IDENTIFICATION OF PROTEINS ASSOCIATED WITH INSECT
DIAPAUSE AND STRESS TOLERANCE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By
Aiqing Li, M.S.
* * * * *
The Ohio State University
2008
Dissertation Committee: Approved by
Professor David L. Denlinger, Advisor
Professor Donald H. Dean ______
Professor Glen R. Needham Advisor Graduate Program in Entomology
Copyright by
Aiqing Li
2008
ABSTRACT
This dissertation focuses on patterns of protein synthesis that are linked to several different stress responses in insects. These include the overwintering dormancy response of insects, known as diapause; the response of insects to rapid drops in temperature, known as rapid cold hardening (RCH), and responses of an
Antarctic insect to desiccation and rehydration.
Proteomics, the comprehensive study of all proteins in an organism, has proven to be a powerful technique for studying changes in protein abundance during various stresses. However, only a few studies have used proteomics to examine diapause and stress responses in insects. The primary focus of this work is to identify proteins associated with diapause, low temperature and desiccation.
The flesh fly, Sarcophaga crassipalpi s, and the midge, Belgica antarctica , were used as model organisms.
A proteomic analysis of pupal brains in S. crassipalpi s revealed that heat shock proteins (Hsp) were among the most conspicuous brain proteins present in higher amounts during diapause. While the mRNAs encoding Hsps were previously known to be associated with diapause, other proteins identified during
ii this study were not known to be linked to diapause, thus suggesting that the proteomic approach nicely supplements work done at the transcript level. A gene encoding neuropeptide like precursor 4 in S. crassipalpis showed close association with diapause, suggesting a potential role for this gene in initiating and maintaining diapause. Changes in brain proteins following pupal diapause termination in S. crassipalpis showed an increase in abundance of myo inositol 1 phosphate synthase (Inos). The elevation of Inos at diapause termination is likely downstream of the physiological regulation that initiates development. During RCH, an increase in ATP synthase suggests that an elevation of ATP is an important component of this response, and a small Hsp increase suggests that at least one of the Hsps is actually mobilized during RCH, rather that after RCH as previously assumed. Proteomics of B. antarctica larvae indicate that both desiccation and rehydration elicit synthesis of contractile and cytoskeletal proteins that are likely to be involved in the body contraction and cytoskeleton rearrangements that are associated with survival of changes in body water content.
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Dedicated to my parents
iv
ACKNOWLEDGMENTS
I am grateful to my advisor, Dr. David L. Denlinger, for his great support and intellectual guidance throughout my 5 year Ph. D. study. His encouragement, enthusiasm and expert editorial contribution make this dissertation possible.
I appreciate my committee members, Drs. Donald H. Dean, David J.
Horn, and Glen R. Needham, for their valuable comments and suggestions to this dissertation.
I would like to thank David Mandich of Plant Microbe Genomics Facility at
The Ohio State University, for his advice on image analysis and Kari
Green Church, OSU Campus Chemical Instrument Center, for assistance with mass spectrometric identification of proteins.
I thank all my current and previous lab colleagues. I appreciate Dr.
Joseph Rinehart and Dr. Rebecca Robich for their constant help during my
v graduate study.
I would like to thank my family for their unconditional love and support during my graduate studies in the Unites States. My special thanks go to my husband. Without his tremendous love and support, this dissertation could not be prepared.
This research is supported by grants from the National Science
Foundation to Dr. David L. Denlinger.
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VITA April 9, 1976…………………………………….…...Born – Jiangxi, China
1999………………….………….…….Bachelor of Science, Plant Protection
Jiangxi Agricultural University
Nanchang, China
2002………………………………....……..…. Master of Science, Zoology
Jiangxi Agricultural University
Nanchang, China
2002 2003……………………………………………………...Staff member
Jiangxi Plant Protection and Quarantine Station
2003 2008………….………….. Graduate Teaching and Research Associate
The Ohio State University
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PUBLICATIONS 1. Rinehart, J. P., Li , A., Yocum, G. D., Robich, R. M., Hayward, S. A. L., Denlinger, D. L., 2007. Upregulation of heat shock proteins is essential for cold survival during insect diapause. Proceedings of the National Academy of Sciences of the United States of America 104, 11130 11137. 2. Li , A., Popova Butler, A., Dean, D. H., Denlinger, D. L., 2007. Proteomics of the flesh fly brain reveals an abundance of upregulated heat shock proteins during pupal diapause. Journal of Insect Physiology 53, 385 391. 3. Li , A., Xue, F., Hua, A., Tang, J., 2003. Photoperiodic clock of diapause termination in Pseudopidorus fasciata (Lepidoptera: Zygaenidae). European Journal of Entomology 100, 287 293. 4. Xue, F., Spieth, H. R., Li A. , Hua A., 2002. The role of photoperiod and temperature in determination of summer and winter diapause in the cabbage beetle, Colaphellus bowringi (Coleoptera: Chrysomelidae). Journal of Insect Physiology 48, 279 286. 5. Wei, X., Xue, F., Li , A., 2001. Photoperiodic clock of diapause induction in Pseudopidorus fasciata (Lepidoptera: Zygaenidae). Journal of Insect Physiology 47, 1367 1375. 6. Xue, F., Li , A., 2002. Diversity in life history of the leaf beetle, Colaphellus bowringi Baly . Acta Entomologica Sinica. 45, 494 498. 7. Li, A., Xue, F., 2002. The effects of temperature and photoperiod on diapause maintenance and termination in both the leaf mining fly, Pegomyia bicolor Wiedemann and its parasitic braconid wasp, Biosteves sp. Acta Agriculturae Universitatis Jiangxiensis 24, 436 440. 8. Xue, F., Li , A., Zhu, X., 2001. The role of temperature during insect diapause. Acta Agriculturae Universitatis Jiangxiensis 23, 62 67.
viii 9. Xue, F., Yang, A., Li , A., Li, F., Zhu, X., 2001. Observation of hibernation and aestivation of Colaphellus bowringi (Col. Chrysomelidae). Jiangxi Plant Protection 24, 1 2. 10. Li , A., 2001. Integrated pest management: the historical review and contemporary development. Jiangxi Plant Protection . 24, 60 65. 11. Li , A., 2001. Integrated pest management: the historical review and contemporary development (continued). Jiangxi Plant Protection . 24, 86 93. FIELDS OF STUDY Major Field: Entomology Study in Molecular Biology
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TABLE OF CONTENTS Abstract...... ii Dedication...... iv Acknowledgements...... v Vita...... vii List of Tables...... xii List of Figures...... xiii Chapters: 1. Chapter 1: Introduction...... 1 References...... 18 2. Chapter 2: Proteomics of the flesh fly brain reveals an abundance of upregulated heat shock proteins during pupal diapause...... 27 Abstract...... 28 Introduction...... 30 Material and methods...... 31 Results...... 36 Discussion...... 38 References...... 44 Illustrations...... 47 3. Chapter 3: Neuropeptide like precursor 4 is uniquely expressed during pupal diapause in the flesh fly...... 49 Abstract...... 50 Introduction...... 51 Material and methods...... 52 Results...... 55 Discussion...... 57 References...... 59 Illustrations...... 61 4. Chapter 4: Rapid elevation of Inos and decreases in abundance of other brain proteins at pupal diapause termination in the flesh fly Sarcophaga crassipalpis ...... 64 Abstract...... 65
x Introduction...... 66 Material and methods...... 67 Results...... 72 Discussion...... 73 References...... 78 Illustrations...... 81 5. Chapter 5: Rapid cold hardening elicits changes in brain protein profiles of the flesh fly...... 85 Abstract...... 86 Introduction...... 87 Results...... 88 Discussion...... 89 Experimental procedures...... 95 References...... 100 Illustrations...... 104 6. Chapter 6: Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration...... 108 Abstract...... 109 Introduction...... 110 Experimental materials and methods...... 111 Results...... 117 Discussion...... 120 References...... 125 Illustrations...... 128 Conclusions...... 139 Appendics...... 139 Appendix A: Diapause associated proteins in the head of the mosquito, Culex pipiens pipiens ...... 146
Appendix B: Ovary proteins involved in a diapause maternal effect in the flesh fly, Sarcophaga bullata...... 154 Bibliography...... 162
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LIST OF TABLES 2.1. Identification of diapause regulated brain proteins in pupae of S. crassipalpis ………….…………………………………………….……………...49 4.1. Identification of brain proteins that change in abundances within 24 h of pupal diapause termination in S. crassipalpis ...... 85 5.1 Identification of differentially regulated brain proteins in pharate adults of S. crassipalpis ...... 109 6.1 Number of proteins that responded to desiccation (75% RH for 12 h at 4 °C) and rehydration (100% RH for 6 h and then in water for 6 h at 4 °C, following desiccation) ...... 135 6.2 Identification of larval proteins in Belgica antarctica that responded to desiccation...... 136 6.3 Identification of larval proteins in Belgica antarctica that responded to rehydration after desiccation...... 139 A.1. Diapause associated proteins identified in heads of Culex pipiens ...... 154
B.1. Differentially regulated ovary proteins in Sarcophaga bullata exposed to a short day history compared to flies exposed to a long day history...... 162
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LIST OF FIGURES 2.1. Two dimensional electrophoresis maps of brain proteins from (A) diapausing and (B) nondiapausing pupae of S. crassipalpis ...... 47 2.2. Selected area of two dimensional gels and corresponding spot volumes for selected spots identified in the nondiapausing (ND) and diapausing (D) flesh flies…………………………………………………………………………….....48 3.1 Nucleotide and deduced amino acid sequence of Nplp4 in Sarcophaga crassipalpis ...... 63 3.2 Alignment of amino acid sequences of S. crassipalpis and D. melanogaster (CG15361 PA) neuropeptide like precursor 4 by ClustalW multiple alignment (http://www.cbs.dtu.dk/clustalw2) ...... 64 3.3 Expression profiles of Nplp4 in diapausing and nondiapausing pupae of S. crassipalpis ...... 65 4.1. Two dimensional electrophoresis master images of pupal brain proteins in S. crassipalpis (A) during diapause and (B) 24 h after diapause was terminated by the application of hexane...... 83 4.2. Enlarged gel images, corresponding 3 D profiles and fold change in abundance of selected proteins identified in brains of S. crassipalpis during diapause (D) and 24 h following diapause termination (DT)...... 84 5.1 A 2 dimensional electrophoresis map of brain proteins from red eye pharate adults of the flesh fly Sarcophaga crassipalpi ...... 106 5.2 Selected 2DE images demonstrating changes in abundance of identified proteins following rapid cold hardening (RCH) for 2 h at 0 °C. Controls (CNT) were held continuously at 20 °C...... 107 5.3 Statistically significant differences in protein abundance in response to RCH...... 108
xiii 6.1 Flow chart of proteomic analysis of hydrated, desiccated and rehydrated B. antarctica larvae...... 130 6.2 Representative 2DE maps of proteins from Belgica antarctica larvae after desiccation at 75% RH for 12 h at 4 °C...... 131 6.3 Representative 2DE maps of proteins from Belgica antarctica larvae after rehydration at 100% RH for 6 h and then in water for 6 h following desiccation at 75% RH for 12 h at 4 °C...... 132 6.4 Quantification of proteins that changed in abundance (A) after desiccation at 75% RH for 12 h and (B) rehydration at 100% RH for 6 h and then in water for 6 h following desiccation at 75% RH for 12 h...... 133 6.5 Functional classification of the identified proteins that changed in abundance in response to (A) desiccation and (B) rehydration...... 134 A.1. A 2 dimensional electrophoresis image of head proteins from female adults of Culex pipiens ...... 152 A.2. Relative abundance of mosquito head proteins...... 153 B.1. A 2 dimensional electrophoresis map of 2 day ovary proteins from Sarcophaga bullata exposed to a short day history...... 159 B.2. A 2 dimensional electrophoresis map of 5 day ovary proteins from Sarcophaga bullata exposed to a short day history...... 160 B.3. Relative abundance of proteins from (A) 2 day and (B) 5 day ovaries in the flesh fly S. bullata ...... 161
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CHAPTER 1
INTRODUCTION
1.1 Diapause
Diapause is an important survival strategy that allows insects to escape harsh weather during cold winters and hot summers, shortage of resources, or other adverse conditions. Diapause is not a static state, but a dynamic developmental process widespread among insects and other invertebrates such as nematodes, crustaceans and earthworms (Denlinger, 2005; Kostal, 2006). It is characterized by arrested development, suppressed metabolism, increased energy reserves, and usually increased resistance to water loss and protection from low temperature
(Denlinger, 2000).
Diapause studies have been gaining increased attention during the last two decades. Denlinger has provided excellent reasons why we need to study diapause
(Denlinger, 2007). Diapause research is essential for understanding population modeling, pest management, biological control, developmental, and even offers insights into aging, obesity, and disease transmission.
1 Environmental regulation of diapause has been comprehensively studied
(Tauber et. al., 1986; Danks, 1987). Diapause is genetically determined and generally occurs at a specific developmental stage for each species. It is induced by environmental factors such as photoperiod, temperature, humidity, food conditions, and population density, among which, photoperiod is the primary cue used to program diapause. Diapause termination of a few species is determined by environmental cues, while in most cases, daylength, or temperature alone work as modulators of the rate of diapause development. Once diapause development has been completed, the insect is capable of breaking diapause and resuming development if favorable conditions such as elevation in temperature and rainfall are available (Denlinger, 2000).
The dynamic processes of insect diapause include three successive phases, pre diapause consisting of induction and preparation, diapause consisting of initiation, maintenance and termination, and post diapause, i.e., quiescence, as proposed by Kostal (2006). During the induction phase, insects perceive environmental signals until a critical level has been attained, and then integrates the information into the decision to enter diapause. During the preparation phase, the insect continues direct development, and decision to enter diapause or not and sometimes diapause duration as well are made. During the initiation phase of diapause, direct development ceases, metabolic rate decreases, physiological preparations for adversity may take place and diapause intensity increases. During
2 the maintenance phase, developmental arrest remains; metabolic rate is low and constant; unknown physiological processes gradually decrease the diapause intensity. During the termination phase, specific environmental conditions decrease diapause intensity to its minimum level and direct development is reinitiated. During the post diapause quiescence phase, development and metabolism are inhibited after diapause termination when environmental conditions are not favorable (Kostal, 2006).
Since photoperiod is the major environmental factor to induce diapause, it is necessary to mention photoreceptor proteins and clock genes linked to photoperiodism. The well documented photoreceptors include the flavin based blue light photoreceptor cryptochrome (Stanewsky, et al., 1998; Goto and
Denlinger, 2002a) and carotenoid/vitamin A associated opsin photoreceptors
(Denlinger, et al., 2005). The molecular study of timekeeping has been well developed in Drosophila malanogaster , which involves interactions of the clock genes period, timeless , clock , cycle are and others (Williams and Sehgal, 2001).
Hormonal control of diapause has been discussed in detail (Denlinger, et al.,
2005). The hormonal basis of diapause differs with both species and diapausing stages. For instance, in Bombyx mori , the neuropeptide diapause hormone plays a role in diapause induction (Yamashita and Hasegawa, 1985), while in Helicoverpa armigera (Zhang, et al., 2004) and Heliothis virescens (Xu and Denlinger, 2003), it is effective in breaking pupal diapause. Prothoracicotropic hormone (PTTH) is
3 the principle hormone controlling larval or pupal diapause in some species such as
Hyalophora cecropia (Agui et al., 1979), and Manduca sexta (Tomioka et al.,
1995); when PTTH is not released, the prothoracic gland fails to produce ecdysone and larval development is halted. Juvenile hormone (JH) is essential for reproduction in most species and plays a major role in regulating adult diapause characterized by suppression of reproduction (de Kort, 1990).
Diapause evokes a specific gene expression pattern, rather than simply shutting down the expression of many genes (Joplin et al., 1990; Flannagan et al.,
1998). Our understanding of the molecular basis of diapause has advanced considerably during the last decade (Denlinger et al., 1995; Flannagan et al., 1998;
Denlinger, 2000; Denlinger, 2002; Robich et al., 2005; Robich et al., 2007). Based on their expression patterns, diapause associated genes can be classified into
(i)genes upregulated throughout diapause, including the 70 kDa heat shock protein
(Hsp70) (Rinehart et al., 2000) and Hsp23 (Yocum et al., 1998), (ii) genes downregulated throughout diapause, including proliferating cell nuclear antigen
(PCNA) (Tammariello and Denlinger, 1998) and Hsp90 (Rinehart and Denlinger,
2000); (iii)genes unchanged during diapause including ecdysone receptor
(Rinehart et al., 2001), heat shock 70 cognate, and 28S ribosomal protein
(Rinehart et al., 2000); (iv)early diapause genes such as one encoding cystatin, a cysteine proteinase inhibitor (Goto and Denlinger, 2002b); (v)late diapause genes such as ultraspiracle (USP) (Rinehart et al., 2001); and (vi) those expressed
4 intermittently through diapause such as a gene encoding 60S ribosomal protein
PO, an apurinic/apyrimidinic endonuclease (Craig and Denlinger, 2000), as described in the flesh fly, Sarcophaga crassipalpis (Denlinger, 2002). Among these diapause associated genes, Hsps are a well known family of proteins upregulated in response to a variety of stresses. However, not all of the Hsps are upregulated in the flesh fly during diapause (Rinehart et al., 2007). Two members of Hsp70 family and at least four members of the small Hsp family are upregulated, while Hsp90 is downregulated during diapause. Upregulation of
Hsps during diapuse is not specific to the flesh fly, but is rather common in many other insect orders with different diapausing stages. Diapause related genes also can be categorized into different functional groups such as genes with regulatory functions, metabolically related genes, stress response genes, cytoskeletal genes, ribosomal genes, transposable elements, as represented in the northern house mosquito, Culex pipiens pipiens (Robich et al., 2007). Several critical diapause regulatory genes underpin the molecular basis of a metabolic switch from blood feeding to sugar gluttony and lipid sequestration in response to diapause in the mosquito (Robich and Denlinger, 2005). Trypsin and a chymotrypsin like protease, genes encoding enzymes for blood meal digestion, remain undetectable during diapause and begin to be expressed in females that are ready to blood feed upon diapause break, while fatty acid synthase, a gene involved in lipid accumulation is highly upregulated during diapause (Robich and Denlinger, 2005).
5 Metabolomics approach has recently been exploited to understand diapause
(Michaud and Denlinger, 2006; Podrabsky et al., 2007). In the flesh fly S. crassipalpis , fatty acids changed due to diapause, and the proportion of oleic acid
(18:1n 9) in diapausing pupae increased from 43% to 58% of the total fatty acid pool. Thus far, little research has been done with respect to diapause using proteomics technology (Joplin et al., 1990), and the work that was done, e. g.
Joplin et al., (1990), was done prior to the advent of modern proteomics technology that is now capable of identifying proteins that can be observed on a two dimensional gel.
1.2 Rapid Cold Hardening
Rapid cold hardening describes the various physiological and biochemical processes that increase an insect’s ability to tolerate low temperatures. Insects usually incur cellular injury, referred to as cold shock, when they are rapidly exposed to low temperatures, even at temperatures above the freezing point for extracellular fluids (Morris et al., 1983; Denlinger, 1991). Injury from cold shock can be significantly reduced by first conditioning insects for a short period at less severe low temperatures. Typically, the hardiness can be rapidly acquired by exposing insects between 0 °C and 10 °C for a short time from a few minutes to a few hours, which enhanced their subsequent survival at sub zero temperatures.
For instance, pharate adults of S. crassipalpis cannot survive direct exposure to
6 –10 °C, which is above the supercooling point, 23 °C (Lee and Denlinger, 1985;
Denlinger et al., 1991). But, if they are first transferred to 0 °C for 2 h, they readily survive exposure to –10 °C (Chen et al., 1987). This rapid cold hardening capability has been observed in several developmental stages within the same species. In S. crassipalpis , the capability was shown in feeding larvae, wandering larvae, diapausing and nondiapausing pupae, and pharate adults (Chen et al., 1987;
Lee et al., 1987). Other species of insects and other arthropods demonstrating such similar response include larvae of the pine needle gall midge, Thecodiplosis japonensis (Li et al, 1999), the kelp fly Paractora dreuxi (Terblanche et al., 2007), the Antarctic midge Belgica antarctica (Lee et al., 2006), the Antarctic mite
Halozetes belgicae (Hawes et al., 2007), pupae and pharate adults of S. bullata , adults of the elm leaf beetle Xanthogaleruca luteola (Lee et al., 1987 ), the milkweed bug Oncopeltus fasciatus (Lee et al., 1987 ), the fruit fly Drosophila melanogaster , (Czajka and Lee, 1990), the olive fruit fly Bactrocera oleae
(Koveos, 2001), the predatory mite Euseius (Amblyseius) finlandicus (Broufas and Koveos, 2001), the migratory locust Locusta migratoria (Wang and Kang,
2003), the grain aphid, Sitobion avenae (Powell and Bale, 2004;), the high Arctic collembolan, Hypogastrura tullbergi (Hawes et al. , 2006), the sub Antarctic springtail Tullbergia antarctica (Worland, 2005), a thrips Frankliniella occidentalis (McDonald et al., 1997), a Karoo beetle, Afrinus sp . (Sinclair and
Chown, 2006); a beetle Chirodica chalcoptera (Terblanche et al., 2005) , and
7 other coleopteran species (Burks and Hagstrum, 1999).
Protection against low temperature injury usually involves a gradual accumulation of a cryoprotectant such as glycerol and other low molecular weight polyhydric alcohols and sugars (Lee et al., 1987). Rapid accumulation of glycerol during rapid cold hardening has provided at least a partial basis for the cryoprotective response. Short term chilling contributes to rapid increase of glycerol in larvae and pharate adults in S. crassipalpis (Chen et al., 1987;
Denlinger et al., 1991). A 2 h exposure to 0 °C induced a two to three fold increase of glycerol in wandering larvae (18 to 43 mM) and pharated adults (28 to
81 mM). Meanwhile, pharate adults also increase hemolymph osmolatity from
309 to 346 mOsm, a response that may be attributed to an increase of glycerol.
Induced accumulation of glycerol due to cold treatment is also noted in larvae of S. bullat , and the brain plays a critical role in initiating glycerol production (Yoder et al., 2006). Interestingly, rapid cold hardening can also protect against other stresses such as desiccation. The concentrations of cryoprotectants including myo inositol, trehalose, mannitol, glycerol, and sorbitol, differ significantly with cooling rates in acclimated eggs of L. migratoria (Wang and Kang, 2005). Several articles illustrated how glycerol can protect against cold injury. Glycerol functions in lowering the supercooling point and prevents protein denaturation and membranes (Chen et al., 1987b; Tsvetkova and Quinn, 1994; Tang and Pikal,
2005; Michaud and Denlinger, 2006). Protection against injury afforded by rapid
8 cold hardening, however, cannot be fully explained by the accumulation of glycerol, because no changes of polyol levels were detected in some rapidly cold hardened insects (Kelty and Lee, 2001).
As temperatures decrease, cellular membranes tend to increase rigidity until the membrane changes from a liquid crystal to gel state and loses its function as a barrier (Cossins, 1983). The membrane can maintain the liquid crystalline state at lower temperature by a process of changing its composition known as homeoviscous adaptation or membrane compositional changes (Sinensky, 1974;
Hazel, 1995). The changes include increasing points of unsaturation along phospholipid fatty acid chains, increasing cholesterol content, and changing phospholipid class distribution (Thompson, 1983; Hazel, 1995; Michaud and
Denlinger, 2006). In the flesh fly S. crassipalpis , the membrane fatty acids change in response to rapid cold hardening, and the proportion of oleic acid in pharate adults increased from 30 to 47% of the total fatty acid pool with declined proportions of almost all the other fatty acids. Oleic acid enhances membrane fluidity and helps maintain a crystalline state of the membrane (Michaud and
Denlinger, 2006).
The physiological mechanisms for insect cold hardiness have been summarized by Michaud and Denlinger (2004). In response to or in preparation for low temperatures, insects produce polyols and other low molecular weight compounds such as the above mentioned glycerol, sorbitol, mannitol, trehalose
9 and proline (Chen and Denlinger, 1990; Lee, 1991; Misener et al., 2001). Insects can also be protected against injury from ice crystal formation by synthesizing antifreeze proteins and ice nucleating agents (Duman et al., 2004; Michaud and
Denlinger, 2004). Heat shock proteins (Hsps) are another diverse family of proteins manufactured by insects to increase cold hardiness. Hsps have been studied in depth in the flesh fly S. crassipalpis (Rinehart et al., 2000; Rinehart and
Denlinger, 2000; Yocum et al., 1998; Rinehart et al., 2007).
