869 CE05514 CYtochrome C cyc-1 0.55048 0.91316 0.60283 0.00075 WBGene00006536 CE16197 TuBulin, Beta tbb-1 0.59018 0.98464 0.59939 0.00003 WBGene00004434 CE04102 60S ribosomal protein L22 rpl-22 0.41374 0.69097 0.59879 0.00133 WBGene00004493 CE40119 Ribosomal Protein, Small subunit rps-24 0.43549 0.72919 0.59722 0.00013 DAF-16/FOXO Controlled, germline Tumor WBGene00010266 CE17924 dct-18 0.39796 0.66666 0.59695 0.00012 affecting WBGene00004439 CE05721 60S ribosomal protein L23a 2 rpl-25.2 0.41585 0.69749 0.59621 0.00005 Probable protein disulfide-isomerase A4 (EC WBGene00015752 CE30601 0.43457 0.73604 0.59041 0.00246 5.3.4.1) (ERp-72 homolog) WBGene00011480 CE06362 Endoplasmin homolog enpl-1 0.50062 0.84827 0.59016 0.00000 WBGene00006914 CE04504 V-type proton ATPase subunit a vha-5 0.64505 1.10495 0.58379 0.00033 WBGene00006537 CE00913 Tubulin beta-2 chain (Beta-2-tubulin) tbb-2 0.57470 0.98448 0.58376 0.00010 WBGene00014095 CE06652 Glutamate dehydrogenase gdh-1 0.45009 0.77198 0.58304 0.00005 WBGene00008506 CE09163 TransKeTolase homolog tkt-1 0.62632 1.07435 0.58297 0.00012 WBGene00004426 CE19677 Ribosomal Protein, Large subunit rpl-14 0.37646 0.64701 0.58184 0.00745 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial (EC 1.2.4.4) (Branched-chain WBGene00012713 CE33866 0.56079 0.96403 0.58171 0.00001 alpha-keto acid dehydrogenase E1 component alpha chain) WBGene00004470 CE00664 40S ribosomal protein S3a rps-1 0.43167 0.74419 0.58005 0.00004 WBGene00004471 CE04237 40S ribosomal protein S2 rps-2 0.40791 0.70573 0.57800 0.01236 WBGene00004450 CE30781 60S ribosomal protein L36 rpl-36 0.44825 0.77706 0.57685 0.00002 WBGene00004487 CE14956 Ribosomal Protein, Small subunit rps-18 0.40891 0.71047 0.57555 0.00028 Ribosomal Protein, Large subunit, 60S WBGene00004414 CE05598 rpl-3 0.44906 0.78446 0.57245 0.00713 ribosomal protein L3 ALdehyde deHydrogenase, Probable methylmalonate-semialdehyde dehydrogenase WBGene00000114 CE02183 [acylating], mitochondrial (MMSDH) alh-8 0.45069 0.78806 0.57189 0.00005 (Malonate-semialdehyde dehydrogenase [acylating]) (EC 1.2.1.18) (EC 1.2.1.27) Fructose-1,6-BiPhosphatase (Fructose-1,6- WBGene00001404 CE21023 fbp-1 0.57109 0.99903 0.57165 0.00002 bisphosphatase) (EC 3.1.3.11) Eukaryotic translation initiation factor 3 WBGene00001228 CE17349 subunit E (eIF3e) (Eukaryotic translation eif-3.E 0.40083 0.70370 0.56961 0.00769 initiation factor 3 subunit 6) WBGene00001694 CE10246 GRounDhog (Hedgehog-like family) grd-5 0.59676 1.04896 0.56891 0.00024 WBGene00007463 CE23530 ViGiLN homolog vgln-1 0.49146 0.86789 0.56627 0.00011 WBGene00004413 CE18478 60S ribosomal protein L8 rpl-8 0.40328 0.71291 0.56569 0.00001 WBGene00001168 CE01270 Elongation factor 1-alpha (EF-1-alpha) eft-3; eft-4 0.54157 0.96038 0.56391 0.00038 WBGene00000822 CE05839 Calsequestrin csq-1 0.50649 0.89865 0.56361 0.00005 WBGene00000214 CE21681 ASpartyl Protease (Aspartic protease 1) asp-1 0.35649 0.63336 0.56285 0.00006 Probable protein disulfide-isomerase A6 (EC WBGene00015168 CE03880 tag-320 0.41999 0.75442 0.55670 0.00001 5.3.4.1) Chaperonin Containing TCP-1, T-complex WBGene00000381 CE01234 protein 1 subunit zeta (TCP-1-zeta) (CCT- cct-6 0.46560 0.83849 0.55528 0.00010 zeta) WBGene00004429 CE22195 60S ribosomal protein L17 rpl-17 0.45200 0.81786 0.55266 0.00040 WBGene00004483 CE00821 40S ribosomal protein S14 rps-14 0.45396 0.82294 0.55163 0.00088 WBGene00004424 CE17986 60S ribosomal protein L12 rpl-12 0.38489 0.70094 0.54911 0.00005 WBGene00002007 CE08177 Heat shock 70 kDa protein C hsp-3 0.44820 0.81716 0.54849 0.00002 WBGene00001774 CE22416 Glutathione S-Transferase gst-26 0.54494 0.99592 0.54718 0.00621 ADP-Ribosylation Factor related (ADP- WBGene00000183 CE17122 arf-3 0.47659 0.87207 0.54650 0.00004 ribosylation factor related (20.5 kD) (Arf-3)) WBGene00004423 CE07033 Ribosomal Protein, Large subunit rpl-11.2 0.38759 0.71096 0.54517 0.00026 WBGene00015344 CE28528 Nicotinic Receptor Associated nra-4 0.50197 0.92090 0.54509 0.00011 WBGene00004480 CE05860 Ribosomal Protein, Small subunit rps-11 0.40978 0.75178 0.54507 0.00149 Nascent polypeptide-associated complex WBGene00022042 CE22740 subunit alpha (NAC-alpha) (Alpha-NAC), icd-2 0.46089 0.84959 0.54248 0.00135 Inhibitor of Cell Death Elongation of very long chain fatty acids protein 6 (EC 2.3.1.199) (3-keto acyl-CoA WBGene00001244 CE10286 elo-6 0.97595 1.80076 0.54197 0.00284 synthase elo-6) (Very-long-chain 3-oxoacyl- CoA synthase 6) DAF-16/FOXO Controlled, germline Tumor WBGene00012615 CE44759 dct-16 0.39219 0.72472 0.54116 0.00016 affecting

99 WBGene00021350 CE26774 Uncharacterized protein 0.35600 0.66153 0.53814 0.00074 WBGene00008132 CE17566 UDP-GALactose 4-Epimerase gale-1 0.58655 1.09002 0.53811 0.00007 Cathepsin B-like cysteine proteinase 5 (EC WBGene00000785 CE14682 cpr-5 0.33756 0.63005 0.53576 0.02233 3.4.22.-) (Cysteine protease-related 5) Dietary Restriction Response (WT but not eat- WBGene00011730 CE16399 drr-2 0.55343 1.03298 0.53576 0.00002 2 lifespan increased) WBGene00004435 CE00778 60S ribosomal protein L23 rpl-23 0.48784 0.91544 0.53290 0.00616 WBGene00004755 CE05594 Yeast SEC homolog sec-24.1 0.47394 0.89008 0.53247 0.00037 T-complex protein 1 subunit epsilon (TCP-1- WBGene00000380 CE02985 epsilon) (CCT-epsilon), Chaperonin cct-5 0.47524 0.89527 0.53083 0.00007 Containing TCP-1 WBGene00004478 CE05849 40S ribosomal protein S9 rps-9 0.33969 0.64183 0.52925 0.00001 Transcription factor BTF3 homolog (Inhibitor WBGene00002045 CE02573 icd-1 0.43893 0.82982 0.52894 0.00163 of cell death 1) WBGene00010605 CE18851 Uncharacterized protein 0.51846 0.98734 0.52511 0.00511 WBGene00004418 CE11024 60S ribosomal protein L7 rpl-7 0.37595 0.71644 0.52474 0.00000 Acetolactate synthase-like protein (EC 2.2.1.-) WBGene00020831 CE26009 0.48794 0.93267 0.52316 0.00303 (IlvB-like protein) WBGene00011128 CE12564 Uncharacterized protein 0.40481 0.77858 0.51993 0.00005 WBGene00004436 CE09047 60S ribosomal protein L24 rpl-24.1 0.37011 0.72198 0.51263 0.00003 WBGene00004433 CE00078 60S ribosomal protein L21 rpl-21 0.43055 0.84072 0.51213 0.02205 Translationally-controlled tumor protein WBGene00009122 CE09656 tct-1 0.37089 0.72495 0.51161 0.00008 homolog (TCTP) WBGene00004473 CE24278 40S ribosomal protein S4 rps-4 0.40407 0.79335 0.50932 0.00004 6-phosphogluconate dehydrogenase, WBGene00012015 CE06508 0.60128 1.18194 0.50872 0.00006 decarboxylating (EC 1.1.1.44) Lysine--tRNA ligase (EC 6.1.1.6) (Lysyl- WBGene00002238 CE04861 kars-1 0.53806 1.05869 0.50823 0.00019 tRNA synthetase) WBGene00001167 CE15900 Elongation factor 2 (EF-2) eef-2 0.38941 0.76652 0.50803 0.00002 WBGene00004456 CE22117 60S ribosomal protein L37a rpl-43 0.34350 0.67996 0.50517 0.00000 WBGene00004476 CE06577 40S ribosomal protein S7 rps-7 0.42693 0.85626 0.49860 0.00001 WBGene00004416 CE02255 60S ribosomal protein L5 rpl-5 0.39505 0.79365 0.49777 0.00022 Eukaryotic translation initiation factor 5A-2 WBGene00002065 CE02249 iff-2 0.40389 0.81235 0.49718 0.02314 (eIF-5A-2) (Initiation factor five protein 2) WBGene00000181 CE03127 Alcohol/Ribitol Dehydrogenase family ard-1 0.46680 0.94038 0.49639 0.00019 WBGene00004485 CE12918 40S ribosomal protein S16 rps-16 0.35949 0.72733 0.49426 0.00024 WBGene00004475 CE24592 40S ribosomal protein S6 rps-6 0.39581 0.80572 0.49125 0.00001 WBGene00004446 CE03709 Ribosomal Protein, Large subunit rpl-32 0.36408 0.74179 0.49081 0.00000 WBGene00004503 CE01253 Probable 26S protease regulatory subunit 6B rpt-3 0.52510 1.07173 0.48996 0.00005 Probable elongation factor 1-beta/1-delta 2 WBGene00012768 CE37568 eef-1B.2 0.40730 0.83199 0.48955 0.00015 (EF-1-beta/delta 2) Chaperonin homolog Hsp-60, mitochondrial WBGene00002025 CE27244 hsp-60 0.40358 0.83099 0.48567 0.00040 (Heat shock protein 60) (HSP-60) WBGene00009342 CE42777 Fatty Acid SyNthase fasn-1 0.37853 0.78022 0.48515 0.00002 WBGene00013228 CE28140 Uncharacterized protein 0.64361 1.33194 0.48321 0.01089 WBGene00003902 CE20412 Polyadenylate-binding protein (PABP) pab-1 0.41547 0.86730 0.47905 0.00001 Cuticle collagen sqt-1 (Protein roller-5) WBGene00005016 CE02104 sqt-1 0.21306 0.44510 0.47867 0.00251 (Protein squat-1) Probable enoyl-CoA hydratase, mitochondrial WBGene00001155 CE00318 ech-6 0.43191 0.90286 0.47838 0.00007 (EC 4.2.1.17) Probable ornithine aminotransferase, WBGene00015814 CE04010 mitochondrial (EC 2.6.1.13) (Ornithine--oxo- 0.31167 0.65232 0.47779 0.00042 acid aminotransferase) WBGene00000216 CE47856 Aspartic protease 3 (EC 3.4.23.-) asp-3 0.35610 0.74601 0.47734 0.00015 WBGene00004754 CE27230 Yeast SEC homolog sec-23 0.42052 0.88119 0.47722 0.00001 WBGene00004490 CE30779 40S ribosomal protein S21 rps-21 0.46214 0.97020 0.47634 0.01594 Probable elongation factor 1-beta/1-delta 1 WBGene00018846 CE00548 eef-1B.1 0.39434 0.82933 0.47549 0.00002 (EF-1-beta/delta 1) WBGene00007605 CE05287 ASpartyl Protease asp-10 0.31288 0.66225 0.47245 0.00145 WBGene00017719 CE17691 Lipid DroPlet localized protein ldp-1 0.83568 1.78512 0.46814 0.00486 WBGene00004417 CE00744 60S ribosomal protein L6 rpl-6 0.32683 0.69979 0.46704 0.00041 WBGene00004469 CE00854 40S ribosomal protein SA rps-0 0.35883 0.76977 0.46615 0.00008 Probable S-adenosylmethionine synthase 1 WBGene00008205 CE08852 (AdoMet synthase 1) (EC 2.5.1.6) (Methionine sams-1 0.41222 0.88974 0.46330 0.00000 adenosyltransferase 1) (MAT 1) CALUmenin (Calcium-binding protein) WBGene00019760 CE12368 calu-1 0.41509 0.90134 0.46052 0.00000 homolog (Calumenin-like protein) WBGene00004419 CE27398 60S ribosomal protein L7a rpl-7A 0.35156 0.76496 0.45958 0.00008 WBGene00004481 CE26896 40S ribosomal protein S12 rps-12 0.33707 0.73517 0.45849 0.00002 WBGene00004412 CE25552 60S ribosomal protein L10a rpl-10a 0.34361 0.75037 0.45792 0.00000 WBGene00000802 CE21562 Calreticulin crt-1 0.38009 0.83463 0.45541 0.00000 WBGene00004447 CE04362 60S ribosomal protein L35a rpl-33 0.35721 0.79057 0.45183 0.00057 WBGene00003982 CE12770 Patterned Expression Site pes-9 0.36051 0.82445 0.43727 0.00000 WBGene00000215 CE33320 ASpartyl Protease asp-2 0.29792 0.68249 0.43652 0.00003 WBGene00004432 CE21392 60S ribosomal protein L18a rpl-20 0.35218 0.81857 0.43024 0.00003 WBGene00004479 CE09041 Ribosomal Protein, Small subunit rps-10 0.34246 0.80264 0.42667 0.00000

