Development of a test to detect & quantify irradiation damage in fruit flies

Dr Richard Glatz

South Australian Research & Development Institute (SARDI)

Project Number: VG09160

VG09160

This report is published by Horticulture Australia Ltd to pass on information concerning horticultural research and development undertaken for the vegetables industry.

The research contained in this report was funded by Horticulture Australia Ltd with the financial support of: Bowen District Growers Assn Inc. the vegetables industry

All expressions of opinion are not to be regarded as expressing the opinion of Horticulture Australia Ltd or any authority of the Australian Government. The Company and the Australian Government accept no responsibility for any of the opinions or the accuracy of the information contained in this report and readers should rely upon their own enquiries in making decisions concerning their own interests.

ISBN 0 7341 3130 5 Published and distributed by: Horticulture Australia Ltd Level 7 179 Elizabeth Street Sydney NSW 2000 Telephone: (02) 8295 2300 Fax: (02) 8295 2399 © Copyright 2013

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VG09160 Final Report 20 May 2013

Development of a test to detect & quantify

irradiation damage in Fruit Flies   

 

 

 

 

 

 

 

 

 

Richard Glatz et al.*

South Australian Research & Development Institute (SARDI)   

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HAL Project: VG09160 Project Leader: Dr Richard Glatz

SARDI Entomology / University of Adelaide 1669a Three Chain Road, MACGILLIVRAY, SA, 5223 PO Box 17, Kingscote, SA, 5223 T: (08) 8553 8294 M: 0419 843 254 E: richard.glatz@sa.gov.au; richard@dess.net.au

Collaborators: Dr Wayne Leifert (CSIRO) Assoc Prof Phil Taylor (Macquarie University) Mr Mohammad Sabbir Siddiqui (CSIRO, University of Adelaide) Dr Sam Collins (Macquarie University) Dr Kelly Hill (SARDI Entomology) Mr Greg Baker (SARDI Entomology) Prof Michael Fenech (CSIRO)

This report summarises all experiments and associated data arising from the HAL-funded project VG09160 Development of a test to detect and quantify irradiation damage in fruit flies.

* Please cite as Leifert, W. R., Glatz, R. V., Siddiqui, M. S., Collins, S. R., Taylor, P. W. and Fenech, M. (2013). Development of a test to detect and quantify irradiation damage in fruit flies. Final Report for Horticulture Australia Ltd. Project VG09160. Pp 53.

This work was supported by Horticulture Australia Limited [project number VG09160] using the vegetable levy, voluntary contributions from industry (Bowen District Growers Association) and matched funds from the Australian Government. Financial assistance was also provided by SARDI Entomology, CSIRO Animal, Food and Health Sciences, and Macquarie University Department of Biological Sciences. Some work was conducted as part of the University of Adelaide PhD project being undertaken by Mohammed Sabbir Siddiqui.

 

HAL Disclaimer

Any recommendations contained in this publication do not necessarily represent current HAL policy. No person should act on the basis of the contents of this publication, whether as to matters of fact or opinion or other content, without first obtaining specific, independent professional advice in respect of the matters set out in this publication.

SARDI Disclaimer

Although all reasonable care has been taken in preparing the information contained in this publication, neither SARDI nor the other contributing authors accept any responsibility or liability for any losses of whatever kind arising from the interpretation or use of the information set out in this publication. Where products and/or their trade names are mentioned, no endorsement of these products is intended, nor is any criticism implied of similar products not mentioned.

 

 

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Contents

List of Figures..................................................................................................................................................... 3

List of Tables ...................................................................................................................................................... 4

Media Summary ................................................................................................................................................. 5

Technical Summary ............................................................................................................................................ 6

1 Introduction .................................................................................................................................................. 8 1.1 Rationale, aims and approach ...................................................................................................... 8 1.2 DNA damage ............................................................................................................................... 8 1.3 DNA damage signalling proteins ................................................................................................. 9 1.4 Histone proteins ........................................................................................................................... 9 1.5 Histone phosphorylation and relevance to QFly ........................................................................ 11

2 Methods ...................................................................................................................................................... 12 2.1 Pupal and adult preparation and irradiation ............................................................................... 12 2.2 Egg collection and irradiation .................................................................................................... 12 2.3 Larvae collection and irradiation ............................................................................................... 13 2.4 Whole pupal lysate preparation for Western blotting ................................................................ 13 2.5 Preparation of isolated nuclei and acid extraction of histone protein from pupae ..................... 13 2.6 Total lysates and histone extracts from individual pupae .......................................................... 14 2.7 Total lysates from irradiated eggs and larvae ............................................................................ 14 2.8 Antibodies .................................................................................................................................. 14 2.9 Western blotting ......................................................................................................................... 14 2.10 Visual scoring of γH2AvB in isolated nuclei ............................................................................ 15 2.11 Immunofluorescence to quantify γH2AvB response in nuclei ................................................. 15 2.12 Laser scanning cytometry ......................................................................................................... 16 2.13 mRNA isolation, cDNA synthesis and 454 sequencing ........................................................... 16 2.14 Sequence analysis and homology search .................................................................................. 17 2.15 Proteomics identification of H2AvB ........................................................................................ 17 2.16 Indirect ELISA protocol ........................................................................................................... 17 2.17 Sandwich ELISA protocol ........................................................................................................ 18 2.18 Preliminary detection of γH2AvB homolog in Mediterranean fruit fly .................................... 18 2.19 Statistical analyses .................................................................................................................... 18

3 Results ........................................................................................................................................................ 19 3.1 Summary of QFly samples prepared .......................................................................................... 19 3.2 Identification of potential candidate protein biomarkers of previous ionising radiation exposure .............................................................................................................................................. 20 3.3 γH2AX, γH2AvD and γH2AvB antibodies ............................................................................... 21 3.4 454 sequencing of transcripts and peptide identification by proteomic analyses ...................... 22 3.5 Investigation of γH2AvB in QFly brain and gonad ................................................................... 25 3.6 Short-term kinetics of H2AvB phosphorylation ........................................................................ 26 3.7 γH2AvB dose-response to previous ionising radiation exposure .............................................. 27 3.8 γH2AvB – long term response ................................................................................................... 28

 

 

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3.9 Visual scoring of IR-induced γH2AvB signal in isolated nuclei ............................................... 32 3.10 Persitent, long-term γH2AvB signal in adult Q-flies ................................................................ 33 3.11 Development of an ELISA to quantify γH2AvB signal ........................................................... 37 3.12 Preliminary detection of γH2AvB homlog in Mediterranean fruit fly (MedFly) ..................... 41

4 Discussion .................................................................................................................................................. 43 4.1 Summary .................................................................................................................................... 43 4.2 Identification of γH2AvB .......................................................................................................... 43 4.3 Kinetics and persistence of γH2AvB signal ............................................................................... 44 4.4 Fluorescence microscopy and laser scanning cytometry ........................................................... 45 4.5 Development of an ELISA for γH2AvB .................................................................................... 45 4.6 Detection of γH2AvB homolog in Mediterranean fruit fly (Medfly) ........................................ 46 4.7 Layperson’s Summary ............................................................................................................... 46

Technology Transfer ........................................................................................................................................ 47

Recommendations ............................................................................................................................................ 48

Acknowledgements .......................................................................................................................................... 49

References ........................................................................................................................................................ 50

 

 

 

 

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List of Figures

Figure 1. Summary of pathways involved in DNA damage/repair .................................................................. 11

Figure 2. Preliminary investigations of DNA damage biomarker proteins in QFly pupae 24 h post IR exposure ............................................................................................................................................................ 21

Figure 3. Amino acid sequence of H2AX homolog (H2AvB) and alignment of H2A histone variants. ......... 22