Both cold hardiness and diapause are essential to survive winter for most temperate insects. Denlinger (1991) clearly illuminated how these two are related.
Cold hardiness is not necessarily associated with diapause and can be acquired independently of diapause. This distinction is evident by the fact that cold hardening occurs in species lacking the genetic capacity for diapause, cold hardiness occurs in nondiapausing stages of diapausing species, and in rapidly cold hardened insects that are not in diapause. However, quite commonly cold hardiness is a component of the diapause syndrome and the expression of diapause enhances the insect’s capacity to cold harden. Flesh flies are good examples showing linkage of cold hardiness and the diapause program. Diapausing pupae have a higher level of cold hardiness than nondiapausing pupae reared at the same temperature (Adedokun and Denlinger, 1984; Lee and Denlinger, 1985). Even reared under a range of photoperiods and temperatures, pupae are consistently cold hardy if they are in diapause and less cold hardy if they are not in diapause
10 (Adekokun and Denlinger, 1984; Lee et al., 1988; Denlinger 1991). There is a tight linkage between cold hardening and diapause since the environmental cues for both are unlikely to be separated in Sarcophaga . Many other examples of interdependence of cold hardiness and diapause include species from different orders and species that diapause in different stages (Denlinger, 1991).
The ecological importance of rapid cold hardening capacity has been reviewed by Danks (2005). The response induced by different cooling rates in nature allows insects to adapt to short term temperature changes and increase their survival (Kelty and Lee, 1999; Powell and Bale, 2004). Furthermore, the rapid cold hardening response also preserves reproductive behaviors such as courtship and mating (Kelty and Lee, 2001; Shreve et al., 2004). Different responses are adaptive to the expected natural temperature at different stages (Wang and Kang,
2003; Powell and Bale, 2004). The response may be considerably important for enabling insects to survive short periods of low temperatures in spring and autumn, although it might not allow them to survive longer term extreme cold in winter
(Lee et al., 1987; Coulson and Bale, 1990).
1.3 The organisms used in this study
1.3.1 The flesh fly Sarcophaga crassipalpis
The flesh flies, Sarcophaga crassipalpis , feed on carrion as larvae. These flies are ovoviviparous, i.e., they lay active larvae of the first instar. The larva leaves
11 the food to pupariate at the late third instar stage. In the temperate regions, the fly exhibits a facultative diapause and photoperiod is the primary environmental cue to induce diapause, with temperature modifying the incidence of diapause
(Denlinger, 1972; Denlinger, 1981; Denlinger, 2005). A short photoperiod less than 13.5 h light/day and 4 day (the last 2 days of embryonic development and the first 2 days of larval life) exposure to these conditions is effective to initiate pupal diapause in the fly (Denlinger, 1971). Temperature plays an important role in diapause regulation, that is, low temperature increases the incidence and duration of diapause. Cold temperature in winter suppresses diapause termination thus ensuring survival of the overwintering period.
The neuroendocrine system plays a critical role in the programming of diapause (Denlinger, 2005). The brain is the primary site of photoreception. It can measure daylength and store the information for diapause initiation, which can be transferred between individuals by brain transplantation (Giebultowicz and
Denlinger, 1986).
Ecdysteroid is important for diapause regulation. Ecdysone synthesis and release by the prothoracic glands is stimulated by prothoracicotropic hormone from the brain. The pupal diapause of flesh flies lack ecdysteroid, with titers quickly dropping at diapause initiation and increasing at diapause termination
(Ohtaki and Takahashi, 1972; Walker and Denlinger, 1980). Ecdysteroid binds to the ecdysone receptor (EcR) and its dimerization partner ultraspiracle (USP).
12 Expression of ecr is not significantly altered throughout diapause. However, USP is affected by diapause, the expression gradually declines at the onset of diapause, rises during the late stages of diapause, and is highly upregulated upon termination of diapause. It is suggested that USP is essential to the maintenance and termination of diapause (Rinehart et al., 2001).
Regulation and the molecular mechanisms of diapause and cold tolerance have been studied fairly extensively in the flesh fly, using a variety of modern molecular techniques including PCR, Northern blotting, Western blotting, DNA microarray, metabolomics, as well as proteomics in our present study (Denlinger,
2002; Denlinger et al., 2005, Kostal, 2006, Michaud and Denlinger, 2006,
Fujiwara and Denlinger, 2007).
1.3.2 The Antarctic midge Belgica antarctica
The Antarctica is a frozen, isolated and inhospitable continent with very few terrestrial animals. The chironomid midge Belgica antarctica is an exception that has been able to colonize this contintent despite its extremely low temperature and low humidity (Convey, 1996; Campbell and Claridge, 1987). B. antarctica is the largest naturally occurring terrestrial animal on the Antarctic continent. Although it is endemic, it has a sporadic distribution on the continent (Usher and Edwards,
1984; Sugg et al., 1983). Such papers describe the life history and ecology of B. antarcitca in detail (Usher and Edwards, 1984; Sugg et al., 1983; Convey and
13 Block, 1996). Briefly, it is a holometabolous species with a 2 year life cycle, and it can overwinter in any of its four edaphic larval stages. Larvae feed on moss, algae, dead plant and animal materials and microorganisms. The larvae can tolerate freezing, desiccation, and all sorts of other environmental stresses.
Wingless adults mate in aggregations within one day of emergence, and females lay eggs within 1 2 days and live for fewer than two weeks in the summer.
How can overwintering larvae survive freezing during winter lows down to
40°C? They are protected against freezing due to thermal buffering provided by ice and snow covering and the surrounding seawater (Baust and Lee, 1981). In contrast to most temperate insects that dramatically increase cold tolerance before temperatures decrease in winter, this species maintains a fairly stable level of freeze tolerance throughout the year while it is sensitive to thermal stress (Baust and Lee, 1987). Rapid cold hardening response of B. antarctica has been investigated under both summer acclimatized and cold acclimated states (Lee et al., 2006). Adults lack the capacity for rapid cold hardening. Cold acclimated larvae have higher supercooling points and are more cold tolerant than summer larvae. Rapid cold hardening allows B. antarctica larvae to increase cold tolerance at the cellular level, even in the frozen state (Lee et al., 2006). Like low temperature extremes during the winter, B. antarctica confronts high temperature stress during the austral summer since the temperature of its habitat can reach 20
°C higher than summer air highs of 5 °C (Sugg et al., 1983). Larvae can maintain
14 a high intrinsic tolerance to heat stress by means of constitutive upregulation of heat shock proteins (Hsp), including small Hsps, Hsp70 and Hsp90, and thus preventing irreversible proteins aggregation resulting from stresses. On the contrary, adults have a low tolerance to high temperatures, but they can thermally activate their Hsps and generate thermoprotection accordingly (Rinehart et al.,
2006).
Other than temperature induced stresses, desiccation poses another common environmental stress to B. antarctica . The Antarctic continent is frozen and is effectively a desert, and water is biologically unavailable in the form of ice
(Campbell and Claridge, 1987; Hayward et al., 2007; Benoit et al., 2007a and b).
B. antarctica larvae are highly permeable with relatively high osmolality and lose water at a extremely high rate (Hayward et al., 2007; Benoit et al., 2007a). At high relative humidity (RH), this species has a slower rate of desiccation and high survival. Rehydration at 100% RH is more beneficial to larvae than direct contact with water. Desiccation enhances freeze tolerance of this polar insect (Hayward et al., 2007). The larvae exhibit a high level of dehydration tolerance and exploit multiple mechanisms to suppress dehydration (Benoit et al., 2007a). Larvae predominantly rely on drinking free water or ingesting food to replenish water stores. Water conservation can be achieved by clustering. The midges significantly increase their overall polysaccharide levels and accumulate trehalose and glycerol in response to slow dehydration (Hayward et al., 2007; Benoit et al., 2007a). A
15 shift from short to long hydrocarbons may contribute to water retention as larvae dehydrate (Michaud and Denlinger, 2007). A decrease in oxygen consumption leads to reduced water loss. The capacity for drought acclimation ameliorates the stress of drying.
The adult midges are very hygric and gain water mainly by free water uptake as observed in larvae (Benoit et al., 2007b). Adults emerge during very brief period only when free water is readily accessible, and moist microhabitats are vital to protect them against desiccation.
1.4 Research goals
Proteomics, the large scale study of proteins, is a young technology and many of the tools for protein purification and identification were developed during the
1980s and early 1990s. But, it has proven to be a powerful technique for monitoring protein changes of cells, tissues or even whole bodies associated with different conditions. Most molecular studies related to diapause done so far are based on the work at the genome and transcript levels. Therefore, we feel it necessary and important to investigate the molecular mechanisms of diapause using proteomic tools because proteins are the final products of gene activation.
My research focuses on exploring diapause associated proteins in the flesh fly,
S. crassipalpis and a study of desiccation and rehydration in the Antarctic midge,
B. antarctica , using a proteomic approach, and characterization of some
16 diapause related genes. The goals of my research are:
1) To determine diapause specific brain proteins and the functional implications of those proteins during pupae diapause in the flesh fly, S. crassipalpis.
2) To identify pupal brain proteins involved in diapause termination in the flesh fly,
S. crassipalpis at select times immediately before and after diapause termination.
3) To study protein changes during rapid cold hardening in pharate adults of the flesh fly, S. crassipalpis .
4) To analyze the protein pattern changes due to desiccation and rehydration in the
Antarctic midge, B. antarctica.
5) To clone and evaluate the role of a potential diapause regulatory gene
Neuropeptide like precursor 4.
17
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21 Kelty, J.D., Lee Jr., R.E., 2001. Rapid cold hardening of Drosophila melanogaster (Diptera: Drosophilidae) during ecologically based thermoperiodic cycles. Journal of Experimental Biology 204, 1659–1666. Kostal, V., 2006. Eco physiological phases of insect diapause. Journal of Insect Physiology 52, 113 127. Koveos, D. S., 2001. Rapid cold hardening in the olive fruit fly Bactrocera oleae under laboratory and field conditions. Entomologia Experimentalis et Applicata 101, 257 263. Lee, R. E., Jr., Denlinger, D. L., 1985. Cold tolerance in diapausing and non diapausing stages of the flesh fly, Sarcophaga crassipalpis . Physiological Entomology 10,309 315. Lee, R. E., Chen, C. P., Denlinger, D. L., 1987. A rapid cold hardening process in insects. Science 238, 1415 1417. Lee, R. E., Denlinger D. L., Chen, C. P. , 1988. Insect cold hardiness and diapause: Regulatory relationships. In: Endocrinological Frontiers in Physiological Insect Ecology (eds. F. Sehnal, A. Zabza and D. L. Denlinger), pp. 243 262. Wroclaw Technical University Press, Wroclaw, Poland. Lee, R. E., 1991. Principles of insect low temperature tolerance. In: Lee RE, Denlinger DL, editors. Insects at low temperature. New York: Chapman and Hall, pp. 17 46. Lee, R. E., Elnitsky, M. A., Rinehart, J. P., Hayward, S. A. L., Sandro, L. H., Denlinger, D. L., 2006. Rapid cold hardening increases the freezing tolerance of the Antarctic midge Belgica antarctica . Journal of Experimental Biology 209, 399 406. Li, Y. P., Gong, H., Park, H. Y., 1999. Characterization of rapid cold hardening response ln the overwintering mature larvae of pine needle gall midge, Thecodiplosis japonensis . Cryo Letters 20, 383 392. McDonald, J. R., Bale J. S.and Walters K. F. A., 1997. Rapid cold hardening in the western flower thrips Frankliniella occidentalis . Journal of Insect Physiology 43,759 766. Michaud, M.R., Denlinger, D.L., 2004. Molecular modalities of insect cold survival: current understanding and future trends. In: Animals and Environments,
22 Morris, S., Vosloo, A, eds. Elsevier, Amsterdam. pp. 32 46. Michaud, M. R., Denlinger, D. L., 2006. Oleic acid is elevated in cell membranes during rapid cold hardening and pupal diapause in the flesh fly, Sarcophaga crassipalpis . Journal of Insect Physiology 52, 1073 1082. Misener, S. R., Chen, C. P., Walker, V. K., 2001. Cold tolerance and proline metabolic gene expression in Drosophila melanogaster . Journal of Insect Physiology 47, 393 400. Morris, G. J., Coulson, G., Meyer, M. A., Mclellan, M. R., Fuller, B. J., Grout, B. W. W., Pritchard, H. W., and Knight S. C., 1983. Cold shock a widespresd cellular reaction. Cryo Letter 4, 179 192. Ohtaki, T., Takahashi, M., 1972. Induction and termination of pupal diapause in relation to the change of ecdysone titer in the fleshfly, Sarcophaga peregrina . Japanese Journal of Medical Sciences and Biology 25, 369–376. Podrabsky, J. E., Lopez, J. P., Fan, T. W. M., Higashi, R., Somero, G. N., 2007. Extreme anoxia tolerance in embryos of the annual killifish Austrofundulus limnaeus : insights from a metabolomics analysis. Journal of Experimental Biology 210, 2253 2266. Powell, S. J., Bale, J. S., 2004. Cold shock injury and ecological costs of rapid cold hardening in the grain aphid Sitobion avenae (Hemiptera : Aphididae) Journal of Insect Physiology 50, 277 284. Powell, S. J., Bale, J. S., 2006. Effect of long term and rapid cold hardening on the cold torpor temperature of an aphid. Physiological Entomology 31, 348 352. Rinehart, J. P., Yocum, G. D., Denlinger, D. L., 2000. Developmental upregualtion of inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh fly, Sarcophaga crassipalpis . Insect Biochem Mol Biol 30, 518 521. Rinehart J. P., Denlinger D. L., 2000. Heat shock protein 90 is downregulated during pupal diapause in the flesh fly, Sarcophaga crassipalpis , but remains responsive to thermal stress. Insect Molecular Biology 9, 641–645 Rinehart, J. P., Yocum, G. D., Denlinger, D. L., 2000. Developmental upregulation of inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh fly, Sarcophaga crassipalpis . Insect Biochemistry and
23 Molecular Biology 30,515 521. Rinehart, J. P., Cikra Ireland, R. A., Flannagan, R. D., Denlinger, D. L., 2001. Expression of ecdysone receptor is unaffected by pupal diapause in the flesh fly, Sarcophaga crassipalpis , while its dimerization partner, USP, is downregulated. Journal of Insect Physiology 47, 915 921. Rinehart, J. P., Hayward, S. A. L., Elnitsky, M. A., Sandro, L. H, Lee, Jr., R. E., Denlinger, D. L., 2006. Continuous up regulation of heat shock proteins in larvae, but not adults, of a polar insect. Proceedings of the National Academy of Sciences of the United States of America 103, 14223 14227. Rinehart, J. P., Li, A., Yocum, G. D., Robich, R. M., Hayward, S. A. L., Denlinger, D. L., 2007. Up regulation of heat shock proteins is essentail for cold survival during insect diapause. Proceedings of the National Academy of Sciences of the United States of America 104, 11130 11137. Robich, R. M., Denlinger, D. L., 2005. Diapause in the mosquito Culex pipiens evokes a metabolic switch from blood feeding to sugar gluttony. Proceedings of the National Academy of Sciences of the United States of America 102, 15912 15917. Robich, R. M., Rinehart, J. P., Kitchen, L. J., Denlinger, D. L., 2007. Diapause specific gene expression in the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization. Journal of Insect Physiology 53, 235 245. Shreve, S. M., Kelty, J. D., Lee Jr., R. E., 2004. Preservation of reproductive behaviors during modest cooling: rapid cold hardening fine tunes organismal response. Journal of Experimental Biology 207, 1797 1802. Sinclair, B. J., Chown, S. L., 2006. Rapid cold hardening in a Karoo beetle, Afrinus sp. Physiological Entomology 31, 98 101. Sinensky, M., 1974. Homeoviscous adaptation—a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli. Proceedings of the National Academy of Sciences, USA 71, 522 525. Sugg, P., Edwards, J. S., Baust, J., 1983. Phenology and life history of Belgica antarctica , an Antarctic midge (Diptera: Chironomidae). Ecol. Entomol. 8, 105 113.
24 Tammariello, S. P., Denlinger, D. L., 1998. G0/G1 cell cycle arrest in the brain of Sarcophaga crassipalpis during pupal diapause and the expression pattern of the cell cycle regulator, proliferating cell nuclear antigen. Insect Biochemistry and Molecular Biology 28,83 89. Tang, X., Pikal, M.J., 2005. The effects of stabilizers and denaturants on the cold denaturation temperatures of proteins and implications for freeze drying. Pharmaceutical Research (Dordrecht) 22, 1167–1175. Tauber, M. J., Tauber, C. A. & Masaki, S. (1986) Seasonal Adaptations of Insects (Oxford Univ. Press, New York). Terblanche, J. S., Sinclair, B. J., Klok, C. J., McFarlane, M. L., Chown,S. L., 2005. The effects of acclimation on thermal tolerance, desiccation resistance and metabolic rate in Chirodica chalcoptera (Coleoptera : Chrysomelidae). Journal of Insect Phyiology 51, 1013 1023. Terblanche, J. S., Marais, E., Chown, S. L., 2007. Stage related variation in rapid cold hardening as a test of the environmental predictability hypothesis. Journal of Insect Phyiology 53, 455 462. Thompson, Jr., G. A., 1983. Mechanisms of homeoviscous adaptation in membranes. In: Cossins, A.R., Sheterline, P. (Eds.), Cellular Acclimatisation to Environmental Change. Cambridge University Press, Cambridge, pp. 33–54. Tomioka, K., Agui, N., Bollenbacher, W. E., 1995. Electrical properties of the cerebral prothoracicotropic hormone cells in diapausing and non diapausing pupae of the tobacco hornworm, Manduca sexta . Zoological Science 12, 165 173. Tsvetkova, N.M., Quinn, P.J., 1994. Compatible solutes modulate membrane lipid phase behaviour. In: Cossins, A.R. (Ed.), Temperature Adaptation of Biological Membranes. Portland Press, London, pp. 49–62. Stanewsky, R., Kaneko, M., Emery, P., Beretta, B., Wager Smith, K., Kay, S. A., Rosbash, M., Hall, J. C., 1998. The cry(b) mutation identifies cryptochrome as a circadian photoreceptor in Drosophila . Cell 95, 681 692. Usher, M. B., Edwards, M., 1984. A dipteran from south of the Antarctic Circle: Belgica antarctica (Chironomidae), with a description of its larva. Biological Journal of the Linnean Society 23, 19 31. Walker, G. P. and Denlinger, D. L., 1980. Juvenile hormone and moulting
25 hormone titres in diapause and non diapause destined flesh flies. Journal of Insect Physiology 26, 661 664. Wang, X. H., Kang, L., 2003. Rapid cold hardening in young hoppers of the migratory locust Locusta migratoria L. (Orthoptera : Acridiidae). Cryoletters 24, 331 340. Wang, H.S., Kang ,L., 2005. Effect of cooling rates on the cold hardiness and cryoprotectant profiles of locust eggs. Cryobiology 51, 220–229. Williams, J. A., Sehgal, A., 2001. Molecular components of the circadian system in Drosophila . Annual Review of Physiology 63, 729 755. Worland, M. R., 2005. Factors that influence freezing in the sub Antarctic springtail Tullbergia Antarctica . Journal of Insect Phyiology 51, 881 894. Xu, W. H., Denlinger, D. L., 2003. Molecular characterization of prothoracicotropic hormone and diapause hormone in Heliothis virescens during diapause, and a new role for diapause hormone. Insect Molecular Biology 12, 509 516. Yocum, G. D., Joplin, K. H., Denlinger, D. L., 1998. Upregulation of a 23 kDa small heat shock protein transcript during pupal diapause in the flesh fly, Sarcophaga crassipalpis . Insect Biochemistry and Molecular Biology 28, 677 682. Yoder, J. A., Benoit, J. B., Denlinger, D. L., Rivers, D. B., 2006. Stress induced accumulation of glycerol in the flesh fly, Sarcophaga bullata : Evidence indicating anti desiccant and cryoprotectant functions of this polyol and a role for the brain in coordinating the response. Journal of Insect Physiology 52, 383 392. Zhang, T. Y., Sun, J. S., Zhang, L. B., Shen, J. L., Xu, W. H., 2004. Cloning and expression of the cDNA encoding the FXPRL family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera . Journal of Insect Physiology 50, 25 33.
26
CHAPTER 2
PROTEOMICS OF THE FLESH FLY BRAIN REVEALS AN ABUNDANCE
OF UPREGULATED HEAT SHOCK PROTEINS DURING PUPAL DIAPAUSE
A.Q. Li 1, A. Popova Butler 2, D. H. Dean 1, 2 , and D. L. Denlinger 1, *
1Department of Entomology, 2Department of Biochemistry, The Ohio State
University, Columbus, OH 43210, USA
*Corresponding author:
Tel: +1 6142928209
Fax: +16142927865
Email: [email protected]
27 Abstract
Most molecular work on insect diapause has focused on the expression of unique diapause transcripts, rather than the protein products. Here we present results from a proteomic comparison of diapausing and nondiapausing pupal brains. Proteins extracted from diapausing pupal brains of the flesh fly
Sarcophaga crassipalpis were separated by two dimensional gel electrophoresis and compared with those from nondiapausing pupal brains. Unique proteins and proteins present at different levels of abundance in diapausing and nondiapausing brains were identified by Nano LC/MS/MS (capillary liquid chromatography nanospray tandem mass spectrometry). With this approach and
Coomassie staining we detected 37 diapause unique or upregulated (≥2x) proteins, and 43 proteins that were downregulated or not present in diapause. Heat shock proteins (Hsp70 and several small Hsps) were among the most conspicuous brain proteins present in higher amounts during diapause. Brain proteins that were less abundant in diapause included phosphoenolpyruvate synthase, fatty acid binding protein, EG0003.7, and an endonuclease. Our 2 D proteome maps included several additional unknown proteins that are more abundant in either the diapause or nondiapause brains. While the mRNAs encoding some of these proteins (e.g.Hsps) were previously known to be associated with diapause, the other proteins were not known to be linked to diapause, thus suggesting that the proteomic approach nicely supplements work done at the transcript level.
28 Key words: proteomics; diapause; nondiapause, brain proteins, Sarcophaga crassipalpis
29 2.1. Introduction
One of the first approaches used by our laboratory to probe molecular changes associated with diapause was the use of two dimensional electrophoresis of radio labeled brain proteins in the flesh fly pupae to determine whether brains from diapausing fly pupae were making different proteins than those produced by nondiapausing pupae (Joplin et al., 1990). Although it was evident from this earlier work that the brains of diapausing pupae are indeed synthesizing distinct proteins, the technology at that time did not allow us to identify such proteins.
With the advent of capillary liquid chromatography nanospray tandem mass spectrometry it is now possible to identify the small quantities of proteins that can be detected with two dimensional gel electrophoresis. The goal of the present study was to exploit this technique to identify the proteins that are more or less abundant in the brains of diapausing pupae.
As in most insects (Denlinger et al., 2005), the brain of the flesh fly presides over the decision to enter and terminate diapause (Giebultowicz and Denlinger,
1986), thus events occurring in the brain are especially critical for unraveling the regulation of this overwintering, developmental arrest. Though a number of genes that are differentially expressed in relation to diapause have now been identified (Denlinger, 2002), most of this work has stopped at the mRNA level.
We usually assume that the presence of transcripts implies that translation is also occurring, but clearly that is not always the case (Gygi et al., 1999; Renaut et al.,
30 2006). In this study, using a proteomic analysis, we augment our genomic database with information on the proteins that are present in association with diapause. Our examination of the brain proteins not only allows us to verify what proteins are present during diapause, but this approach can also suggest the possibility that certain of those proteins may be post translationally modified. We report the presence of a preponderance of heat shock proteins (Hsps) during diapause, an observation that supports our previous mRNA results (Yocum et al.,
1998, Rinehart et al., 2000), yet goes far beyond our previous results by revealing the presence of many additional Hsps that are extremely abundant during diapause.