100 WBGene00044294 CE38583 Uncharacterized protein 0.32880 0.78176 0.42060 0.01832 WBGene00004420 CE01380 60S ribosomal protein L9 rpl-9 0.33830 0.81276 0.41624 0.00003 WBGene00001775 CE22417 Glutathione S-Transferase gst-27 0.27979 0.68167 0.41045 0.00446 WBGene00012097 CE18971 ABC transporter, class F abcf-2 0.29647 0.73002 0.40611 0.00054 WBGene00004492 CE05747 40S ribosomal protein S23 rps-23 0.30286 0.75157 0.40297 0.00015 WBGene00017691 CE04442 Invertebrate LYSozyme ilys-5 0.23938 0.59889 0.39971 0.00025 WBGene00004259 CE41886 PYRimidine biosynthesis pyr-1 0.24402 0.61322 0.39794 0.00014 WBGene00002077 CE26971 IMportin Beta family imb-3 0.27613 0.70011 0.39441 0.00007 WBGene00004431 CE08034 60S ribosomal protein L19 rpl-19 0.30975 0.79185 0.39118 0.00002 WBGene00003963 CE03972 Protein Disulfide Isomerase pdi-2 0.30332 0.77742 0.39016 0.00002 DumPY: shorter than wild-type (Thioredoxin- WBGene00001073 CE20819 dpy-11 0.31303 0.80328 0.38969 0.00001 like protein DPY-11) Probable N-acetylgalactosaminyltransferase 8 (EC 2.4.1.-) (Protein-UDP acetylgalactosaminyltransferase 8) (UDP- WBGene00001633 CE15691 gly-8 0.27506 0.72395 0.37994 0.00262 GalNAc:polypeptide N- acetylgalactosaminyltransferase 8) (pp- GaNTase 8) WBGene00004474 CE06360 40S ribosomal protein S5 rps-5 0.28775 0.75891 0.37916 0.00003 WBGene00004425 CE08526 60S ribosomal protein L13 rpl-13 0.29677 0.78515 0.37798 0.00008 Putative phospholipase B-like 2 (EC 3.1.1.-) WBGene00008607 CE31469 (LAMA-like protein 2) (Lamina ancestor 0.31706 0.85185 0.37220 0.00372 homolog 2) WBGene00004484 CE09945 40S ribosomal protein S15 rps-15 0.33376 0.90441 0.36904 0.00007 Adenosylhomocysteinase (AdoHcyase) (EC WBGene00019322 CE17154 3.3.1.1) (Protein dumpy-14) (S-adenosyl-L- ahcy-1 0.32882 0.89936 0.36561 0.00003 homocysteine hydrolase) Probable hydroxyacid-oxoacid WBGene00012608 CE24222 transhydrogenase, mitochondrial (HOT) (EC 0.35237 0.96422 0.36544 0.00271 1.1.99.24) WBGene00004427 CE12148 60S ribosomal protein L15 rpl-15 0.36534 1.00675 0.36289 0.00326 WBGene00004491 CE10884 Ribosomal Protein, Small subunit rps-22 0.42844 1.18746 0.36081 0.00331 Homologous to Drosophila SQD (Squid) WBGene00022235 CE29008 sqd-1 0.30007 0.83741 0.35832 0.00000 protein Serine hydroxymethyltransferase (SHMT) (EC 2.1.2.1) (Glycine hydroxymethyltransferase) WBGene00003214 CE01130 mel-32 0.36821 1.04844 0.35120 0.00001 (Glycosylation-related protein 1) (Maternal effect lethal protein 32) (Serine methylase) WBGene00004452 CE20485 60S ribosomal protein L38 rpl-38 0.33909 0.97004 0.34956 0.00001 WBGene00019682 CE32696 Putative serine protease K12H4.7 (EC 3.4.-.-) 0.18205 0.53291 0.34161 0.00051 WBGene00001430 CE08912 Peptidyl-prolyl cis-trans isomerase fkb-5 0.24249 0.71555 0.33888 0.00006 WBGene00004449 CE00450 60S ribosomal protein L35 rpl-35 0.31837 0.95461 0.33351 0.00000 WBGene00016943 CE09015 Acyl CoA DeHydrogenase acdh-1 0.14129 0.96193 0.14689 0.00004

101 Table-2.8: GO-MF terms for the proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm GO terms Wornbase Gene ID Protein name Protein class WBGENE00004473 40S ribosomal protein S4;rps-4;ortholog ribosomal protein(PC00171) WBGENE00004474 40S ribosomal protein S5;rps-5;ortholog ribosomal protein(PC00171) WBGENE00004488 40S ribosomal protein S19;rps-19;ortholog ribosomal protein(PC00171) Probable elongation factor 1-beta/1-delta 1;eef- WBGENE00018846 translation elongation factor(PC00171) 1B.1;ortholog Eukaryotic translation initiation factor 3 subunit B;eif- WBGENE00001225 translation initiation factor(PC00171) 3.B;ortholog WBGENE00004423 Protein RPL-11.2;rpl-11.2;ortholog ribosomal protein(PC00171) WBGENE00004420 60S ribosomal protein L9;rpl-9;ortholog ribosomal protein(PC00171) Eukaryotic translation initiation factor 3 subunit E;eif- WBGENE00001228 translation initiation factor(PC00171) 3.E;ortholog WBGENE00004408 60S acidic ribosomal protein P0;rpa-0;ortholog ribosomal protein(PC00171) WBGENE00004414 60S ribosomal protein L3;rpl-3;ortholog ribosomal protein(PC00171) translation elongation factor(PC00171);translation WBGENE00001168 Elongation factor 1-alpha;eft-3;ortholog initiation factor(PC00031);hydrolase(PC00223);G- protein(PC00222) translation elongation factor(PC00171);translation WBGENE00001167 Elongation factor 2;eef-2;ortholog initiation factor(PC00031);hydrolase(PC00223);G- protein(PC00222) WBGENE00000802 Calreticulin;crt-1;ortholog calcium-binding protein(PC00060) WBGENE00000197 Alanine--tRNA ligase, cytoplasmic;aars-2;ortholog RNA binding protein(PC00171) WBGENE00004439 60S ribosomal protein L23a 2;rpl-25.2;ortholog ribosomal protein(PC00171) Acidic leucine-rich nuclear phosphoprotein 32-related WBGENE00009367 phosphatase inhibitor(PC00095) protein 1;F33H2.3;ortholog transporter(PC00227);transfer/carrier WBGENE00002077 Protein IMB-3;imb-3;ortholog protein(PC00219);G-protein modulator(PC00095) WBGENE00004416 60S ribosomal protein L5;rpl-5;ortholog ribosomal protein(PC00171) WBGENE00004431 60S ribosomal protein L19;rpl-19;ortholog ribosomal protein(PC00171) WBGENE00004479 Protein RPS-10;rps-10;ortholog ribosomal protein(PC00171) WBGENE00004418 60S ribosomal protein L7;rpl-7;ortholog ribosomal protein(PC00171) Eukaryotic translation initiation factor 3 subunit D;eif- WBGENE00001227 translation initiation factor(PC00171) 3.D;ortholog WBGENE00012344 Obg-like ATPase 1;tag-210;ortholog G-protein(PC00095) WBGENE00004413 60S ribosomal protein L8;rpl-8;ortholog ribosomal protein(PC00171) WBGENE00004425 60S ribosomal protein L13;rpl-13;ortholog ribosomal protein(PC00171) transcription cofactor(PC00218);nucleic acid WBGENE00006626 Protein TSN-1;tsn-1;ortholog binding(PC00217) WBGENE00011730 Protein DRR-2;drr-2;ortholog translation initiation factor(PC00171) WBGENE00004424 60S ribosomal protein L12;rpl-12;ortholog ribosomal protein(PC00171) WBGENE00006924 Protein VIG-1, isoform a;vig-1;ortholog RNA binding protein(PC00171) WBGENE00006914 Protein VHA-5;vha-5;ortholog ATP synthase(PC00227);hydrolase(PC00068) ATP-binding cassette (ABC) WBGENE00012097 Protein ABCF-2;abcf-2;ortholog transporter(PC00227);translation elongation factor(PC00003);hydrolase(PC00171) WBGENE00021350 Protein Y37E3.8, isoform a;CELE_Y37E3.8;ortholog ribosomal protein(PC00171) WBGENE00004452 60S ribosomal protein L38;rpl-38;ortholog ribosomal protein(PC00171) binding WBGENE00004435 60S ribosomal protein L23;rpl-23;ortholog ribosomal protein(PC00171) (GO:0005488) WBGENE00000822 Calsequestrin;csq-1;ortholog calsequestrin(PC00060) Eukaryotic translation initiation factor 5A-2;iff- WBGENE00002065 translation initiation factor(PC00171) 2;ortholog WBGENE00002045 Transcription factor BTF3 homolog;icd-1;ortholog transcription factor(PC00218) RNA binding protein(PC00171);aminoacyl-tRNA WBGENE00002238 Lysine--tRNA ligase;krs-1;ortholog synthetase(PC00031) WBGENE00019760 Calumenin-like protein;calu-1;ortholog calmodulin(PC00060) amino acid transporter(PC00227);mitochondrial carrier protein(PC00046);transfer/carrier WBGENE00006439 Protein ANT-1.1, isoform a;ant-1.1;ortholog protein(PC00158);ribosomal protein(PC00219);calmodulin(PC00171) WBGENE00004438 60S ribosomal protein L23a 1;rpl-25.1;ortholog ribosomal protein(PC00171) acyltransferase(PC00220);G-protein WBGENE00001558 Protein GDI-1, isoform b;gdi-1;ortholog modulator(PC00042) Probable elongation factor 1-beta/1-delta 2;eef- WBGENE00012768 translation elongation factor(PC00171) 1B.2;ortholog Translationally-controlled tumor protein homolog;tct- WBGENE00009122 non-motor microtubule binding protein(PC00085) 1;ortholog WBGENE00004428 60S ribosomal protein L13a;rpl-16;ortholog ribosomal protein(PC00171) WBGENE00004417 60S ribosomal protein L6;rpl-6;ortholog ribosomal protein(PC00171) WBGENE00004754 Protein SEC-23;sec-23;ortholog G-protein modulator(PC00095) ribonucleoprotein(PC00171);ribosomal WBGENE00004485 40S ribosomal protein S16;rps-16;ortholog protein(PC00031) WBGENE00004419 60S ribosomal protein L7a;rpl-7A;ortholog ribosomal protein(PC00171) WBGENE00004436 60S ribosomal protein L24;rpl-24.1;ortholog ribosomal protein(PC00171) WBGENE00004483 40S ribosomal protein S14;rps-14;ortholog ribosomal protein(PC00171) ADP-ribosylation factor related (20.5 kD) (Arf-3);arf- WBGENE00000183 small GTPase(PC00095) 3;ortholog

102 binding (GO:0005488)