Figure 4. Species distribution of homologous sequences ................................................................................. 23

Figure 5. Gene ontology analyses of Bactrocera tryoni transcriptome data. Transcript sequence distribution: biological processes ..................................................................................................................... 23

Figure 6. Gene ontology analyses of Bactrocera tryoni transcriptome data. Transcript sequence distribution: molecular function ....................................................................................................................... 24

Figure 7. Gene ontology analyses of Bactrocera tryoni transcriptome data. Transcript sequence distribution: cellular component ....................................................................................................................... 24

Figure 8. Complete amino acid sequence of QFly H2AvB .............................................................................. 25

Figure 9. Comparison of two anatomical regions showing γH2AvB response ................................................ 26

Figure 10. Short-term kinetics of H2AvB phosphorylation ............................................................................. 27

Figure 11. γH2AvB dose-response following IR ............................................................................................. 28

Figure 12. γH2AvB signal after 5 days. ........................................................................................................... 29

Figure 13. γH2AvB response following 70 Gy exposure at different time post IR ......................................... 31

Figure 14. Visual scoring of the frequency of γH2AvB foci in pupae 24 h post IR ........................................ 32

Figure 15. Visual scoring of the frequency of γH2AvB foci in 1st instar larvae 2 days post IR ...................... 33

Figure 16. Bleaching of the scutellum of adult Q-flies following 70 Gy IR .................................................... 34

Figure 17. Quantification of γH2AvB signal in isolated nuclei by laser scanning cytometry .......................... 36

Figure 18. Concept of the γH2AvB Indirect ELISA assay ............................................................................... 37

Figure 19. Indirect ELISA of histone extracts .................................................................................................. 38

Figure 20. Concept of the γH2AvB Sandwich ELISA assay ........................................................................... 39

Figure 21. Anti H2A.Z antibody binds to histone H2A proteins independent of previous IR exposure .......... 39

Figure 22. Representative Western blot showing a result using QFly specific γH2AvB antibody .................. 40

Figure 23. Sandwich ELISA of histone extracts .............................................................................................. 41

Figure 24. Preliminary detection of γH2AvB homolog in Mediterranean fruit fly .......................................... 42

 

 

 

 

 

 

 

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List of Tables

Table 1. Collection of QFly specimens from Macquarie University at various life stages and ionising radiation doses .................................................................................................................................................. 19

Table 2. Antibodies used for the preliminary investigation of DNA damage/response protein biomarkers of previous ionising radiation ........................................................................................................................... 20

Table 3. Total weight of 10 randomly selected adult Q-flies; 0, 70, 160 Gy ................................................... 35 

 

 

 

 

 

 

 

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Media Summary

Cost-effective options for post-harvest disinfestation of horticultural produce are being severely depleted by withdrawal of key chemical treatments. This could result in significant negative impact on international markets requiring consistent delivery of high quality, pest-free produce. Meanwhile, approval of irradiation as a disinfestation technique by regulatory bodies (e.g. FSANZ, APVMA, IAEA) will likely see it become the method of choice for many primary products. Despite clear advantages of irradiation being an effective and established technology with no withholding period, there is no way to test if live pests have been irradiated if they are found in traded produce, which has quarantine/market access implications. HAL Project VG09160 aimed to address this technological shortfall with a proof-of-concept study aiming to use the commercially important pest Queensland fruit fly (QFly; Bactrocera tryoni) as a model to find a marker of irradiation exposure in insects, which could be measured in tests to detect, and perhaps quantify, prior irradiation exposure. Such a marker would also have application for testing of irradiated flies released during Sterile Insect Technique (SIT) eradication programs.

Researchers from SARDI, CSIRO, Macquarie University and University of Adelaide, collaborated in discovering a QFly protein that was modified due to irradiation, with the amount of modified protein increasing with increasing irradiation doses. Doses tested included those approved for disinfestation and SIT, and the protein occurs in all insects. The findings will soon be published in the international science journal Mutagenesis*.

Highly specific antibodies were produced against the QFly protein allowing its sensitive detection using standard commercial technologies such as ELISA. As far as we are aware, this is the first retrospective test for irradiation exposure and it has the potential to provide Australian producers with an advantage in facilitating broad use and confidence in irradiated produce.

Future R&D will largely aim to facilitate practical application of the test for industry. This could include validation of the test under commercial/quarantine conditions, incorporation of the test into the respective facilities, broadening pest target range, and investigating other useful markers. Some of these issues are currently being assessed as an Expression of Interest for HAL Transformational Funding.

* Mohammad Sabbir Siddiqui, Erika Filomeni, Maxime François, Samuel R. Collins, Tamara Cooper, Richard V. Glatz, Phillip W. Taylor, Michael Fenech and Wayne R. Leifert (2013). Exposure of insect cells to ionising radiation in vivo induces persistent phosphorylation of a H2AX homolog (H2AvB). Mutagenesis (in press)  

 

 

 

 

 

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Technical Summary

This project (VG09160; Development of a test to detect and quantify irradiation damage in fruit flies) was undertaken to address potential market failure associated with withdrawal of standard post-harvest insect disinfestation chemicals, fenthion and dimethoate. One of the only other commercially applicable disinfestation methods that is well validated and safe, is the use of irradiation. Irradiation is currently used to disinfest a relatively small number of high value crops (e.g. seed weevil disinfestation in mangoes). While the regulatory framework exists for broad use of irradiation for commercial disinfestation of primary produce, the lack of a reliable test to retrospectively assess radiation reduces market confidence in the situation where live pests are detected in exported/imported produce. Currently, certification by the treatment facility is the only means of assessing prior treatment of traded fruit/vegetables. A retrospective test for irradiation would allow Australia to assess quarantine intercepts or imported produce from regions where certification is less reliable. It would also facilitate/improve commercial irradiation treatments within Australia, giving Australian producers potential production and market access advantages. Additionally, retrospective assessment of irradiation in flies used for Sterile Insect Technique (SIT) eradication programs is another key challenge for government, and a test delivering this capacity reliably would be of great benefit.

The project set out to use a key target of horticultural disinfestation in Australia, Queensland fruit fly (QFly; Bactrocera tryoni) as a model species to achieve two broad aims:

assess potential biomarkers of irradiation exposure

incorporate the best biomarker into a test capable of use in commercial disinfestation

To achieve these aims, batches of QFly were irradiated under controlled conditions and then were subjected to molecular analyses. Deep sequencing identified the presence of a QFly histone which we have termed H2AvB. A linear dose-response of the phosphorylated form (γH2AvB) to 0–400 Gy ionising radiation was observed in whole QFly pupal lysates 24 h post-ionising radiation and was detected at doses as low as 20 Gy. γH2AvB signal peaked at approximately 20 min after irradiation and at 24 h post irradiation the signal remained elevated but declined significantly by 5 days. Persistent and dose-dependent γH2AvB signal could be detected and quantified either by Western blot or Laser Scanning Cytometry up to 17 days post irradiation in histone extracts or isolated nuclei from adult QFly. We conclude that ionising radiation exposure in QFly at an early life-stage leads to persistent γH2AvB signals that can easily be detected by Western blot, ELISA or quantitative immunofluorescence techniques. These approaches have potential as the basis for detection and quantification of previous ionising radiation exposure in pest fruit flies (imports and exports) and to confirm the identity of unmarked flies captured in monitoring traps during SIT releases. This provides a technological capacity that will aid greatly in facilitating broad uptake and acceptance of irradiation as the preferred method of disinfestation due to its many safety and commercial advantages.