In addition, we identify a number of down regulated proteins not previously associated with diapause.
2.2. Material and methods
2.2.1 Fly rearing
The colony of S. crassipalpis was maintained as previously described
(Denlinger, 1972). Nondiapausing pupae were produced by rearing the adults under long day conditions (15h light: 9h dark) at 25°C and the larvae and pupae at
20°C under the same long daylength. Diapausing pupae were produced by rearing the adults under short day conditions (12h light: 12h dark) at 25°C and their progeny at 20°C under the same short daylength.
31
2.2.2 Protein preparation
Twenty pupal brains were dissected 5 days after pupariation for nondiapause samples or 20 days after pupariation for diapause samples. On day 5, the nondiapausing pupae are at a developmental stage equivalent to diapause. The brains were directly placed in a cell lysis buffer consisting of 50 mM Tris Cl pH7.5, 100 mM NaCl, 1% (v/v) Triton X 100, 1 mM EDTA, 1 mM EGTA and 50 mM β glycerophosphate. Fresh 1 mM dithiothertol (DTT) and a protease inhibitor mixture containing 1 mM PMSF, 2 mM sodium orthovanadate, 10 g/ml leupeptin, 10 g/ml pepstatin A, and 10 g/ml aprotinin (Sigma, St. Louis) were added to all buffers prior to use. Samples were homogenized for 2 min using a motor driven pestle, cleaned using a ReadyPrepTM 2D cleanup kit, and rehydrated in ReadyPrep 2 D starter kit rehydration/sample buffer (8M urea, 2%
CHAPS, 50mM DTT, 0.2% Bio Lyte 3/10 ampholyte, 0.001% Bromophenol
Blue). Protein concentrations in the samples were determined by a RC/DC TM protein assay after rehydration. All chemicals and kits were supplied by BioRad
(Hercules, CA) unless otherwise specified.
2.2.3 Two dimensional gel electrophoresis
The manufacturer’s recommended volume (185 l, containing approximately
32 400 g protiens) of recovered proteins was applied to 11 cm IPG strips (BioRad), pH 3 10, for passive overnight rehydration. The IPG strips were then subjected to isoelectric focusing using a Protean IEF Cell. Focusing was performed as follows: 400 V for 20 min, 8,000 V for 2.5 h and then up to 20,000 V hr for approximately 3 h. Current did not exceed 50 A per strip.
After isoelectric focusing, the IPG strips were equilibrated for 15 min in equilibration buffer I (6 M urea, 2% (w/v) SDS, 0.375 M Tris HCl (pH 8.8), 20%
(v/v) glycerol and 2% (w/v) 1,4 dithiothreitol (DTT)) followed by 15 min in 6 M urea in buffer II (same as buffer I but containing 2.5% iodacetamide instead of
DTT).
For the second dimension, IPG strips were placed across a Criterion Precast
Gel, overlayed with agarose. Electrophoresis was run with a constant voltage,
200V, for 1 hr in Tris glycine buffer (25mMTris, 192 mM glycine, 0.1 % SDS. pH
8.3).
Gels were fixed overnight in a solution of 50 % (v/v) ethanol and 10% (v/v) acetic acid, stained with Bio safe Coomassie blue stain (BioRad) for at least one hour and washed in water.
At least three independent samples were analyzed for each treatment.
Gel images were scanned with a BioRad VersaDoc model 1000 imaging system and analyzed with PDQuest software according to the protocols provided
33 by BioRad. Protein spots were matched automatically using three gels from nondiapausing brains and three gels from diapausing brains. Spot intensities were normalized to make the total density in each gel image equal, and analysis was performed using quantitative and qualitative modes. A spot was detected when its intensity was 10 fold or more above background. Following reproducibility tests for up and down regulation of proteins in samples from diapausing pupae, the confidence threshold was set at 2 fold below or above the spot intensity observed in nondiapause samples.
2.2.5 In Gel Digestion
Gels were digested with sequencing grade trypsin from Promega (Madison
WI), using the Montage In Gel Digestion Kit from Millipore (Bedford, MA) following the manufacturer’s recommended protocols. Briefly, bands were trimmed as close as possible to minimize background polyacrylamide material.
The gels were washed in 50% methanol/5% acetic acid for several hours. The gel bands were dried with acetonitrile and reconstituted with DTT solution to reduce the cysteines. Iodoacetamide was added to alkylate the cysteines, and the gel was washed again with cycles of acetonitrile and ammonium bicarbonate. Trypsin was added and digested at room temperature overnight. The peptides were extracted from the polyacrylamide with 50% (v/v) acetonitrile and 5% (v/v) formic acid
34 several times, and the extracted pools were concentrated in a SpeedVac (Savant) to ~25 L.
2.2.6 Protein Identification
Capillary liquid chromatography nanospray tandem mass spectrometry
(Nano LC/MS/MS) was performed on a Thermo Finnigan LTQ mass spectrometer equipped with a nanospray source operated in a positive ion mode. The LC system was an UltiMate™ Plus system from LC Packings A Dionex Co (Sunnyvale, CA) with a Famos autosampler and Switchos column switcher. Solvent A was water containing 50 mM acetic acid, and solvent B was acetonitrile. Five microliters of each sample were first injected into the trapping column (LC Packings A Dionex
Co, Sunnyvale, CA) and washed with 50 mM acetic acid. The peptides were eluted off the trap onto the column. A 5 cm 75 m ID ProteoPep II C18 column
(New Objective, Inc. Woburn, MA) packed directly in the nanospray tip was used for chromatographic separations. Peptides were eluted directly off the column into the LTQ system using a gradient of 2 80% solvent B over 30 min, with a flow rate of 300 nl/min and a total run time of 58 min. The scan sequence of the mass spectrometer was based on the TriplePlay™ method; briefly the analysis was programmed for a full scan, a zoom scan to determine the charge of the peptide and a MS/MS scan to generate product ion spectra to determine amino acid
35 sequence in consecutive instrument scans of the most abundant peak in the spectrum. Dynamic exclusion was used to exclude multiple MS/MS of the same peptide.
2.2.7 Database Search
Sequence information from the MS/MS data was processed using a Turbo
SEQUEST algorithm in BioWorks 3.1 Software. Data processing was performed following the guidelines by Carr et al. (2004). Assigned peaks have a minimum of
10 counts (S/N of 3). The mass accuracy of the precursor ions was set to 1.5 Da to accommodate accidental selection of the C13 ion, and the fragment mass accuracy was set to 0.5 Da. Considered modifications were methionine oxidation (variable) and carbamidomethyl cysteine (fixed).
2.3. Results
2.3.1 Protein maps of brains from diapausing and nondiapausing pupae
Approximately 440 individual spots (ND 449 and D 434) with molecular masses from 15 180kDa were detected in both the diapausing and nondiapausing brains with 2DE separations using Coomassie stain (Figs. 2.1A and 2.1B). To confirm the protein expression profiles, a second set of gels loaded with 100 g of proteins was stained with Sypro Ruby (BioRad). The Sypro Ruby staining yielded
36 approximately 1.5X more spots due to its higher sensitivity, and nearly all of the spots evident with Coomassie staining were also observed in the Sypro
Ruby stained gels (data not shown).
2.3.2 Protein differences between diapausing and nondiapausing brains
Qualitative and quantitative comparisons were made between the brain proteins from diapausing and nondiapausing flies using PDQuest software. A comparison of the location and volumes of each spot showed that the majority of brain proteins remained virtually unchanged between diapausing and nondiapausing flies (Fig.2.1). Eighty spots out of 449 total spots in
Coomassie stained samples showed 2 fold or higher differences between the two samples. Samples of diapausing brains contained 3 unique and 34 upregulated protein spots, and 5 missing and 38 downregulated protein spots.
Only the relatively abundant proteins, those stained with Coomassie and having at least a 2 fold difference between diapause and nondiapause in all three replicates, were selected for identification. These spots, 14 from diapause and 4 from nondiapause, are indicated in Figures 2.1A and 2.1B.
2.3.3 Identification of regulated proteins
Regulated spots from Coomassie stained gels were excised and analyzed by mass spectrometry. Spot volume comparisons of the selected spots (Fig. 2.2)
37 indicated pronounced differences in the amounts of specific proteins present in the brains of diapausing and nondiapausing pupae. All of the diapause upregulated proteins that have been identified show highest identity to heat shock proteins: two show highest identity to the Hsp70 family and six show highest identities to the small Hsp family (Table 2.1). Five (spot 10 14) of the diapause unique or upregulated proteins showed no identity to other known proteins, and one protein, identified as keratin (spot 9), is assumed to be a contaminant. The diapause downregulated proteins show highest identity to phosphoenolpyruvate synthase, fatty acid binding protein, EG: EG 0003.7 and an endonuclease (Table 2.1).
2.4. Discussion
The appeal of a proteomics analysis is, of course, that it is one step closer to functionality than a study based on mRNA. While mRNA based studies are enormously useful, one cannot be certain that the transcripts are actually translated unless the work is complemented with protein studies (e.g., Gygi et al., 1999;
Renaut et al., 2006). By working at the protein level it is also possible to detect multiple forms of a protein that may be the consequence of families of closely related proteins or post translational modifications.
Though we previously reported the presence of diapause specific proteins present in the brains of flesh fly pupae (Joplin et al., 1990), at that time the technology did not allow us to identify the proteins of interest. We report slightly
38 different numbers of diapause unique or upregulated proteins in these two studies,
14 in Joplin et al. (1990) and 37 in the current paper, but the two studies are not identical. The previous paper used radio labeled methionine to identify proteins that were currently being made by the brain, while in the current study we used
Coomassie staining to identify proteins that are present, not simply those that are being made currently. Our earlier study that used radiolabeling demonstrated that far fewer proteins are being synthesized in the brain during diapause, but in this study we detected nearly the same number of proteins in the brains of diapausing and nondiapausing pupae. A comparison of the results of these two protocols suggests that many of the proteins present in the brain may be synthesized before the onset of diapause or may be synthesized only periodically.
Both are likely. There is indeed evidence that at least some proteins are synthesized periodically during pupal diapause in flesh flies; peak periods of protein synthesis coincide with peaks in the infradian cycles of oxygen consumption that are characteristic of this diapause (Joplin and Denlinger, 1989).
The protein database for S. crassipalpis and other flesh flies is poor, thus the task of identifying the proteins with certainty remains a challenge. In this study, 2
(spot 10 and 11) of the diapause unique and 3 (spot 12 14) of the diapause upregulated proteins that were quite abundant could not be identified.
Eight of the diapause abundant proteins could be identified with a fair level of confidence, and all showed highest identity to heat shock proteins. Two of these
39 showed highest identities to Hsp70 and the other 6 were most identical to Hsp23, one of the small heat shock proteins previously identified in S. crassipalpis . The two spots identified as Hsp70s (spot 1 and 2) are directly beside each other on the gel, suggesting that they represent one protein with different post translational modifications such as phosphorylation or glycosylation, but we cannot eliminate the possibility that they are two distinct proteins. By contrast, the spots with high identity to Hsp23 (spot 3 8) are not present in “chains” that typically represent one protein but are widely distributed across the gel, suggesting it is more likely that they are distinct members of the small Hsp family. Though Hsp23 is the only small Hsp previously reported in S. crassipalpis (Yocum et al., 1998), we have unpublished evidence that additional small Hsp transcripts are present in this species, as is well known for other organisms (Taylor and Benjamin, 2005; Sun and MacRae, 2005).
The presence of the Hsps in the brains of diapausing pupae nicely supports our previous evidence that the mRNAs encoding Hsp70 (Rinehart et al., 2000;
Hayward et al., 2005) and Hsp23 (Yocum et al., 1998; Hayward et al., 2005) are highly upregulated during diapause. This is our first evidence that these transcripts are indeed translated. The Hsps are upregulated in several other diapausing insects as well (Denlinger et al., 2001), and in dormant stages of some other organisms such as the brine shrimp Artemia franciscana (MacRae, 2003). We have proposed that the upregulated Hsps serve a dual function during diapause,
40 both conferring cold tolerance and helping to suppress development (Denlinger et al., 2001). Our unpublished results using RNA interference (RNAi) demonstrate a significant loss of cold tolerance when RNAi is used to knock down expression of
Hsp70. We thus speculate that Hsp70, and possibly other Hsps, play an essential role in enabling the diapausing fly pupae to survive for long periods at low temperature. The developmental upregulation of Hsps during the many months of diapause is an unusual pattern of expression that contrasts to the better known rapid response of these chaperone molecules to heat stress and other environmental insults (Feder and Hoffman, 1999). The fact that the expression of
Hsps is incompatible with the progression of development (Krebs and Feder, 1997) suggests that the presence of Hsps during diapause may also function in arresting development. What is clear from the present study is that there are many more
Hsps or post translationally modified Hsps present during diapause than we had anticipated. Whether these different Hsps are playing distinct roles in diapause or simply represent redundancy in function is an intriguing question, but what is clear is that this result portends an unexpected complexity in the diapause response.
Three of the identified proteins shown to be less abundant in brains of diapausing pupae , i.e. more abundant in brains from nondiapausing pupae, are related to metabolic processes that are most likely less active during diapause.
These include phosphoenolpyruvate (PEP) synthase, an enzyme involved in
41 gluconeogenesis, a fatty acid binding protein, and an endonuclease. Another downregulated protein has high identity to a protein known from Drosophila melanogaster (EG: EG0003.7) but the function of this protein is unknown.
Diapause is characterized by a major shut down in metabolism and thus it is not surprising that proteins associated with metabolic activity would be less abundant.
Proteins that are less abundant in diapause could potentially be just as important as proteins that are unique to diapause or are more abundant in diapause. At the transcript level, for example, the downregulation of the cell cycle regulator,
Proliferating Cell Nuclear Antigen, appears to be a key feature shutting down the cell cycle during diapause in S. crassipalpis (Tammariello and Denlinger, 1998;
Hayward et al., 2005). But, none of the proteins identified in this study are likely to exert a regulatory role.
We assume the presence of keratin in our samples is a contaminant, as is common in proteomic studies, but we are intrigued with the fact that it consistently was more abundant in our diapausing samples, and its closest identity was to a type I microfibrillar 48 kDa keratin from sheep, rather than humans.
The fact that there are reports of a keratin like protein in the taenidia of insect trachea (Baccetti et al., 1984a) and spermatozoa (Baccetti et al., 1984b) suggests that perhaps we should not summarily dismiss the possibility that such a protein is present in the diapausing brain.
Although the proteins observed in this study represent only the most abundant
42 proteins, we assume that the number of diapause upregulated or unique proteins observed in this study (37 out of 449 total proteins, 8.2%) is indicative of the proportion of upregulated proteins that would be present in a more complete profile of brain proteins. This estimate is slightly higher than a previous calculation suggesting that approximately 4% of the transcripts are diapause upregulated (Flannagan et al., 1998). What is perhaps remarkable is that so few differences are evident between these two developmental states that are so profoundly different.
Acknowledgements
The authors thank David Mandich of the Plant Microbe Genomics Facility for assistance with image analysis and Kari Green Church of the Campus Chemical
Instrument Center for MS protein identification. This project was supported in part by NSF grant IOB 0416720.
43 References Baccetti, B., Burrini, A.G., Gabbiani, G., Leoncini, P., 1984a. Insect tracheal taenidia contain a keratin like protein. Physiological Entomology 9, 239 245. Baccetti, B., Burrini, A.G., Gabbiani, G., Leoncini, P., Runggerbrandle, E., 1984b. Filamentous structures containing a keratin like protein in spermatozoa of an insect, Bacillus rossius . Journal of Ultrastructure Research 86, 86 92. Carr, S., Aebersold, R., Baldwin, M., Burlingame, A., Clauser, K., Nesvizhskii, A., 2004. The need for guidelines in publication of peptide and protein identification data. Molecular and Cellular Proteomics 3, 531 533. Denlinger, D.L., 1972. Induction and termination of pupal diapause in Sarcophaga (Diptera: Sarcophagidae). Biological Bulletin, Woods Hole 142, 11 24. Denlinger, D.L., 2002. Regulation of diapause. Annual Review of Entomology 47, 93 122. Denlinger, D.L., Rinehart, J.P., Yocum, G.D., 2001. Stress proteins: a role in insect diapause? In: Denlinger, D. L., Giebultowicz, J. M., Saunders, D. S. (Eds.), Insect Timing: Circadian Rhythmicity to Seasonality. Elsevier Science, Amsterdam, pp. 155 171. Denlinger, D.L., Yocum, G.D., Rinehart, J.P., 2005. Hormonal control of diapause. In: Gilbert, L. I., Iatrou, K., Gill, S. (Eds.), Comprehensive Molecular Insect Science, Vol.3. Elsevier, Amsterdam, pp. 615 650. Feder, M.E., Hoffmann, G.E., 1999. Heat shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annual Review of Physiology 61,243 282. Flannagan, R.D., Tammariello, S.P., Joplin, K.H., Cikra Ireland, R.A., Yocum, G.D., Denlinger, D.L., 1998. Diapause specific gene expression in pupae of the flesh fly Sarcophaga crassipalpis. Proceedings of the National Academy of Sciences of the United States of America 95, 5616 5620. Giebultowicz, J.M. Denlinger, D.L., 1986. Role of the brain and ring gland in relation to pupal diapause in the flesh fly, Sarcophaga crassipalpis . Journal of Insect Physiology 32,161 166.
44 Gygi, S.P., Rochon, Y., Franza, B.R., Aebersold, R., 1999. Correlation between protein and mRNA abundance in yeast. Moleculae and Cellulae Biology 19, 1720 1730. Hayward, S.A.L., Pavlides, S.C., Tammariello, S.P., Rinehart, J.P., Denlinger, D.L., 2005. Temporal expression patterns of diapause associated genes in the flesh fly pupae from the onset of diapause through post diapause quiescence. Journal of Insect Physiology 51, 631 640. Joplin, K.H., Denlinger, D.L., 1989. Cycles of protein synthesis during pupal diapause in the flesh fly, Sarcophaga crassipalpis . Archives of Insect Biochemistry and Physiology 12, 111 122. Joplin, K.H., Yocum, G.D., Denlinger, D.L., 1990. Diapause specific proteins expressed by the brain during the pupal diapause of the flesh fly, Sarcophaga crassipalpis . Journal of Insect Physiology 36, 775 783. Krebs, R.A., Feder, M. E., 1997. Deleterious consequences of Hsp70 overexpression in Drosophila melanogaster larvae. Cell Stress & Chaperones 2, 60 71. MacRae, T.H., 2003. Molecular chaperones, stress resistance and development in Artemia franciscana . Seminars in Cell and Development Biology 14, 251 258. Renaut, J., Hausman, J.F., Wisniewski, M.E., 2006. Proteomics and low temperature studies: bridging the gap between gene expression and metabolism. Physiologia Plantarum.126, 97 109. Rinehart, J.P., Yocum, G.D., Denlinger, D.L., 2000. Developmental upregulation of inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh fly, Sarcophaga crassipalpis . Insect Biochemistry and Molecular Biology 30, 515 521. Sun, Y., MacRae, T.H., 2005. Small heat shock proteins: molecular structure and chaperone function. Cellular and Molecular Life Sciences 62, 2460 2476. Tammariello, S.P., Denlinger, D.L., 1998. G0/G1 cell cycle arrest in the brain of Sarcophaga crassipalpis during pupal diapause and the expression pattern of the cell cycle regulator, proliferating cell nuclear antigen. Insect Biochemistry and Molecular Biology 28, 83 89. Taylor, R.P., Benjamin, I.V., 2005. Small heat shock proteins: a new classification
45 scheme in mammals. Journal of Molecular and Cellular Cardiology 38, 433 444. Yocum, G.D., Joplin, K.H., Denlinger, D.L., 1998. Upregulation of a 23 kDa small heat shock protein transcript during pupal diapause in the flesh fly, Sarcophaga crassipalpis . Insect Biochemistry and Molecular Biology 28, 677 682.
46
Fig. 2.1. Two dimensional electrophoresis maps of brain proteins from (A) diapausing and (B) nondiapausing pupae of S. crassipalpis . Proteins were separated by IEF in the first dimension, pH 3 10, 11cm, then by size (15 180kDa) in the second dimension and stained with Coomassie Blue. Maps were analyzed with PDQuest software. Selected spots were numbered 1 18.
47 Fig. 2.2. Selected area of two dimensional gels and corresponding spot volumes for selected spots identified in the nondiapausing (ND) and diapausing (D) flesh flies. Protein spot migration patterns were detected in Coomassie stained gels using BioRad VersaDoc model 1000 imaging system. Image pairs and detection of protein spots with relative spot volumes were achieved using PDQuest software (BioRad). 2 DE, two dimensional gel electrophoresis.
48 Predicted Mowse Spot No. Accession No. Species Protein ID mass(kDa)/PI Score* Diapause up-regulated proteins
1 13560880 Drosophila melanogaster heat shock protein Hsp70Bc 70.1/5.5 192 1037174 Eimeria maxima immunoglobulin heavy chain binding protein 38.1/5.4 91 2 13560880 Drosophila melanogaster heat shock protein Hsp70Bc 70.1/5.5 130 5570 Aplysia californica BiP/GRP78 73.7/4.8 90 1037174 Eimeria maxima immunoglobulin heavy chain binding protein 38.1/5.4 90 3 2058737 Sarcophaga crassipalpis 23kDa heat shock protein ScHSP23 22.9/6.0 553 4 2058737 Sarcophaga crassipalpis 23kDa heat shock protein ScHSP23 23.0/6.0 435
49 5 2058737 Sarcophaga crassipalpis 23kDa heat shock protein ScHSP23 23.0/6.0 142 66548185 Apis mellifera PREDICTED: similar to thiol peroxiredoxin 18.1/5.6 114 67083335 Ixodes scapularis thioredoxin dependent peroxide reductase 25.8/9.1 98 6 2058737 Sarcophaga crassipalpis 23kDa heat shock protein ScHSP23 23.0/6.0 517 7 2058737 Sarcophaga crassipalpis 23kDa heat shock protein ScHSP23 23.0/6.0 817 8 2058737 Sarcophaga crassipalpis 23kDa heat shock protein ScHSP23 23.0/6.0 134 Table 2.1(continued) Table 2.1. Identification of diapause regulated brain proteins in pupae of S. crassipalpis . * Mowse score>54 indicates identity or extensive homology (P<0.05); higher scores indicate higher confidence of identity.
49 Table 2.1(continued) Predicted Mowse Spot No. Accession No. Species Protein ID mass(kDa)/PI Score* 9 125090 Ovis aries Keratin,type I microfibrillar 48 kDa, 48.1/4.77 285 component 8C 1(assumed: contaminant) 10 14 Unknowns Diapause down-regulated proteins
15 1591246 Methanocaldococcus Phosphoenolpyruvate synthase 134.6/6.2 59 jannaschii DSM 2661
50 16 160843 Schistocerca gregaria Fatty acid binding protein 15.0/6.2 179 17 3757564 Drosophila melanogaster EG:EG00037 24.3/4.4 214 18 74310551 Epidermophyton floccosum Endonuclease 34.2/9.53 74
50
CHAPTER 3
NEUROPEPTIDE LIKE PRECURSOR 4 IS UNIQUELY EXPRESSED
DURING PUPAL DIAPAUSE IN THE FLESH FLY
Aiqing Li, Joseph P. Rinehart and David. L. Denlinger *
Department of Entomology, Ohio State University, 318 W. 12 th Avenue, Columbus,
OH 43210, USA.
*Corresponding author. Tel.: 614 292 6425; fax: 614 292 2180.
E mail: [email protected] (D.L. Denlinger).
51 Abstract
Suppression subtractive hybridization comparing brains from diapausing and nondiapausing pupae of the flesh fly, Sarcophaga crassipalpis, suggested that the gene encoding neuropeptide like precursor 4 (Nplp4) was uniquely expressed during diapause. We have sequenced the full length cDNA encoding Nplp4 and used northern blots to further evaluate linkage to diapause. The open reading frame of this cDNA encodes a 61 amino acid residue precursor protein containing a predicted 18 residue signal peptide, one 22 amino acid and one 2 amino acid propeptides, and a 19 amino acid neuropeptide. The amino acid sequence of the precursor protein shows 64% identity to Drosophila melanogaster Nplp4; homologues of this precursor protein are not known from species other than these two flies. Nplp4 mRNA levels were quite low in nondiapausing (long day) pupae, but in contrast the gene was highly upregulated in diapausing (short day) pupae. Expression increased at the onset of diapause, remained high throughout diapause, and then decreased 2 days after diapause was terminated. Although the function of this precursor protein and the neuropeptide it yields remain unknown, this close association with diapause suggests a potential role for Nplp4 in initiating and maintaining diapause in the flesh fly.