WBGENE00004450 60S ribosomal protein L36;rpl-36;ortholog ribosomal protein(PC00171) glycosyltransferase(PC00220);ribosomal WBGENE00004449 60S ribosomal protein L35;rpl-35;ortholog protein(PC00111) WBGENE00020915 Protein NOL-5;nol-5;ortholog ribonucleoprotein(PC00171) WBGENE00016493 60S acidic ribosomal protein P2;rpa-2;ortholog ribosomal protein(PC00171) WBGENE00004492 40S ribosomal protein S23;rps-23;ortholog ribosomal protein(PC00171) WBGENE00004434 60S ribosomal protein L22;rpl-22;ortholog ribosomal protein(PC00171) WBGENE00004447 60S ribosomal protein L35a;rpl-33;ortholog ribosomal protein(PC00171) amino acid transporter(PC00227);mitochondrial Phosphate carrier protein, carrier protein(PC00046);transfer/carrier WBGENE00008505 mitochondrial;F01G4.6;ortholog protein(PC00158);ribosomal protein(PC00219);calmodulin(PC00171) WBGENE00004427 60S ribosomal protein L15;rpl-15;ortholog ribosomal protein(PC00171) WBGENE00004412 60S ribosomal protein L10a;rpl-10a;ortholog ribosomal protein(PC00171) WBGENE00004481 40S ribosomal protein S12;rps-12;ortholog ribosomal protein(PC00171) WBGENE00004478 40S ribosomal protein S9;rps-9;ortholog ribosomal protein(PC00171) isomerase(PC00135);chaperone(PC00072);calcium- WBGENE00001430 Peptidyl-prolyl cis-trans isomerase;fkb-5;ortholog binding protein(PC00060) WBGENE00004491 Protein RPS-22, isoform a;rps-22;ortholog ribosomal protein(PC00171) aspartic protease(PC00190);aspartic WBGENE00007605 Protein ASP-10, isoform a;asp-10;ortholog protease(PC00053) transferase(PC00220);dehydrogenase(PC00176);oxi WBGENE00016943 Protein ACDH-1, isoform a;acdh-1;ortholog dase(PC00092) WBGENE00000181 Protein ARD-1;ard-1;ortholog dehydrogenase(PC00176);reductase(PC00092) WBGENE00003962 Protein disulfide-isomerase 1;pdi-1;ortholog aspartic protease(PC00190);aspartic WBGENE00000214 Aspartic protease 1;asp-1;ortholog protease(PC00053) Probable protein disulfide-isomerase WBGENE00015752 A4;C14B9.2;ortholog transketolase(PC00220);dehydrogenase(PC00221);l WBGENE00008506 Protein TKT-1;tkt-1;ortholog yase(PC00176) translation elongation factor(PC00171);translation WBGENE00001168 Elongation factor 1-alpha;eft-3;ortholog initiation factor(PC00031);hydrolase(PC00223);G- protein(PC00222) WBGENE00022170 Protein Y71H2AM.5;CELE_Y71H2AM.5;ortholog oxidase(PC00176) translation elongation factor(PC00171);translation WBGENE00001167 Elongation factor 2;eef-2;ortholog initiation factor(PC00031);hydrolase(PC00223);G- protein(PC00222) WBGENE00000197 Alanine--tRNA ligase, cytoplasmic;aars-2;ortholog RNA binding protein(PC00171) Acidic leucine-rich nuclear phosphoprotein 32-related WBGENE00009367 phosphatase inhibitor(PC00095) protein 1;F33H2.3;ortholog WBGENE00007836 Protein C31C9.2;C31C9.2;ortholog dehydrogenase(PC00176) transporter(PC00227);transfer/carrier WBGENE00002077 Protein IMB-3;imb-3;ortholog protein(PC00219);G-protein modulator(PC00095) Probable 4-aminobutyrate aminotransferase, WBGENE00001794 transaminase(PC00220) mitochondrial;gta-1;ortholog WBGENE00000881 Peptidyl-prolyl cis-trans isomerase 5;cyn-5;ortholog isomerase(PC00135) carbohydrate kinase(PC00220);nucleotide WBGENE00011128 Protein R07H5.8;CELE_R07H5.8;ortholog kinase(PC00137);carbohydrate kinase(PC00065);nucleotide kinase(PC00172) Elongation of very long chain fatty acids protein 6;elo- WBGENE00001244 acyltransferase(PC00220) 6;ortholog carbohydrate phosphatase(PC00181);carbohydrate WBGENE00001404 Fructose-1,6-bisphosphatase;fbp-1;ortholog phosphatase(PC00066) Probable 26S protease regulatory subunit 6B;rpt- WBGENE00004503 hydrolase(PC00121) 3;ortholog WBGENE00002152 Isoleucine--tRNA ligase, cytoplasmic;irs-1;ortholog aminoacyl-tRNA synthetase(PC00142) oxidoreductase(PC00176);dehydratase(PC00144);ep WBGENE00008132 Protein GALE-1, isoform b;gale-1;ortholog imerase/racemase(PC00091) WBGENE00019322 Adenosylhomocysteinase;ahcy-1;ortholog hydrolase(PC00121) WBGENE00012344 Obg-like ATPase 1;tag-210;ortholog G-protein(PC00095) 2-oxoisovalerate dehydrogenase subunit alpha, WBGENE00012713 dehydrogenase(PC00176) mitochondrial;Y39E4A.3;ortholog acetyltransferase(PC00220);acyltransferase(PC0003 Probable enoyl-CoA hydratase, mitochondrial;ech- WBGENE00001155 8);dehydrogenase(PC00042);hydratase(PC00176);li 6;ortholog gase(PC00092);epimerase/racemase(PC00144) aspartic protease(PC00190);aspartic WBGENE00000216 Aspartic protease 3;asp-3;ortholog protease(PC00053) Probable ornithine aminotransferase, WBGENE00015814 transaminase(PC00220) mitochondrial;C16A3.10;ortholog Probable N-acetylgalactosaminyltransferase 8;gly- WBGENE00001633 glycosyltransferase(PC00220) 8;ortholog acyltransferase(PC00220);methyltransferase(PC000 WBGENE00009342 Protein FASN-1;fasn-1;ortholog 42);dehydrogenase(PC00155);ligase(PC00176);ester ase(PC00092) metalloprotease(PC00190);reductase(PC00153);este WBGENE00012158 Protein UCR-2.1, isoform b;ucr-2.1;ortholog rase(PC00176);metalloprotease(PC00198) WBGENE00014095 Glutamate dehydrogenase;gdh-1;ortholog dehydrogenase(PC00176) WBGENE00006914 Protein VHA-5;vha-5;ortholog ATP synthase(PC00227);hydrolase(PC00068)

103 catalytic activity (GO:0003824)

ATP-binding cassette (ABC) WBGENE00012097 Protein ABCF-2;abcf-2;ortholog transporter(PC00227);translation elongation factor(PC00003);hydrolase(PC00171) Probable methylmalonate-semialdehyde dehydrogenase WBGENE00000114 dehydrogenase(PC00176) [acylating], mitochondrial;alh-8;ortholog Probable S-adenosylmethionine synthase 1;sams- WBGENE00008205 nucleotidyltransferase(PC00220) 1;ortholog cysteine protease(PC00190);cysteine WBGENE00000542 Calpain clp-1;clp-1;ortholog protease(PC00081);annexin(PC00121);calmodulin(P C00190) RNA binding protein(PC00171);aminoacyl-tRNA WBGENE00002238 Lysine--tRNA ligase;krs-1;ortholog synthetase(PC00031) WBGENE00019682 Putative serine protease K12H4.7;K12H4.7;ortholog serine protease(PC00190);serine protease(PC00203) Probable hydroxyacid-oxoacid transhydrogenase, WBGENE00012608 dehydrogenase(PC00176) mitochondrial;Y38F1A.6;ortholog acyltransferase(PC00220);G-protein WBGENE00001558 Protein GDI-1, isoform b;gdi-1;ortholog modulator(PC00042) WBGENE00001073 Protein DPY-11;dpy-11;ortholog WBGENE00004754 Protein SEC-23;sec-23;ortholog G-protein modulator(PC00095) cysteine protease(PC00190);cysteine WBGENE00000785 Cathepsin B-like cysteine proteinase 5;cpr-5;ortholog protease(PC00081) 6-phosphogluconate dehydrogenase, WBGENE00012015 dehydrogenase(PC00176) decarboxylating;T25B9.9;ortholog aspartic protease(PC00190);aspartic WBGENE00000215 Protein ASP-2, isoform a;asp-2;ortholog protease(PC00053) WBGENE00003214 Serine hydroxymethyltransferase;mel-32;ortholog methyltransferase(PC00220) ADP-ribosylation factor related (20.5 kD) (Arf-3);arf- WBGENE00000183 small GTPase(PC00095) 3;ortholog metalloprotease(PC00190);deacetylase(PC00153);m WBGENE00003982 Protein PES-9;pes-9;ortholog etalloprotease(PC00121) glycosyltransferase(PC00220);ribosomal WBGENE00004449 60S ribosomal protein L35;rpl-35;ortholog protein(PC00111) WBGENE00004259 Protein PYR-1;pyr-1;ortholog transferase(PC00220);ligase(PC00142) WBGENE00003963 Protein disulfide-isomerase 2;pdi-2;ortholog Probable protein disulfide-isomerase A6;tag- WBGENE00015168 320;ortholog WBGENE00003964 CeERp57;pdi-3;ortholog WBGENE00044294 Protein C01B10.11;C01B10.11;ortholog acetyltransferase(PC00220) transferase(PC00220);dehydrogenase(PC00176);dec WBGENE00020831 Acetolactate synthase-like protein;T26C12.1;ortholog arboxylase(PC00092) isomerase(PC00135);chaperone(PC00072);calcium- WBGENE00001430 Peptidyl-prolyl cis-trans isomerase;fkb-5;ortholog binding protein(PC00060) WBGENE00004473 40S ribosomal protein S4;rps-4;ortholog ribosomal protein(PC00171) WBGENE00004474 40S ribosomal protein S5;rps-5;ortholog ribosomal protein(PC00171) WBGENE00004488 40S ribosomal protein S19;rps-19;ortholog ribosomal protein(PC00171) WBGENE00006537 Tubulin beta-2 chain;tbb-2;ortholog tubulin(PC00085) WBGENE00004423 Protein RPL-11.2;rpl-11.2;ortholog ribosomal protein(PC00171) WBGENE00004420 60S ribosomal protein L9;rpl-9;ortholog ribosomal protein(PC00171) WBGENE00004408 60S acidic ribosomal protein P0;rpa-0;ortholog ribosomal protein(PC00171) WBGENE00004414 60S ribosomal protein L3;rpl-3;ortholog ribosomal protein(PC00171) WBGENE00004487 Protein RPS-18;rps-18;ortholog WBGENE00004439 60S ribosomal protein L23a 2;rpl-25.2;ortholog ribosomal protein(PC00171) WBGENE00004471 40S ribosomal protein S2;rps-2;ortholog ribosomal protein(PC00171) WBGENE00004416 60S ribosomal protein L5;rpl-5;ortholog ribosomal protein(PC00171) WBGENE00004431 60S ribosomal protein L19;rpl-19;ortholog ribosomal protein(PC00171) WBGENE00004479 Protein RPS-10;rps-10;ortholog ribosomal protein(PC00171) WBGENE00004418 60S ribosomal protein L7;rpl-7;ortholog ribosomal protein(PC00171) WBGENE00004413 60S ribosomal protein L8;rpl-8;ortholog ribosomal protein(PC00171) WBGENE00004425 60S ribosomal protein L13;rpl-13;ortholog ribosomal protein(PC00171) WBGENE00004469 40S ribosomal protein SA;rps-0;ortholog WBGENE00005016 Cuticle collagen sqt-1;sqt-1;ortholog extracellular matrix structural protein(PC00102) WBGENE00004477 40S ribosomal protein S8;rps-8;ortholog WBGENE00004424 60S ribosomal protein L12;rpl-12;ortholog ribosomal protein(PC00171) WBGENE00004470 40S ribosomal protein S3a;rps-1;ortholog ribosomal protein(PC00171) WBGENE00021350 Protein Y37E3.8, isoform a;CELE_Y37E3.8;ortholog ribosomal protein(PC00171) WBGENE00004452 60S ribosomal protein L38;rpl-38;ortholog ribosomal protein(PC00171) WBGENE00004435 60S ribosomal protein L23;rpl-23;ortholog ribosomal protein(PC00171) amino acid transporter(PC00227);mitochondrial carrier protein(PC00046);transfer/carrier WBGENE00006439 Protein ANT-1.1, isoform a;ant-1.1;ortholog protein(PC00158);ribosomal structural protein(PC00219);calmodulin(PC00171) molecule WBGENE00003829 NUD-1;nud-1;ortholog microtubule family cytoskeletal protein(PC00085) activity WBGENE00004438 60S ribosomal protein L23a 1;rpl-25.1;ortholog ribosomal protein(PC00171) Translationally-controlled tumor protein homolog;tct- (GO:0005198) WBGENE00009122 non-motor microtubule binding protein(PC00085) 1;ortholog WBGENE00004428 60S ribosomal protein L13a;rpl-16;ortholog ribosomal protein(PC00171) WBGENE00004417 60S ribosomal protein L6;rpl-6;ortholog ribosomal protein(PC00171)

104 structural molecule activity (GO:0005198)

ribonucleoprotein(PC00171);ribosomal WBGENE00004485 40S ribosomal protein S16;rps-16;ortholog protein(PC00031) WBGENE00006536 Protein TBB-1;tbb-1;ortholog tubulin(PC00085) WBGENE00004419 60S ribosomal protein L7a;rpl-7A;ortholog ribosomal protein(PC00171) WBGENE00004480 Protein RPS-11;rps-11;ortholog ribosomal protein(PC00171) WBGENE00004436 60S ribosomal protein L24;rpl-24.1;ortholog ribosomal protein(PC00171) WBGENE00004483 40S ribosomal protein S14;rps-14;ortholog ribosomal protein(PC00171) WBGENE00004450 60S ribosomal protein L36;rpl-36;ortholog ribosomal protein(PC00171) glycosyltransferase(PC00220);ribosomal WBGENE00004449 60S ribosomal protein L35;rpl-35;ortholog protein(PC00111) WBGENE00016493 60S acidic ribosomal protein P2;rpa-2;ortholog ribosomal protein(PC00171) WBGENE00004492 40S ribosomal protein S23;rps-23;ortholog ribosomal protein(PC00171) WBGENE00004434 60S ribosomal protein L22;rpl-22;ortholog ribosomal protein(PC00171) WBGENE00004447 60S ribosomal protein L35a;rpl-33;ortholog ribosomal protein(PC00171) amino acid transporter(PC00227);mitochondrial Phosphate carrier protein, carrier protein(PC00046);transfer/carrier WBGENE00008505 mitochondrial;F01G4.6;ortholog protein(PC00158);ribosomal protein(PC00219);calmodulin(PC00171) WBGENE00004490 40S ribosomal protein S21;rps-21;ortholog ribosomal protein(PC00171) WBGENE00004427 60S ribosomal protein L15;rpl-15;ortholog ribosomal protein(PC00171) WBGENE00004412 60S ribosomal protein L10a;rpl-10a;ortholog ribosomal protein(PC00171) WBGENE00004481 40S ribosomal protein S12;rps-12;ortholog ribosomal protein(PC00171) WBGENE00004478 40S ribosomal protein S9;rps-9;ortholog ribosomal protein(PC00171) WBGENE00004484 40S ribosomal protein S15;rps-15;ortholog ribosomal protein(PC00171) WBGENE00004491 Protein RPS-22, isoform a;rps-22;ortholog ribosomal protein(PC00171) Probable elongation factor 1-beta/1-delta 1;eef- WBGENE00018846 translation elongation factor(PC00171) 1B.1;ortholog translation elongation factor(PC00171);translation WBGENE00001168 Elongation factor 1-alpha;eft-3;ortholog initiation factor(PC00031);hydrolase(PC00223);G- protein(PC00222) translation elongation factor(PC00171);translation WBGENE00001167 Elongation factor 2;eef-2;ortholog initiation factor(PC00031);hydrolase(PC00223);G- translation protein(PC00222) regulator Eukaryotic translation initiation factor 3 subunit D;eif- WBGENE00001227 translation initiation factor(PC00171) activity 3.D;ortholog (GO:0045182) WBGENE00011730 Protein DRR-2;drr-2;ortholog translation initiation factor(PC00171) ATP-binding cassette (ABC) WBGENE00012097 Protein ABCF-2;abcf-2;ortholog transporter(PC00227);translation elongation factor(PC00003);hydrolase(PC00171) Eukaryotic translation initiation factor 5A-2;iff- WBGENE00002065 translation initiation factor(PC00171) 2;ortholog Probable elongation factor 1-beta/1-delta 2;eef- WBGENE00012768 translation elongation factor(PC00171) 1B.2;ortholog transporter(PC00227);transfer/carrier WBGENE00002077 Protein IMB-3;imb-3;ortholog protein(PC00219);G-protein modulator(PC00095) WBGENE00006914 Protein VHA-5;vha-5;ortholog ATP synthase(PC00227);hydrolase(PC00068) ATP-binding cassette (ABC) WBGENE00012097 Protein ABCF-2;abcf-2;ortholog transporter(PC00227);translation elongation transporter factor(PC00003);hydrolase(PC00171) amino acid transporter(PC00227);mitochondrial activity carrier protein(PC00046);transfer/carrier (GO:0005215) WBGENE00006439 Protein ANT-1.1, isoform a;ant-1.1;ortholog protein(PC00158);ribosomal protein(PC00219);calmodulin(PC00171) amino acid transporter(PC00227);mitochondrial Phosphate carrier protein, carrier protein(PC00046);transfer/carrier WBGENE00008505 mitochondrial;F01G4.6;ortholog protein(PC00158);ribosomal protein(PC00219);calmodulin(PC00171) Acidic leucine-rich nuclear phosphoprotein 32-related WBGENE00009367 phosphatase inhibitor(PC00095) enzyme protein 1;F33H2.3;ortholog transporter(PC00227);transfer/carrier regulator WBGENE00002077 Protein IMB-3;imb-3;ortholog protein(PC00219);G-protein modulator(PC00095) activity acyltransferase(PC00220);G-protein (GO:0030234) WBGENE00001558 Protein GDI-1, isoform b;gdi-1;ortholog modulator(PC00042) WBGENE00004754 Protein SEC-23;sec-23;ortholog G-protein modulator(PC00095) nucleic acid transcription cofactor(PC00218);nucleic acid WBGENE00006626 Protein TSN-1;tsn-1;ortholog binding binding(PC00217) transcription WBGENE00002045 Transcription factor BTF3 homolog;icd-1;ortholog transcription factor(PC00218) protein binding transcription transcription cofactor(PC00218);nucleic acid WBGENE00006626 Protein TSN-1;tsn-1;ortholog factor activity binding(PC00217) (GO:0000988)