To our knowledge, this is the only available test capable of retrospectively assessing prior irradiation exposure of living organisms. We have four broad recommendations with regards to further work to exploit the outputs of this project:

 

 

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1. Improve understanding of the biomarker’s response. Future studies should extend the time course following IR exposure and examine tissue-specificity of γH2AvB signals in QFly. Furthermore, understanding the kinetics of γH2AvB phosphorylation/dephosphorylation in different life stages of QFly would also be of benefit. As the biomarker is associated with DNA-damage, its response to other environmental challenges (such as toxins) also needs to be assessed.

2. Broadening the pests targeted by the irradiation test. The biomarker occurs in most organisms (all insects) where it is involved in similar DNA-repair mechanisms. Therefore, the opportunity exists to adapt the test to a suite of insects of market access and biosecurity concern.

3. Incorporation of the test into commercial irradiation facilities and/or quarantine facilities. Applications could include providing a stronger certification “brand” and/or confirming exported produce was treated if pests are found after export. Additionally, incorporation into quarantine facilities would allow Australia to confidently assess prior irradiation of imported produce.

4. Promotion of the availability and benefits of irradiation to industry and the public. Many producers may be unaware of its applicability/advantages for their particular product. Similarly, the public needs to be informed of the broad use of irradiation as a sterilization technique, its advantages for food disinfestation, and its high level of safety in terms of food quality, particularly in comparison to the chemical options being withdrawn due to health concerns.

 

 

 

 

 

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1 Introduction

1.1 Rationale, aims and approach

This project was undertaken to address potential market failure associated with withdrawal of standard post-harvest disinfestation chemicals. While the regulatory framework exists for broad use of irradiation for commercial disinfestation of primary produce, the lack of a reliable test to retrospectively assess radiation reduces confidence in the context of live pests being detected in exported/imported fruit. Such a test would allow Australia to assess quarantine intercepts or imported produce from regions where certification is less reliable. It would also facilitate/improve commercial irradiation treatments within Australia, giving Australian producers potential production and market access advantages. Additionally, retrospective assessment of irradiation in flies used for Sterile Insect Technique (SIT) eradication programs is another key challenge for government, and a test delivering this capacity reliably would be of great benefit. Therefore, this project (VG09160; Development of a test to detect and quantify irradiation damage in fruit flies) was developed in response to this technological requirement to assess prior irradiation exposure.

The project set out to use a key target of horticultural disinfestation in Australia, Queensland fruit fly (QFly; Bactrocera tryoni) as a model species to achieve two broad aims:

assess potential biomarkers of irradiation exposure

incorporate the best biomarker into a test capable of use in commercial disinfestation

To achieve these aims, batches of QFly were irradiated under controlled conditions and then were subjected to molecular analyses. The molecular analyses involved sequencing of QFly genes, standard protein analyses such as Western blot, and more advanced protein analyses such as Laser Scanning Cytometry. These analyses ultimately revealed that levels of a phosphorylated form a histone protein occurring in QFly, were increased in response to irradiation and were proportional to dose received. This response was also persistent, making it a good candidate for a commercial test.

The following report provides a description of relevant scientific background, the methodology used, the data obtained, and a discussion of the data. Recommendations for future research are also presented.

1.2 DNA damage

Cells encounter genotoxic stress on a regular basis, and are protected by the DNA damage response invoked after damage occurs. The most deleterious lesions caused by DNA damaging agents are DNA double strand breaks (DSBs). In response to DNA DSBs, cells activate evolutionarily conserved cell-cycle checkpoint pathways to arrest cell cycle progression and activate DNA damage repair machinery at break sites in order to repair lesions (Rouse, Jackson. 2002; Sancar, et al. 2004). Ionising radiation (IR) exposure induces highly lethal DNA DSBs in all phases of the cell cycle. After DNA DSBs are detected by the cellular machinery, these breaks are repaired by either of two mechanisms: (1) nonhomologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination (van Gent, van der Burg. 2007). Among the major factors influencing DNA organization are specific histone and nonhistone proteins that form chromatin. During the process of DNA DSB repair, several chromatin alterations are required to sense damage and facilitate accessibility of the repair machinery (Mendez-Acuna, et al. 2010). The DNA DSB

 

 

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response is also facilitated by hierarchical signalling networks that orchestrate chromatin structural changes that may coordinate cell-cycle checkpoints involving multiple protein activities to repair broken DNA ends. During DNA damage sensing and repair, histones and other regulator proteins undergo a series of post-translational modifications. Such modifications represent a code that directs the recruitment of proteins involved in DNA damage sensing and repair processes to the lesion.

1.3 DNA damage signalling proteins

Upon DNA damage or replication block, eukaryotic cells activate checkpoint pathways that delay cell cycle progression. In mammals, the main transducers of the cell cycle checkpoint pathways are ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), Chk1, and Chk2 (Cds1) (Sancar, et al. 2004; Riches, et al. 2008; Savic, et al. 2009). ATM controls G1 and G2 arrest through Chk2. ATM also phosphorylates several key proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. Chk2 is phosphorylated on Thr68 by ATM kinase when exposed to ionising radiation (Matsuoka, et al. 2000). In turn Chk2 phosphorylates Cdc25C on Ser216, which interferes with Cdc25C’s ability to activate Cdc2 and cause G2 arrest (Matsuoka, et al. 1998). Chk2 also stabilises the transcription factor p53 by phosphorylation on Ser20 and consequently causes G1 arrest (Chehab, et al. 2000). p53 has been described as "the guardian of the genome" because of its role in conserving genomic stability by preventing genome mutation (Lane. 1992). Checkpoint- and DNA repair-associated proteins such as Rad50, Rad51, and Brca1 as well as the p53 binding protein 1 (53BP1) co-localize with the histone protein γH2AX (Furuta, et al. 2003; Huyen, et al. 2004). Rad6 is an evolutionary conserved ubiquitin-conjugating enzyme required for post-replication repair of damaged DNA. Rad6 acts on stalled replication forks in the presence of DNA damage, allowing repair and resumption of DNA replication (Prakash, et al. 1993). Some of the DNA damage/repair pathways are summarised in a schematic as shown in Figure 1.

1.4 Histone proteins

Histones consist structurally of a globular core domain with flexible tail regions at both the N-terminal and C-terminal regions. The globular core domain interacts with other histones while the termini contain highly conserved, charged lysine and arginine amino acid residues involved in DNA–histone interaction. In addition to histones, non-histone proteins are also involved in developing special chromatin structures. The known histone modifications induced following ionising radiation exposure are phosphorylation, acetylation, methylation, and ubiquitination (Pandita, Richardson. 2009; Deem, et al. 2012). Among these major chromatin modifications, post-damage-induced histone phosphorylation has been the most thoroughly studied (Mendez-Acuna, et al. 2010). Post-translational modifications of histone proteins are critical for cellular recognition of DNA damage and subsequent recruitment of repair protein complexes to the damage sites.

The SQ motif in H2AX is highly conserved among animals, plants, and fungi (Downs, et al. 2000; Friesner, et al. 2005; Lang, et al. 2012). This evolutionary conservation of the phosphorylation of the core histone protein H2AX suggests the DNA DSB damage-response mechanism is a fundamental process in DNA repair that arose prior to the evolutionary divergence of fungi, plants and animals. This is partly evidenced by the fact that SQ-specific antibodies raised against the mammalian γH2AX sequence can recognize DNA DSBs in the frog Xenopus laevis, vinegar fly Drosophila melanogaster and bread/wine yeast Saccharomyces cerevisiae, after exposure to IR or

 

 

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other genotoxic agents (Rogakou, et al. 1999; Redon, et al. 2002). Antibodies that recognise phosphorylated H2AX in mammals have also been shown to recognise IR-induced H2Av (H2AX variant) in D. melanogaster (H2AvD) and binding has been shown to be dependent on the presence of the SQ motif (Rogakou, et al. 1999; Madigan, et al. 2002).