Key words: Diapause; overwintering; neuropeptide like precursor 4; Sarcophaga crassipalpis
52 Introduction
Neuropeptide like precursor 4 (Nplp4) has no known function. A peptidomics study of the larval central nervous system in Drosophila melanogaster first identified the novel YSY peptide that is a component of NPLP4
(Baggerman et al., 2002). There are no other reports of Nplp4 as far as we know.
Three other novel peptide precursors, designated as neuropeptide like precursors 1,
2 and 3, respectively, were also identified in D. melanogaster central nervous system (Baggerman et al., 2002), but these peptide precursors appear to be not related. Regulatory neuropeptides are synthesized as components of larger precursor proteins that are subsequently cleaved into the smaller active molecules.
Signal peptides present within these precursor proteins direct the cleavage of the peptides from their precursors (Canaff et al., 1999). The well known roles of neuropeptides in influencing a wide range of functions including behavior, development, immunity, and indeed most physiological processes (Liu et al.,
2006), highlights the importance of linking unknown peptides to potential functions or developmental stages.
Through the use of suppressive subtractive hybridization (SSH) to identify diapause specific genes in the flesh fly Sarcophaga crassipalpis we isolated a potentially diapause upregulated clone with high identity to Nplp4. This prompted us to further investigate this clone by obtaining its full length sequence and to use
Northern blot hybridization to verify the association with the overwintering pupal diapause of the flesh fly.
Diapause, a form of developmental arrest used by most temperate zone insects, is characterized by a major shutdown of many genes but also by the
53 upregulation of a select set of genes (Denlinger, 2002). Among the diapause upregulated genes known from S. crassipalpis are genes that encode several heat shock proteins (Rinehart et al., 2007), a cystatin (Goto and Denlinger,
2002) and an AP endonuclease (Craig and Denlinger, 2000). Genes that are specifically upregulated during diapause are also known from several other species (e.g. Yocum, 2001; Lee et al., 2002; Robich et al., 2007).
In this study, we provide evidence that the gene encoding Nplp4 is another gene with an expression pattern finely linked to diapause. The fact that peptides play much important roles as regulatory molecules suggests that Nplp4 may deserve close attention as a potential regulator of developmental arrest in S. crassipalpis .
2. Material and methods
2.1. Insects
The colony of S. crassipalpis Macquart was maintained as decribed
(Denlinger, 1972). Nondiapausing adult flies were held at long daylength (15 h light: 9 h dark), 25 °C, and the resultant nondiapause destined larvae and pupae were reared at 20 °C under the same long day conditions. Diapausing flies were reared at short daylengths (12 h light: 12 h dark, 25 °C for adults and 20 °C for larvae and pupae). Termination of 20 day old diapausing pupae was achieved by directly applying 5 l hexane to the heads of the pupae (Denlinger et al., 1980).
2.2 Suppressive subtractive hybridization
Total RNA from 20 diapause and 20 nondiapause pupae was isolated using
54 Trizol reagent (Chomczynski and Sacchi, 1987), each of which was then pooled for use in suppressive subtractive hybridization (SSH) following a protocol previously described (Robich et al, 2007). First, the pools of total RNA were used as templates for cDNA synthesis using the BD SMART PCR Synthesis Kit
(BD Biosciences). SSH was performed using the Clontech PCR Select cDNA
Subtraction Kit, with the diapause sample as the tester and the nondiapause sample as the driver. Finally, subtracted libraries were constructed using the
TOPO TA Cloning Kit (Invitrogen), and randomly selected clones were sequenced at the Ohio State University Plant Microbe Genomics Facility using a Applied
Biosystems 3730 DNA Analyzer with Big Dye Terminator Cycle Sequencing
(Applied Biosystems).
2.3 Cloning
The initial Nplp4 clone (449 bp) including the 3′ end was obtained by SSH.
For 5′ RACE, the adaptor added first strand cDNA was synthesized using 1 g
RNA from diapausing pupae, with 5' CDS Primer A (5'–(T)25V N–3', N = A, C,
G, or T; V = A, G, or C) and SMART II™ A Oligonucleotide (5'–AAG CAG
TGG TAT CAA CGC AGA GTA CGC GGG–3') from SMART RACE cDNA amplification kits (Clontech, BD). The 5' sequence was amplified using the specific reverse primer (5' TGG CAG GCA CAC ACA TCA CAC TA 3'), and
Universal Primer A Mix (5' CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG
GTA TCA CGC AGA GT 3'). Thermal cycling was run with the following program: (i) 94°C for 5 min, (ii) 94°C for 30 s, (iii) 65°C for 30 s, (iv) 72°C for 3 min, (v) repetition of steps ii to iv for 35 cycles, and (vi) 70°C for 7 min. The
55 amplified cDNA product, separated in a 1% (w/v) low melting point agarose gel was isolated from the gel and cloned into a pCR2.1 TOPO TA vector (Invitrogen).
The TOPO clones were transformed into TOP 10 competent cells (Invitrogen) and grown overnight on Luria Bertani plates supplemented with X Gal and ampicillin.
Bacterial colonies were picked and purified using Qiaprep Spin Minipreps
(QIAGEN). The purified DNA was then sequenced with the M13 reverse primer at the Ohio State University Plant Microbe Genomics Facility on an Applied
Biosystems 3730 DNA Analyzer using Big Dyes Terminator Cycle Sequencing chemistry (Applied Biosystems) according to the manufacturer’s instructions.
2.4 Northern blot hybridization
For Northern blot analysis, 10 g total RNA was separated on a 1.4% (w/v) agarose gel containing 0.41 M formaldehyde and ethidium bromide. The RNA was subsequently transferred onto a Hybond nylon positive membrane
(Amersham Biosciences) by downward capillary action using a Schleicher and
Schull Turboblotter system and alkaline transfer buffer. The nylon membrane was neutralized in 0.2 M phosphate buffer and RNA was immobilized using a UV crosslinker (Fisher Scientific). The membrane was stored at 20 °C for later prehybridization.
The Nplp4 fragment was amplified by rt PCR from 5'RACE cDNA. The primers were NPF1 (5' TTA TTG CTG CCT TGT TCG CTG TCA 3') and NPR1
(5' CAT CAG TTG TTG TTG GGG GAC AGA 3'). Amplication was achieved with a preheating step at 94 °C for 3 min and 35 cycles of 30 s at 94 °C, 30 s at 62
°C and 30 s at 72 °C , followed by a 7 min extension at 72 °C. Identity of the PCR
56 product was confirmed by DNA sequencing. The product was electrophored on a
1% agarose TAE gel, the band was excised and purified using a QIAquick gel
Extraction kit (QIAGEN, USA) and used as a template for the probe. Probe labeling, hybridization and detection were performed using the Dig High Prime
DNA Labeling and Detection Starter Kit II (Roche Applied Sciences) following the manufacturer’s instructions. Briefly, the membrane was pre hybridized in hybridization buffer for 30 min. After overnight hybridization at 37 °C with the
DIG labeled probe, the blot was subjected to stringency washes, immunological detection and exposure to chemilluminescence film (Kodak Biomax).
DIG labeled 28S cDNA was used as a control for Northern hybridization to confirm equal loading of total RNA.
3. Results
3.1 Clone identification
The full length cDNA of Nplp4 obtained by RACE is a 533 bp sequence
(GenBank accession no. EU526381 ) that encodes 61 amino acid residues (Fig.
3.1). The open reading frame (ORF) of Nplp4, terminated by a TAA stop codon, is
183 bp, from nucleotides 64 to 249. The 5'untranslated region is 63 bp and the
3'untranslated region, including the poly A tail, is 284 bp. The putative polyadenylation signal (ATTAAA) was identified at nucleotide positions 444 449.
A sequence alignment of the deduced Nplp4 amino acid sequence with the sequence of Nplp4 from Drosophila melanogaster is shown in Fig. 3.2. The ORF of Nplp4 from S. crassipalpis shares 64% identity and 76% positives with Nplp4 from D. melanogaster . No homologues of this precursor protein have thus far
57 been found in other organisms.
Both precursor proteins contain a predicted 18 residue signal peptide, according to Nielsen et al. (1997). While the coded neuropeptide in D. melanogaster , YSY peptide, is 22 amino acid residues (Baggerman et al., 2002), the putative neuropeptide in S. crassipalpis , a YSY peptide, consists of 19 amino acid residues (Fig. 3.2).
3.2 Transcription of Nplp4 associated with diapause
To generate a DIG labeled probe for Northern blot hybridization, we cloned a partial Nplp4 cDNA that produced a 273 bp band. Northern blot hybridization confirmed a diapause specific expression pattern for Nplp4 (Fig.
3.3). Nplp4 was expressed at a low level in nondiapause destined wandering larvae and was nearly undetectable in nondiapausing pupae (5 d after pupariation at 20 °C). Expression was likewise displayed at a low level in wandering larvae programmed for pupal diapause, but expression of Nplp4 was highly upregulated at the very onset of pupal diapause, i.e. , 5 d after pupariation at 20 °C (Fig. 3.3).
Expression remained at a high level in early diapause, declines a bit in mid and late diapause and then dropped sharply 2 d after diapause was terminated with a topical application of hexane, a chemical tool that readily breaks diapause
(Denlinger et al., 1980). In all three replicates of the experiment, Nplp4 was highly expressed from the beginning to the end of pupal diapause (short daylength) but was not present in nondiapausing (long daylength) pupae reared at the same temperature.
58 4. Discussion
In this study, we have identified the gene that encodes Nplp4 in the flesh fly and determined that its expression is developmentally upregulated upon entry into pupal diapause. NPLP4 encodes a signal peptide, according to Nielsen et al.
(1997), and a YSY neuropeptide as seen in D. melanogaster (Baggerman et al.,
2002). Thus far, NPLP4 and the YSY peptide it contains have not been reported from other insects.
Although a peptidomics study showed the presence of NPLP4 peptide fragments in the fly’s central nervous systems (Baggerman et al., 2002), no additional information about NPLP4 has been obtained (Baggerman et al., 2005;
Taraszka et al., 2005). To further investigate whether NPLP4 is present in central nervous system, we investigated the tissue specific expression of Nplp4 mRNA by Northern blot hybridization (data not shown). Intriguingly, no expression was detected in the brain, thorax abdomen ganglion, midgut, malpighian tubules or in fat body, and lower abundance detected in epidermis than in whole body of diapausing pupae. The absence of Nplp4 transcript in the nerve tissues brain and thorax abdomen ganglion can be explained in two ways. One is that, Nplp4, as a neuropeptide hormone, is likely to be synthesized in the brain and released into haemolymph upon entry into diapause, similar to Nplp2 as suggested by Verleyen et al. (2006). The other is that, Nplp4 might be present in other tissues such as subesophageal ganglion and imaginal discs of central nervous system. Thus, the tissue distribution of Nplp4 needs further investigation.
Our results show a close relationship between the expression of Nplp4 and pupal diapause in S. crassipalpis . While long day nondiapausing pupae at a
59 developmental stage equivalent to diapause (young phanerocephalic pupae, 5 days after pupariation at 20 °C) fail to express Nplp4, short day (diapause programmed) pupae of the same age (5 d after pupariation at 20 °C) highly express this gene.
Expression remains high throughout pupal diapause and then expression drops sharply 2 d after diapause is artificially terminated. Thus tight linkage with diapause suggests a potential role for Nplp4 in diapause regulation, but exactly what this role may be remains to be determined. The Nplp4 diapause expression pattern does differ slightly from that of the heat shock proteins which are also upregulated throughout diapause (Hayward et al., 2005; Rinehart et al., 2007). The difference is in the decline in expression at the end of diapause. While Hsp70 and
Hsp23 are shut off within 6 12 h after diapause is artificially terminated with hexane (Hayward et al., 2005; Rinehart et al., 2007), Nplp4 continues to be expressed for an additional 12 18 h. This slight difference in temporal pattern of expression implies that different components of the diapause program may be turned off at different time, hence suggesting a finely tuned orchestration of the genes regulating insect diapause.
Acknowledgements
We thank Giancarlo Lopez Martinez for helpful advice during the course of this study. This work was supported in part by NSF grant IOB 0416720.
60 References
Baggerman, G., Cerstiaens, A., De Loof, A., Schoofs, L., 2002.Peptidomics of the larval Drosophila melanogaster central nervous system. Journal of Biological Chemistry 277, 40368–40374. Baggerman, G., Boonen, K., Verleyen, P., De Loof, A., Schoofs, L., 2005. Peptidomic analysis of the larval Drosophila melanogaster central nervous system by two dimensional capillary liquid chromatography quadrupole time of flight mass spectrometry. Journal of Mass Spectrometry 40, 250–260. Canaff, L., Bennett, H. P. J., Hendy, G. N., 1999. Peptide hormone precursor processing: getting sorted? Molecular and Cellular Endocrinology 156, 1–6. Chomczynski, P., Sacchi, N., 1987. Single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Analytical Biochemistry 162,156–159. Craig, T.L., Denlinger, D.L., 2000. Sequence and transcription patterns of 60S ribosomal protein P0, a diapause regulated AP endonuclease in the flesh fly, Sarcophaga crassipalpis . Gene 255, 381–388. Denlinger, D.L., 1972. Induction and termination of pupal diapause in Sarcophaga (Diptera: Sarcophagidae). Biological Bulletin, Woods Hole 142, 11–24. Denlinger, D.L., Campbell, J.J., Bradfield, J.Y., 1980. Stimulatory effect of organic solvents on initiating development in diapausing pupae of the flesh fly, Sarcophaga crassipalpis , and the tobacco hornworm Manduca sexta . Physiological Entomology 5, 7–15. Denlinger, D.L., 2002. Regulation of diapause. Annual Review of Entomology 47, 93–122. Goto, S. G., Denlinger, D. L., 2002. Genes encoding two cystatins in the flesh fly Sarcophaga crassipalpis and their distinct expression patterns in relation to pupal diapause. Gene 292, 121–127. Hayward, S. A. L., Pavlides, S. C., Tammariello, S. P., Rinehart, J. P., Denlinger, D. L., 2005. Temporal expression patterns of diapause associated genes in flesh fly pupae from the onset of diapause through post diapause quiescence. Journal of Insect Physiology 51, 631–640. Lee, K. Y., Horodyski, F.M., Valaitis, A.P., Denlinger, D.L., 2002. Molecular characterization of the insect immune protein hemolin and its high induction during embryonic diapause in the gypsy moth, Lymantria dispar . Insect Biochemistry and Molecular Biology 32, 1457–1467.
61 Liu, F., Baggerman, G., D'Hertog, W., Verleyen, P., Schoofs, L., Wets, G., 2006. In Silico identification of new secretory peptide genes in Drosophila melanogaster . Molecular & Cellular Proteomics 5, 510–522. Nielsen, H., Engelbrecht, J., Brunak, S., von Heijne, G., 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10, 1–6. Robich, R.M., Rinehart, J.P., Kitchen, L.J., Denlinger, D.L., 2007. Diapause specific gene expression in the northern mosquito, Culex pipiens L. , identified by suppressive substractive hybridization. Journal of Insect Physiology 53, 235–245 Taraszka, J.A., Kurulugama, R., Sowell, R.A., Valentine, S.J., Koeniger, S.L., Arnold, R.J., Miller, D.F., Kaufman, T.C., Clemmer, D.E., 2005. Mapping the proteome of Drosophila melanogaster : analysis of embryos and adult heads by LC IMS MS methods. Journal of Proteome Research 4, 1223–1237. Verleyen, P., Baggerman, G., D'Hertog,W., Vierstraete, E., Husson, S. J., Schoofs, L., 2006. Identification of new immune induced molecules in the haemolymph of Drosophila melanogaster by 2D nanoLC MS/MS. Journal of Insect Physiology 52, 379–388. Yocum, G.D., 2001. Differential expression of two HSP70 transcripts in response to cold shock, thermoperiod, and adult diapause in the Colorado potato beetle. Journal of Insect Physiology 47, 1139–1145.
62
Fig. 3.1 Nucleotide and deduced amino acid sequence of Nplp4 in Sarcophaga crassipalpis . The nucleotide sequence of the 5' and 3' UTR flanking ORF was determined by 5' and 3'RACE. The nucleotide sequence is numbered from the 5' to 3' direction. Key features of the gene include the suggested start ATG and stop TAA codons, indicated in boxes, and the putative polyadenylation signal shown in bold letters. The deduced amino acid sequence of the ORF is shown below the nucleotide sequence.
63
Fig. 3.2 Alignment of amino acid sequences of S. crassipalpis and D. melanogaster (CG15361 PA) neuropeptide like precursor 4 by ClustalW multiple alignment (http://www.cbs.dtu.dk/clustalw2). The signal peptide predicted by software analysis (SignalP 3.0 Server, http://www.cbs.dtu.dk/services/SignalP/, using neural networks) is shown in italics. Predicted neuropeptide like 4 is in bold, and the propeptide is boxed. * = identical amino acids, and : = conserved amino acids.
64
Fig. 3.3 Expression profiles of Nplp4 in diapausing and nondiapausing pupae of S. crassipalpis . RNA samples were extracted from whole larvae or pupae and 10 g was loaded onto each lane. wl=wandering larvae. Numbers refer to days after puparium formation or after diapause termination. 28s RNA expression was used as a loading control. The experiment was replicated 3 times, and a representative blot is shown.
65
CHAPTER 4
RAPID ELEVATION OF INOS AND DECREASES IN ABUNDANCE OF
OTHER PROTEINS AT PUPAL DIAPAUSE TERMINATION IN THE
FLESH FLY SARCOPHAGA CRASSIPALPIS
Aiqing Li, M. Robert Michaud and David L. Denlinger *
Department of Entomology, The Ohio State University, Columbus, OH 43210,
USA
*Corresponding author:
Tel: +1 6142928209
Fax: +1 6142927865
Email: [email protected]
66 Abstract
We analyzed changes in brain proteins following pupal diapause termination in Sarcophaga crassipalpis by a combination of 2 dimensional gel electrophoresis and mass spectrometry. The proteome analysis revealed significant changes in 20 proteins, 11 of which represented 2.5 fold changes. Three proteins were present only in the brains of diapausing pupae. Among the most abundant proteins that showed a change, 1 was more abundant, 7 were less abundant, and 2 were absent following diapause termination. The protein that increased in abundance following diapause termination showed highest identity to myo inositol 1 phosphate synthase (Inos). Proteins that decreased at diapause termination include those showing highest identity to fatty acid binding protein,
CG2331 PA, twinstar, catalase, and a histone. Proteins absent at diapause termination included ribosomal protein L17A and one unnamed protein. Attempts to terminate diapause by injection of several Inos related metabolites failed, thus suggesting that the elevation of Inos at diapause termination is downstream of the physiological regulation that initiates development.
Key words: proteomics; diapause; diapause termination; brain proteins;
Sarcophaga crassipalpis
67 4.1. Introduction
Changes in brain proteins that occur at diapause termination may prove useful for understanding critical events involved in transition from the arrested state of diapause to the initiation of development. In a previous proteomics study, we compared proteins in the brains of diapausing pupae of the flesh fly
Sarcophaga crassipalpis with proteins present in the brains of pupae that did not enter diapause (Li et al., 2007), but in this study we compare brains from diapausing pupae with brains from pupae that were triggered to artificially break diapause with an application of hexane 24 h earlier. Though several previous studies have monitored mRNA expression associated with diapause (e.g.
Flannagan et al., 1998; Hayward et al., 2005; Robich et al., 2007), few studies have examined changes in proteins. Proteomics has proved to be a powerful tool for understanding physiological changes and cellular processes and can detect post translational modifications as well (Elrick et al., 2006). However, to the best of our knowledge, this approach has not previously been used to examine diapause termination, with the exception of a recent study reporting protein changes in response to diapause termination in the brine shrimp Artemia franciscana (O'Connell et al., 2006). Diapause termination in the brine shrimp was linked to shifts in several proteins involved with metabolism (fructose 1,
6 bisphosphate aldolase and glyceraldehyde 3 phosphate dehydrogenase), molecular chaperones (heat shock protein 70, small heat shock protein precursor), protein synthesis (40S ribosomal protein S12), and structure (beta tubulin)
(O'Connell et al., 2006).
In this paper we use two dimensional gel electrophoresis (2 DE) and mass
68 spectrometry to analyze the proteome of brains in S. crassipalpis during diapause and 24 h after diapause has been terminated with hexane. Within this time frame we report a major increase in the abundance of one identified protein, Inos, and a decline in abundance of several others. In addition, we test the possibility that Inos metabolites may be involved as regulators of diapause termination.
4.2. Materials and methods
4.2.1 Flies
Our colony of S. crassipalpis was maintained at 25 °C with 15 h light and
9 h dark as previously described (Denlinger, 1972). To induce pupal diapause, the adults were reared under short day conditions with 12 h light and 12 h dark at 25
°C, and the larvae and pupae were reared at 20 °C under the same short daylength.
To terminate diapause, 5 l hexane was applied directly to the heads of diapausing pupae, 20 days after pupariation; this treatment immediately initiates development in diapausing pupae of the flesh fly (Denlinger et al., 1980).
4.2.2 Sample preparation of brains for two-dimensional gel electrophoresis
Diapause samples consisted of 30 pupal brains dissected 20 days after pupariation. Brains at diapause termination were collected 24 h after hexane was applied to 20 day old diapausing pupae. Brains were homogenized in triplicate in a cell lysis buffer consisting of 50 mM Tris Cl pH 7.5, 100 mM NaCl, 1% Triton
X 100, 1 mM EDTA, 1 mM EGTA, 50 mM β glycerophosphate, 1 mM dithiothertol, and protease inhibitors including 1 mM PMSF, 2 mM sodium orthovanadate, 10 g/ml leupeptin, 10 g/ml pepstatin A, and 10 g/ml aprotinin
69 (Sigma, St. Louis). Proteins were extracted using the ReadyPrepTM 2D cleanup kit according to the manufacturer’s instructions. All chemicals and kits were supplied by BioRad (Hercules, CA) unless otherwise specified.
4.2.3 Two dimensional gel electrophoresis
The protein extracts (200 l containing 300 500 g proteins) were used to rehydrate 11 cm IPG strips (pH 3 10 NL from BioRad) overnight at room temperature. Isoelectric focusing (IEF) was performed at 400 V for 20 min, 8,000
V for 2.5 h and then up to 30,000 V h for approximately 6 h at 20 °C in a Protean
IEF Cell (BioRad). The maximum current was set as 50 A per strip. After IEF, the IPG strips were equilibrated for 20 min in equilibration buffer I [6 M urea, 2%
(w/v) SDS, 0.375 M Tris HCl (pH 8.8), 20% (v/v) glycerol and 2% (w/v)
1,4 dithiothreitol (DTT)] followed by 20 min in buffer II (same as buffer I except that DTT was replaced with 2.5% iodacetamide). For the second dimension, IPG strips were overlayed with 0.5% agarose at the top of 8 16% Criterion Tris HCl gels. Tris glycine buffer (25 mM Tris, 192 mM glycine, 0.1% SDS. pH 8.3) was used and a constant voltage for 2 dimensional electrophoresis was set at 200 V for approximately 1 h. The gels were then fixed in a mixture of 50 % ethanol, 10% acetic acid and 40% water overnight and stained with Bio safe Coomassie blue stain (BioRad). Protein concentrations were estimated using the BioRad protein assay.
4.2.4 Protein visualization and image analysis
The gels were scanned using a BioRad VersaDoc imaging system. Spot
70 detection and evaluation were carried out using the computer assisted image analysis software PDQuest (BioRad). 2 D gel processing procedures included image creating and Gaussian modeling, spot detection, normalization, gel matching, and data analysis. A matchset of 3 diapause and 3 nondiapause sample images was created to compare and analyze all the spots in each gel. The matchset contained the raw 2 D scan, Gaussian and filtered images of the 6 gels. During the matching process, common landmark spots were chosen from each of the gels, and manual matching was used. Quantitative and statistical analyses were performed on the Gaussian image by comparing the average volume of resolved spots.