105 Table-2.9: GO-BP terms for the proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm.

GO terms Wornbase Gene ID Protein name Protein class WBGENE00004473 40S ribosomal protein S4;rps-4;ortholog ribosomal protein(PC00171) aspartic protease(PC00190);aspartic WBGENE00007605 Protein ASP-10, isoform a;asp-10;ortholog protease(PC00053) transferase(PC00220);dehydrogenase(PC00176);oxida WBGENE00016943 Protein ACDH-1, isoform a;acdh-1;ortholog se(PC00092) WBGENE00004474 40S ribosomal protein S5;rps-5;ortholog ribosomal protein(PC00171) WBGENE00002005 Heat shock 70 kDa protein A;hsp-1;ortholog Hsp70 family chaperone(PC00072) WBGENE00004488 40S ribosomal protein S19;rps-19;ortholog ribosomal protein(PC00171) Probable elongation factor 1-beta/1-delta 1;eef- WBGENE00018846 translation elongation factor(PC00171) 1B.1;ortholog WBGENE00000181 Protein ARD-1;ard-1;ortholog dehydrogenase(PC00176);reductase(PC00092) Eukaryotic translation initiation factor 3 subunit B;eif- WBGENE00001225 translation initiation factor(PC00171) 3.B;ortholog WBGENE00003962 Protein disulfide-isomerase 1;pdi-1;ortholog WBGENE00004423 Protein RPL-11.2;rpl-11.2;ortholog ribosomal protein(PC00171) WBGENE00004420 60S ribosomal protein L9;rpl-9;ortholog ribosomal protein(PC00171) Eukaryotic translation initiation factor 3 subunit E;eif- WBGENE00001228 translation initiation factor(PC00171) 3.E;ortholog aspartic protease(PC00190);aspartic WBGENE00000214 Aspartic protease 1;asp-1;ortholog protease(PC00053) Probable protein disulfide-isomerase WBGENE00015752 A4;C14B9.2;ortholog transketolase(PC00220);dehydrogenase(PC00221);lyas WBGENE00008506 Protein TKT-1;tkt-1;ortholog e(PC00176) WBGENE00004408 60S acidic ribosomal protein P0;rpa-0;ortholog ribosomal protein(PC00171) WBGENE00004414 60S ribosomal protein L3;rpl-3;ortholog ribosomal protein(PC00171) translation elongation factor(PC00171);translation WBGENE00001168 Elongation factor 1-alpha;eft-3;ortholog initiation factor(PC00031);hydrolase(PC00223);G- protein(PC00222) WBGENE00022170 Protein Y71H2AM.5;CELE_Y71H2AM.5;ortholog oxidase(PC00176) translation elongation factor(PC00171);translation WBGENE00001167 Elongation factor 2;eef-2;ortholog initiation factor(PC00031);hydrolase(PC00223);G- protein(PC00222) WBGENE00000802 Calreticulin;crt-1;ortholog calcium-binding protein(PC00060) WBGENE00000197 Alanine--tRNA ligase, cytoplasmic;aars-2;ortholog RNA binding protein(PC00171) WBGENE00002008 Heat shock 70 kDa protein D;hsp-4;ortholog Hsp70 family chaperone(PC00072) WBGENE00004487 Protein RPS-18;rps-18;ortholog WBGENE00004439 60S ribosomal protein L23a 2;rpl-25.2;ortholog ribosomal protein(PC00171) Acidic leucine-rich nuclear phosphoprotein 32-related WBGENE00009367 phosphatase inhibitor(PC00095) protein 1;F33H2.3;ortholog WBGENE00007836 Protein C31C9.2;C31C9.2;ortholog dehydrogenase(PC00176) transporter(PC00227);transfer/carrier WBGENE00002077 Protein IMB-3;imb-3;ortholog protein(PC00219);G-protein modulator(PC00095) Probable 4-aminobutyrate aminotransferase, WBGENE00001794 transaminase(PC00220) mitochondrial;gta-1;ortholog WBGENE00000881 Peptidyl-prolyl cis-trans isomerase 5;cyn-5;ortholog isomerase(PC00135) WBGENE00004471 40S ribosomal protein S2;rps-2;ortholog ribosomal protein(PC00171) WBGENE00000381 T-complex protein 1 subunit zeta;cct-6;ortholog chaperonin(PC00072) carbohydrate kinase(PC00220);nucleotide WBGENE00011128 Protein R07H5.8;CELE_R07H5.8;ortholog kinase(PC00137);carbohydrate kinase(PC00065);nucleotide kinase(PC00172) WBGENE00004416 60S ribosomal protein L5;rpl-5;ortholog ribosomal protein(PC00171) WBGENE00004431 60S ribosomal protein L19;rpl-19;ortholog ribosomal protein(PC00171) Elongation of very long chain fatty acids protein 6;elo- WBGENE00001244 acyltransferase(PC00220) 6;ortholog carbohydrate phosphatase(PC00181);carbohydrate WBGENE00001404 Fructose-1,6-bisphosphatase;fbp-1;ortholog phosphatase(PC00066) WBGENE00004479 Protein RPS-10;rps-10;ortholog ribosomal protein(PC00171) Probable 26S protease regulatory subunit 6B;rpt- WBGENE00004503 hydrolase(PC00121) 3;ortholog WBGENE00002152 Isoleucine--tRNA ligase, cytoplasmic;irs-1;ortholog aminoacyl-tRNA synthetase(PC00142) oxidoreductase(PC00176);dehydratase(PC00144);epim WBGENE00008132 Protein GALE-1, isoform b;gale-1;ortholog erase/racemase(PC00091) WBGENE00019322 Adenosylhomocysteinase;ahcy-1;ortholog hydrolase(PC00121) WBGENE00004418 60S ribosomal protein L7;rpl-7;ortholog ribosomal protein(PC00171) Eukaryotic translation initiation factor 3 subunit D;eif- WBGENE00001227 translation initiation factor(PC00171) 3.D;ortholog WBGENE00012344 Obg-like ATPase 1;tag-210;ortholog G-protein(PC00095) WBGENE00004413 60S ribosomal protein L8;rpl-8;ortholog ribosomal protein(PC00171) 2-oxoisovalerate dehydrogenase subunit alpha, WBGENE00012713 dehydrogenase(PC00176) mitochondrial;Y39E4A.3;ortholog WBGENE00004476 40S ribosomal protein S7;rps-7;ortholog ribosomal protein(PC00171) acetyltransferase(PC00220);acyltransferase(PC00038); Probable enoyl-CoA hydratase, mitochondrial;ech- WBGENE00001155 dehydrogenase(PC00042);hydratase(PC00176);ligase( 6;ortholog PC00092);epimerase/racemase(PC00144) WBGENE00004425 60S ribosomal protein L13;rpl-13;ortholog ribosomal protein(PC00171) WBGENE00004469 40S ribosomal protein SA;rps-0;ortholog

106 WBGENE00004477 40S ribosomal protein S8;rps-8;ortholog transcription cofactor(PC00218);nucleic acid WBGENE00006626 Protein TSN-1;tsn-1;ortholog binding(PC00217) WBGENE00011730 Protein DRR-2;drr-2;ortholog translation initiation factor(PC00171) aspartic protease(PC00190);aspartic WBGENE00000216 Aspartic protease 3;asp-3;ortholog protease(PC00053) Probable ornithine aminotransferase, WBGENE00015814 transaminase(PC00220) mitochondrial;C16A3.10;ortholog Probable N-acetylgalactosaminyltransferase 8;gly- WBGENE00001633 glycosyltransferase(PC00220) 8;ortholog acyltransferase(PC00220);methyltransferase(PC00042) WBGENE00009342 Protein FASN-1;fasn-1;ortholog ;dehydrogenase(PC00155);ligase(PC00176);esterase(P C00092) WBGENE00004424 60S ribosomal protein L12;rpl-12;ortholog ribosomal protein(PC00171) 26S proteasome non-ATPase regulatory subunit 6;rpn- WBGENE00004463 metabolic 7;ortholog process WBGENE00004470 40S ribosomal protein S3a;rps-1;ortholog ribosomal protein(PC00171) (GO:0008152) metalloprotease(PC00190);reductase(PC00153);esteras WBGENE00012158 Protein UCR-2.1, isoform b;ucr-2.1;ortholog e(PC00176);metalloprotease(PC00198) WBGENE00014095 Glutamate dehydrogenase;gdh-1;ortholog dehydrogenase(PC00176) WBGENE00006914 Protein VHA-5;vha-5;ortholog ATP synthase(PC00227);hydrolase(PC00068) ATP-binding cassette (ABC) WBGENE00012097 Protein ABCF-2;abcf-2;ortholog transporter(PC00227);translation elongation factor(PC00003);hydrolase(PC00171) WBGENE00021350 Protein Y37E3.8, isoform a;CELE_Y37E3.8;ortholog ribosomal protein(PC00171) Probable methylmalonate-semialdehyde dehydrogenase WBGENE00000114 dehydrogenase(PC00176) [acylating], mitochondrial;alh-8;ortholog WBGENE00000380 T-complex protein 1 subunit epsilon;cct-5;ortholog chaperonin(PC00072) WBGENE00006889 Probable prefoldin subunit 3;pfd-3;ortholog chaperone(PC00072) WBGENE00004452 60S ribosomal protein L38;rpl-38;ortholog ribosomal protein(PC00171) WBGENE00004435 60S ribosomal protein L23;rpl-23;ortholog ribosomal protein(PC00171) Probable S-adenosylmethionine synthase 1;sams- WBGENE00008205 nucleotidyltransferase(PC00220) 1;ortholog cysteine protease(PC00190);cysteine WBGENE00000542 Calpain clp-1;clp-1;ortholog protease(PC00081);annexin(PC00121);calmodulin(PC 00190) Eukaryotic translation initiation factor 5A-2;iff- WBGENE00002065 translation initiation factor(PC00171) 2;ortholog WBGENE00011480 Protein ENPL-1, isoform a;enpl-1;ortholog Hsp90 family chaperone(PC00072) WBGENE00002045 Transcription factor BTF3 homolog;icd-1;ortholog transcription factor(PC00218) RNA binding protein(PC00171);aminoacyl-tRNA WBGENE00002238 Lysine--tRNA ligase;krs-1;ortholog synthetase(PC00031) amino acid transporter(PC00227);mitochondrial carrier protein(PC00046);transfer/carrier WBGENE00006439 Protein ANT-1.1, isoform a;ant-1.1;ortholog protein(PC00158);ribosomal protein(PC00219);calmodulin(PC00171) WBGENE00019682 Putative serine protease K12H4.7;K12H4.7;ortholog serine protease(PC00190);serine protease(PC00203) Probable hydroxyacid-oxoacid transhydrogenase, WBGENE00012608 dehydrogenase(PC00176) mitochondrial;Y38F1A.6;ortholog WBGENE00004438 60S ribosomal protein L23a 1;rpl-25.1;ortholog ribosomal protein(PC00171) acyltransferase(PC00220);G-protein WBGENE00001558 Protein GDI-1, isoform b;gdi-1;ortholog modulator(PC00042) Probable elongation factor 1-beta/1-delta 2;eef- WBGENE00012768 translation elongation factor(PC00171) 1B.2;ortholog WBGENE00001073 Protein DPY-11;dpy-11;ortholog WBGENE00004428 60S ribosomal protein L13a;rpl-16;ortholog ribosomal protein(PC00171) WBGENE00004417 60S ribosomal protein L6;rpl-6;ortholog ribosomal protein(PC00171) WBGENE00004754 Protein SEC-23;sec-23;ortholog G-protein modulator(PC00095) WBGENE00001775 Protein GST-27;gst-27;ortholog ribonucleoprotein(PC00171);ribosomal WBGENE00004485 40S ribosomal protein S16;rps-16;ortholog protein(PC00031) WBGENE00016250 Uncharacterized protein C30C11.4;C30C11.4;ortholog Hsp70 family chaperone(PC00072) cysteine protease(PC00190);cysteine WBGENE00000785 Cathepsin B-like cysteine proteinase 5;cpr-5;ortholog protease(PC00081) WBGENE00004419 60S ribosomal protein L7a;rpl-7A;ortholog ribosomal protein(PC00171) WBGENE00004480 Protein RPS-11;rps-11;ortholog ribosomal protein(PC00171) 6-phosphogluconate dehydrogenase, WBGENE00012015 dehydrogenase(PC00176) decarboxylating;T25B9.9;ortholog aspartic protease(PC00190);aspartic WBGENE00000215 Protein ASP-2, isoform a;asp-2;ortholog protease(PC00053) WBGENE00004436 60S ribosomal protein L24;rpl-24.1;ortholog ribosomal protein(PC00171) WBGENE00004483 40S ribosomal protein S14;rps-14;ortholog ribosomal protein(PC00171) WBGENE00003214 Serine hydroxymethyltransferase;mel-32;ortholog methyltransferase(PC00220) WBGENE00000915 Heat shock protein 90;daf-21;ortholog Hsp90 family chaperone(PC00072) ADP-ribosylation factor related (20.5 kD) (Arf-3);arf- WBGENE00000183 small GTPase(PC00095) 3;ortholog metalloprotease(PC00190);deacetylase(PC00153);meta WBGENE00003982 Protein PES-9;pes-9;ortholog lloprotease(PC00121) WBGENE00004450 60S ribosomal protein L36;rpl-36;ortholog ribosomal protein(PC00171)