Phosphorylation of the C-terminal tails of H2AX histones in nucleosomes which are located in the vicinity of the break (Rogakou, et al. 1998; Savic, et al. 2009), is one of the earliest known responses to DNA DSB formation in cells. The nucleosome complex comprises DNA wrapped around eight histone proteins, two from each of the four core histone families (H4, H3, H2B, H2A), and is essential for genome health in terms of normal regulation of gene expression as well as genome maintenance and replication (Rogakou, et al. 1999; Goll, Bestor. 2002; Mendez-Acuna, et al. 2010). Induction of DNA DSBs in live mammalian cells triggers the phosphorylation of Ser139 contained in the SQ motif near the carboxy-terminus of H2AX, resulting in the formation of phosphorylated H2AX, termed γH2AX (Redon, et al. 2002; Kinner, et al. 2008). Whilst H2AX is distributed uniformly throughout chromatin, only H2AX molecules located in close vicinity to DNA DSBs become phosphorylated (Rogakou, et al. 1998; Rogakou, et al. 1999; Savic, et al. 2009). Several kinase proteins are known to phosphorylate H2AX including phosphatidylinositol-3-OH serine/threonine protein kinase-like kinases (PIKKs), ataxia telangiectasia mutated (ATM), ATM and Rad-3-related (ATR) and DNA-dependent protein kinase (DNA-PK). However, only ATM and DNA-PKs have been shown to phosphorylate H2AX in response to ionising radiation (IR) (Rogakou, et al. 1998; Burma, et al. 2001; Redon, et al. 2002; Park, et al. 2003; Fernandez-Capetillo, et al. 2004; Olive, Banath. 2004; Stiff, et al. 2004), and γH2AX is a sensitive target for identifying DNA DSBs in cells.

Shortly after induction of DNA DSBs by ionising radiation, the appearance of γH2AX in chromatin can be detected immunocytochemically in the form of discrete nuclear foci (Rogakou, et al. 1999; Sedelnikova, et al. 2002), each focus being presumed to represent a single DNA DSB (Sedelnikova, et al. 2002). Thus, the frequency of foci per nucleus is considered to reflect the incidence of DNA DSBs. Checkpoint and DNA repair proteins such as Rad50, Rad51, and Brca1 co-localize with γH2AX (Paull, et al. 2000). In addition, the translocation of the p53 binding protein 1 (53BP1) to irradiation-induced foci is mediated by H2AX (Anderson, et al. 2001; Park, et al. 2003).

 

 

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Figure 1. Summary of pathways involved in DNA damage/repair. Recognition and signalling of DNA damage is mediated by ATM and ATR, which bind to broken DNA ends (e.g. following exposure to ionising radiation). Upon activation of their intrinsic kinase activity, ATM and/or ATR phosphorylate H2AX or Chk1 and Chk2, which in turn phosphorylate p53 and CDc25, thus provoking cell cycle arrest. In addition, ATM and/or ATR phosphorylate several other proteins. Schematic was from (Christmann, et al. 2003).

 

1.5 Histone phosphorylation and relevance to QFly

In the present study we identified the sequence of a H2AX protein variant from deep sequencing analysis of QFly transcripts and mass spectrometry of the irradiation-induced protein (we have termed this variant H2AvB and the sequence has been deposited into the NCBI Short Read Archive; BankIt1580860 isotig00988 KC161252). We found that H2AvB amino acid sequence is 96.4% similar to the homolog found in the genetic model D. melanogaster, 54.8% similar to human H2AX, and identical in comparison with Glossina morsitans morsitans (the Savannah tsetse fly). Using Western blotting and laser scanning cytometry (LSC) techniques we demonstrate an irradiation-induced short-term rapid increase in γH2AvB followed by a long-term (persistent) and dose-dependent γH2AvB response in QFly. These assays have practical application to confirm irradiation status of live QFly found in exported fruits and to confirm the identity of unmarked flies captured in monitoring traps during SIT releases.

 

 

 

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2 Methods

2.1 Pupal and adult preparation and irradiation

Bactrocera tryoni (QFly) pupae were obtained from the NSW Department of Primary Industries Fruit Fly Production Facility at Elizabeth Macarthur Agricultural Institute (EMAI, New South Wales, Australia). Pupae from this facility are routinely sent to the Australian Nuclear Science and Technology Organisation (ANSTO, Lucas Heights, NSW, Australia) for irradiation as part of the SIT control program to suppress outbreak populations of wild Q-flies. Individual ‘zip-lock’ plastic bags (100 x 150 mm) containing approximately 8,000 pupae were sealed and packed at EMAI, and transported directly to ANSTO in an air-conditioned vehicle. All pupae were packed on the day of pupation and all irradiated pupae were treated one-day post the onset of pupation. Bags of control and test pupae were packed together at all times during transport and storage to ensure that all pupae received similar conditions. To achieve a hypoxic atmosphere prior to irradiation, the sealed bags were held overnight at ANSTO in a temperature-controlled room at approximately 18oC. The following day, pupae were treated with IR using ANSTO’s 60Co GATRI facility delivering final doses of 0-400 Gy at a dose rate of 5 Gy/min. We investigated doses greater than the standard disinfestations dose of 150 Gy up to 400 Gy, since Bactrocera fruit flies appear to be considerably more tolerant to IR compared with other fruit fly genera such as Ceratitis, Anastrepha and Rhagoletis (Follett, et al. 2011).

After irradiation, pupae were immediately transported in a closed styrofoam box in an air-conditioned vehicle to a laboratory at Macquarie University, Sydney, where they were housed to emerge in 5 L plastic cages, each with a large mesh-covered ventilation hole in the top. Pupae were held in a laboratory maintained at 25 ± 1oC and 70 ± 5% relative humidity, on a 14:10 day:night cycle including one hour dawn and dusk periods during which the lights turned on and off intermittently. At one and five days post IR, a sample of QFly pupae was frozen and stored at -80°C until assays were performed. Other IR-treated pupae were allowed to emerge as adults, then collected using an aspirator and frozen at -80oC at 17 days post IR. Adult flies were maintained on a standard diet of granular sucrose and yeast hydrolysate, with water provided in soaked cotton wool.

2.2 Egg collection and irradiation

Adult Q-flies were housed in 5 L plastic cages with one side replaced with mesh screen for ventilation. Approximately 150 flies were kept per cage. After observed mating (post 10 days of age), each cage was provided with an egging dish comprising of a 55 mm Petri dish containing a solution of lemon essence and water in a 140:1 ratio, covered with a layer of parafilm. The parafilm was pierced 5-6 times with an entomological pin to release the odour of lemon. After 2 days the egging dishes were collected and a plastic 5 ml pipette was used to transfer eggs to a 10 ml vial of water. Each vial contained approximately 500 eggs. Vials were then exposed to either 0 or 150 Gy ionising radiation and then frozen at -80oC 2 h post IR.

 

 

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2.3 Larvae collection and irradiation

Adult Q-flies were housed in 5 L plastic cages with one side replaced with mesh screen for ventilation. Approximately 150 flies were kept per cage. After observed mating (post 10 days of age), each cage was provided a collection of fresh organic chillies resting on a 15 cm plate. After 4 days the chillies were inspected for the presence of larvae. All chillies were then left a further 4 days to allow larvae to mature to 3rd instar. Chillies were placed into separate ‘zip-lock’ bags and then exposed to 0 or 150 Gy ionising radiation and maintained at 25 ± 1oC and 70 ± 5% relative humidity for 24 h. Chillies were then sliced longitudinally in half and larvae were gently removed using a pair of forceps. Collected larvae were frozen at -80oC in 10 ml vials containing water.