4.2.5 Mass spectrometric protein identification
Protein spots were cored automatically by a spot cutter with robotic arm and hollow drill bit (BioRad). Excised spots were subjected to in gel digestion with sequencing grade trypsin from Promega (Madison WI), using the Montage In Gel
Digestion Kit from Millipore (Bedford, MA) according to the manufacturer’s protocols. Gel pieces were washed in a mixture of 50% (v/v) methanol and 5%
(v/v) acetic acid for several hours. The gel bands were dried with acetonitrile and reduced with DTT solution. Iodoacetamide was added to alkylate the cysteines, the gel was washed again with cycles of acetonitrile and ammonium bicarbonate, trypsin was added, and the samples were incubated at room temperature overnight.
Subsequently, the peptides were extracted from the polyacrylamide with 50% (v/v) acetonitrile and 5% (v/v) formic acid several times and pooled. The extracted pools were concentrated in a speed vac to approximately 25 L.
71 Capillary liquid chromatography nanospray tandem mass spectrometry was performed on a Micromass hybrid quadrupole time of flight Q Tof(tm) II
(Micromass, Wythenshawe, UK) mass spectrometer equipped with an orthogonal nanospray source from New Objective, Inc. (Woburn, MA) operated in positive ion mode. The liquid chromatography (LC) system was an UltiMateTM Plus system from LC Packings with a Famos autosampler and Switchos column switcher. Solvent A was water containing 50 mM acetic acid and solvent B was acetonitrile. A sample volume of 2.5 l was first injected onto the trapping column, and then washed with 50 mM acetic acid. The injector port was switched to inject, and the peptides were eluted off of the trap onto the column. A 5 cm 75 mM ID
ProteoPep C18 column packed directly in the nanospray tip was used for chromatographic separations. Peptides were eluted directly off the column into the
Q TOF system using a gradient of 2 80% B over 30 minutes, with a flow rate of
40 l/min with a pre column split to approximately 500 nl/min. Total run time was
55 min. The nanospray capillary voltage was set at 3.0 kV and the cone voltage at
55 V. The source temperature was maintained at 100°C. Mass spectra were recorded using MassLynx 4.0 with automatic switching functions. Mass spectra were acquired from mass 400 2,000 daltons every 1 sec. with a resolution of
8,000 (FWHM: full width at half maximum). When the desired peak was detected at a minimum of 15 ion counts, the mass spectrometer automatically switched to acquire CID MS/MS spectrum of the individual peptide. Collision energy was set dependent on charge state recognition properties.
72 4.2.6 Database search
Sequence information from the MS/MS data was processed using Mascot
Distiller from Matrix Science to form a peaklist (mgf file). Data processing was performed following the guidelines specified in Carr et al. (2004). Briefly, data were minimally processed, a 3 point smooth was applied, and the centroid was calculated from the top 80% of the peak height. The charge state of each ion selected for MS/MS was calculated, however the peaks were not deisotoped.
Assigned peaks have a minimum of 5 counts (S/N of 3) and must show the corresponding C13 ion to be considered valid. The mass accuracy of the precursor ions was set to 1.2 Da to accommodate accidental selection of the C13 ion, and fragment mass accuracy was set to 0.3 Da. Considered modifications (variable) were methionine oxidation and carbamidomethyl cysteine. The data was acquired several times to ensure reproducibility.
4.2.7 Injection of myo-inositol and other metabolites
To determine if Inos metabolites such as myo inositol, myo inositol 1, 4,
5 trisphosphate, or phosphatidylinositol could terminate diapause, solutions of these compound were injected into 20 day old diapausing pupae. The chemicals
(Sigma Aldrich, St. Louis, MO) were dissolved in saline, and 1 l volumes were injected using a finely drawn glass capillary. Doses injected included 10, 25, 50,
75, 100, 125 and 150 g myo inositol, 0.001, 0.01, 0.1, 1, 5 g myo inositol 1, 4,
5 trisphosphate, and 0.1, 1, 5, 10 g phosphatidylinositol. The injected pupae were then held at 20 °C under short day conditions for 2 months, and checked daily for development, as indicated by migration and elongation of the antennal discs
73 (Fraenkel and Hsiao, 1968).
4.3. Results
4.3.1 Differences in abundance of specific proteins
An average of 523 spots (range 461 to 595 spots) with molecular masses of
10 250 kDa were detected in the Coomassie stained gels by computer assisted image analysis. The 2D maps of brains from diapause and diapause termination showed good reproducibility, and a representative of both groups is shown in Fig.
4.1. Quantitative and statistical comparisons, using PDQuest software, revealed several distinct differences in the two types of brain samples. Quantitative analysis indicated statistically significant changes (≥ 98% confidence) in 20 proteins at the termination of diapause; the changes in 11 proteins were 2.5 fold or greater. In addition, 3 proteins were present only in the brains of diapausing pupae.
The most abundant proteins, those showing at least 2.5 fold changes or significant changes with 98% statistical confidence were selected for MS identification. Those spots are indicated in Fig. 4.1A and B: Spot 1 was more abundant in the brains 24 h after diapause termination, while spots 2 10 decreased in abundance at diapause termination.
Spot volume comparisons of these numbered spots (Fig. 4.2) show pronounced differences in the amounts of specific brain proteins in the two samples.
Quantification of spots revealed that spot 1 increased 4.3 fold following diapause termination, while spots 2 7 and 9 deceased 3.0 , 4.5 , 2.6 , 2.0 , 2.5 , 2.9 , and
3.6 fold, respectively. Spots 8 and 10 were unique to diapause (Fig. 4.2).
74 4.3.2 Protein identification
The selected regulated spots were excised and identified by mass spectrometry as indicated in Table 4.1. Several of the spots contained mixtures of comigrating proteins. Some proteins were present in more than one spot, ( e.g. the protein similar to Drosophila melanogaster tsr was present in spots 5 and 6). The protein that increased in abundance at diapause termination (spot 1) showed highest identity to myo inositol 1 phosphate synthase (Inos CG11143 PA). Proteins that decreased in abundance at diapause termination included proteins showing highest identity to fatty acid binding protein, protein CG2331 PA, twinstar, catalase
CG6871 PA, and histone HIST2H3C. Proteins unique to diapause included ribosomal protein L17A and one unnamed protein.
4.3.3 Can an injection of myo-inositol or its metabolites terminate diapause?
The protein Inos that increased in abundance at diapause termination has myo inositol 1 phosphate synthase activity and is also associated with biosynthesis of myo inositol and the turnover of phosphoinositides. To evaluate the possibility that myo inositol or its metabolites might elicit diapause termination, we injected a range of doses, indicated in Materials and Methods, of myo inositol, myo inositol 1, 4, 5 trisphosphate, and phosphatidylinositol into diapausing pupae. None of the 3 compounds had a significant effect on diapause termination (data not shown).
4.4. Discussion
We previously presented proteome maps of diapausing and nondiapausing
75 brain of S. crassipalpis (Li et al., 2007), but this study yielded slightly more proteins due to modifications of sample preparation and handling. In our previous study we compared the brains of diapausing pupae with brains of pupae that had never entered diapause. By contrast, this study examines the changes in the protein profiles of brain from diapausing pupae and those in which diapause was terminated by hexane 24 h earlier.
Our previous work revealed 8 diapause abundant proteins with highest identities to heat shock proteins (Hsp) and a small heat shock protein precursor which declines 12 h post diapause in the brine shrimp Artemia franciscana
(O'Connell et al., 2006). Although the transcripts encoding Hsp23 (Yocum et al.,
1998) and Hsp70 (Rinehart et al., 2000) begin to decline 6 h after diapause termination in S. crassipalpis , no decrease of the Hsp proteins was noted in this study. Presumably, this 24 h post diapause termination period is simply too short to detect the decline in presence of the heat shock proteins. The approach we have taken here is most effective for revealing early changes in proteins that are prompted by diapause termination.
In the present study, a phosphoinositide synthase, Inos CG11143 PA (spot 1), was the only upregulated protein we observed in the pupal brains of S. crassipalpis following diapause termination. This protein catalyzes the rate limiting step of inositol biosynthesis (Ju and Greenberg, 2004). Inositol is rate limiting for the synthesis of phosphatidylinositol 4, 5 bisphosphate (PIP2).
In the Wnt Ca 2+ cGMP signaling pathway (Wang and Malbon, 2003), Wnt5A binds and activates Rfz2, thus activating G protein β/γ subunits (G β/γ) which then activates phospholipase C, PLC. PLC hydrolyzed membrane PIP2 into two second
76 messengers, inositol 1, 4, 5 triphosphate (IP3) and diacylglycerol (DAG).
IP3catalyzes calcium release from its intracellular stores, activating the
Ca 2+ sensitive protein phosphatase calcineurin. DAG released from PIP2 is the physiological activator of protein kinase C. Both calcineurin and protein kinase C can influence the activity of various transcription factors, thus influencing gene expression. Therefore, the elevation of Inos can lead to enhanced activity of IP3 and DAG and direct agene expression. This important function of Inos in signal transduction pathways is compatible with its rapid upregulation within 24 h of diapause termination, suggesting that Inos may function in initiating development.
However, injection of Inos related metabolites including myo inositol, myo inositol 1, 4, 5 trisphosphate, and phosphatidylinositol have thus far failed to support this hypothesis, but experiments with additional derivatives would appear to be merited. The other possibility is that the injection of these molecules into hemalymph does not ensure they can be delivered to proper sites and bound by receptors. Of course, there is also a possibility that elevation we see represents, not a signaling event, but an early downstream response to diapause termination.
Among the nine proteins that are less abundant following the break of diapause, two (spots 5 and 6) match the protein twinstar (tsr) from Drosophila melanogaster . Twinstar, which encodes a Cofilin/ADF (actin depolymerization factor) homolog, is a component of the cytoskeleton and regulator of actin dynamics (Bamburg, 1999; Blair et al., 2006). Structural features show that twinstar retains the conserved domains of actin binding and depolymerizing
(Gunsalus et al., 1995). In vivo studies of the twinstar protein suggest that it is involved in centrosome migration and separation, as well as cytokinesis (Gunsalus
77 et al., 1995). The polymerization state of actin controls processes involved in the determination of cell shape, oogenesis, hair and bristle formation, growth cone morphology, and lamellipodial extension (Rogers et al., 2005). Twinstar’s involvement in depolymerizing actin may contribute to the shutdown of key cellular and developmental events during diapause. Previous studies in mosquitoes indicate important shifts in polymerized actin during diapause (Kim et al., 2006).
Two proteins similar to histone HIST2H3C (spots 8 and 9) also decreased following diapause termination. Histones are involved in DNA binding control processes such as chromosome organization, biogenesis, and nucleosome assembly. Histones control gene expression by modulating the structure of chromatin and the accessibility of regulatory DNA sequences to transcriptional activators and repressors (Backs and Olson, 2006). The drop in histone abundance is likely critical for this developmental switch that allows the onset of development.
Another two identified proteins expressed less abundantly in brains following diapause termination have highest identities to Drosophila erecta protein
CG2331 PA, a protein with high similarity to the known valosin containing protein (VCP/TER94/Cdc48p) ATPase sequences, members of the AAA
(ATPase associated with various cellular activities) ATPase family. TER 94 displays an apoptosis inducing activity and plays a key role as a cell death effector molecule in polyglutamine induced neurodegeneration (Higashiyama et al., 2002).
RNAi of VCP indicates the effect of VCP on knocking down function of the ubiquitin proteasome system (UPS) and thereby inhibiting UPS dependent proteolysis (Wojcik et al., 2004). Thus, high abundance of VCP during diapause
78 may result in a depressed rate of proteolysis. This is consistent with the suppression of protein degradation noted during hypometabolic states such as diapause (Storey and Storey, 2004).
Another protein that is less abundant at diapause termination shows highest identity to catalase from Drosophila melanogaster . Catalase is an important antioxidant enzyme, and several other defensive type proteins are known to be elevated during diapause (Denlinger, 2002; Robich et al., 2007). Catalase activity is higher in nondiapausing than diapausing larvae of the European corn borer,
Ostrinia nubilalis (Jovanovic´ Galovic´ et al., 2004), thus there are previous results suggesting catalase is associated with diapause, although it remains unknown how catalase activity is related its abundance and whether catalase is more abundant during diapause in O. nubilalis .
Two proteins present only in the brains of diapausing pupae were ribosomal protein L17A and an unnamed protein. As yet, we can propose no function for these potentially interesting proteins.
Acknowledgements
This work was supported by NSF grant IOB 0416720. We thank David Mandich,
Ohio State University’s Plant Microbe Genomics Facility, for assistance with image analysis and Dr. Kari Green Church, OSU’s Campus Chemical Instrument
Center, for assistance with MS protein identification.
79 References
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81 Proceedings of the National Academy of Sciences of the United States of America 102, 15912 15917. Robich, R. M., Rinehart, J.P., Ketchen, L. J., and Denlinger, D. L., 2007. Diapause specific gene expression in the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization. Journal of Insect Physiology 53, 235 245. Rogers, E. M., Hsiung, F., Rodrigues, A. B., and Roses, K., 2005. Slingshot cofilin phosphatase localization is regulated by Receptor Tyrosine Kinases and regulates cytoskeletal structure in the developing Drosophila eye. Mechanisms of Development 122, 1194 1205. Storey, K. B., and Storey, J. M., 2004. Metabolic rate depression in animals: transcriptional and translational controls. Biological Reviews 79, 207 233. Wang, H. and Malbon C. C., 2003. Wnt Signaling, Ca2_, and Cyclic GMP: Visualizing Frizzled Functions. Science 300, 1529 1530. Wójcik, C., Yano, M., and DeMartino, G.N., 2004. RNA interference of valosin containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome dependent proteolysis. Journal of Cell Science 117, 281 292. Yocum, G. D., Joplin, K. H., and Denlinger, D. L., 1998. Upregulation of a 23 kDa small heat shock protein transcript during pupal diapause in the flesh fly, Sarcophaga crassipalpis . Insect Biochemistry and Molecular Biology 28, 677 682.
82 Fig. 4.1. Two dimensional electrophoresis master images of pupal brain proteins in S. crassipalpis (A) during diapause and (B) 24 h after diapause was terminated by the application of hexane. The proteins were separated by isoelectric focusing, pH range 3 10, in the first dimension and by molecular weight in the second dimension. Gels were stained with Coomassie blue and protein spots identified by MS are numbered.
83 Fig. 4.2. Enlarged gel images, corresponding 3 D profiles and fold change in abundance of selected proteins identified in brains of S. crassipalpis during diapause (D) and 24 h following diapause termination (DT). Images pairing relative spot volumes were generated from PDQuest software (BioRad). 2 DE, two dimensional electrophoresis.
84
Predicted Spot Accession Mowse Sequence Species Protein ID mass No. No. Score* coverage (kDa)/PI More abundant following diapause termination 1 17137626 Drosophila melanogaster Inos CG11143 PA 62.5/5.82 225 6% Less abundant or absent following diapause termination 2 160843 Schistocerca gregaria fatty acid binding protein 15.0/6.19 144 9% 3 27374245 Drosophila erecta CG2331 PA 89.5/5.20 1049 30% 85 55238432 Anopheles gambiae ENSANGP00000022801 89.9/5.15 889 22% 83423461 Bombyx mori transitional endoplasmic reticulum ATPase TER94 89.8/5.30 261 20% 4 27374245 Drosophila erecta CG2331 PA 89.5/5.20 211 7% 3869204 Drosophila melanogaster Cortactin 61.1/5.03 137 4% 5 38047865 Drosophila yakuba similar to Drosophila melanogaster tsr 17.4/6.74 1370 70% 6 38047865 Drosophila yakuba similar to Drosophila melanogaster tsr 17.4/6.74 555 68% 7 17981717 Drosophila melanogaster catalase CG6871 PA 57.6/8.39 210 7% Table 4.1 (continued) Table 4.1. Identification of brain proteins that change in abundances within 24 h of pupal diapause termination in S. crassipalpis . * Mowse score>54 indicates identity or extensive homology (P<0.05); higher scores indicate higher confidence of identity.
85 Table 4.1 (continued) Predicted Spot Accession Mowse Sequence Species Protein ID mass No. No. Score* coverage (kDa)/PI 8 7442 Drosophila hydei unnamed protein 13.7/10/34 304 42% 9 55626038 Pan troglodytes PREDICTED: similar to HIST2H3C protein 61.0/10.50 360 12% 10 158330 Drosophila melanogaster ribosomal protein L17A 15.0/10.83 135 17% 86
86
CHAPTER 5
RAPID COLD HARDENING ELECITS CHANGES IN BRAIN PROTEIN
PROFILES OF THE FLESH FLY
Aiqing Li and David L. Denlinger *
Department of Entomology, The Ohio State University, 318 West 12 th Avenue,
Columbus, OH 43210, USA.
*Corresponding author. Tel.: 614 292 6245; fax: 614 292 2180.
E mail: [email protected] (D.L. Denlinger).
Running title: Proteomics of rapid cold hardening
87 Abstract
Rapid cold hardening (RCH) refers to the enhanced cold tolerance acquired by a brief exposure to a moderately low temperature. Although ecological aspects of this response have been well documented in insects less is known about the physiological and biochemical mechanisms elicited by RCH. In this study we used two dimensional electrophoresis to detect potential difference in brain protein abundance in response to a 2 h RCH exposure at 0 °C. Fourteen proteins showing the greatest differences were selected for mass spectrometric identification. Three proteins that increased in abundance during RCH included
ATP synthase subunit alpha, a small heat shock protein (smHsp), and tropomyosin 1 isoforms 33/34. Eleven proteins that decreased in abundance or were missing following RCH included several proteins involved in energy metabolism, protein degradation, transcription, actin binding, and cytoskeleton organization. That several proteins increased in abundance during RCH underscores the dynamics of the RCH mechanism and points to several physiological responses that likely contribute to RCH. The increase in ATP synthase suggests an elevation of ATP during RCH, and the smHsp increase suggests that at least one of the Hsps is actually mobilized during RCH, rather that after RCH as previously assumed.
Key words: Proteomics, Rapid cold hardening, Heat shock proteins, ATP synthase, Sarcophaga crassipalpis .
88 Introduction
Insects have a remarkable ability to adjust their physiology in response to daily cycles of temperature change. Brief exposure to a moderately low temperature promotes survival at an even lower temperature, a response referred to as rapid cold hardening (RCH) by Lee et al. (1987). RCH is distinct from the long term cold acclimation commonly associated with an overwintering diapause
(Denlinger, 1991) and can occur at any developmental stage. The physiological adjustments associated with RCH take place within minutes or hours and enable an insect to quickly respond to drops in temperature that may occur on a daily basis.
Although the ecological basis for this response is clear, the physiological mechanisms involved in RCH are less well understood. The initial description of
RCH, based on pharate adults of Sarcophaga crassipalpis (Chen et al., 1987, Lee et al., 1987), documented a modest elevation of glycerol associated with RCH, but it was clear even then that other adjustments were likely. More recently, changes in membrane composition that lead to increased fluidity were documented during RCH (Overgaard et al., 2005; Michaud and Denlinger, 2006), and metabolomic profiling points to major shifts in pools of carbohydrates, polyols and amino acids (Michaud and Denlinger, 2007). These results thus suggest that RCH is not a simple response that relies on a single determinant, but instead is a complex response involving several metabolic pathways and
89 alterations in the chemical composition of the hemolymph, cell metabolism, and cell membranes.
In this study we further probe mechanisms involved in RCH by using proteomic profiling to identify changes in brain proteins that may be associated with RCH. This study uses pharate adults of the flesh fly, S. crassipalpis, and seeks to identify changes in protein abundance that occur during RCH (2 h exposure to 0 °C). Such an exposure at 0 °C was previously shown to enable this species to survive a 2 h exposure to 10 °C, a low temperature stress it could not survive without prior exposure to 0 °C (Chen et al., 1987). Protein profiles that compare brain proteins in flies held at 20 °C with those exposed to 0 °C for 2 h reveal three proteins that increased in abundance during RCH and 11 proteins that decreased in abundance or were absent after RCH.
Results
Comparison of proteomic patterns
A representative 2D gel image of brain proteins from a control group of flies
held at 20 °C is shown in Fig. 5.1. Both the control gels and those from the RCH
treatment contained approximately 370 dominant spots, and the majority (330)
could be matched between the two groups. A correlation analysis of the gels
indicated a spot to spot correlation coefficient of 0.94. Most proteins showed no
differences between the control and RCH gels, but 68 proteins showed a
90 ≥1.5 fold difference and 38 of the proteins were significantly different in abundance (Student’s t test, p<0.05). The 14 proteins showing the greatest differences were selected for mass spectrometric identification. 2DE images demonstrated differences in abundance of the 14 selected proteins (Fig. 5.2), and
PDQuest software was used to define statistically significant differences based on 3 gel replicates (Fig. 5.3). In response to RCH, 3 of the 14 proteins increased in abundance, 10 decreased, and 1 was totally missing.
LC-MS/MS and protein identification
Positive identifications were obtained for all 14 proteins (Table 5.1). The three proteins that increased in abundance during RCH included ATP synthase subunit alpha, a small heat shock protein, and tropomyosin 1 isoforms 33/34. The ten proteins that decreased in abundance included heat shock protein 90, T complex protein 1 subunit gamma, F actin capping protein subunit alpha, tropomyosin 1 isoforms 9A/A/B, 14 3 3 protein epsilon, 26S protease regulatory subunit 7, isocitrate dehydrogenase subunit alpha, aldehyde dehydrogenase 2B7, cysteine desulfurase, and trans activator protein BZLF1. A protein that was absent following RCH was identified as zinc finger protein on ecdysone puffs.
Discussion
We identified 14 brain proteins from S. crassipalpis that changed in abundance
91 in response to a 2 h RCH exposure to 0 °C. Three of these proteins increased in abundance, while the other 11 were either less abundant or disappeared entirely.
We emphasize that the changes we note here focus strictly on protein changes that occur during RCH, rather than changes that may occur during a later recovery period.
Of particular note was an increase in abundance of a small heat shock protein having high identity to Hsp26 from Drosophila melanogaster. Although there was previous evidence that several Hsps, including Hsp70, increase in abundance when a cold shocked insect is returned to a more moderate temperature (Petersen et al., 1990; Joplin et al., 1990), there was little evidence to suggest that any of the Hsps are actually expressed during low temperature exposure. In fact, Hsp70 antibodies failed to detect Hsp70 during RCH in D. melanogaster (Kelty and Lee, 2001), leading to the assumption that Hsps are involved only in recovery from cold stress. But, there are many different members of the major Hsp families, and it is becoming more clear that they can have diverse functions in chaperoning, maintaining the integrity of proteins in the cell, and signaling select proteins for removal (Feder and Hoffmann, 1999;
Sun and MacRae, 2005). Our RCH experiments also failed to show elevated abundance for Hsp70, but the observed increase in a smHsp points to a potential role for this stress protein during RCH. This observation is consistent with our unpublished observations (Michaud and Denlinger) demonstrating that RNAi
92 directed against Hsp70 has no effect on RCH, but RNAi directed against one of the smHsps, Hsp23, blocks RCH. Several small Hsps and Hsp70 are highly upregulated during pupal diapause in S. crassipalpis (Yocum et al., 1998,
Rinehart et al., 2000, Rinehart et al., 2007, Li et al., 2007), and knock down experiments indicate a role for Hsps in generating cold tolerance in this overwintering stage (Rinehart et al., 2007). These results thus suggest that S. crassipalpis uses a suite of Hsps to counter low temperature stress during its overwintering diapause but uses only a limited number of Hsps, perhaps only a single smHsp, to participate in RCH.
The other Hsp that changed in abundance during RCH was Hsp90, but in this case abundance of the protein decreased. Hsp90 is also the one Hsp that is down regulated during pupal diapause in S. crassipalpis (Rinehart and
Denlinger, 2000, Rinehart et al., 2007), thus in both RCH and diapause, a smHsp is upregulated while Hsp90 is downregulated, suggesting that this species may exploit similar mechanisms to increase cold hardiness during RCH and diapause.