107 glycosyltransferase(PC00220);ribosomal WBGENE00004449 60S ribosomal protein L35;rpl-35;ortholog protein(PC00111) WBGENE00020915 Protein NOL-5;nol-5;ortholog ribonucleoprotein(PC00171) WBGENE00016493 60S acidic ribosomal protein P2;rpa-2;ortholog ribosomal protein(PC00171) WBGENE00004492 40S ribosomal protein S23;rps-23;ortholog ribosomal protein(PC00171) WBGENE00004259 Protein PYR-1;pyr-1;ortholog transferase(PC00220);ligase(PC00142) WBGENE00004434 60S ribosomal protein L22;rpl-22;ortholog ribosomal protein(PC00171) WBGENE00003963 Protein disulfide-isomerase 2;pdi-2;ortholog WBGENE00004447 60S ribosomal protein L35a;rpl-33;ortholog ribosomal protein(PC00171) amino acid transporter(PC00227);mitochondrial carrier Phosphate carrier protein, protein(PC00046);transfer/carrier WBGENE00008505 mitochondrial;F01G4.6;ortholog protein(PC00158);ribosomal protein(PC00219);calmodulin(PC00171) Probable protein disulfide-isomerase A6;tag- WBGENE00015168 320;ortholog WBGENE00003964 CeERp57;pdi-3;ortholog WBGENE00004490 40S ribosomal protein S21;rps-21;ortholog ribosomal protein(PC00171) WBGENE00004427 60S ribosomal protein L15;rpl-15;ortholog ribosomal protein(PC00171) WBGENE00001774 Protein GST-26;gst-26;ortholog WBGENE00004475 40S ribosomal protein S6;rps-6;ortholog WBGENE00004412 60S ribosomal protein L10a;rpl-10a;ortholog ribosomal protein(PC00171) WBGENE00044294 Protein C01B10.11;C01B10.11;ortholog acetyltransferase(PC00220) WBGENE00004481 40S ribosomal protein S12;rps-12;ortholog ribosomal protein(PC00171) WBGENE00004478 40S ribosomal protein S9;rps-9;ortholog ribosomal protein(PC00171) transferase(PC00220);dehydrogenase(PC00176);decar WBGENE00020831 Acetolactate synthase-like protein;T26C12.1;ortholog boxylase(PC00092) isomerase(PC00135);chaperone(PC00072);calcium- WBGENE00001430 Peptidyl-prolyl cis-trans isomerase;fkb-5;ortholog binding protein(PC00060) WBGENE00002007 Heat shock 70 kDa protein C;hsp-3;ortholog Hsp70 family chaperone(PC00072) WBGENE00004491 Protein RPS-22, isoform a;rps-22;ortholog ribosomal protein(PC00171) WBGENE00004266 Protein RAB-1;rab-1;ortholog WBGENE00006537 Tubulin beta-2 chain;tbb-2;ortholog tubulin(PC00085) Eukaryotic translation initiation factor 3 subunit B;eif- WBGENE00001225 translation initiation factor(PC00171) 3.B;ortholog WBGENE00003962 Protein disulfide-isomerase 1;pdi-1;ortholog Eukaryotic translation initiation factor 3 subunit E;eif- WBGENE00001228 translation initiation factor(PC00171) 3.E;ortholog Probable protein disulfide-isomerase WBGENE00015752 A4;C14B9.2;ortholog WBGENE00004487 Protein RPS-18;rps-18;ortholog WBGENE00000881 Peptidyl-prolyl cis-trans isomerase 5;cyn-5;ortholog isomerase(PC00135) WBGENE00004471 40S ribosomal protein S2;rps-2;ortholog ribosomal protein(PC00171) WBGENE00002152 Isoleucine--tRNA ligase, cytoplasmic;irs-1;ortholog aminoacyl-tRNA synthetase(PC00142) WBGENE00019322 Adenosylhomocysteinase;ahcy-1;ortholog hydrolase(PC00121) WBGENE00004476 40S ribosomal protein S7;rps-7;ortholog ribosomal protein(PC00171) WBGENE00004469 40S ribosomal protein SA;rps-0;ortholog WBGENE00005016 Cuticle collagen sqt-1;sqt-1;ortholog extracellular matrix structural protein(PC00102) WBGENE00004477 40S ribosomal protein S8;rps-8;ortholog 26S proteasome non-ATPase regulatory subunit 6;rpn- WBGENE00004463 7;ortholog WBGENE00011629 Protein T08G11.1, isoform a;CELE_T08G11.1;ortholog WBGENE00004470 40S ribosomal protein S3a;rps-1;ortholog ribosomal protein(PC00171) cellular process WBGENE00006914 Protein VHA-5;vha-5;ortholog ATP synthase(PC00227);hydrolase(PC00068) (GO:0009987) WBGENE00000822 Calsequestrin;csq-1;ortholog calsequestrin(PC00060) WBGENE00019760 Calumenin-like protein;calu-1;ortholog calmodulin(PC00060) WBGENE00003829 NUD-1;nud-1;ortholog microtubule family cytoskeletal protein(PC00085) acyltransferase(PC00220);G-protein WBGENE00001558 Protein GDI-1, isoform b;gdi-1;ortholog modulator(PC00042) WBGENE00001073 Protein DPY-11;dpy-11;ortholog WBGENE00004754 Protein SEC-23;sec-23;ortholog G-protein modulator(PC00095) WBGENE00006536 Protein TBB-1;tbb-1;ortholog tubulin(PC00085) WBGENE00004480 Protein RPS-11;rps-11;ortholog ribosomal protein(PC00171) ADP-ribosylation factor related (20.5 kD) (Arf-3);arf- WBGENE00000183 small GTPase(PC00095) 3;ortholog WBGENE00020915 Protein NOL-5;nol-5;ortholog ribonucleoprotein(PC00171) WBGENE00004259 Protein PYR-1;pyr-1;ortholog transferase(PC00220);ligase(PC00142) WBGENE00003963 Protein disulfide-isomerase 2;pdi-2;ortholog Probable protein disulfide-isomerase A6;tag- WBGENE00015168 320;ortholog WBGENE00003964 CeERp57;pdi-3;ortholog WBGENE00004490 40S ribosomal protein S21;rps-21;ortholog ribosomal protein(PC00171) WBGENE00004475 40S ribosomal protein S6;rps-6;ortholog WBGENE00004478 40S ribosomal protein S9;rps-9;ortholog ribosomal protein(PC00171) WBGENE00004484 40S ribosomal protein S15;rps-15;ortholog ribosomal protein(PC00171) isomerase(PC00135);chaperone(PC00072);calcium- WBGENE00001430 Peptidyl-prolyl cis-trans isomerase;fkb-5;ortholog binding protein(PC00060) WBGENE00002005 Heat shock 70 kDa protein A;hsp-1;ortholog Hsp70 family chaperone(PC00072) WBGENE00006537 Tubulin beta-2 chain;tbb-2;ortholog tubulin(PC00085) WBGENE00002008 Heat shock 70 kDa protein D;hsp-4;ortholog Hsp70 family chaperone(PC00072)

cellular component organization or 108 biogenesis (GO:0071840) WBGENE00004487 Protein RPS-18;rps-18;ortholog WBGENE00000381 T-complex protein 1 subunit zeta;cct-6;ortholog chaperonin(PC00072) WBGENE00004476 40S ribosomal protein S7;rps-7;ortholog ribosomal protein(PC00171) WBGENE00004469 40S ribosomal protein SA;rps-0;ortholog cellular WBGENE00005016 Cuticle collagen sqt-1;sqt-1;ortholog extracellular matrix structural protein(PC00102) component WBGENE00004477 40S ribosomal protein S8;rps-8;ortholog organization or WBGENE00006914 Protein VHA-5;vha-5;ortholog ATP synthase(PC00227);hydrolase(PC00068) biogenesis WBGENE00000380 T-complex protein 1 subunit epsilon;cct-5;ortholog chaperonin(PC00072) (GO:0071840) WBGENE00006889 Probable prefoldin subunit 3;pfd-3;ortholog chaperone(PC00072) WBGENE00006536 Protein TBB-1;tbb-1;ortholog tubulin(PC00085) WBGENE00016250 Uncharacterized protein C30C11.4;C30C11.4;ortholog Hsp70 family chaperone(PC00072) WBGENE00020915 Protein NOL-5;nol-5;ortholog ribonucleoprotein(PC00171) WBGENE00004490 40S ribosomal protein S21;rps-21;ortholog ribosomal protein(PC00171) WBGENE00004475 40S ribosomal protein S6;rps-6;ortholog WBGENE00004484 40S ribosomal protein S15;rps-15;ortholog ribosomal protein(PC00171) WBGENE00002007 Heat shock 70 kDa protein C;hsp-3;ortholog Hsp70 family chaperone(PC00072) Probable elongation factor 1-beta/1-delta 1;eef- WBGENE00018846 translation elongation factor(PC00171) 1B.1;ortholog WBGENE00003962 Protein disulfide-isomerase 1;pdi-1;ortholog translation elongation factor(PC00171);translation WBGENE00001168 Elongation factor 1-alpha;eft-3;ortholog initiation factor(PC00031);hydrolase(PC00223);G- protein(PC00222) translation elongation factor(PC00171);translation WBGENE00001167 Elongation factor 2;eef-2;ortholog initiation factor(PC00031);hydrolase(PC00223);G- protein(PC00222) Acidic leucine-rich nuclear phosphoprotein 32-related WBGENE00009367 phosphatase inhibitor(PC00095) protein 1;F33H2.3;ortholog transporter(PC00227);transfer/carrier WBGENE00002077 Protein IMB-3;imb-3;ortholog protein(PC00219);G-protein modulator(PC00095) Eukaryotic translation initiation factor 3 subunit D;eif- WBGENE00001227 translation initiation factor(PC00171) 3.D;ortholog biological regulation WBGENE00011730 Protein DRR-2;drr-2;ortholog translation initiation factor(PC00171) (GO:0065007) WBGENE00011629 Protein T08G11.1, isoform a;CELE_T08G11.1;ortholog WBGENE00006914 Protein VHA-5;vha-5;ortholog ATP synthase(PC00227);hydrolase(PC00068) ATP-binding cassette (ABC) WBGENE00012097 Protein ABCF-2;abcf-2;ortholog transporter(PC00227);translation elongation factor(PC00003);hydrolase(PC00171) Eukaryotic translation initiation factor 5A-2;iff- WBGENE00002065 translation initiation factor(PC00171) 2;ortholog WBGENE00002045 Transcription factor BTF3 homolog;icd-1;ortholog transcription factor(PC00218) acyltransferase(PC00220);G-protein WBGENE00001558 Protein GDI-1, isoform b;gdi-1;ortholog modulator(PC00042) Probable elongation factor 1-beta/1-delta 2;eef- WBGENE00012768 translation elongation factor(PC00171) 1B.2;ortholog WBGENE00004754 Protein SEC-23;sec-23;ortholog G-protein modulator(PC00095) WBGENE00004478 40S ribosomal protein S9;rps-9;ortholog ribosomal protein(PC00171) WBGENE00004266 Protein RAB-1;rab-1;ortholog WBGENE00006537 Tubulin beta-2 chain;tbb-2;ortholog tubulin(PC00085) transporter(PC00227);transfer/carrier WBGENE00002077 Protein IMB-3;imb-3;ortholog protein(PC00219);G-protein modulator(PC00095) WBGENE00004469 40S ribosomal protein SA;rps-0;ortholog WBGENE00011629 Protein T08G11.1, isoform a;CELE_T08G11.1;ortholog WBGENE00006914 Protein VHA-5;vha-5;ortholog ATP synthase(PC00227);hydrolase(PC00068) ATP-binding cassette (ABC) WBGENE00012097 Protein ABCF-2;abcf-2;ortholog transporter(PC00227);translation elongation factor(PC00003);hydrolase(PC00171) amino acid transporter(PC00227);mitochondrial carrier localization protein(PC00046);transfer/carrier WBGENE00006439 Protein ANT-1.1, isoform a;ant-1.1;ortholog (GO:0051179) protein(PC00158);ribosomal protein(PC00219);calmodulin(PC00171) WBGENE00004755 Protein SEC-24.1;sec-24.1;ortholog vesicle coat protein(PC00150) acyltransferase(PC00220);G-protein WBGENE00001558 Protein GDI-1, isoform b;gdi-1;ortholog modulator(PC00042) WBGENE00004754 Protein SEC-23;sec-23;ortholog G-protein modulator(PC00095) WBGENE00006536 Protein TBB-1;tbb-1;ortholog tubulin(PC00085) ADP-ribosylation factor related (20.5 kD) (Arf-3);arf- WBGENE00000183 small GTPase(PC00095) 3;ortholog amino acid transporter(PC00227);mitochondrial carrier Phosphate carrier protein, protein(PC00046);transfer/carrier WBGENE00008505 mitochondrial;F01G4.6;ortholog protein(PC00158);ribosomal protein(PC00219);calmodulin(PC00171) WBGENE00002005 Heat shock 70 kDa protein A;hsp-1;ortholog Hsp70 family chaperone(PC00072) WBGENE00003962 Protein disulfide-isomerase 1;pdi-1;ortholog Probable protein disulfide-isomerase WBGENE00015752 A4;C14B9.2;ortholog WBGENE00002008 Heat shock 70 kDa protein D;hsp-4;ortholog Hsp70 family chaperone(PC00072) WBGENE00011480 Protein ENPL-1, isoform a;enpl-1;ortholog Hsp90 family chaperone(PC00072) response to stimulus (GO:0050896)