2.4 Whole pupal lysate preparation for Western blotting

Whole pupae were thawed from -80oC at room temperature (RT) for 5 min. 10 pupae of each IR dose being investigated were placed in cold (4°C) TBS solution (50 mM Trizma Base, 150 mM NaCl, pH 8.0) in a Petri dish on ice. The pupae were then added to 1 ml lysis buffer comprising RIPA buffer (Sigma) with additional 0.9 % SDS, phosphatase inhibitors (25 mM NaF, 0.25 mM sodium orthovanadate, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol) and a protease inhibitor cocktail (Sigma), and their tissues disrupted in a glass tissue homogenizer on ice until a clear suspension was achieved (usually 15 passes). Lysates were centrifuged at 4°C for 5 min at 300 xg to remove debris. Total protein from the pupal samples was quantified using the QuantiProTM BCA Assay kit (Sigma) as per manufacturer’s instructions, using bovine serum albumin (BSA) as a standard. Sample concentrations were adjusted to the same total protein concentration prior to gel electrophoresis. Samples were stored at -20°C until used for Western blotting. Various amounts of total protein were added depending on the assay conducted as indicated in relevant figures.

2.5 Preparation of isolated nuclei and acid extraction of histone protein from pupae

To obtain histone proteins from pupal samples, an acid extraction technique was performed as previously described (Shechter, et al. 2007) with some modification. Pupae were washed twice with TBS and placed in 3 ml of hypotonic lysis buffer (10 mM Trizma Base pH 8.0, 1 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol), a commercial protease inhibitor cocktail and other phosphatase inhibitors (as above), in a glass homogenizer on ice. Pupae were then homogenized until a clear suspension was produced, followed by filtration with nylon net filters (filter type: 100 µm NY1H) and then incubation for 30 min (on a rotator at 4°C) to allow hypotonic swelling and lysis of cells. The crude extract was then centrifuged at 15000 xg for 10 min at 4°C to separate the pellet (containing nuclei) from the soluble cytosol. The pellet was then resuspended in 400 µl of 0.8 M H2SO4 and vortexed thoroughly until aggregates were dispersed in the solution. This solution was vortexed gently overnight at 4°C using a minishaker. After centrifugation at 15000 xg for 10 min at 4°C the pellet was discarded and the acid-soluble histone proteins in the supernatant were then precipitated with a 33% trichloroacetic acid solution. The solution containing precipitated histones was mixed several times producing a milky suspension. Subsequently, the histone solution was incubated at 4oC overnight and then again centrifuged at 15000 xg for 10 min at 4°C; the supernatant was then carefully discarded. The pellet of precipitated histones was washed 3 times with 1 ml ice-cold acetone to remove the acid from the protein sample. The acetone supernatant was removed and the protein pellet was air dried for 30 min at RT and then dissolved in 150 µl of purified H20. Finally, the histone extract was stored at -20°C for subsequent analyses. In some

 

 

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experiments, dephosphorylation of the purified proteins was achieved by dissolving the extracted protein pellet in 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol (pH 7.9) and incubated with (or without for negative control) 1000 U/ml calf intestinal alkaline phosphatase (New England Biolabs, USA) overnight at 37oC.

2.6 Total lysates and histone extracts from individual pupae

Total lysates or histone extracts were prepared from individual pupae by a modification of the above method. For total lysates, the lysis volume was decreased to 150 µl of RIPA buffer (final volume), and for histone extracts of single pupae the hypotonic buffer was decreased to 150 µl. For the single pupae total lysates, 180 µg total protein was used for SDS-PAGE and analysed by Western blotting, while 1.3 µg total protein was loaded for the histone extracts from individual pupae.

2.7 Total lysates from irradiated eggs and larvae

Samples of irradiated QFly eggs were homogenised in liquid nitrogen and subsequently lysed in 150 µl RIPA buffer giving a final protein concentration of approximately 400 µg/ml. 3rd instar larvae (collected from 0 or 150 Gy irradiated chillies) were lysed (using the same method as for pupae) giving a final total protein concentration of approximately 7 mg/ml.

2.8 Antibodies

Anti γH2AX was prepared by Biosensis Pty Ltd. (Thebarton, South Australia, Australia). The affinity purified KKAATQA[PSer]QEY (human sequence) peptide conjugated with KLH was used as antigen to generate high titer polyclonal antiserum in rabbit against γH2AX and this antibody was used in preliminary studies. Drosophila anti-histone H2AvD pS137 (γH2AvD) rabbit polyclonal antibody (Rockland Immunochemicals Inc. Gilbertsville, PA, USA) (Madigan, et al. 2002) was routinely used to detect IR-induced histone in QFly. Both antibodies (γH2AX and H2AvD pS137) recognised a 15 kDa protein in Western blot analyses. In later studies (both Western blot and ELISA) when the transcript sequence for the Bactrocera tryoni H2AX homolog (termed “γH2AvB”) was known, a specific antibody directed to Bactrocera tryoni (QFly) protein sequence QDPQRKNTVILS*QGY was prepared by Biosensis. In ELISA, the capture antibody was from Abcam (catalog number ab18263). This antibody recognised the N-terminal AGGKAGKDSGKAKAKA which is a conserved histone variant H2A.Z. Cytochrome C oxidase subunit II and β-actin antibodies were from Abcam. Alexa Fluor 488 (green) or Alexa 633 (red)-conjugated goat IgG was from Invitrogen (Victoria, Australia) and horseradish peroxidase-labelled secondary antibodies were from Perkin Elmer (Victoria, Australia).

2.9 Western blotting

Whole and histone-extracted lysates were diluted in Laemmli buffer (1:2 vol:vol) containing β-mercaptoethanol followed by heating at 95°C for 5 min, before being loaded on a CriterionTM-TGXTM precast polyacrylamide gels (BioRad) and subjected to electrophoresis. Gels were then stained with Coomassie Blue to ensure the electrophoresis had been successful and that similar amounts of protein were loaded in each well. A separate (duplicate) gel was used for Western

 

 

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blotting onto a 0.2 µm pore nitrocellulose membrane (BioRad) for 1 h at 100 V in chilled transfer buffer (25 mM Trizma base, 190 mM glycine, 20% methanol, pH 8.5). The membrane was washed 3 times (5 min each) in TBST (TBS containing 0.5% Tween-20) and then blocked for 1 h at RT in TBST containing 5% BSA. Membranes were then incubated overnight at 4°C in primary antibody solution diluted 1:1000 in TBST containing 5% BSA. Membranes were then thoroughly washed 3 times (5 min each time in TBST), then incubated with anti–rabbit horseradish peroxidase–linked secondary antibody (PerkinElmer) at a dilution of 1:2000 in TBST containing 5% BSA for 2 h at RT. Probed membranes were then finally washed 3 times with TBST prior to imaging by enhanced chemiluminescence (ECL) (Western Lightning® Plus-ECL, PerkinElmer) using an ImageQuant LAS 4000 imager (GE Health Care). Images were saved as 8-bit TIFF files and band intensities (as integrals) were quantified with ImageJ software (Abramoff, et al. 2004). Data were normalised to β-actin (loading controls) where possible i.e. in histone extracts this was not possible since cytoplasmic actin was removed during the processing of the samples. In Western blots showing histone extracts containing γH2AvB where β-actin could not be used, we also show Coomassie-stained gel bands at approximately 15 kDa to demonstrate similar loading of histone proteins.

2.10 Visual scoring of γH2AvB in isolated nuclei

Microscope slides were visualised using an Olympus fluorescence microscope. Nuclei from 1st instar larvae 2 days post IR were used and nuclei were stained with DAPI (blue) and the γH2AvD antibody (with Alexa 488 (green) secondary antibody).