The increased abundance of ATP synthase subunit alpha suggests an important role for generating and mobilizing ATP (Talamillo et al., 2004) during
RCH. ATP synthase subunits also increased in a parasitic wasp during fluctuating thermal regimes and at constant low temperatures (Colinet et al.,
2007). Likewise, in fish, an increase in transcription and translation of
93 mitochondrial ATP synthase subunits was observed during cold acclimation
(Kikuchi et al., 1999, Itoi et al., 2003). Low temperature reduces the catalytic activity of ATP synthase, and the animal thus compensates by increasing the concentration of the enzyme (Itoi et al., 2003), a scenario that is also quite likely during RCH in insects.
Levels of two different tropomyosins changed in response to RCH: tropomyosin 1, isoforms 33/34 was elevated, while tropomyosin 1, isoforms
9A/9B decreased in abundance. Tropomyosin 1, a major isoform of tropomyosin, binds to and stabilizes actin cables and filaments. Its downregulation has previously been linked to the reorganization of microfilaments in mammalian cell cultures (e.g. Boyd et al., 1995), and the affinity of tropomyosin for actin microfilaments is know to result in alterations in actin (Lazarides, 1976;
Matsumura et al. 1983; Bretscher 1986) that can lead to loss of cytoplasmic actin cables and perturbations of cellular growth (Liu and Bretshcer, 1989).
Gunning (2008) has nicely documented that tropomyosin regulates actin filament dynamics and organization.
Several other proteins that were less abundant in response to RCH are also linked to cytoskeletal functions. T complex protein (TCP) contributes to microtubule elongation (Ursic and Culbertson, 1991; Ursic et al., 1994, Liang and MacRae, 1997). In the onion maggot, Delia antiqua, the gene encoding
TCP 1 is upregulated in response to cold (Kayukawa et al., 2005), and in S.
94 crassipalpis , TCP 1 is also modestly upregulated during pupal diapause
(Rinehart et al., 2007), but in response to RCH, we note that this protein is less abundant. Capping protein was also less abundant during RCH. Capping protein binds to the barbed ends of actin filaments and regulates depolymerization of the actin (Amatruda et al., 1990; Nakano and Mabuchi, 2006). The gene encoding this protein is also altered in expression by fluctuating thermal regimes in a parasitic wasp (Colinet et al., 2007). Redistribution of polymerized actin is triggered by low temperature (and diapause) in mosquitoes (Kim et al., 2006), thus there is growing evidence that a number of low temperature responses evoke changes in the organization of the cytoskeleton.
Most of the other protein changes we noted can likely be attributed to the decreased metabolic activity associated with low temperature exposure.
Isocitrate dehydrogenase, for example, is an important enzyme in the TCA cycle and is one of the three control points in the cycle. A decrease in this enzyme may reflect the lower metabolism expected at low temperature. Cysteine desulfurase, the enzyme that provides sulfur for Fe S cluster synthesis, was also less abundant during RCH, as was protease. The decreases in both of these enzymes during RCH likely reflect an overall decrease in protein synthesis at low temperature.
Aldehyde dehydrogenase, another protein that decreased during RCH, is a member of a superfamily that converts aldehydes by oxidation to their
95 corresponding carboxylic acids (Kirch et al., 2004). mRNAs encoding this protein are induced by cold in Arabidopsis (Kursteiner et al., 2003), but during
RCH in S. crassipalpis the protein declined in abundance.
14 3 3 proteins are involved in the RAS1 signaling cascade and bind to several critical molecules that regulate the cell cycle and control cell growth, differentiation, apoptosis and migration (Aitken, 1995; Mhawech, 2004). A decrease in this protein is consistent with a cold induced halt in development. In
D. melanogaster , Zinc finger protein on ecdysone puffs appears to communicate between the hormonally activated transcription complexes and the RNA packaging and processing machinery, possibly contributing to early and late gene activation or possibly to RNA processing (Amero et al., 1990).
Suppression of this protein in RCH possibly reduces the rate of gene activation or RNA processing, consequently leading to the decreased protein synthesis and slow development at low temperatures. Zinc finger protein on ecdysone puffs was completely absent in all of our RCH gels, suggesting a major shut down in the production of this protein.
In summary, this proteomics approach points to several physiological responses that may be important components of the RCH mechanism. Our results imply that at least one of the sHsps is upregulated during RCH and that a mechanism to generate ATP is likely to be an essential component of RCH.
Likewise, at least one of the many tropomyosins is implicated in responding to
96 low temperature by increasing in abundance, thus suggesting a role for cytoskeletal modification during RCH.
Experimental procedures
Insect rearing
The colony of S. crassipalpis was maintained in our laboratory as described
(Denlinger, 1972). The flies were kept under long day conditions (15:9; light/dark) at 25 °C until larviposition, then maintained under the same long day conditions at 20 °C until they developed to the red eye pharate adult stage, at which time they were used for experiments. Under these conditions, none of the flies entered diapause.
Rapid cold hardening
Groups of 20 red eye pharate adult flies that had been maintained at 20 °C were placed in thin walled, 13 x 100 mm cotton plugged Pyrex test tubes, and the tubes were held at 0°C in a Lauda model RM20 glycerol bath for 2 h, after which the brains were immediately dissected for protein extraction.
Protein extraction
Brains were homogenized in 20mM Tris buffer (pH 8.0) supplemented with complete protease inhibitor cocktail tablets (Roche, Mannheim, Germany),
97 using a PowerGen homogenizer (Fisher Scientific) in an ice bath. The resulting homogenates were sonicated using a microtip followed by 60 min of centrifugation at 16000 g at 4°C. Supernatants were collected and processed using the ReadyPrepTM 2D cleanup kit (BioRad) according to the manufacturer’s instructions. Protein concentrations were assayed using an
RC/DC TM protein assay (BioRad) prior to two dimensional gel electrophoresis.
Two-dimensional gel electrophoresis
Protein samples were subjected to two dimensional gel electrophoresis (2DE) and Coomassie blue staining. In brief, the first dimensional isoelectric focusing was performed using IPG strips (pH 3 10 non linear, 11 cm) in the Protean system (BioRad). Focusing conditions were 20 min at 400 V, 2.5 h at 8000 V, and then at 8000 V until a total of at least 30 000 V h was reached at 20°C. After isoelectric focusing, the focused IPG strip was equilibrated and subjected to the second dimension using an 8 16% polyacrylamide SDS gel (BioRad). Each sample was run in triplicate. Gels were stained with Bio safe Coomassie blue and scanned using a BioRad VersaDoc imaging system.
In-gel digestion
Coomassie blue stained 2D gels were analyzed using Bio Rad software
PDQuest. Relatively abundant differentially displayed protein spots were
98 automatically excised from the 2D gels by a spot cutter (BioRad). The excised gels were subjected to in gel digestion with sequencing grade trypsin from
Promega (Madison, WI) according to standard protocols provided by Millipore
(Bedford, MA). Gel pieces were washed in 50% methanol/5% acetic acid for 1 h.
The wash step was repeated once, and then gel pieces were dehydrated in acetonitrile. The gel bands were rehydrated and incubated with dithiothreitol
(DTT) solution (5 mg/ml in 100 mM ammonium bicarbonate) for 30 min prior to the addition of 15 mg/ml iodoacetamide in 100 mM ammonium bicarbonate solution. Iodoacetamide was incubated with the gel bands in darkness for 30 min before being removed. The gel bands were washed again with cycles of acetonitrile and ammonium bicarbonate (100 mM) in 5 min increments. After the gels were dried in a speed vac, the protease was driven into the gel pieces by rehydrating them in 50 l sequencing grade modified trypsin or chymotrypsin at
20 g/mL in 50 mM ammonium bicarbonate for 10 min. Twenty microliters of
50 mM ammonium bicarbonate were added to the gel bands, and the mixture was incubated at room temperature overnight. Peptides were extracted from the polyacrylamide with 50% (v/v) acetonitrile and 5% (v/v) formic acid several times, pooled and concentrated in a speed vac to 25 l.
Mass spectrometric identification of proteins
Capillary liquid chromatography nanospray tandem mass spectrometry
99 (Nano LC/MS/MS) was performed on a Micromass Hybrid Quadrupole time of flight Q Tof (tm) II mass spectrometer (Micromass, Wythenshawe, UK) equipped with an orthogonal nanospray source from New Objective (Woburn,
MA) operated in a positive ion mode. The LC system was an UltiMate TM Plus system from LC Packings with a Famos autosampler and Switchos column switcher. Solvent A was water containing 50 mM acetic acid, and solvent B was acetonitrile. Five microliters of each sample was first injected onto the trapping column and then washed with 50 mM acetic acid. The injector port was switched to inject and the peptides were eluted from the trap onto the column. A
10 cm 50 mM ID BioBasic C18 column packed directly in the nanospray tip was used for chromatographic separations. Peptides were eluted directly off the column into the Q TOF system using a gradient of 2–80% B over 30 min, with a flow rate of 300 nl/min using a pre column split to approximately 500 nl/min.
Total run time was 55 min. The nanospray capillary voltage was set at 2.8 kV and the cone at 55 V. Source temperature was maintained at 100 °C. Mass spectra were recorded using MassLynx 4.0 with automatic switching functions.
Mass spectra were acquired from mass 400–2,000 Da/s with a resolution of
8,000 (FWHM: full width at half maximum). When the desired peak was detected at a minimum of 8 ion counts, the mass spectrometer automatically switched to acquire the CID MS/MS spectrum of the individual peptide.
Collision energy was set dependent on charge state recognition properties.
100 Sequence information from the MS/MS data was processed using Mascot
Distiller to form a peaklist (.mgf file) and by using the MASCOT MS/MS search engine and Turbo SEQUEST algorithm in BioWorks 3.1 Software. Data processing was performed following the guidelines of Carr et al. (2004).
Assigned peaks had a minimum of 10 counts (S/N of 3). The mass accuracy of the precursor ions were set to 1.5 Da to accommodate accidental selection of the
C13 ion, and the fragment mass accuracy was set to 0.5 Da. Considered modifications (variables) were methionine oxidation and carbamidomethyl cysteine.
Acknowledgments
We thank M. Robert Michaud, Ohio State University, for his helpful comments, David Mandich, OSU Plant Microbe Genomics Facility, for technical assistance with image analysis, and Kari Green Church, OSU Campus
Chemical Instrument Center, for assistance with mass spectrometric identification of proteins. This work was supported NSF grant IOB 0416720.
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104 Rinehart, J. P., Yocum, G. D. and Denlinger, D. L. (2000) Developmental upregulation of inducible hsp70 transcripts, but not the cognate form, during pupal diapause in the flesh fly, Sarcophaga crassipalpis . Insect Biochem Mol Biol 30 : 515–521. Rinehart, J. P., Li, A., Yocum, G. D., Robich, R. M., Hayward, S. A. L. and Denlinger, D. L. (2007) Up regulation of heat shock proteins is essential for cold survival during insect diapause. Proc Natl Acad Sci USA 104 : 11130–11137. Sun, Y. and MacRae, T. H. (2005) Small heat shock proteins: molecular structure and chaperone function. Cell Mol Life Sci 62 : 2460–2476. Talamillo, A., Fernandes Moreno, M. A., Martinez Azorin, F., Bornstein, B., Ochoa, P. and Garesse, R. (2004) Expression of the Drosophila melanogaster ATP synthase alpha subunit gene is regulated by a transcriptional element containing GAF and Adf 1 binding sites. Eur J Biochem 271 : 4003–4013. Ursic, D. and Culbertson, M. R. (1991) The yeast homolog to mouse Tcp-1 affects microtubule mediated processes. Mol Cell Biol 11 : 2629–2640. Ursic, D., Sedbrook, J. C., Himmel, K. L. and Culbertson, M. R. (1994) The essential yeast Tcp1 protein affects actin and microtubules. Mol Cell Biol 5: 1065–1080. Yocum, G. D., Joplin, K. H. and Denlinger, D. L. (1998) Upregulation of a 23 kDa small heat shock protein transcript during pupal diapause in the flesh fly, Sarcophaga crassipalpis . Insect Biochem Mol Biol 28 : 677–682.
105 Fig. 5.1 A 2 dimensional electrophoresis map of brain proteins from red eye pharate adults of the flesh fly Sarcophaga crassipalpis . Numbers refer to proteins that are identified in Table 5.1. pI= isoelectric point, MM= molecular markers.
106
Fig. 5.2 Selected 2DE images demonstrating changes in abundance of identified proteins following rapid cold hardening (RCH) for 2 h at 0 °C. Controls (CNT) were held continuously at 20 °C. Arrows indicate corresponding spots with significant differences. See Table 5.1 for further information on protein identification.
107
1.2 CNT RCH 1
0.8
0.6
0.4 RelativeRelative volume volume RelativeRelative volume volume 0.2
0 1 2 3 4 5 6 7 8 9 1011121314 Spot No.
Fig. 5.3 Statistically significant differences in protein abundance in response to RCH. PDQuest software 8.0.1 (BioRad) was used for spot detection, quantification and statistical analysis. See Fig. 5.2 for protein identification and experimental treatments.
108 Calculated Mowse Spot No. Entry name Species Protein ID mass (kDa)/PI score a Upregulated proteins in response to rapid cold hardening ATP synthase subunit alpha, 1 ATPA_DROME Drosophila melanogaster 59.6/9.09 480 mitochondrial precursor 10 HSP26_DROME Drosophila melanogaster Heat shock protein 26 23.2/7.12 58 12 TPM4_DROME Drosophila melanogaster Tropomyosin 1, isoforms 33/34 54.7/4.39 1359 Downregulated proteins in response to rapid cold hardening 2 BZLF1_EBV Epstein-Barr virus Trans activator protein BZLF1 27.1/5.25 86 Probable cysteine desulfurase, 3 NFS1_DROME Drosophila melanogaster 51.4/8.40 84 mitochondrial precursor 4 Aldehyde dehydrogenase 2B7,
109 AL2B7_ARATH Arabidopsis thaliana 58.5/6.88 61 mitochondrial precursor 5 TCPG_DROME Drosophila melanogaster T complex protein 1 subunit gamma 59.9/6.38 362 Probable isocitrate dehydrogenase 6 IDH3A_DROME Drosophila melanogaster subunit alpha, mitochondrial 41.2/6.96 433 precursor 7 PRS7_XENLA Xenopus laevis 26S protease regulatory subunit 7 49.0/ 5.72 123 9 CAPZA_DROME Drosophila melanogaster F actin capping protein subunit alpha 32.9/5.61 108 11 TPM1_DROME Drosophila melanogaster Tropomyosin 1, isoforms 9A/A/B 39.4/4.93 482 13 1433E_DROME Drosophila melanogaster 14 3 3 protein epsilon 30.0/4.68 721 Heat shock protein 83 14 HSP83_DROAV Drosophila auraria 82.0/4.89 418 (=Hsp90 in S. crassipalpis ) Proteins missing after rapid cold hardening 8 PEP_DROME Drosophila melanogaster Zinc finger protein on ecdysone puffs 78.6/5.46 90 Table 5.1 Identification of differentially regulated brain proteins in pharate adults of S. crassipalpis a Mowse score ＞54 indicates identity or extensive homology (P ＜0.05); higher scores indicate higher confidence of identity.
109
CHAPTER 6
DISTINCT CONTRACTILE AND CYTOSKELETAL PROTEIN PATTERNS IN
THE ANTARCTIC MIDGE ARE ELICITED BY DESICCATION AND
REHYDRATION
Aiqing Li 1, Joshua B. Benoit 1, Giancarlo Lopez Martinez 1, Michael A. Elnitsky 2,
Richard E. Lee Jr 2, and David L. Denlinger 1*
1 Department of Entomology, The Ohio State University, Columbus, OH 43210
2 Department of Zoology, Miami University, Oxford, OH 45056
*Corresponding author. Tel.: 614 292 6245; fax: 614 292 2180.
E mail: [email protected] (D.L. Denlinger).
110 ABSTRACT
Desiccation is well recognized as a major challenge for the Antarctic midge,
Belgica antarctica , but this stress is also exploited by the midge as a strategy to enhance freeze tolerance. In this study, midge larvae were desiccated at 75% relative humidity (RH) for 12 h to obtain a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration.
Rehydraton was performed at 100% RH for 6 h and in water for 6 h after desiccation, yielding fully hydrated larvae. Proteins were extracted from controls held at 100% RH, desiccated, and rehydrated larvae. Protein analysis was performed using 2 dimensional electrophoresis and nanoscale capillary
LC/MS/MS. Among the 26 identified proteins that changed in abundance in response to desiccation, 18 were upregulated and 8 were downregulated. Twenty of the 26 proteins (76%) were contractile and cytoskeletal proteins, including 6 actins, 10 myosin heavy chains, 1 myosin light chain, and 3 tropomyosins. A total of 13 rehydration regulated proteins were identified: 8 were upregulated and 5 were downregulated. Interestingly, nine of the 13 proteins (69%) were also contractile and cytoskeletal proteins, including 1 actin, 2 myosin heavy chains and
6 tropomyosins. Additional proteins responsive to desiccation and rehydration were functionally involved with a variety of responses including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major responses to both desiccation and rehydration elicit synthesis of a distinct pattern of contractile and cytoskeletal proteins that are likely to be involved in body contraction and cytoskeleton rearrangements.
111 INTRODUCTION
The Antarctic midge Belgica Antarctica (around 1.1 mg at 4 th instar larvae) is the largest free living terrestrial animal that lives year round in Antarctica, and it is the southern most free living holometabolous insect (Sugg et al., 1983; Usher and
Edwards, 1984). During its two year life cycle, larvae feed on moss, terrestrial algae, plant and animal debris and microorganisms, and overwinter in any of the four larval instars (Convey and Block, 1996; Sugg et al., 1983). Wingless adults emerge, mate in aggregations, lay eggs and die within a short two week period.
Desiccation stress is commonly encountered by B. antarctica larvae. On one hand, its habitat, the Antarctica continent, is frozen and is effectively a desert with water biologically unavailable in the form of ice (Campbell and Claridge,
1987). On the other hand, the larvae are highly permeable, with relatively high hemolymph osmolality, and lose water at an extremely high rate (Hayward et al.,
2007; Benoit et al., 2007a). To suppress desiccation, multiple mechanisms are exploited (Benoit et al., 2007a, 2007b). Behaviorally, water conservation can be achieved by clustering. Physiologically, the midges significantly increase their overall polysaccharide levels and accumulate trehalose and glycerol in response to slow dehydration (Hayward et al., 2007; Benoit et al., 2007a). Other physiological changes include a shift from short to long cuticular hydrocarbons, a decrease in oxygen consumption, elevated metabolites from pathways of central carbohydrate metabolism, and a shift in the free amino acid pool (Benoit et al., 2007a; Michaud et al., 2008). Water stores can be replenished only by intake of liquid water. A novel form of dehydration, cryoprotective dehydration, appears to be critical for winter survival (Elnitsky et al., 2008), thus bouts of dehydration and rehydration
112 appear to be a normal component of the midge’s seasonal cycle.
Proteomics, the comprehensive study of all proteins encoded by the genome of an organism, is a powerful technique for studying changes in protein abundance in response to various stresses. However, only a few studies have used proteomics to examine desiccation and rehydration responses of animals
(nematodes by Chen et al., 2006; rats by Gouraud et al., 2007). In the present study, a comparative proteome analysis was performed on B. antarctica larvae under hydrated conditions and in response to desiccation and rehydration stress to gain insights into the molecular mechanisms that control osmotic stress. Our results indicate that the pattern of contractile and cytoskeletal proteins changes dramatically in response to both desiccation as well as rehydration, thus suggesting that elements involved in body contraction and cytoskeleton rearrangements are among the functions most significantly altered by desiccation and recovery.
EXPERIMENTAL MATERIALS AND METHODS
Insects Third and fourth instar larvae of Belgica antarctica Jacobs used in these experiments were collected on Cormorant Island, Torgersen Island and Bonaparte
Point, near Palmer Station on Anvers Island (64°46'S, 64°40 'W) on the Antarctic
Peninsula in January 2006 and 2007. Substrate containing the larvae was brought into the Palmer Station laboratory and held at 4 °C. Larvae were hand picked from the substrate in ice cold water and stored at 4°C for 1–2 days prior to experimental use.
The experimental procedures are illustrated as a flow chart presented in
113 Fig. 6.1.
Desiccation and Rehydration Relative humidities (%RH) were generated in glass desiccators as described by Benoit et al. (2007a). Control larvae were held at
100% RH for 12 h. For the desiccation treatment, larvae were maintained at 75%
RH for 12 h. For the rehydration treatment, larvae were first desiccated at 75%
RH for 12 h and then transferred to 100% RH for 6 h and then to water for 6 h. All experiments were performed at 4 °C.
Extraction of Proteins Whole larvae were homogenized in 20 mM Tris HCl at pH
8.0, with complete protease inhibitor cocktail tablets (Roche, Mannheim,
Germany). The homogenates were sonicated using a microtip, centrifuged (16,000 g for 60 min at 4°C), and fractionated into soluble (supernatants) and insoluble
(pellets) proteins.
Protein in the supernatants were precipitated using the ReadyPrepTM 2D cleanup kit (BioRad) according to the manufacturer’s instructions and resolved in general purpose rehydration buffer [8 M urea, 2% (w/v) CHAPS. The protein solution in rehydration buffer was supplemented with 0.2% (w/v) Bio Lyte 3/10 ampholyte, 0.002% (w/v) bromophenol blue, and 50 mM dithiothreitol (DTT)], that formed soluble fractions for two dimensional polyacrylamide gel electrophoresis (2D PAGE).
The protein pellets obtained from centrifugation of the homogenates were sequentially washed three times in Tris HCl buffer (20 mM, pH 8.0) containing protease inhibitors, and centrifuged (13, 000 g for 30 min at 4 °C) after each wash.
114 The supernatants were removed after each spin. The pellets were taken up in strongly chaotropic 2 D rehydration buffer [7 M urea, 2 M thiourea, 4% (w/v)
CHAPS. The protein solution in rehydration buffer was supplemented with 0.2%
(w/v) Bio Lyte 3/10 ampholyte, 0.002% (w/v) bromphenol blue, 2% (w/v) SB
3 10 (N decyl N,N dimethyl 3 ammonio 1 propanesulfonate), and 50 mM DTT] and kept for 1 h at room temperature. These samples were then centrifuged
(16,000 g for 60 min at 25°C) and supernatants containing the proteins were defined as insoluble fractions used for 2D PAGE. Concentrations of soluble and insoluble proteins were determined using an RC/DC TM protein assay (BioRad) prior to 2D PAGE.
2D-PAGE The resulting protein samples were electrofocused on IPG strips (pH
3 10 non linear, 11 cm, BioRad) in a PROTEAN IEF Cell (BioRad). The IPG strips were rehydrated for 12 h at room temperature. The IEF running conditions were: 400 V for 20 min, 8,000 V for 2.5 h and 8,000 V for 35,000 V h for comparison of hydrated and desiccation soluble fractions with a pH range of
3 10; 250 V for 1h, 500 V for 1 h, 8,000 V for 2.5 h and 8,000 V for 40,000
V h for comparison of hydrated and desiccation insoluble fractions with pH ranges of 3 10 and 3 6; 400 V for 20 min, 8,000 V for 2.5 h and 8,000 V for 35,000
V h for comparison of desiccation and rehydration soluble fractions with a pH range of 3 10; 250 V for 20 min, 8,000 V for 1 h and 8,000 V for 45,000 V h for separation of desiccation and rehydration insoluble fractions with pH ranges of
3 10 and 3 6 at 20°C, respectively. Fifty A current was used per strip. The IPG strips were incubated for 20 min in equilibration buffer [6 M urea, 1.5 M Tris HCl,
115 pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS] containing 2% (w/v) DTT, followed by 20 min incubation in equilibration buffer containing 2.5% (w/v) iodoacetamide.
Second dimension electrophoresis was performed on an 8 16% polyacrylamide SDS gel in the BioRad Criterion Cell. Electrophoresis was carried out for 60 min at a steady voltage of 200 V. For each condition, namely normal hydration, desiccation and rehydration, at least 3 2D gels were run.