109 response to WBGENE00001073 Protein DPY-11;dpy-11;ortholog stimulus WBGENE00016250 Uncharacterized protein C30C11.4;C30C11.4;ortholog Hsp70 family chaperone(PC00072) (GO:0050896) WBGENE00000915 Heat shock protein 90;daf-21;ortholog Hsp90 family chaperone(PC00072) WBGENE00003963 Protein disulfide-isomerase 2;pdi-2;ortholog Probable protein disulfide-isomerase A6;tag- WBGENE00015168 320;ortholog WBGENE00003964 CeERp57;pdi-3;ortholog WBGENE00002007 Heat shock 70 kDa protein C;hsp-3;ortholog Hsp70 family chaperone(PC00072) WBGENE00006537 Tubulin beta-2 chain;tbb-2;ortholog tubulin(PC00085) developmental WBGENE00003962 Protein disulfide-isomerase 1;pdi-1;ortholog process WBGENE00003902 Protein PAB-1, isoform a;pab-1;ortholog (GO:0032502) WBGENE00005016 Cuticle collagen sqt-1;sqt-1;ortholog extracellular matrix structural protein(PC00102) WBGENE00006536 Protein TBB-1;tbb-1;ortholog tubulin(PC00085) multicellular WBGENE00001775 Protein GST-27;gst-27;ortholog organismal WBGENE00001774 Protein GST-26;gst-26;ortholog apoptotic process WBGENE00003962 Protein disulfide-isomerase 1;pdi-1;ortholog (GO:0006915) biological adhesion WBGENE00005016 Cuticle collagen sqt-1;sqt-1;ortholog extracellular matrix structural protein(PC00102) (GO:0022610) immune system process WBGENE00002008 Heat shock 70 kDa protein D;hsp-4;ortholog Hsp70 family chaperone(PC00072) (GO:0002376)

110 a

111 b

112

Figure 2.11: Pathway association for 1.5-fold down regulated proteins in dauer. Proteins mapped to (a) the REACTOME pathways (b) KEGG pathways. (a) REACTOME pathways graph showing mapped proteins to pathway in light pink and significantly overrepresented pathways are highlighted in dark brown color (see Table - 2.11). (b) KEGG pathways mapped are highlighted in red on the background of different metabolic pathways denoted in different colors (see Table-2.10) The color coding for different metabolic pathways are as follows: fatty acid metabolism (green); Amino acid metabolism (orange); Purine and pyrimidine metabolism (red); oxidative phosphorylation (violet); TCA cycle and glyoxylate dicarboxylate metabolism (blue). .

113 Pathway Search Result

Following object(s) was/were not found cel:CELE_B0491.2 cel:CELE_C01B10.11 cel:CELE_C02E11.1 cel:CELE_C06G4.2 cel:CELE_C07G2.3 cel:CELE_C08H9.2 cel:CELE_C15C8.3 cel:CELE_C30C11.4 cel:CELE_C39F7.4 cel:CELE_C50F2.6 cel:CELE_C53D5.6 cel:CELE_C55B7.4 cel:CELE_C56C10.8 cel:CELE_E04A4.8 cel:CELE_F01F1.8 cel:CELE_F01G4.6 cel:CELE_F09B12.3 cel:CELE_F10G7.2 cel:CELE_F22A3.6 cel:CELE_F22F7.1 cel:CELE_F25H2.11 cel:CELE_F25H5.4 cel:CELE_F31C3.1 cel:CELE_F33H2.3 cel:CELE_F40E10.3 cel:CELE_F41E6.2 cel:CELE_F41H10.8 cel:CELE_F43E2.8 cel:CELE_F46E10.9 cel:CELE_F53A2.4 cel:CELE_F54C9.1 cel:CELE_F54H12.6 cel:CELE_F56D12.5 cel:CELE_F57H12.1 cel:CELE_F58G1.4 cel:CELE_K06G5.1 cel:CELE_K12H4.7 cel:CELE_M02D8.1 cel:CELE_M03F4.7 cel:CELE_R11H6.1 cel:CELE_T06G6.9 cel:CELE_T08G11.1 cel:CELE_T12D8.2 cel:CELE_T18H9.2 cel:CELE_T26C12.1 cel:CELE_T27E9.7 cel:CELE_W02D3.5 cel:CELE_W08E3.3 cel:CELE_Y38F1A.6 cel:CELE_Y38H6C.1 cel:CELE_Y39B6A.20 cel:CELE_Y41E3.10 cel:CELE_Y53F4B.29 cel:CELE_Y53F4B.30 cel:CELE_Y56A3A.7 cel:CELE_Y57G11C.10 cel:CELE_Y65B4BR.5 cel:CELE_Y66A7A.6

Sort by the pathway list Table-2.10: KEGG pathway for the proteins for which the level of expression is lower Hidethan all objects 1.5-fold in dauer larvae compared to L3 worm

cel03010 Ribosome - Caenorhabditis elegans (nematode) (52)

cel:CELE_B0250.1 rpl-2; 60S ribosomal protein L8 cel:CELE_B0336.10 rpl-23; 60S ribosomal protein L23 cel:CELE_B0393.1 rps-0; 40S ribosomal protein SA cel:CELE_C04F12.4 rpl-14; Ribosomal Protein, Large subunit cel:CELE_C06B8.8 rpl-38; 60S ribosomal protein L38 cel:CELE_C09D4.5 rpl-19; 60S ribosomal protein L19 cel:CELE_C14B9.7 rpl-21; 60S ribosomal protein L21 cel:CELE_C27A2.2 rpl-22; 60S ribosomal protein L22 cel:CELE_C32E8.2 rpl-13; 60S ribosomal protein L13 cel:CELE_C37A2.7 C37A2.7; 60S acidic ribosomal protein P2 cel:CELE_C49H3.11 rps-2; 40S ribosomal protein S2 cel:CELE_D1007.12 rpl-24.1; 60S ribosomal protein L24 cel:CELE_D1007.6 rps-10; Ribosomal Protein, Small subunit cel:CELE_F07D10.1 rpl-11.2; Ribosomal Protein, Large subunit cel:CELE_F10E7.7 rpl-33; 60S ribosomal protein L35a cel:CELE_F13B10.2 rpl-3; 60S ribosomal protein L3 cel:CELE_F25H2.10 rla-0; 60S acidic ribosomal protein P0 cel:CELE_F28D1.7 rps-23; 40S ribosomal protein S23 cel:CELE_F36A2.6 rps-15; 40S ribosomal protein S15 cel:CELE_F37C12.11 rps-21; 40S ribosomal protein S21 cel:CELE_F37C12.4 rpl-36; 60S ribosomal protein L36 cel:CELE_F37C12.9 rps-14; 40S ribosomal protein S14 cel:CELE_F40F11.1 rps-11; Ribosomal Protein, Small subunit cel:CELE_F40F8.10 rps-9; 40S ribosomal protein S9 cel:CELE_F42C5.8 rps-8; 40S ribosomal protein S8 cel:CELE_F52B5.6 rpl-25.2; 60S ribosomal protein L23a 2 cel:CELE_F53A3.3 rps-22; Ribosomal Protein, Small subunit cel:CELE_F53G12.10 rpl-7; 60S ribosomal protein L7 cel:CELE_F54C9.5 rpl-5; 60S ribosomal protein L5 cel:CELE_F54E7.2 rps-12; 40S ribosomal protein S12 cel:CELE_F55D10.2 rpl-25.1; 60S ribosomal protein L23a 1 cel:CELE_F56F3.5 rps-1; 40S ribosomal protein S3a cel:CELE_JC8.3 rpl-12; 60S ribosomal protein L12 cel:CELE_K11H12.2 rpl-15; 60S ribosomal protein L15 cel:CELE_M01F1.2 rpl-16; 60S ribosomal protein L13a cel:CELE_R13A5.8 rpl-9; 60S ribosomal protein L9 cel:CELE_R151.3 rpl-6; 60S ribosomal protein L6 cel:CELE_T01C3.6 rps-16; 40S ribosomal protein S16 cel:CELE_T05E11.1 rps-5; 40S ribosomal protein S5 cel:CELE_T05F1.3 rps-19; 40S ribosomal protein S19 cel:CELE_T07A9.11 rps-24; Ribosomal Protein, Small subunit cel:CELE_T24B8.1 rpl-32; Ribosomal Protein, Large subunit cel:CELE_Y24D9A.4 rpl-7A; 60S ribosomal protein L7a cel:CELE_Y37E3.8 Y37E3.8; Uncharacterized protein cel:CELE_Y43B11AR.4 rps-4; 40S ribosomal protein S4 114 cel:CELE_Y48B6A.2 rpl-43; 60S ribosomal protein L37a cel:CELE_Y48G8AL.8 rpl-17; 60S ribosomal protein L17 cel:CELE_Y57G11C.16 rps-18; Ribosomal Protein, Small subunit Pathway Search Result

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cel03010 Ribosome - Caenorhabditis elegans (nematode) (52)

cel:CELE_B0250.1 rpl-2; 60S ribosomal protein L8 cel:CELE_B0336.10 rpl-23; 60S ribosomal protein L23 cel:CELE_B0393.1 rps-0; 40S ribosomal protein SA cel:CELE_C04F12.4 rpl-14; Ribosomal Protein, Large subunit cel:CELE_C06B8.8 rpl-38; 60S ribosomal protein L38 cel:CELE_C09D4.5 rpl-19; 60S ribosomal protein L19 cel:CELE_C14B9.7 rpl-21; 60S ribosomal protein L21 cel:CELE_C27A2.2 rpl-22; 60S ribosomal protein L22 cel:CELE_C32E8.2 rpl-13; 60S ribosomal protein L13 cel:CELE_C37A2.7 C37A2.7; 60S acidic ribosomal protein P2 cel:CELE_C49H3.11 rps-2; 40S ribosomal protein S2 cel:CELE_D1007.12 rpl-24.1; 60S ribosomal protein L24 cel:CELE_D1007.6 rps-10; Ribosomal Protein, Small subunit cel:CELE_F07D10.1 rpl-11.2; Ribosomal Protein, Large subunit cel:CELE_F10E7.7 rpl-33; 60S ribosomal protein L35a cel:CELE_F13B10.2 rpl-3; 60S ribosomal protein L3 cel:CELE_F25H2.10 rla-0; 60S acidic ribosomal protein P0 cel:CELE_F28D1.7 rps-23; 40S ribosomal protein S23 cel:CELE_F36A2.6 rps-15; 40S ribosomal protein S15 cel:CELE_F37C12.11 rps-21; 40S ribosomal protein S21 cel:CELE_F37C12.4 rpl-36; 60S ribosomal protein L36 cel:CELE_F37C12.9 rps-14; 40S ribosomal protein S14 cel:CELE_F40F11.1 rps-11; Ribosomal Protein, Small subunit cel:CELE_F40F8.10 rps-9; 40S ribosomal protein S9 cel:CELE_F42C5.8 rps-8; 40S ribosomal protein S8 cel:CELE_F52B5.6 rpl-25.2; 60S ribosomal protein L23a 2 cel:CELE_F53A3.3 rps-22; Ribosomal Protein, Small subunit cel:CELE_F53G12.10 rpl-7; 60S ribosomal protein L7 cel:CELE_F54C9.5 rpl-5; 60S ribosomal protein L5 cel:CELE_F54E7.2 rps-12; 40S ribosomal protein S12 cel:CELE_F55D10.2 rpl-25.1; 60S ribosomal protein L23a 1 cel:CELE_F56F3.5 rps-1; 40S ribosomal protein S3a cel:CELE_JC8.3 rpl-12; 60S ribosomal protein L12 cel:CELE_K11H12.2 rpl-15; 60S ribosomal protein L15 cel:CELE_M01F1.2 rpl-16; 60S ribosomal protein L13a cel:CELE_R13A5.8 rpl-9; 60S ribosomal protein L9 cel:CELE_R151.3 rpl-6; 60S ribosomal protein L6 cel:CELE_T01C3.6 rps-16; 40S ribosomal protein S16 cel:CELE_T05E11.1 rps-5; 40S ribosomal protein S5 cel:CELE_T05F1.3 rps-19; 40S ribosomal protein S19 cel:CELE_T07A9.11 rps-24; Ribosomal Protein, Small subunit cel:CELE_T24B8.1 rpl-32; Ribosomal Protein, Large subunit cel:CELE_Y24D9A.4 rpl-7A; 60S ribosomal protein L7a cel:CELE_Y37E3.8 Y37E3.8; Uncharacterized protein cel:CELE_Y43B11AR.4 rps-4; 40S ribosomal protein S4 cel:CELE_Y48B6A.2 rpl-43; 60S ribosomal protein L37a cel:CELE_Y48G8AL.8 rpl-17; 60S ribosomal protein L17 cel:CELE_Y57G11C.16 rps-18; Ribosomal Protein, Small subunit cel:CELE_Y71A12B.1 rps-6; 40S ribosomal protein S6 cel:CELE_Y71F9AL.13 rpl-1; 60S ribosomal protein L10a cel:CELE_ZC434.2 rps-7; 40S ribosomal protein S7 cel:CELE_ZK652.4 rpl-35; 60S ribosomal protein L35