QFly pupae nuclei 24 h post IR were stained with DAPI (Blue) and γH2AvB antibody (with Alexa 633 (red) secondary antibody). The frequency of γH2AvB foci were scored for approximately 100 nuclei per IR dose (0, 90, 240, 400 Gy). The scoring of foci were based on the following 4 criteria: (1) no foci, (2) a single focus, (3) >1 foci or (4) a large group of foci occupying at least one third of the nuclear area. Foci were classified as a single spot when they appeared less than 1/20th of the nuclear area.

2.11 Immunofluorescence to quantify γH2AvB response in nuclei

Cell nuclei obtained from adult QFly were extracted using a similar protocol as described above with the following modifications: adult Q-flies (17 days post-IR) were thawed from -80oC at RT for 5 min and suspended in 1.5 ml of hypotonic lysis buffer containing 10 mM Tris-HCl pH 8.0, 1 mM KCl, 1.5 mM MgCl2, phosphatase inhibitors (as above) and protease inhibitor cocktail, in a glass tissue homogenizer. Tissues were homogenized on ice until a clear suspension was achieved (usually 5 passes). The suspension was filtered using nylon net filters (filter type: 100 µm NY1H) to remove most of the particles and then incubated for 30 min on a rotator at 4°C to allow the hypotonic swelling and lysis of cells, which were subsequently fixed in 1% formaldehyde in the same tube for 15 min at RT. Nuclei were then spotted on slides (using 10 µl of the suspension) and air-dried for 20 min at RT. Spotted nuclei were re-hydrated in phosphate-buffered saline (PBS) for 15 min. Slides were then incubated in pre-chilled 70% ethanol for at least 20 min and washed in PBS for 15 min. Cell nuclei were “blocked” using TBST containing 5% BSA for 30 min at RT, and slides were then washed once in PBS. Primary antibody (anti-H2AvB) was added at 1:500 dilution in TBST containing 5% BSA and covered with parafilm and incubated overnight at 4°C. Slides were then washed three times in PBS for 5 min each to remove unbound antibody, and then incubated with secondary antibody (Alexa Fluor 488-conjugated) at a dilution of 1:500 in TBST containing 5% BSA for 1 h at RT. Slides were then washed three times in PBS for 5 min each to remove unbound, or non-specifically bound, antibody. Nuclei staining was achieved using 4',6-

 

 

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diamidino-2-phenylindole (DAPI) at a concentration of 0.2 µg/ml for 7 min at RT and then washed in a solution containing 300 mM NaCl and 30 mM trisodium citrate (pH 7.0). Spotted, DAPI-stained nuclei were subsequently mounted under a cover slip using mounting medium consisting of PBS and glycerol (1:1). Cover slip edges were sealed with nail polish to prevent desiccation prior to analysis by laser scanning cytometry.

2.12 Laser scanning cytometry

Laser scanning cytometry (LSC) is a very accurate cytometric method to colocalise and quantify fluorescent events in thousands of nuclei (Zhao, et al. 2009a; Zhao, et al. 2009b) (which is not practical with visual scoring techniques), therefore we used LSC to quantify the γH2AvB signal in nuclei on microscope slides. QFly pupae were exposed to 0, 20 or 240 Gy IR and allowed to emerge as adults. At 17 days post-IR the adult Q-flies were frozen at -80oC. Nuclei were subsequently extracted after hypotonic lysis and then fixed and stained on microscope slides. LSC was performed using an iCyte® Automated Imaging Cytometer (CompuCyte Corporation, Westwood, MA, USA) with full autofocus function and an inverted fluorescence microscope with laser excitation (Argon 488 nm, and Violet 405 nm) for quantitation of green and blue emission, respectively. A total of 2656 (0 Gy), 3078 (20 Gy) or 3571 (240 Gy) nuclei were examined using iCyte cytometric analysis software version 3.4.10. The ‘‘CompuColor’’ feature in iCyte was used to observe nuclear staining as blue and γH2AX signal as green. The slides were scanned using a 40x objective and a 0.25 µm resolution step. Two lasers (405 nm and 488 nm) were used to excite the dyes DAPI and Alexa Fluor 488, respectively. The two lasers were scanned over the samples in separate passes, one immediately following the other, to prevent any overlapping (thus compensation) of fluorescence signals. The emitted and filtered fluorescence was then detected by photomultiplier tubes in separate channels (blue and green). The nuclei and γH2AvB events were contoured using empirically determined thresholds to exclude the scoring of false positives (e.g. small fluorescent debris). Any small debris or larger blue-emitting particulate matter (which was rarely observed) was excluded from the analyses. Individual data points for each nuclear event were automatically generated using the iCyte® software and transferred to statistical analysis software (see below).

2.13 mRNA isolation, cDNA synthesis and 454 sequencing

Frozen pupae (10-11 pupae) that had been irradiated with 150 Gy were divided into 3 replicate groups. mRNA was purified using a GenEluteTM Direct mRNA miniprep kit (Sigma) according to the manufacturer’s directions. Briefly, tissues were homogenized and lysed using liquid nitrogen with mortar and pestle and 1 ml of lysis solution containing proteinase K. mRNA extraction proceeded using oligo(dT) beads and eluted mRNA was precipitated overnight at -20oC using 1 µl of 20 µg/µl glycogen, 0.1 volumes of 3 M sodium acetate pH 5.2, and 3 volumes of ice cold ethanol. Precipitated mRNA was centrifuged and the pellet washed in 70% ethanol. mRNA was then resuspended in 19 µl of elution buffer and checked for quantity and quality using a NanoDrop1000 spectrophotometer (Thermo Fisher, USA) and gel electrophoresis. The cDNA library was then generated according to the cDNA Rapid Library Preparation Method Manual (Roche). Each replicate group was ligated with different MID adaptors (RL 13, 14, 15; manufactured by Integrated DNA Technologies). Following library quantitation using a FLURO Star Optima (BMG Labtech, Germany), 20 µl of each replicate was then pooled together and the combined library diluted to a final concentration of 1x106 molecules/µl. Emulsion PCR and bead enrichment was performed as per the emPCR amplification method manual –Lib-L (Roche Applied Science, USA) using 2 library molecules per bead. Approximately 500,000 of the enriched beads were loaded onto a PicoTiter-Plate (Roche Applied Science, USA) and pyrosequencing was

 

 

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performed using a 454 GS Junior (Roche Applied Science, USA) according to the manufacturer’s sequencing method manual (Roche) using the default parameters for cDNA.

2.14 Sequence analysis and homology search

454 sequencing of the cDNA library generated 3,166,947 bases from 91,349 reads. These reads were assembled into 2,512 contigs, 2,258 isotigs and 21,950 singletons using de novo assembly by Newbler version 2.0.1 (Roche Applied Science). Isotig sequences were compared to sequences in the NCBI database by BLASTn using Blast2goPro (www.Blast2GO.org) (Gotz, et al. 2008). E-values lower than 1.0E-3 were considered significant. Isotig00988 (GenBank Acc No. KC161252) was found to be most similar to H2A of Glossina morsitans. Isotig00988 contained 748 bp and the nucleotide sequence was submitted to the ORF finder at NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The longest ORF was found to be the candidate H2A protein coding region. Clustal Omega (accessed through http://www.uniprot.org/) was used to compare the resulting amino acid sequence to Drosophila (accession no.P0895), Human (accession no. P16104) and Glossina (accession no. D3PTWO) H2A sequences.