Staining of 2D-PAGE and Image Analysis Gels were fixed in 100 ml 40% ethanol and 10% acetic acid overnight. The fixed gels were then washed 3 times with 200 ml distilled water for 5 min. Proteins separated by 2D gels were visualized after staining with Bio safe Coomassie blue. The stained 2D gels were scanned using a
BioRad VersaDoc imaging system. Images of the scanned 2D gels were processed by PDQuest (version 7.4 for analysis between normal and desiccation, version 8.0 for analysis between desiccation and rehydration). The computer analysis included automatic detection, normalization, quantification of protein spots, and matching between control and treatment gels. The quantitative and statistical analyses were performed using suitable functions within the PDQuest software. The relative change in protein abundance for each protein spot was calculated by quantitative comparisons of the averaged normalized spot quantity between two conditions. A two tailed nonpaired Student’s t test was performed to determine if the relative change was statistically significant. Protein spots of interest were subjected to
LC MS/MS.
116 In-gel Trypsin Digestion Protein spots of interest were automatically excised from the 2D gels by a spot cutter (BioRad) and in gel digested with sequencing grade trypsin from Promega (Madison, WI) according to standard protocols provided by
Millipore (Bedford, MA). Gel pieces were twice washed in 50% methanol/5% acetic acid for 1 h and then dehydrated in acetonitrile. The gel bands were rehydrated and incubated with DTT solution (5mg/ml in 100 mM ammonium bicarbonate) for 30 min prior to the addition of 15mg/ml iodoacetamide in 100 mM ammonium bicarbonate solution. Iodoacetamide was incubated with the gel bands in darkness for 30 min before being removed. The gel bands were washed again with cycles of acetonitrile and ammonium bicarbonate (100mM) in 5 min increments. The gels were then dried in a speed vac and reconstituted in 50 l sequencing grade modified trypsin or chymotrypsin at 20 g/mL in 50 mM ammonium bicarbonate for 10 min. Twenty microliters of 50 mM ammonium bicarbonate were added to the gel bands, and the mixture was incubated at room temperature overnight. Peptides were extracted from the polyacrylamide with 50% acetonitrile and 5% formic acid several times, combined and dried in a speed vac to 25 l.
Nano-LC-MS/MS Capillary liquid chromatography nanospray tandem mass spectrometry (Nano LC/MS/MS) was performed by coupling an UltiMate TM Plus system (LC Packings) to a Micromass Hybrid Quadrupole time of flight Q Tof
(tm) II mass spectrometer (Micromass, Wythenshawe, UK), operating in a positive ion mode and equipped with an orthogonal nanospray source from New Objective
(Woburn, MA). The LC system was equipped with a Famos autosampler and
117 Switchos column switcher. Solvent A was water containing 50 mM acetic acid, and solvent B was acetonitrile. Five microliters of each sample was first injected onto the trapping column and then washed with 50 mM acetic acid. The injector port was switched to inject and the peptides were eluted from the trap onto the column. A BioBasic C18 column (10 cm 50 M ID) packed directly in the nanospray tip was used for chromatographic separations. Peptides were eluted directly off the column into the Q TOF system using a gradient of 2–80% B over
30 min, with a flow rate of 300 nl/min using a pre column split to approximately
500 nl/min. Total run time was 55 min. The nanospray capillary voltage was set at
2.8 kV and the cone at 55 V. Source temperature was maintained at 100 °C. Mass spectra were recorded using MassLynx 4.0 with automatic switching functions.
Mass spectra were acquired from mass 400–2,000 Da/s with a resolution of 8,000
(FWHM: full width at half maximum). When the desired peak was detected at a minimum of 8 ion counts, the mass spectrometer automatically switched to acquire the CID MS/MS spectrum of the individual peptide. Collision energy was set dependent on charge state recognition properties.
Protein Identification Sequence information from the MS/MS data was processed using Mascot Distiller to form a peaklist (.mgf file) and by using the MASCOT
MS/MS search engine and Turbo SEQUEST algorithm in BioWorks 3.1 Software.
Data processing was performed following the guidelines of Carr et al. (2004).
Assigned peaks had a minimum of 10 counts (S/N of 3). The mass accuracy of the precursor ions were set to 1.5 Da to accommodate accidental selection of the C13 ion, and the fragment mass accuracy was set to 0.5 Da. Methionine oxidation and
118 carbamidomethyl cysteine were variable modifications.
RESULTS
2D-PAGE Analysis of Desiccated Larvae
To identify desiccation responsive proteins, total proteins were extracted from control larvae held at 100% RH for 12 h and larvae desiccated at 75% RH for 12 h.
To enhance the relative abundance of low solubility proteins, we fractionated the proteins into soluble and insoluble proteins. Both soluble and insoluble proteins were run on replicate 2D gels (8 16%, pH 3 10 nonlinear) and representative images are shown in Fig. 6.2 A and B, respectively. When insoluble proteins, regardless of whether they were extracted from hydrated or desiccated larvae, were subjected to IEF in a 3 10 pH gradient, a nonrandom distribution was observed because many spots clustered in the 3 6 pH range. Therefore, in subsequent experiments, we fractionated insoluble proteins from the two conditions on a 3 6 pH gradient in the first dimension (Fig. 6.2C).
Quantitative analysis based on PDQuest revealed that 107 proteins were regulated by as least 1.5 fold in response to desiccation, including 30 from the soluble fraction and 48 from the insoluble fraction on a 3 10 pH gradient and 29 from the insoluble fraction on a 3 6 pH gradient. Analysis by Student’s t test showed that the proteins with significant differences in abundance included 21 from the soluble fraction, 25 from the insoluble fraction on a 3 10 pH gradient, and 6 from the insoluble fraction on a 3 6 pH gradient (Table 6.1). Relatively abundant protein spots with an average ratio value greater than 1.5 fold and a t test p value <0.05 were selected for mass spectrometric identification. A total of
119 27 selected proteins (13 soluble, and 14 insoluble) are shown in Fig. 6.2, and their corresponding identities are summarized in Table 6.2.
Identification of Proteins that Changed in Response to Desiccation
The relative abundance of 27 differentially expressed proteins in control and desiccated larvae was determined based on PDQuest (Fig. 6.4 A): 18 were upregulated and 9 were downregulated upon desiccation. Quantities of the 27 proteins were normalized and divided by the quantity of the most abundant spot
(spot 5). Changes in abundance of these proteins ranged from 1.5 fold (spot 19) to
2.2 fold (spot 13).
Twenty six of the 27 proteins were successfully identified and could be functionally classified as cytoskeletal and contractile proteins (76%), proteins involved in energy metabolism (12%), stress responses (8%) and membrane transport (4%) (Fig. 6.5A). The majority of proteins that showed differences (20 proteins) were contractile and cytoskeletal proteins, including 6 actins, myosin light chain, 10 myosin heavy chains and 3 tropomyosins. Among these 20 cytoskeletal and contractile protein, 2 isoforms of actins and 5 isoforms of myosin heavy chains were less abundant, but the remainder were more abundant following desiccation. The three proteins involved in energy metabolism (arginine kinase, glyceraldehyde phosphate dehydrogenase, enolase) were all more abundant following desiccation. The two stress related proteins included catalase, which was more abundant, and ubiquitin like protein, which was less abundant after desiccation. Porin, a protein functionally involved in membrane transport, increased in abundance when the larvae were desiccated.
120
2D-PAGE Analysis of Larvae that were Desiccated, then Rehydrated
To investigate changes in protein abundance during larval recovery from desiccation, total proteins were extracted from larvae desiccated at 75% RH for 12 h and then rehydrated at 100% RH for 6 h and in water for 6 additional hours following desiccation. As above, proteins were separated into soluble and insoluble fractions, and run on 2D gels (8 16%, pH 3 10 nonlinear).
Representative images for the soluble and insoluble fractions are shown in Fig. 6.3
A and B, respectively. Insoluble proteins from both desiccation and rehydration treatments were also focused on a 3 6 pH gradient in the first dimension because clustered proteins were observed in the 3 6 pH range (Fig 6.3C).
Based on quantitative analysis, 176 proteins were regulated by as least
1.5 fold due to rehydration, including 76 from the soluble fraction and 57 from the insoluble fraction on a 3 10 pH gradient and 43 from the insoluble fraction on a
3 6 pH gradient. Statistical analysis using Student’s t test showed that proteins with significant differences between the 2 treatments included 23 from the soluble fraction, 26 from the insoluble fraction on a 3 10 pH gradient and 6 from the insoluble fraction on a 3 6 pH gradient (Table 6.1). Relatively abundant protein spots with an average ratio value greater than 1.5 fold and a t test p value <0.05 were selected for identification (Fig. 6.3) and their corresponding identities are displayed in Table 6.3.
Identification of Proteins that Changed in Response to Rehydration
PDQuest based image analyses were used to quantify the relative abundance of all
121 25 desiccation and rehydration regulated proteins (Fig. 6.4B). The quantity of the spots were normalized and divided by that of the most abundant spot (spot 8). The extent of differential abundance of these proteins ranged from 1.5 fold (spot 9) to
4.4 fold (spot 24).
Thirteen of the 25 proteins were successfully identified by LC MS/MS
(Table 6.3). These 13 proteins were classified according to their functions (Fig.
6.5B). Nine proteins (69% of the total) were contractile or cytoskeletal proteins: actin, 3 tropomyosins and 2 myosin heavy chains were more abundant and 3 tropomyosins were less abundant. Two proteins were stress related: heat shock protein Hsp70, which increased in abundance and anterior fat body protein like, which was less abundant after rehydration. One protein that was more abundant, elongation factor 1 alpha, is involved in the regulation of protein synthesis. Malate dehydrogenase, which is involved in glucogenesis, was less abundant following rehydration.
DISCUSSION
We compared the proteomic profiles of larval B. antarctica during their normal hydrated state, following desiccation, and after rehydration. One might anticipate that rehydration would simply be the reverse of desiccation, and for some proteins, this was the case. For example, one of the actins and two myosin heavy chains that decreased in abundance during desiccation increased again during rehydration, and by contrast, three tropomyosins that increased during desiccation declined again after rehydration. But such reversals were not consistently observed, indicating that rehydration is not simply the reverse of
122 desiccation but elicits distinct changes in many contractile and cytoskeletal proteins.
Tropomoysin plays a central role in regulating muscle contraction in skeletal muscle. In non muscle cells, it is associated with contractile actomyosin structures as well (Lin et al., 1997). Actin, tropomyosin and myosin are functionally linked in the myofibrils (Corsi and Perry, 1958). Muscle contraction is driven by interactions of the actin myosin cross bridge, where the binding of myosin to actin is regulated by a complex of thin filament associated proteins, tropomyosin and troponin (Sliwinska et al., 2008). Our results, showing that 11 myosin proteins change during desiccation and 2 myosins during recovery, suggests that contraction modulates myosin synthesis, as suggested by previous research (McDermott and Morgan, 1989; Qi et al., 1997). Tropomyosin, a core component of the actin linked regulatory system for contraction, stabilizes the actin filament and modulates muscle contraction (Yu and Ono, 2006). The isoforms perform unique functions and are not redundant (Gunning et al., 2008).
Animals contract when external water decreases. Thus, the contracted animals decrease their surface to volume ratio as they lose internal water loss during desiccation (Ricci et al., 2003). Changes of many different contractile proteins
(actins, tropomyosins and myosins, including light chains and heavy chains) support the idea that active contraction helps prevent mechanical damage during desiccation (Ricci et al., 2003; Alpert, 2005). During contraction, the actin skeleton is dynamic and actin remodeling occurs (Gerthoffer, 2005). Tropomyosin regulates actin myosin interactions in the cytoskeleton as well as in muscle, and the isoforms differ in their ability both to interact with myosin and to inhibit
123 translocation of actin filaments by myosin (Gunning et al., 2008).
Body contraction may be a key feature for recovery from desiccation
(Ricci et al., 2003). The cytoskeleton plays a significant role in the desiccation response and recovery in moss (Proctor et al., 2007). The cytoskeleton is more complex than the contractile systems in terms of composition and function: the contractile systems use 4 actins and 5 tropomyosins, while the cytoskeleton uses 2 actins and over 40 tropomyosins (Gunning et al., 2008). But, how the different isoforms contribute to body contraction and the cytoskeleton dynamics remains unknown.
Another group of proteins that increased in response to desiccation is involved in energy metabolism. Arginine kinase plays a role in maintenance of
ATP levels by producing phosphoarginine, which can rapidly replenish ATP.
Two other proteins upregulated in response to desiccation are glyceraldehyde 3 phosphate dehydrogenase and enolase, enzymes involved in the energy yielding phase of the glycolysis pathway. An increase in abundance of all three of these enzymes suggests an increase in ATP production and enhanced ability of maintaining ATP levels during desiccation.
Catalase also increased in response to desiccation stress. This protein response is consistent with the elevation of mRNA encoding catalase that was also noted in response to desiccation (Lopez Martinez et al., 2008). A similar elevation in catalase was observed in response to dehydration in yeast (Franca et al., 2005).
An increase in catalase is frequently linked to oxidative stress, and desiccation possibly contributes to oxidative stress in the midge larvae.
Porin is a non specific channel that is permeable to hydrophilic solutes and
124 is involved in maintenance of cell surface structure in bacteria (Nikaido and Vaara,
1985). In B. antarctica , an increase in porin during desiccation may be involved in the efflux of water or other solutes during this time. Ubiquitin is well known to play a role in controlling protein turnover, and it appears to play a role in combating dehydration induced stresses in a eubacterium (Durner and Boger,
1995). But, during desiccation in B. antarctica this protein declined in abundance.
Heat shock proteins play an important chaperone role in a variety of cellular stress responses (Feder and Hofmann, 1999). In this system we found that
Hsp70 was not present in increased abundance during desiccation, but it did increase during recovery from desiccation, i.e., rehydraton. This result is consistent with the observation that Hsp transcripts remained unchanged in response to desiccation stress (Hayward et al., 2007). Distinct Hsp responses were also noted in response to desiccation and rehydration in flesh fly pupae (Hayward et al ., 2004), but in the fly pupae Hsp23 and Hsp70 were upregulated by desiccation and Hsp90 and Hsc70 were upregulated by rehydration. This suggests important roles for Hsps in these responses but also suggests that different species may respond differently.
Anterior fat body protein like protein decreases during rehydration. This protein, which previously has been associated with stress responses, is restricted to the anterior fat body (Nakajima and Natori, 2000) and is involved in regulation of endocytosis of hexamerin by fat body cells (Hansen et al., 2002). It is not at all clear what role it may play in association with desiccation and rehydration.
Another protein that is less abundant during rehydration is malate dehydrogenase, an enzyme involved in carbohydrate metabolism. The decrease of
125 this protein during rehydration may be associated with depression of the gluconeogenesis pathway, a pathway that is highly activated during osmotic stress
(Dihazi et al., 2005), but switches off as the insect reacts to its normal hydrated state.
Elongation factor 1 alpha is a key factor in the elongation process of protein synthesis. Besides its role in translation, it is also implicated in cytoskeletal remodeling (Condeelis, 1995). The increase of elongation factor 1 alpha during rehydration suggests increased protein synthesis. The regulation of this protein together with the cytoskeletal proteins described earlier further illustrate that reorganization of the cytoskeleton is essential to desiccation stress tolerance and recovery.
Our results clearly show that both desiccation and rehydration elicit dramatic changes in protein abundance within a short period of time. The majority of proteins that are altered in abundance are associated with contraction of the body and cytoskeleton rearrangements. It is also clear from this study that rehydration is not simply the reverse of dehydration, but instead generates a distinct protein profile.
Acknowledgments We thank the support staff at Palmer Station for their assistance in Antarctica, David Mandich of Plant Microbe Genomics Facility at
The Ohio State University, for technical assistance with image scanning, and Kari
Green Church, OSU Campus Chemical Instrument Center, for assistance with mass spectrometric identification of proteins. This research was supported by NSF grants OPP 0337656 and OPP 0413786.
126 REFERENCES
Alpert, P. (2005) The Limits and Frontiers of Desiccation Tolerant Life. Interg. Comp. Biol. 45 , 685–695 Benoit, J.B., Lopez Martinez, G., Michaud, M. R., Elnitsky, M. A., Lee, R. E., and Denlinger, D.L. (2007a) Mechanisms to reduce dehydration stress in larvae of the Antarctic midge, Belgica antarctica . J. Insect Physiol. 53 , 656–667 Benoit, J. B., Lopez Martineza, G., Elnitsky, M. A., Lee Jr, R. E., Denlinger, D. L. ( 2007b) Moist habitats are essential for adults of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae), to avoid dehydration. Eur. J. Entomol. 104 , 9–14 Campbell, I. B., and Claridge, G. G. C. (1987) Antarctica: Soils, Weathering Processes and Environment . Amsterdam: Elsevier. Carr, S., Aebersold, R., Baldwin, M., Burlingame, A., Clauser, K. and Nesvizhskii, A. (2004) The need for guidelines in publication of peptide and protein identification data. Mol and Cell Proteomics 3, 531–533 Chen , S., Glazer, I., Gollop, N., Cash, P., Argo, E., Innes, A., Stewart, E., Davidson, I., Wilson, M. J. (2006) Proteomic analysis of the entomopathogenic nematode Steinernema feltiae IS 6 IJs under evaporative and osmotic stresses. Mol. Biochem. Parasitol. 145 , 195–204 Condeelis, J. (1995) Elongation factor 1a, translation and the cytoskeleton. Trends Biochem. Sci. 20 , 169–170 Convey, P., and Block, W. (1996) Antarctic Diptera: ecology, physiology and distribution. Eur. J. Entomol. 93 , 1–13 Corsi, A., and Perry, S. V. (1958) Some observations on the localization of myosin, actin and tropomyosin in the rabbit myofibril. Biochem J 68 , 12–17 Dihazi, H., Asif, A. R., Agarwal, N. K., Doncheva, Y., and Muller, G. A. (2005) Proteomic analysis of cellular response to osmotic stress in thick ascending limb of Henle’s loop (TALH) Cells. Mol Cell Proteomic 4, 1445–1458 Durner, J. and Boger, P. (1995) Ubiquitin in the prokaryote Anabaena variabilis. J. Biol. Chem 270 , 3720–3725 Elnitsky, M. A., Hayward, S. A. L., Rinehart, J. P., Denlinger, D. L., and Lee, R. E. (2008) Cryoprotective dehydration and the resistance to inoculative freezing in the Antarctic midge, Belgica antarctica . J. Exp. Biol. 211 , 524–530
127 Franca, M. B., Panek, A. D., and Eleutherio, E. C. A. 92005) The role of cytoplasmic catalase in dehydration tolerance of Saccharomyces cerevisiae . Cell Stress Chaperones 10 , 167–170 Gerthoffer, W. T. (2005) Actin cytoskeletal dynamics in smooth muscle contraction. Can. J. Physiol. Pharmacol. 83 , 851–856 Gouraud, S.S., Yao, S. T., Heesom, K. J., Paton, J. F. R., and Murphy, D. (2007) 14 3 3 proteins within the hypothalamic neurohypophyseal system of the osmotically stressed rat: transcriptomic and proteomic studies. J Neuroendocrinol 19 , 913–922 Gunning, P., Schevzov, G., Kee, A., and Hardeman, E. (2005) Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends Cell Biol. 15 , 333–341 Gunning, P., O'Neill, G., Hardeman, E. (2008) Tropomyosin based regulation of the actin cytoskeleton in time and space. Physiol. Rev. 88 , 1–35 Hansen, I. A., Meyer, S. R., Schafer, I., and Scheller, K. (2002) Interaction of the anterior fat body protein with the hexamerin receptor in the blowfly Calliphora vicina. Eur. J. Biochem. 269 , 954–960 Hayward, S. A. L., Rinehart, J. P., and Denlinger, D. L. (2004) Desiccation and rehydration elicit distinct heat shock protein transcript responses in flesh fly pupae. J. Exp. Biol. 207 , 963–971 Hayward, S. A. L., Rinehart, J. P., Sandro, L. H., Lee, Jr, R. E., and Denlinger, D. L. (2007) Slow dehydration promotes desiccation and freeze tolerance in the Antarctic midge Belgica antarctica . J. Exp. Biol. 210 , 836–844 Lin, J. J., Warren, K. S., Wamboldt, D. D., Wang, T., and Lin, J. L. (1997) Tropomyosin isoforms in nonmuscle cells. Int. Rev. Cytol. 170 , 1–38 McDermott, P.J., and Morgan, H.E. (1989) Contraction modulates the capacity for protein synthesis during growth of neonatal heart cells in culture. Circ. Res. 64 , 542–553 Michaud, M. R., Benoit, J. B., Lopez Martinez, G., Elnitsky, M. A., Lee, R. E., and Denlinger, D. L. (2008) Metabolomics reveals unique and shared metabolic changes in response to heat shock, freezing, and desiccation in the Antarctic midge, Belgica antarctica. J. Insect Physiol. 54 , 653–663 Nakajima, Y., and Natori, S. (2000) Identification and characterization of an anterior fat body protein in an insect. J. Biochem. 127 , 901–908 Proctor, M. C. F., Ligrone, R., and Duckett, J. G. (2007) Desiccation tolerance in
128 the moss Polytrichum formosum : physiological and fine structural changes during desiccation and recovery. Ann. Bot. 99, 75–93 Qi, M., Puglisi, J.L., Byron, K.L., Ojamaa, K., Klein, L., Bers, D.M., and Samarel, A.M. (1997) Myosin heavy chain gene expression in neonatal rat heart cells: effects of [Ca2+]i and contractile activity. Am. J. Physiol. 273 , C394–C403. Ricci, C., Melone, G., Santo, N., and Caprioli., M. (2003) Morphological response of a bdelloid rotifer to desiccation. J. Morphol. 257 , 246–253 Sinclair, B. J., Gibbs, A. G., and Roberts, S. P. (2007) Gene transcription during exposure to, and recovery from, cold and desiccation stress in Drosophila melanogaster . Insect Mol. Biol. 16 , 435–443 Sliwinska, M., Skorzewski, R., and Moraczewska, J. (2008) Role of actin C terminus in regulation of striated muscle thin filament. Biophys. J. 94 , 1341–1347 Sugg, P., Edwards, J.S., and Baust, J. (1983) Phenology and life history of Belgica antarctica , an Antarctic midge (Diptera: Chironomidae). Ecol. Entomol. 8, 105–113 Usher, M.B., Edwards, M., (1984) A dipteran from south of the Antarctic Circle: Belgica antarctica (Chironomidae), with a description of its larva. Biol. J. Linn. Soc. 23 , 19–31 Yu, R. and Ono, S. (2006) Dual roles of tropomyosin as an F actin stabilizer and a regulator of muscle contraction in Caenorhabditis elegans body wall muscle. Cell Motil. Cytoskeleton 63 , 659–672
129
Fig. 6.1 Flow chart of proteomic analysis of hydrated, desiccated and rehydrated B. antarctica larvae.
130
Fig. 6.2 Representative 2DE maps of proteins from Belgica antarctica larvae after desiccation at 75% RH for 12 h at 4 °C. (A) Soluble proteins were resolved onto IPG3 10 strips (nonlinear); (B) insoluble proteins resolved onto IPG3 10 strips (nonlinear) and (C) IPG3 6 strips in the first dimension, and 8 16% polyacrylamide SDS PAGE gels in the second dimension. Protein spots were then visualized by Bio safe Coomassie staining. Maps were analyzed with PDQuest software. The identified spots are annotated by numbers according to Table 6.2.
131
Fig. 6.3 Representative 2DE maps of proteins from Belgica antarctica larvae after rehydration at 100% RH for 6 h and then in water for 6 h following desiccation at 75% RH for 12 h at 4 °C . (A) Soluble proteins were resolved onto IPG3 10 strips (nonlinear); (B) insoluble proteins resolved onto IPG3 10 strips (nonlinear) and (C) IPG3 6 strips in the first dimension, and 8 16% polyacrylamide SDS PAGE gels in the second dimension. Protein spots were then visualized by Bio safe Coomassie staining. Maps were analyzed with PDQuest software. The identified spots are annotated by numbers according to Table 6.3.
132 1.2 A Hydrated state Desiccation 1
0.8
0.6
0.4 Relative volume . 0.2
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Spot number
1.2 B Desiccation Rehydration 1
0.8
0.6
0.4 Relative volume . 0.2
0 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425 Spot number
Fig. 6.4 Quantification of proteins that changed in abundance (A) after desiccation at 75% RH for 12 h and (B) rehydration at 100% RH for 6 h and then in water for 6 h following desiccation at 75% RH for 12 h. All protein spots were quantified by scanning Coomassie stained images by PDQuest image analysis software. The numbers denoting various proteins on the x axis are the same as shown in Tables 6.2 3 and Figs. 6.2 3.