cel01100 Metabolic pathways - Caenorhabditis elegans (nematode) (24)

cel:CELE_C05D11.11 mel-32; Serine hydroxymethyltransferase cel:CELE_C16A3.10 C16A3.10; Uncharacterized protein cel:CELE_C31C9.2 C31C9.2; Uncharacterized protein cel:CELE_C47B2.6 gale-1; UDP-GALactose 4-Epimerase cel:CELE_C49F5.1 sams-1; Probable S-adenosylmethionine synthase 1 cel:CELE_C54G4.8 cyc-1; CYtochrome C cel:CELE_D2085.1 pyr-1; PYRimidine biosynthesis cel:CELE_F01G10.1 tkt-1; TransKeTolase homolog cel:CELE_F01G4.2 ard-1; Alcohol/Ribitol Dehydrogenase family cel:CELE_F13D12.4 alh-8; Probable methylmalonate-semialdehyde dehydrogenase [acylating], mitochondrial cel:CELE_F32H2.5 fasn-1; Fatty Acid SyNthase cel:CELE_F35H10.4 vha-5; Vacuolar H ATPase cel:CELE_F47B10.1 suca-1; Probable succinyl-CoA ligase [ADP-forming] subunit beta, mitochondrial cel:CELE_F59B8.2 idh-1; Isocitrate dehydrogenase [NADP] cel:CELE_K02F2.2 ahcy-1; Adenosylhomocysteinase cel:CELE_K04D7.3 gta-1; Probable 4-aminobutyrate aminotransferase, mitochondrial cel:CELE_K07A3.1 fbp-1; Fructose-1,6-BiPhosphatase cel:CELE_R07H5.8 R07H5.8; Uncharacterized protein cel:CELE_T05G5.6 ech-6; Probable enoyl-CoA hydratase, mitochondrial cel:CELE_T25B9.9 T25B9.9; 6-phosphogluconate dehydrogenase, decarboxylating cel:CELE_VW06B3R.1 ucr-2.1; Ubiquinol-Cytochrome c oxidoReductase complex cel:CELE_Y39E4A.3 Y39E4A.3; Uncharacterized protein cel:CELE_Y71H2AM.5 Y71H2AM.5; Uncharacterized protein cel:CELE_ZK829.4 gdh-1; Glutamate dehydrogenase

cel04141 Protein processing in endoplasmic reticulum - Caenorhabditis elegans (nematode) (14)

cel:CELE_B0403.4 pdi-6; Probable protein disulfide-isomerase A6 cel:CELE_C06A1.1 cdc-48.1; Transitional endoplasmic reticulum ATPase homolog 1 cel:CELE_C07A12.4 pdi-2; Protein disulfide-isomerase 2 cel:CELE_C14B1.1 pdi-1; Protein disulfide-isomerase 1 cel:CELE_C14B9.2 C14B9.2; Probable protein disulfide-isomerase A4 cel:CELE_C15H9.6 hsp-3; Heat shock 70 kDa protein C cel:CELE_C41C4.8 cdc-48.2; Transitional endoplasmic reticulum ATPase homolog 2 cel:CELE_C47E8.5 daf-21; Heat shock protein 90 115 cel:CELE_F12F6.6 sec-24.1; yeast SEC homolog cel:CELE_F26D10.3 hsp-1; Heat shock 70 kDa protein A cel:CELE_H06O01.1 pdi-3; Protein Disulfide Isomerase cel:CELE_T05E11.3 enpl-1; ENdoPLasmin homolog cel:CELE_Y113G7A.3 sec-23; yeast SEC homolog cel:CELE_Y38A10A.5 crt-1; Calreticulin

cel01200 Carbon metabolism - Caenorhabditis elegans (nematode) (10)

cel:CELE_C05D11.11 mel-32; Serine hydroxymethyltransferase cel:CELE_C31C9.2 C31C9.2; Uncharacterized protein cel:CELE_F01G10.1 tkt-1; TransKeTolase homolog cel:CELE_F13D12.4 alh-8; Probable methylmalonate-semialdehyde dehydrogenase [acylating], mitochondrial cel:CELE_F47B10.1 suca-1; Probable succinyl-CoA ligase [ADP-forming] subunit beta, mitochondrial cel:CELE_F59B8.2 idh-1; Isocitrate dehydrogenase [NADP] cel:CELE_K07A3.1 fbp-1; Fructose-1,6-BiPhosphatase cel:CELE_T05G5.6 ech-6; Probable enoyl-CoA hydratase, mitochondrial cel:CELE_T25B9.9 T25B9.9; 6-phosphogluconate dehydrogenase, decarboxylating cel:CELE_ZK829.4 gdh-1; Glutamate dehydrogenase

cel03013 RNA transport - Caenorhabditis elegans (nematode) (6)

cel:CELE_B0511.10 eif-3.E; Eukaryotic translation initiation factor 3 subunit E cel:CELE_F31E3.5 eef-1A.1; Elongation factor 1-alpha cel:CELE_R03G5.1 eef-1A.2; Elongation factor 1-alpha cel:CELE_R08D7.3 eif-3.D; Eukaryotic translation initiation factor 3 subunit D cel:CELE_Y106G6H.2 pab-1; PolyA Binding protein cel:CELE_Y54E2A.11 eif-3.B; Eukaryotic translation initiation factor 3 subunit B cel:CELE_Y71A12B.1 rps-6; 40S ribosomal protein S6 cel:CELE_Y71F9AL.13 rpl-1; 60S ribosomal protein L10a cel:CELE_ZC434.2 rps-7; 40S ribosomal protein S7 cel:CELE_ZK652.4 rpl-35; 60S ribosomal protein L35 cel01100 Metabolic pathways - Caenorhabditis elegans (nematode) (24) cel:CELE_C05D11.11 mel-32; Serine hydroxymethyltransferase cel:CELE_C16A3.10 C16A3.10; Uncharacterized protein cel:CELE_C31C9.2 C31C9.2; Uncharacterized protein cel:CELE_C47B2.6 gale-1; UDP-GALactose 4-Epimerase cel:CELE_C49F5.1 sams-1; Probable S-adenosylmethionine synthase 1 cel:CELE_C54G4.8 cyc-1; CYtochrome C cel:CELE_D2085.1 pyr-1; PYRimidine biosynthesis cel:CELE_F01G10.1 tkt-1; TransKeTolase homolog cel:CELE_F01G4.2 ard-1; Alcohol/Ribitol Dehydrogenase family cel:CELE_F13D12.4 alh-8; Probable methylmalonate-semialdehyde dehydrogenase [acylating], mitochondrial cel:CELE_F32H2.5 fasn-1; Fatty Acid SyNthase cel:CELE_F35H10.4 vha-5; Vacuolar H ATPase cel:CELE_F47B10.1 suca-1; Probable succinyl-CoA ligase [ADP-forming] subunit beta, mitochondrial cel:CELE_F59B8.2 idh-1; Isocitrate dehydrogenase [NADP] cel:CELE_K02F2.2 ahcy-1; Adenosylhomocysteinase cel:CELE_K04D7.3 gta-1; Probable 4-aminobutyrate aminotransferase, mitochondrial cel:CELE_K07A3.1 fbp-1; Fructose-1,6-BiPhosphatase cel:CELE_R07H5.8 R07H5.8; Uncharacterized protein cel:CELE_T05G5.6 ech-6; Probable enoyl-CoA hydratase, mitochondrial cel:CELE_T25B9.9 T25B9.9; 6-phosphogluconate dehydrogenase, decarboxylating cel:CELE_VW06B3R.1 ucr-2.1; Ubiquinol-Cytochrome c oxidoReductase complex cel:CELE_Y39E4A.3 Y39E4A.3; Uncharacterized protein cel:CELE_Y71H2AM.5 Y71H2AM.5; Uncharacterized protein cel:CELE_ZK829.4 gdh-1; Glutamate dehydrogenase cel04141 Protein processing in endoplasmic reticulum - Caenorhabditis elegans (nematode) (14) cel:CELE_B0403.4 pdi-6; Probable protein disulfide-isomerase A6 cel:CELE_C06A1.1 cdc-48.1; Transitional endoplasmic reticulum ATPase homolog 1 cel:CELE_C07A12.4 pdi-2; Protein disulfide-isomerase 2 cel:CELE_C14B1.1 pdi-1; Protein disulfide-isomerase 1 cel:CELE_C14B9.2 C14B9.2; Probable protein disulfide-isomerase A4 cel:CELE_C15H9.6 hsp-3; Heat shock 70 kDa protein C cel:CELE_C41C4.8 cdc-48.2; Transitional endoplasmic reticulum ATPase homolog 2 cel:CELE_C47E8.5 daf-21; Heat shock protein 90 cel:CELE_F12F6.6 sec-24.1; yeast SEC homolog cel:CELE_F26D10.3 hsp-1; Heat shock 70 kDa protein A cel:CELE_H06O01.1 pdi-3; Protein Disulfide Isomerase cel:CELE_T05E11.3 enpl-1; ENdoPLasmin homolog cel:CELE_Y113G7A.3 sec-23; yeast SEC homolog cel:CELE_Y38A10A.5 crt-1; Calreticulin cel01200 Carbon metabolism - Caenorhabditis elegans (nematode) (10) cel:CELE_C05D11.11 mel-32; Serine hydroxymethyltransferase cel:CELE_C31C9.2 C31C9.2; Uncharacterized protein cel:CELE_F01G10.1 tkt-1; TransKeTolase homolog cel:CELE_F13D12.4 alh-8; Probable methylmalonate-semialdehyde dehydrogenase [acylating], mitochondrial cel:CELE_F47B10.1 suca-1; Probable succinyl-CoA ligase [ADP-forming] subunit beta, mitochondrial cel:CELE_F59B8.2 idh-1; Isocitrate dehydrogenase [NADP] cel:CELE_K07A3.1 fbp-1; Fructose-1,6-BiPhosphatase cel:CELE_T05G5.6 ech-6; Probable enoyl-CoA hydratase, mitochondrial cel:CELE_T25B9.9 T25B9.9; 6-phosphogluconate dehydrogenase, decarboxylating cel:CELE_ZK829.4 gdh-1; Glutamate dehydrogenase cel03013 RNA transport - Caenorhabditis elegans (nematode) (6) cel:CELE_B0511.10 eif-3.E; Eukaryotic translation initiation factor 3 subunit E cel:CELE_F31E3.5 eef-1A.1; Elongation factor 1-alpha cel:CELE_R03G5.1 eef-1A.2; Elongation factor 1-alpha cel:CELE_R08D7.3 eif-3.D; Eukaryotic translation initiation factor 3 subunit D cel:CELE_Y106G6H.2 pab-1; PolyA Binding protein cel:CELE_Y54E2A.11 eif-3.B; Eukaryotic translation initiation factor 3 subunit B