2.15 Proteomics identification of H2AvB

24 h post-irradiated pupae nuclear extracts were subjected to Polyacrylamide Gel Electrophoresis (as above). Bands were excised corresponding to approximately 15 kDa and provided to the Adelaide Proteomics Centre (University of Adelaide). Samples were further treated for data acquisition by liquid chromatography – electrospray ionisation tandem mass spectrometry. Post acquisition, acquired spectra were subjected to peak detection and de-convolution using DataAnalysis (Version 4.0 SP4 Build 281, Bruker Daltonics). Processed MS/MS spectra were then exported to Mascot generic format (mgf) and submitted to Mascot (Version 2.3.02) for identification. Search parameters as follows:

The name of the identified protein and the organism from which the protein was identified The accession number of the protein in the database. The combined ion score for all queries (i.e. MS/MS spectra) matched to a single protein. The number of queries that were matched to a single protein and the total number of queries

resulting from that LC-ESI-MS/MS run. The sequences of the identified peptides. The independent ion scores for each of the matched peptides and the cut off score, whereby

peptides with ion scores above this threshold indicate identity or extensive homology. The exponentially modified protein abundance index (emPAI value) as an approximate

measure of relative quantitation. This is calculated from the number of observed peptides relative to that predicted from the matched sequence. In general, higher scores indicate greater relative abundance.

The predicted molecular weight (MW) and isoelectric point (pI) of the matched protein based on the values provided in the MASCOT Summary Report.

2.16 Indirect ELISA protocol

Wells of a 96-well plate were treated with Poly-L-lysine (Sigma) and incubated at room temperature for 15 min followed by 2 washes in PBS. Whole pupal lysates and extracted histones were diluted in carbonate-bicarbonate buffer (Sigma) to 5, 25, 50 and 100 μg/ml and added to each

 

 

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well and incubated overnight at 4oC. Wells were thoroughly washed 3 times with PBS buffer (100 μl) and then blocked for 1 h at RT in PBS (100 μl) containing 1% BSA. Each well was then thoroughly washed 3 times in PBS (100 μl) for 5 min each time, then incubated with yH2AVB primary antibody (100 μl) diluted 1:500 in PBS containing 1% BSA. After 3 more washes wells were incubated with anti-rabbit horseradish peroxidise-linked secondary antibody (100 μl) at a dilution of 1:500 in PBS containing 1% BSA for 2 h at RT. Wells were then finally washed 3 times with PBS and added 100 μl of colouring reagent (SIGMAFAST –OPD) was added. Absorbance was measured at 492 nm using a 96-well plate spectrophotometer with Spectra MAX software (Molecular Devices, USA).

2.17 Sandwich ELISA protocol

Wells of a 96-well plate were treated with Poly-L-lysine (Sigma) and incubated at room temperature for 15 min followed by washes in PBS. Wells were then coated with the capture antibody histone H2A.Z at a dilution of 1:400 in carbonate-bicarbonate buffer for 2 h at room temperature. Each well was then washed 3 times in PBS for 5 min each time and then blocked for 1 h at RT in PBS (100 μl) containing 1% BSA. Wells were thoroughly washed 3 times with PBS buffer (100 μl) and then samples (50 μl) diluted in carbonate-bicarbonate buffer (5, 25, 50 and 100 μg/ml) were added to each well and incubated at 4oC overnight. Each well was thoroughly washed 3 times in PBS buffer (100 μl) for 5 min each time, then incubated with yH2AvB primary antibody (100 μl) diluted at 1:500 in PBS containing 1% BSA. After 3 more washes wells were incubated with anti-rabbit horseradish peroxidise-linked secondary antibody (100 μl) at a dilution of 1:500 in PBS containing 1% BSA for 2 h at RT. Wells were finally washed 3 times with PBS and 100 μl of colouring reagent (SIGMAFAST –OPD) added. Absorbance was measured at 492 nm using a 96-well plate spectrophotometer with Spectra MAX software (Molecular Devices, USA).

2.18 Preliminary detection of γH2AvB homolog in Mediterranean fruit fly

Mediterranean fruit fly (Ceratitis capitata) pupae irradiated at doses of 40 Gy, 80 Gy and 160 Gy were obtained from the Western Australian Department of Agriculture and Food’s sterile Medfly production facility (at South Perth) and frozen 4 days post irradiation. Whole pupal lysates were collected and 70 g of each was subjected to Western blot analysis using Drosophila-based primary γH2AX antibody and alkaline phosphatase-conjugated anti-rabbit antibody as described above (combined with -actin antibody as a housekeeping control). The histone:actin ratio was calculated for each dose. Image J software was used to compare band intensities.

2.19 Statistical analyses

GraphPad Prism 5 was used to analyse data using the student’s t-test and to determine the correlation coefficients. Data were expressed as mean ± standard error of the mean. GraphPad InStat 3.1 was used for other statistical analyses.

 

 

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3 Results

3.1 Summary of QFly samples prepared

Table 1 summarizes QFly specimens that were reared or collected through Macquarie University. All samples were irradiated at the doses indicated, mostly under hypoxic conditions as described in the table. Samples were then frozen at -80oC at various times after irradiation and stored, prior to shipping to CSIRO Animal, Food and Health Sciences (Adelaide) to investigate the biomarker(s) of irradiation.

 

Table 1. Collection of QFly specimens from Macquarie University at various life stages and ionising radiation doses.

Sample type  Irradiation dose (Gy)  Condition Freezing time

One‐day old pupae  20,  30,  40,  50,  60,  70,  80,  160, 240, 320 and 400 

Hypoxic  1 day post‐irradiation including control  (0 Gy) 

One‐day old pupae  20,  30,  40,  50,  60,  70,  80,  160, 240, 320 and 400 

Hypoxic  2 days post‐irradiation including control (0 Gy) 

One‐day old pupae  20,  30,  40,  50,  60,  70,  80,  160, 240, 320 and 400 

Hypoxic  5 days post‐irradiation including control (0 Gy) 

One‐day old pupae  20,  30,  40,  50,  60,  70,  80,  160, 240, 320 and 400 

Hypoxic  17 days  (adult) post‐irradiation  including  control (0 Gy) 

Eggs  10, 20, 30, 60, 90, 120, 150, 200, 250, 300, 350, 400 

Hypoxic  2 h post‐irradiation including control 

(0 Gy) 

1st instar larvae  10, 20, 30, 60, 90, 120, 150, 200, 250, 300, 350, 400 

Hypoxic  same day post‐irradiation including control (0 Gy) 

1st instar larvae  10, 20, 30, 60, 90, 120, 150, 200, 250, 300, 350, 400 

Hypoxic  1 day post‐irradiation including control (0 Gy) 

1st instar larvae  10, 20, 30, 60, 90, 120, 150, 200, 250, 300, 350, 400 

Hypoxic  Frozen when reached to 3rd instar post‐irradiation 

3rd instar larvae   10, 20, 30, 60, 90, 120, 150, 200, 250, 300, 350, 400 

Hypoxic  2 day post‐irradiation including control (0 Gy) 

One‐day old pupae  20,  30,  40,  50,  60,  70,  80,  160, 240, 320 and 400 

Air  From 5, 10, 20, 30, 60, 120 min  to 1 and 2 days post‐irradiation including control (0 Gy) 

Nine  –  days  old pupae (for SIT) 

0, 50, 60, 70  Hypoxic  Frozen  at  1,  2,  10  and  20  days  post‐irradiation including control (0 Gy). Adults emerged on day 2 post‐irradiation so samples frozen from 2‐20 days post‐irradiation  were  frozen  adults  (pupae  only frozen at 1 day post‐irradiation). 