133 B. Proteins responding to rehydration A. Proteins responding to desiccation
Membrane transport Protein synthesis 4% Stress responses 8% 8% Glucogenesis 8%
Energy metabolism 12%
Stress responses 15%
Cytoskeletal and contractile Contractile and cytoskeletal protein protein 76% 69%
Fig. 6.5 Functional classification of the identified proteins that changed in abundance in response to (A) desiccation and (B) rehydration.
134 No. Responding to desiccation No. Responding to rehydration Analysis Soluble Insoluble Insoluble Soluble Insoluble Insoluble pH 3 10 pH 3 10 pH 3 6 pH 3 10 pH 3 10 pH 3 6 Proteins ≥1.5 30 48 29 76 57 43 fold change Significant change 21 25 6 23 26 6 (t test) Significant and ≥1.5 fold 14 10 4 10 18 3 change Table 6. 1 Number of proteins that responded to desiccation (75% RH for 12 h at 4 °C) and rehydration (100% RH for 6 h and then in water for 6 h at 4 °C, following desiccation). Signifiance based on Student’s t test ( p <0.05).
135 Predicted Spot Accession Mowse Species Protein ID mass(kDa)/ No. No. Score* PI Desiccation upregulated proteins 2 46909285 Enallagma aspersum catalase 32.2/7.26 184 PREDICTED: similar to 3 66521459 Apis mellifera 30.6/8.25 108 porin glyceraldehydephosphate 4 62616 Coturnix coturnix 35.9/8.71 87 dehydrogenase
136 5 86450230 Blattella germanica enolase 47.4/5.91 245 6 551380 Marsupenaeus japonicus arginine kinase 40.3/6.36 306 7 7321108 Chironomus kiiensis tropomyosin 32.6/4.74 1059 PREDICTED: similar to 8 66522386 Apis mellifera 32.3/4.73 674 tropomyosin 1 Table 6.2 (continued) Table 6.2 Identification of larval proteins in Belgica antarctica that responded to desiccation. * Mowse score>54 indicates identity or extensive homology (P<0.05); higher scores indicate higher confidence of identity.
136 Table 6.2 (continued) Spot Predicted Accession Mowse No. Species Protein ID mass(kDa)/ No. Score* PI PREDICTED: similar to 9 66522386 Apis mellifera 32.3/4.73 730 tropomyosin 1 10 797290 Molgula citrina muscle actin 41.9/5.31 379 11 72256864 Culex pipiens pallens putative myosin light chain 2 22.9/ 4.65 243 137 12 71616 Bos taurus actin, aortic smooth muscle 42.1/5.24 172 13 15553463 Oryctolagus cuniculus beta actin 8.5/5.50 307 18 157891 Drosophila melanogaster myosin heavy chain 225.4/5.86 554 21 2546937 Drosophila melanogaster muscle myosin heavy chain 135.6/5.46 2022 22 2546937 Drosophila melanogaster muscle myosin heavy chain 135.6/5.46 1382 24 2546937 Drosophila melanogaster muscle myosin heavy chain 135.6/ 5.46 1185 25 2546937 Drosophila melanogaster muscle myosin heavy chain 135.6/ 5.46 810
137 Table 6.2 (continued) Predicted Spot Accession Mowse Species Protein ID mass(kDa)/ No. No. Score* PI 27 156765 Drosophila melanogaster actin 42.2/5.45 1022 Desiccation downregulated proteins 1 Autographa californica 332489 ubiquitin like protein 8.7/ 5.76 90 nucleopolyhedrovirus 138 14 17975540 Drosophila melanogaster Actin 79B CG7478 PA 42.2/5.30 236 15 2546936 Drosophila melanogaster muscle myosin heavy chain 135.8/5.49 92 17 157891 Drosophila melanogaster myosin heavy chain 225.4/5.86 106 19 17975540 Drosophila melanogaster Actin 79B CG7478 PA 42.2/5.30 134 20 2546936 Drosophila melanogaster muscle myosin heavy chain 135.8/5.49 361 23 2546936 Drosophila melanogaster muscle myosin heavy chain 135.8/5.49 253 26 2546936 Drosophila melanogaster muscle myosin heavy chain 135.8/5.49 379 16 - unknown
138 Predicted Accession Mowse Spot No. Species Protein ID mass(kDa)/ No. Score* PI Rehydration upregulated proteins 7 157131813 Aedes aegypti tropomyosin invertebrate 32.6/4.83 300 10 2921217 Beroe ovata heat shock protein Hsp70 51.8/ 121 19 157110721 Aedes aegypti myosin heavy chain 222.4/5.76 132 20 46909337 Stylochus sp. elongation factor 1 alpha 45.7/ 76 139 21 157110721 Aedes aegypti myosin heavy chain 222.4/5.76 215 Strongylocentrotus 22 47551039 cytoskeletal actin IIIa 42.0/5.46 317 purpuratus similar to tropomyosin 2 24 66522386 Apis mellifera 32.2/4.73 390 CG4843 PB, isoform B similar to tropomyosin 1 25 48094441 Apis mellifera CG4898 PD, isoform D 33.0/ 4.74 490 isoform 1 8,9,23 - unknown Table 6.3 (continued) Table 6.3 Identification of larval proteins in Belgica antarctica that responded to rehydration after desiccation.
139 Table 6.3 (continued) Predicted Accession Mowse Spot No. Species Protein ID mass(kDa)/ No. Score* PI Rehydraton downregulated proteins similar to mitochondrial 1 91085015 Tribolium castaneum malate dehydrogenase 35.5/9.42 319 precursor 32.8/4.72 4 51979106 Myzus persicae tropomyosin 532 140 tropomyosin 2 CG4843 PB, 5 24647095 Drosophila melanogaster 32.9/4.74 454 isoform B 6 53148459 Plutella xylostella tropomyosin I 32.6/4.74 282 11 104531117 Belgica antarctica anterior fat body protein like 150.0/5.23 210 2,3, 12 18 - unknown * Mowse score>54 indicates identity or extensive homology (P<0.05); higher scores indicate higher confidence of identity.
140
CONCLUSIONS
This dissertation reports on insect diapause and stress responses at the protein level. The flesh fly, S. crassipalpi s, and the Antarctica midge, B. antarctica , were chosen as model organisms. Using two dimensional gel electrophoresis and mass spectrometry, we have identified proteins upregulated, downregulated, missing and uniquely present during diapause, rapid cold hardening, desiccation and rehydration. When possible, we have assigned functions to these proteins. In addition, a diapause associated gene encoding neuropeptide like precursor 4 in S. crassipalpis was cloned and characterized.
Ⅰ. Proteomics of the flesh fly brain reveals an abundance of upregulated heat shock proteins during pupal diapause
1. We detected 37 diapause unique or upregulated (≥2x) proteins, and 43
proteins that were downregulated or not present in diapause.
2. Hsps (Hsp70 and several small Hsps) were among the most conspicuous
brain proteins present in higher amounts during diapause. The presence of
141 the Hsps in the brains of diapausing pupae nicely supports our previous
evidence that the mRNAs encoding Hsp70 and Hsp23 are highly
upregulated during diapause. We speculate that Hsps play an essential
role in enabling the diapausing fly pupae to survive for long periods at
low temperature.
3. Three of the identified proteins shown to be less abundant in brains of
diapausing pupae are related to metabolic processes that are most likely
less active during diapause. These include phosphoenolpyruvate synthase,
an enzyme involved in gluconeogenesis, a fatty acid binding protein, and
an endonuclease.
4. While the mRNAs encoding some of these proteins (e.g.Hsps) were
previously known to be associated with diapause, the other proteins were
not known to be linked to diapause, thus suggesting that the proteomic
approach nicely supplements work done at the transcript level.
Ⅱ. Neuropeptide like precursor 4 (Nplp4) is uniquely expressed during pupal diapause in the flesh fly
1. Suppressive subtractive hybridization comparing brains from diapausing
142 and nondiapausing pupae of the flesh fly, S. crassipalpis, suggested that
the gene encoding Nplp4 was uniquely expressed during diapause. We
have sequenced the full length cDNA encoding Nplp4 and used northern
blots to further evaluate linkage to diapause.
2. The open reading frame of this cDNA encodes a 61 amino acid residue
precursor protein containing a predicted 18 residue signal peptide, two
22 amino acid and 2 amino acid propeptides, and a 19 amino acid
neuropeptide. The amino acid sequence of the precursor protein shows
64% identity to Drosophila melanogaster Nplp4.
3. Nplp4 mRNA levels were quite low in nondiapausing (long day) pupae,
but in contrast the gene was highly upregulated in diapausing (short day)
pupae. Expression increased at the onset of diapause, remained high
throughout diapause, and then decreased 2 days after diapause was
terminated. This close association with diapause suggests a potential role
for Nplp4 in initiating and maintaining diapause in the flesh fly.
Ⅲ. Rapid elevation of Inos and decreases in abundance of other brain proteins at pupal diapause termination in the flesh fly Sarcophaga crassipalpis
143 1. The proteome analysis of brains following pupal diapause termination in
S. crassipalpis showed that among the most abundant proteins that
showed a significant change, 1 was more abundant, 7 were less abundant,
and 2 were absent following diapause termination.
2. The protein that increased in abundance following diapause termination
showed highest identity to myo inositol 1 phosphate synthase (Inos).
3. Proteins that decreased at diapause termination include those showing
highest identity to fatty acid binding protein, CG2331 PA, twinstar,
catalase, and a histone. Proteins absent at diapause termination included
ribosomal protein L17A and one unnamed protein.
4. Attempts to terminate diapause by injection of several Inos related
metabolites failed, thus suggesting that the elevation of Inos at diapause
termination is downstream of the physiological regulation that initiates
development.
Ⅳ. Rapid cold hardening elicits changes in brain protein profiles of the flesh fly
1. Fourteen proteins showing the greatest differences in response to rapid
144 cold hardening (RCH) were selected for mass spectrometric identification.
Three proteins that increased in abundance during RCH included ATP
synthase subunit alpha, a small heat shock protein (smHsp), and
tropomyosin 1 isoforms 33/34. Eleven proteins that decreased in
abundance or were missing following RCH included several proteins
involved in energy metabolism, protein degradation, transcription, actin
binding, and cytoskeleton organization.
2. That several proteins increased in abundance during RCH underscores
the dynamics of the RCH mechanism and points to several physiological
responses that likely contribute to RCH.
3. Our results imply that at least one of the sHsps is upregulated during
RCH and that a mechanism to generate ATP is likely to be an essential
component of RCH.
4. At least one of the many tropomyosins is implicated in responding to low
temperature by increasing in abundance, thus suggesting a role for
cytoskeletal modification during RCH.
Ⅴ. Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are
145 elicited by desiccation and rehydration
1. Proteins from controls held at 100% RH, desiccated, and rehydrated
larvae were analyzed using 2 dimensional electrophoresis and nanoscale
capillary LC/MS/MS.
2. Among the 26 identified proteins that changed in abundance in response
to desiccation, 18 were upregulated and 8 were downregulated. A total of
13 rehydration regulated proteins were identified: 8 were upregulated and
5 were downregulated.
3. The major responses to both desiccation and rehydration elicit synthesis
of a distinct pattern of contractile and cytoskeletal proteins that are likely
to be involved in body contraction and cytoskeleton rearrangements.
4. Additional proteins responsive to desiccation and rehydration were
functionally involved with a variety of responses including stress
responses, energy metabolism, protein synthesis, glucogenesis and
membrane transport.
5. Our results clearly show that both desiccation and rehydration elicit
146 dramatic changes in protein abundance within a short period of time. It is
also clear from this study that rehydration is not simply the reverse of
dehydration, but instead generates a distinct protein profile.
147
APPENDICES
APPENDIX A
DIAPAUSE ASSOCIATED PROTEINS IN THE HEAD OF THE MOSQUITO,
CULEX PIPIENS PIPIENS
Mosquitoes, including Culex pipiens, have attracted much attention because they play important roles as vectors of disease transmission. Many mosquito vectors enter an overwinter dormancy, diapause. The diapausing stage is critical for successful disease transmission since pathological agents, such as West Nile
Virus, can survive cold winter in diapausing mosquitoes and reinitiate disease the following spring. Thus, studying the diapausing stage is extremely important for understanding the seasonal occurrence of insect borne diseases and regulation of pathogens. Some aspects of the molecular basis for diapause in C. pipiens have been documented by our laboratory (Robich, et al., 2007). Most of the work focused on identification and characterization of genes involved in the programming and expression of diapause. Diapause in this species has not been studied at the protein level. Some proteomics work has been done with other
148 mosquitoes including Anopheles gambiae ( Francischetti et al., 2002 ) and Aedes aegypt (Biron et al, 2005), but no such work has been done in association with diapause.
An anautogenous colony of C. pipiens was maintained at 25°C, 75% RH
(relative humidity), with a daily cycle of 15h light: 9h dark. Eggs and first instar larvae were kept under the same conditions as the colony. From the second instar, larvae were transferred to 18°C, 75% RH, and 15L: 9D for nondiapause or 9L:
15D under the same temperature and humidity for diapause. Larvae were fed a diet of ground fish food, and adults were provided with water and honey.
Nondiapausing and early diapausing female mosquitoes held at 18°C were collected 7 days after adult eclosion. Mid diapausing female mosquitoes were collected 30 days after adult eclosion.
This work focuses on the head proteins that are differentially expressed during the adult diapause of C. pipiens . Head proteins were extracted and used for
2 D gel electrophoresis. Gels were stained with Bio safe Coomassie blue
(BioRad), imaged and analyzed with software PDQuest. Differentially expressed proteins with an average fold change greater than 3 fold and a t test p value <0.05 were subsequently subjected to identification by mass spectrometric techniques.
A representative 2 dimentional electrophoresis image of mosquito head proteins was shown in Fig. A.1. The relative abundance of a total of 13 selected proteins was determined based on PDQuest (BioRad) (Fig. A. 2) and their
149 corresponding identities are summarized in Table A.1. Three proteins increased in abundance during early diapause and then declined during mid diapause, all of which showed high identity to pupal cuticle protein (D2, E2 and F2). Compared with the nondiapause condition, 3 proteins decreased in abundance in only early diapause but not mid diapause: aspartate ammonia lyase (B1), venom allergen 5
(F1) and heat shock 70 kDa protein cognate 4 (B2). Four proteins decreased in abundance in both early diapause and mid diapause: isocitrate dehydrogenase
(C1), arginine kinase (D1 and E1), and malic enzyme (G1). Three other proteins decreased in abundance during mid diapause but not early diapause: actin (H1), vacuolar ATP synthase alpha subunit (A2) and profiling (C2). How these proteins are functionally associated with diapause needs further study.
150 REFERENCES Biron D. G, Agnew P, Marche L, Renault L, Sidobre C, Michalakis Y, 2005. Proteorne of Aedes aegypti larvae in response to infection by the intracellular parasite Vavraia culicis . International Journal for Parasitology 35, 1385 1397. Francischetti I. M. B., Valenzuela J. G., Pham V. M., Garfield M. K., Ribeiro J. M. C., 2002. Toward a catalog for the transcripts and proteins (sialome) from the salivary gland of the malaria vector Anopheles gambiae . Journal of Experimental Biology 205, 2429 2451. Robich R. M., Rinehart J. P., Kitchen L. J., Denlinger D. L., 2007. Diapause specific gene expression in the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization. Journal of Insect Physiology 53, 235 245.
151
Fig. A.1. A 2 dimensional electrophoresis image of head proteins from female adults of Culex pipiens . Numbers refer to proteins that are identified in Table A.1. pI= isoelectric point, MM= molecular markers.
152
b 1 ND7 D7 0.9 D30
0.8 b
0.7
0.6
0.5
Relative volume 0.4 a
0.3 a b a a a a a a 0.2 a a a a b a a a a 0.1 b b b b b b b b b b b b b 0 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2 F2 Spot number
Fig. A.2. Relative abundance of mosquito head proteins. PDQuest software 8.0.1 (BioRad) was used for spot detection, quantification and statistical analysis. ND7= proteins from nondiapause females 7 days after adult eclosion. D7= proteins from early diapause females 7 days after adult eclosion. D30= proteins from mid diapause females 30 days after adult eclosion. Abundances marked by the same lower case letters were not significantly different.
153 Spot Calculated Mowse Entry name Species Protein ID No. mass (kDa)/PI score * More abundant proteins in early diapause than both nondiapause and mid-diapause D2 170056109 Culex pipiens quinquefasciatus pupal cuticle protein 31.4/5.67 299 E2 170056109 Culex pipiens quinquefasciatus pupal cuticle protein 31.4/5.67 431 F2 170056109 Culex pipiens quinquefasciatus pupal cuticle protein 31.4/5.67 299 Less abundant proteins in early or mid-diapause than nondiapause B1 170044493 Culex pipiens quinquefasciatus aspartate ammonia lyase 60.0/8.72 1405 154 C1 170033046 Culex pipiens quinquefasciatus isocitrate dehydrogenase 39.1/8.30 959 D1 170044007 Culex pipiens quinquefasciatus arginine kinase 45.5/5.55 1476 E1 170044007 Culex pipiens quinquefasciatus arginine kinase 45.5/5.55 1417 F1 170059015 Culex pipiens quinquefasciatus venom allergen 5 29.6/6.15 581 G1 170046750 Culex pipiens quinquefasciatus malic enzyme 68.8/ 6.35 1317 H1 68270850 Culex pipiens pipiens actin 42.1/5.30 887 Table A.1 (continued) Table A.1. Diapause associated proteins identified in heads of Culex pipiens * Mowse score>54 indicates identity or extensive homology (P<0.05); higher scores indicate higher confidence of identity. 154 Table A.1 (continued) Spot Calculated Mowse Entry name Species Protein ID No. mass (kDa)/PI score * ATP synthase alpha subunit A2 170043178 Culex pipiens quinquefasciatus 68.4/5.27 936 vacuolar heat shock 70 kDa protein B2 170045895 Culex pipiens quinquefasciatus 71.8/5.36 1199 cognate 4 C2 170030306 Culex pipiens quinquefasciatus profilin 13.9/5.63 439 155
155
APPENDIX B
OVARY PROTEINS INVOLVED IN A DIAPAUSE MATERNAL EFFECT IN
THE FLESH FLY, SARCOPHAGA BULLATA
Previous research indicated a maternal effect that prevents pupal diapause in progeny of the flesh fly, Sarcophaga bullata (Henrich & Denlinger, 1982; Rockey et al., 1989). In this fly, the mother’s photoperiodic history determines whether the progeny can respond to short daylength by entering diapause. Diapause can not be induced in progeny whose mother has undergone pupal diapause, even if the progeny are maintained in short day (diapause inducing condition). Diapause can be induced only in the progeny of mothers reared under long daylength. Similarly, a maternal influence that controls embryonic diapause in Bombyx mori has been reported (Yamashita, 1996). However, little is known about how maternal effects control diapause in insects and what proteins play critical roles. Ovarian transplants done by Rockey et al (1989) suggested that the information regulating the maternal effect is transferred to the ovary sometime between the end of larval life and the third day of adult life. Therefore, the maternal effect has already been transferred to the eggs before they are fertilized to become embryos.
We report that eggs from mothers reared in long daylength and short daylength synthesize different protein products or produce different levels of certain proteins. Two dimensional electrophoresis was performed to separate total
156 proteins from the ovaries 2 days and 5 days after adult eclosion from the two groups of mothers. The 5 day eggs were fully formed but had not yet been ovulated and fertilized. Nineteen proteins that had different levels of abundance with an average fold change greater than 1.5 fold and a t test p value <0.05 were identified using Nano LC/MS/MS.
Representative images for 2 day and 5 day ovary proteins are shown in Fig.
B.1 and B.2. The relative abundance of 19 differentially expressed in flies exposed to a long day history and short day history was determined based on PDQuest (Fig.
B.3). The identified proteins are shown in Table B.2. Seven proteins increased in abundance in 2 day ovaries dissected from flies that had been exposed to short day history, while 5 proteins decreased in abundance. In 5 day ovaries from flies that were exposed to short day history, 6 proteins increased and 1 protein decreased in abundance.
157 REFERENCES Henrich VC, Denlinger DL, 1982. A maternal effect that eliminates pupal diapause in progeny of the flesh fly, Sarcophaga bullata . Journal of Insect Physiology 28, 881 884. Rockey SJ, Miller BB, Denlinger DL, 1989. A diapause maternal effect in the flesh fly, Sarcophaga bullata : transfer of information from mother to progeny. Journal of Insect Physiology 35, 553 558.
Yamashita O, 1996. Diapause hormone of the silkworm, Bombyx mori , structure, gene expression and function. Journal of Insect Physiology 42, 669 679.
158 Fig. B.1. A 2 dimensional electrophoresis map of 2 day ovary proteins from Sarcophaga bullata exposed to a short day history. Numbers refer to proteins that are identified in Table B.1. pI= isoelectric point, MM= molecular markers.
159
Fig. B.2. A 2 dimensional electrophoresis map of 5 day ovary proteins from Sarcophaga bullata exposed to a short day history. Numbers refer to proteins that are identified in Table B.1. pI= isoelectric point, MM= molecular markers.
160
1 A d2 CNT 0.9 d2 SDH 0.8 0.7 0.6 0.5 0.4 0.3
Relative volume Relative 0.2 0.1 0 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2 Spot number
1 B d5 CNT 0.9 d5 SDH 0.8 0.7 0.6 0.5 0.4 0.3
Relative volume Relative 0.2 0.1 0 F2 G2 H2 A3 B3 C3 D3 Spot number Fig. B.3. Relative abundance of proteins from (A) 2 day and (B) 5 day ovaries in the flesh fly S. bullata . PDQuest software 8.0.1 (BioRad) was used for spot detection, quantification and statistical analysis. CNT= proteins from flies exposed to a long day history . SDH= proteins from flies exposed to a short day history
161 Calculated Mowse Spot No. Entry name Species Protein ID mass (kDa)/PI score a Proteins more abundant in 2-day ovaries from short-day history female Anoplophora B1 67527227 muscle protein 20 like protein 20.4/7.77 253 glabripennis Drosophila C1 125977990 GA13413 PA 19.7/7.60 329 pseudoobscura Polysaccharide biosynthesis D1 4100597 Campylobacter jejuni 76.0/8.14 91 protein homolog F1 17136986 Drosophila melanogaster twinstar CG4254 PA 17.4/6.74 559 162 B2 24644504 Drosophila melanogaster Gasp CG10287 PA, isoform A 29.4/4.73 386 Ribosomal protein LP1 D2 17136320 Drosophila melanogaster 11.6/4.33 191 CG4087 PA G1 - - Unknown Proteins less abundant in 2-day ovaries from short-day history female E1 28461261 Bos taurus fatty acid binding protein 1 14.3/7.77 276 H1 287945 Drosophila melanogaster ATP synthase beta subunit 53.5/5.19 1087 Table B.1 (continued) Table B.1. Differentially regulated ovary proteins in Sarcophaga bullata exposed to a short day history compared to flies exposed to a long day history * Mowse score>54 indicates identity or extensive homology (P<0.05); higher scores indicate higher confidence of identity.
162 Table B.1 (continued) Calculated Mowse Spot No. Entry name Species Protein ID mass (kDa)/PI score a A2 464020 Drosophila melanogaster La/SS B 44.9/6.64 89 C2 157105484 Aedes aegypti fk506 binding protein 23.5/4.70 201 E2 984655 Sarcophaga peregrina storage protein binding protein 133.5/6.05 119 Proteins more abundant in 5-day ovaries from short-day history female F2 7726 Drosophila melanogaster chorion protein S36 30.1/8.62 82 uncultured bacterium predicted dimethylallyltransferase G2 68304991 31.8/8.79 69 BAC13K9BAC FN1327
163 adventurous gliding motility A3 27804872 Myxococcus xanthus 281.1/5.21 63 protein K hypothetical protein C3 31432042 Oryza sativa 43.6/5.14 95 LOC_Os10g27180 D3 20126677 Sarcophaga crassipalpis sarcocystatin A 11.9/5.35 173 B3 - - Unknown Proteins less abundant in 5-day ovaries from short-day history female Methanococcoides protein of unknown function H2 91773981 45.8/6.49 89 burtonii DSM 6242 DUF651
163
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