116 Table-2.11: REACTOME overrepresentation pathway for the proteins for which the level of expression is lower than 1.5-fold in dauer larvae compared to L3 worm. Wormbase Gene ID Pathway name p Value FDR WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00004414;WBGene00004413;WBGeneGTP hydrolysis and joining of the 60S ribosomal subunit 1.11E-16 1.22E-15 00004490;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00004429;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00004414;WBGene00004413;WBGeneSRP-dependent cotranslational protein targeting to membrane 1.11E-16 1.22E-15 00004490;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00004429;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00001225;WBGene00004414;WBGeneFormation of a pool of free 40S subunits 1.11E-16 1.22E-15 00001228;WBGene00004413;WBGene00004490;WBGene00001227;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00004429;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 Nonsense Mediated Decay (NMD) independent of the Exon Junction WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00004414;WBGene00004413;WBGene1.11E-16 1.22E-15 00004490;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00003902;WBGene00004429;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 Complex (EJC) WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00001225;WBGene00004414;WBGeneL13a-mediated translational silencing of Ceruloplasmin expression 1.11E-16 1.22E-15 00001228;WBGene00004413;WBGene00004490;WBGene00001227;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00003902;WBGene00004429;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00004414;WBGene00004413;WBGeneNonsense-Mediated Decay (NMD) 1.11E-16 1.22E-15 00004490;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00003902;WBGene00004429;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 Nonsense Mediated Decay (NMD) enhanced by the Exon Junction WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00004414;WBGene00004413;WBGene1.11E-16 1.22E-15 00004490;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00003902;WBGene00004429;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 Complex (EJC) WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00001225;WBGene00004414;WBGeneTranslation 1.11E-16 1.22E-15 00001228;WBGene00004413;WBGene00004490;WBGene00001227;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00001168;WBGene00001167;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00003902;WBGene00004429;WBGene00018846;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00001225;WBGene00004414;WBGeneEukaryotic Translation Initiation 1.11E-16 1.22E-15 00001228;WBGene00004413;WBGene00004490;WBGene00001227;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00003902;WBGene00004429;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00001225;WBGene00004414;WBGeneCap-dependent Translation Initiation 1.11E-16 1.22E-15 00001228;WBGene00004413;WBGene00004490;WBGene00001227;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00004429;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 WBGene00004481;WBGene00004488;WBGene00004480;WBGene00004471;WBGene00004470;WBGene00004483;WBGene00004485;WBGene00004476;WBGeneFormation of the ternary complex, and subsequently, the 43S complex 1.11E-16 1.22E-15 00004484;WBGene00004475;WBGene00004487;WBGene00004474;WBGene00004473;WBGene00001225;WBGene00001228;WBGene00004490;WBGene00001227;WBGene00004477;WBGene00004491;WBGene00004478;WBGene00004492;WBGene00004469;WBGene00004479;WBGene00004493 WBGene00004488;WBGene00004420;WBGene00004266;WBGene00000183;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGeneMetabolism of proteins 1.11E-16 1.22E-15 00001225;WBGene00001228;WBGene00004414;WBGene00001227;WBGene00004490;WBGene00004413;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00001633;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00001168;WBGene00001167;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00002065;WBGene00003964;WBGene00004487;WBGene00020915;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00000802;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004754;WBGene00004755;WBGene00000380;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00000381;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00004424;WBGene00004423;WBGene00003902;WBGene00004429;WBGene00004428;WBGene00018846;WBGe!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! WBGene00004488;WBGene00004420;WBGene00004416;WBGene00004418;WBGene00004417;WBGene00004412;WBGene00001225;WBGene00004414;WBGeneGene Expression 1.11E-16 1.22E-15 00001228;WBGene00004413;WBGene00004490;WBGene00001227;WBGene00004491;WBGene00004492;WBGene00004493;WBGene00004456;WBGene00004419;WBGene00004452;WBGene00004450;WBGene00021350;WBGene00016493;WBGene00001168;WBGene00001167;WBGene00004449;WBGene00004446;WBGene00004447;WBGene00004408;WBGene00004481;WBGene00004480;WBGene00004483;WBGene00004485;WBGene00004484;WBGene00004487;WBGene00004434;WBGene00004433;WBGene00004436;WBGene00004435;WBGene00004438;WBGene00004439;WBGene00004477;WBGene00004478;WBGene00004479;WBGene00004471;WBGene00004470;WBGene00004476;WBGene00004475;WBGene00004474;WBGene00004473;WBGene00004431;WBGene00004432;WBGene00004425;WBGene00002005;WBGene00004424;WBGene00004423;WBGene00003902;WBGene00004429;WBGene00018846;WBGene00004428;WBGene00004427;WBGene00004426;WBGene00004469 WBGene00004481;WBGene00004488;WBGene00004480;WBGene00004471;WBGene00004470;WBGene00004483;WBGene00004485;WBGene00004476;WBGeneRibosomal scanning and start codon recognition 3.11E-15 3.11E-14 00004484;WBGene00004475;WBGene00004487;WBGene00004474;WBGene00004473;WBGene00001225;WBGene00001228;WBGene00004490;WBGene00001227;WBGene00004477;WBGene00004491;WBGene00004478;WBGene00004492;WBGene00004469;WBGene00004479;WBGene00004493 WBGene00010317 NADPH regeneration 4.87E-04 4.87E-03 WBGene00001168;WBGene00018846;WBGene00001167Eukaryotic Translation Elongation 1.11E-03 9.13E-03 WBGene00001168;WBGene00000915;WBGene00008053;WBGene00007352HSF1 activation 1.14E-03 9.13E-03 WBGene00000114;WBGene00003214;WBGene00016943;WBGene00000181;WBGene00012713Branched-chain amino acid catabolism 1.70E-03 1.36E-02 WBGene00004266 Golgi Cisternae Pericentriolar Stack Reorganization 5.36E-03 4.28E-02 WBGene00006439;WBGene00009342;WBGene00008506Integration of energy metabolism 4.20E-02 3.36E-01 WBGene00001167 Peptide chain elongation 4.85E-02 3.39E-01

117 Gene ontology and pathway association of unique proteins present in dauer larvae

There were peptides corresponding to 69 proteins observed that were unique to dauer larvae and not observed at all in the L3 stage C. elegans, of which 19 proteins were found in at least two replicate samples were also subjected to bioinformatics analysis

(Table-2.12). The gene ontology and pathway mapping for these 19 proteins (Table 2.13 and 2.14) showed that most of the proteins belong to catalytic and metabolic processes.

These proteins were muscle related proteins (ttn-1 (ortholog of titin), myo-6 (ortholog of human myosin with motor activity), pfn-3 (muscle thin filament assembly), npp-21

(ortholog of human TPR, nuclear basket protein, participate in locomotion)), chromatin organization proteins (histones (his-48; his-58; his-62; his-66; hil-2)), stress related (ctl-1

(catalase, an antioxidant enzyme contributes to extended life span), ttr-46 (Transthyretin- like protein 46), paxt-1 (ortholog of CDK, cell cycle), cco-1 (cytochrome c oxidase, involve in life span regulation and unfolding protein response (UPR)), glrx-10 ( encodes a glutaredoxin, oxidoreductases of the thioredoxin family)).

The statistical overrepresentation test (PANTHER) found oxidoreductase activity

(GO-MF term) and ‘respiratory electron transport chain’ and ‘generation of precursor metabolites and energy’ (GO-BP term) as significant overrepresented GO terms.

The top five pathways mapped to KEGG pathway was shown in Table-2.15 and

Figure 2.11b. Only two proteins, ctl-1 and npp-21 mapped to different KEGG pathways.

The catalase (ctl-1) is associated with longevity (FoxO pathway), oxidative phosphorylation, and glyoxylate and dicarboxylate pathway and npp-21 is associated

118 with RNA transport. Interestingly, the uncharacterized proteins, which are not recognized by PANTHER and KEGG mapping tools, were found to be associated with significantly overrepresented pathways in the REACTOME overrepresentation test

(Table-2.16, Figure 2.11a). These overrepresented pathways were senescence related pathways, MAPK and JNK pathways, and innate immune response pathways (TLR signaling).

These results indicate that this distinct expressed set of proteins that are unique to the dauer stage are involved in stress response, innate immune response, and muscle physiology. These results indicate that these pathways might be beneficial for survival and protection from extreme environment of dauer larvae.

119 Table-2.12: Unique proteins present in dauer Dauer Wormbase Gene ID Wormpep ID Protein name Gene name (Ratio- light/heavy) Phosphatidylethanolamine-binding protein WBGene00018218 CE38516 0.64794 homolog F40A3.3 WBGene00006436 CE36725 Titin homolog (EC 2.7.11.1) ttn-1 1.85985 his-48; his-58; WBGene00001922 CE05165 Probable histone H2B 4 0.86762 his-62; his-66 WBGene00016435 CE16903 Uncharacterized protein 1.05454 WBGene00019940 CE37543 Nuclear Pore complex Protein npp-21 2.72222 WBGene00000830 CE30713 Catalase-2 (EC 1.11.1.6) ctl-1 2.27853 WBGene00008032 CE27838 Uncharacterized protein 1.09301 WBGene00017098 CE08610 Uncharacterized protein 0.94847 WBGene00018610 CE36546 Uncharacterized protein 1.22173 WBGene00003991 CE07332 Profilin-3 pfn-3 0.99814 WBGene00011034 CE32473 PArtner of Xrn-2 (Two) paxt-1 1.91731 WBGene00012853 CE19160 Uncharacterized protein 1.09460 WBGene00001853 CE47059 Histone H1.2 (Histone H1-like protein 2) hil-2 1.75941 WBGene00000371 CE09693 Cytochrome C Oxidase cco-1 1.31882 WBGene00009730 CE28727 MYOsin heavy chain structural genes myo-6 1.93911 WBGene00011571 CE06389 Transthyretin-like protein 46 ttr-46 0.86301 WBGene00012126 CE24022 Uncharacterized protein 0.72665 WBGene00021331 CE25238 GLutaRedoXin glrx-10 1.03735 WBGene00014016 CE28193 Uncharacterized protein ZK632.9 2.32343

120 Table-2.13: GO-MF terms for the unique proteins in dauer. GO term Wormbase Gene ID Protein name Protein class G-protein modulator(PC00095);actin binding motor WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog protein(PC00022);cell junction protein(PC00085) Protein E02D9.1, isoform transcription cofactor(PC00218);mRNA splicing WBGene00017098 a;CELE_E02D9.1;ortholog factor(PC00217) WBGene00000830 Catalase-2;ctl-1;ortholog peroxidase(PC00176) catalytic activity WBGene00021331 Protein GLRX-10;glrx-10;ortholog oxidoreductase(PC00176) (GO:0003824) Protein T28D6.6, isoform WBGene00012126 small GTPase(PC00095) a;CELE_T28D6.6;ortholog WBGene00000371 Protein CCO-1;cco-1;ortholog oxidase(PC00176) WBGene00018610 Protein F48E8.3;CELE_F48E8.3;ortholog dehydrogenase(PC00176);oxidase(PC00092) WBGene00016435 Protein C35B1.5;C35B1.5;ortholog oxidoreductase(PC00176) G-protein modulator(PC00095);actin binding motor WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog protein(PC00022);cell junction protein(PC00085) Protein E02D9.1, isoform transcription cofactor(PC00218);mRNA splicing WBGene00017098 binding a;CELE_E02D9.1;ortholog factor(PC00217) (GO:0005488) WBGene00001853 Histone H1.2;hil-2;ortholog histone(PC00171) WBGene00003991 Profilin-3;pfn-3;ortholog non-motor actin binding protein(PC00085) Protein T28D6.6, isoform WBGene00012126 small GTPase(PC00095) a;CELE_T28D6.6;ortholog G-protein modulator(PC00095);actin binding motor structural molecule WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog protein(PC00022);cell junction protein(PC00085) activity (GO:0005198) WBGene00003991 Profilin-3;pfn-3;ortholog non-motor actin binding protein(PC00085) nucleic acid binding transcription factor Protein E02D9.1, isoform transcription cofactor(PC00218);mRNA splicing WBGene00017098 activity a;CELE_E02D9.1;ortholog factor(PC00217) (GO:0001071) enzyme regulator activity WBGene00012853 Protein Y44A6D.2;CELE_Y44A6D.2;ortholog (GO:0030234) antioxidant activity G-protein modulator(PC00095);actin binding motor WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog (GO:0016209) protein(PC00022);cell junction protein(PC00085) protein binding transcription factor Protein E02D9.1, isoform transcription cofactor(PC00218);mRNA splicing WBGene00017098 activity a;CELE_E02D9.1;ortholog factor(PC00217) (GO:0000988)

121 Table-2.14: GO-BP terms for the unique proteins in dauer GO term Wormbase Gene ID Protein name Protein class G-protein modulator(PC00095);actin binding WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog motor protein(PC00022);cell junction protein(PC00085) Protein E02D9.1, isoform transcription cofactor(PC00218);mRNA WBGene00017098 a;CELE_E02D9.1;ortholog splicing factor(PC00217) WBGene00001853 Histone H1.2;hil-2;ortholog histone(PC00171) metabolic process (GO:0008152) WBGene00000830 Catalase-2;ctl-1;ortholog peroxidase(PC00176) WBGene00021331 Protein GLRX-10;glrx-10;ortholog oxidoreductase(PC00176) Protein T28D6.6, isoform WBGene00012126 small GTPase(PC00095) a;CELE_T28D6.6;ortholog WBGene00000371 Protein CCO-1;cco-1;ortholog oxidase(PC00176) WBGene00018610 Protein F48E8.3;CELE_F48E8.3;ortholog dehydrogenase(PC00176);oxidase(PC00092) WBGene00016435 Protein C35B1.5;C35B1.5;ortholog oxidoreductase(PC00176) G-protein modulator(PC00095);actin binding WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog motor protein(PC00022);cell junction cellular process (GO:0009987) protein(PC00085) WBGene00001853 Histone H1.2;hil-2;ortholog histone(PC00171) WBGene00003991 Profilin-3;pfn-3;ortholog non-motor actin binding protein(PC00085) G-protein modulator(PC00095);actin binding WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog motor protein(PC00022);cell junction cellular component organization protein(PC00085) or biogenesis (GO:0071840) WBGene00001853 Histone H1.2;hil-2;ortholog histone(PC00171)

WBGene00003991 Profilin-3;pfn-3;ortholog non-motor actin binding protein(PC00085) immune system process WBGene00000830 Catalase-2;ctl-1;ortholog peroxidase(PC00176) (GO:0002376) G-protein modulator(PC00095);actin binding developmental process WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog motor protein(PC00022);cell junction (GO:0032502) protein(PC00085) G-protein modulator(PC00095);actin binding multicellular organismal process WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog motor protein(PC00022);cell junction (GO:0032501) protein(PC00085) G-protein modulator(PC00095);actin binding biological regulation WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog motor protein(PC00022);cell junction (GO:0065007) protein(PC00085) G-protein modulator(PC00095);actin binding localization (GO:0051179) WBGene00009730 Protein MYO-6, isoform a;myo-6;ortholog motor protein(PC00022);cell junction protein(PC00085)

122 a

123 b

124 Figure 2.12: Pathway association for unique proteins of dauer. Proteins mapped to (a) the REACTOME pathways (b) KEGG pathways. (a) REACTOME pathways graph showing proteins mapped to pathway (in dark pink) and significantly overrepresented pathways (p <0.05) are highlighted in dark brown color (see also Table-2.16). All known pathways are shown in background as light pink and labeled. (b) KEGG pathways mapped are highlighted in red on the background of different metabolic pathways denoted in different colors (see also Table-2.15 for proteins mapped to KEGG metabolic pathway map). The color coding for different metabolic pathways in each figure (a, b, and c) are as follows: fatty acid metabolism (green); Amino acid metabolism (orange); Purine and pyrimidine metabolism (red); oxidative phosphorylation (violet); TCA cycle and glyoxylate dicarboxylate metabolism (blue).

125 Pathway Search Result

Following object(s) was/were not found cel:CELE_B0035.8 cel:CELE_C35B1.5 cel:CELE_C39E9.8 cel:CELE_E02D9.1 cel:CELE_F45G2.2 cel:CELE_F48E8.3 cel:CELE_F54E12.4 cel:CELE_F55G1.3 cel:CELE_H02I12.6 cel:CELE_K03E6.6 cel:CELE_R05D11.6 cel:CELE_T07C12.7 cel:CELE_T28D6.6 cel:CELE_W06H8.8 cel:CELE_Y34D9A.6 cel:CELE_Y44A6D.2 cel:CELE_Y73B6BL.9 cel:CELE_ZK632.9

Sort by the pathway list Table-2.15: KEGG pathway of the unique proteins in dauer Hide all objects

cel00380 Tryptophan metabolism - Caenorhabditis elegans (nematode) (1)