3rd  instar  larvae collected  from  fresh organic chillies 

0, 70, 150, 250, 400  Hypoxic  1 day post‐irradiation including control (0 Gy) 

Organic  chillies infected with larvae 

0, 70, 150, 250, 400  Hypoxic  1 day post‐irradiation including control (0 Gy) 

 

 

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3.2 Identification of potential candidate protein biomarkers of previous ionising radiation exposure

Various candidate cell markers that can be correlated with irradiation dosages were investigated; including Histone H2AX, Histone H3, Rad6, Chk1, Chk2 and p53. These nuclear proteins have been well characterized in terms of their activity following DNA double strand breaks following exposure to ionising radiation. Table 2 summarises the antibodies used in the initial pre-screening assay to identify ideal biomarkers of previous ionising radiation exposure.

Table 2. Antibodies used for the preliminary investigation of DNA damage/response protein biomarkers of previous ionising radiation.

DNA  damage/response protein 

Mechanism  Antibody used 

H2AX (S139)  phosphorylation  triggered  by  DNA DSB 

Biosensis; custom made phospho‐H2AX (Ser 139) 

Histone H3 (S28)  involved  in  DNA  damage  response and mitosis/meiosis 

Abcam; ab4178 Histone H3 (phospho S28) 

 

Rad6    involved  in DNA  repair, ubiquitylates H2B 

Abcam; ab31917  

Chk1 (S345)  involved  in DNA  damage  checkpoint response to γ‐IR 

Abcam; ab47318 Chk1 (phospho S345) 

Chk2 (T68)  is  phosphorylated  by  ATM  following ionising radiation 

Abcam; ab3501 Chk2 (phospho T68) 

p53 (S15)  is phosphorylated by Chk2  Abcam; ab1431 p53 (phospho S15) 

Abbreviations: DSB, double strand breaks; IR, ionising radiation.

 

Figure 2 shows the results of Western blots using the DNA damage/response proteins in QFly pupae 24 h post IR exposure. Of the six antibodies investigated Rad6, Chk1, Chk2 and p53 yielded little to no IR dose effects. However, the two histone protein antibodies; Histone 3 (Ser28) and Histone H2AX (Ser 139) showed dose-dependent increases in signal 24 h after IR exposure on Western blots. Therefore, we used the H2AX (Ser 139) antibody in further studies as it yielded the most intense signal at both 160 and 240 Gy.

 

 

 

 

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Figure 2. Preliminary investigations of DNA damage biomarker proteins in QFly pupae 24 h post IR exposure. Whole pupae lysates were prepared and subjected to Western blot analyses. Comparison of Western blots using antibodies to identify Histone 3 (S28), Rad6, Chk1 (S345), Chk2 (T68), p53 (S15) and histone H2AX (S139) for 0, 160 and 240 Gy are shown.

 

 

3.3 γH2AX, γH2AvD and γH2AvB antibodies

Some of the preliminary studies used an antibody that was prepared based on the human γH2AX sequence KKAATQA[PSer]QEY (as shown in Figure 2 “Histone H2AX (S139)”). The antibody clearly identified a nuclear protein of approximately 15 kDa that was evident in irradiated pupal samples and is consistent with the molecular weight of γH2AX as observed in other species (Rogakou, et al. 1999; Redon, et al. 2002). Although the (human) antibody provided a clear band at approximately 15 kDa, there was some non-specific binding detected. Since there was no available “γH2AX” antibody specific to B. tryoni at that time (as the sequence was not known) we tested an antibody that was specific to the Drosophila melanogaster γH2AX sequence (called γH2AvD) which also resulted in a single band of approximately 15 kDa in irradiated QFly samples. Interestingly, both these antibodies recognized a 15 kDa protein that was responsive to IR exposure in Bactrocera tryoni. This suggested that the antibodies likely recognized the conserved SQ motif

 

 

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(phosphorylated) in the C-terminal of the putative QFly histone H2A protein as it has been shown for many other species (Downs, et al. 2000; Friesner, et al. 2005; Lang, et al. 2012). Although these antibodies (human and Drosophila) were useful at recognizing the γH2AvB protein in QFly, we aimed to identify the (phosphorylated) H2A sequence of B. tryoni using transcriptomics. Once the full sequence was known (as described in the next section) we then successfully developed an antibody that recognized the C-terminal region of the H2A sequence of B. tryoni; QDPQRKNTVILS*QGY. We termed this phosphorylated protein in QFly “γH2AvB”. For the majority of this study the γH2AvD was used until the γH2AvB antibody became available. In later Western blot and ELISA assays the γH2AvB antibody was routinely used.

 

3.4 454 sequencing of transcripts and peptide identification by proteomic analyses

Figure 3 shows that 454 sequencing revealed a H2AX protein sequence that was identical to that found in G. mortisans, was 96.4% similar to D. melanogaster, and only 54.8% similar to human H2AX. We have termed the B. tryoni H2AX homolog “H2AvB” and the phosphorylated form “γH2AvB”. The SQ motif of H2AvB was conserved as for all other species in which the histone has been sequenced. Furthermore, 2D-gel electrophoresis and subsequent Western blots showed a band of expected molecular weight (not shown).

 

 

 

 

Figure 3. Amino acid sequence of H2AX homolog (H2AvB) and alignment of H2A histone variants. The conserved SQ motif is highlighted in red text. The sequence of a H2AX homolog protein was identified from deep sequencing transcript analyses and mass spectrometry of QFly (B. tryoni). The QFly H2A variant is termed H2AvB (GenBank Accession #KC161252). We found that H2AvB is 96.4% similar to that of the vinegar fly (genetic model species) D. melanogaster (H2AvD), 54.8% similar to human H2AX, and identical to G. morsitans (the Savannah tsetse fly). The numbers in parentheses represents the UniProtKB accession numbers for each sequence. Numbers to the left of sequences represent the first amino acid position of each line.

 

 

 

 

 

 

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As described in methods, isotig sequences were compared to sequences in the NCBI database by BLASTn using Blast2goPro (www.Blast2GO.org) (Gotz, et al. 2008). The species distribution was analysed and shown summarized in Figure 4. The species distribution of the top BLAST hits for the Bactrocera tryoni transcriptome showed that Bactrocera genes had the greatest number of matches with several species of Drosophila as well as Glossina morsitans genes. Gene ontology assignment programs were used for functional characterization of annotated genes. Three categories were used including biological processes (Figure 5), molecular function (Figure 6), and cellular component (Figure 7).

 

 

 

Figure 4. Species distribution of homologous sequences.

 

 

 

 

Figure 5. Gene ontology analyses of Bactrocera tryoni transcriptome data. Transcript sequence distribution: biological processes.

 

 

 

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Figure 6. Gene ontology analyses of Bactrocera tryoni transcriptome data. Transcript sequence distribution: molecular function.

 

 

 

Figure 7. Gene ontology analyses of Bactrocera tryoni transcriptome data. Transcript sequence distribution: cellular component.

 

 

 

   

 

 

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Proteomic analyses were performed on 24 h post-irradiated pupae nuclear extracts. Samples were subjected to 2D-gel electrophoresis and Western blotting. Peptide sequences were identified in trypsin digests according to the criteria in the Methods section and matched identical regions are shown on the 454 sequenced data in Figure 8.

 

 

 

 

Figure 8. Complete amino acid sequence of QFly H2AvB. Complete amino acid sequence of QFly H2AvB is shown with trypsin digested peptide sequences identified from mass spectrometry (shown in red parentheses above the amino acid sequence). The identified peptide sequences (red) overlapped but did not cover the entire sequence of H2AvB. The site of phosphorylation at the SQ motif is shown in bold.

 

 

3.5 Investigation of γH2AvB in QFly brain and gonad

Figure 2 lower right panel labelled “Histone H2AX (S139)” and several other preliminary Western blot assays (not shown) routinely demonstrated a consistent elevated level of γH2AvB response to IR. We questioned whether two important anatomical regions (brain and gonad) of the pupae were showing different responses of H2AvB phosphorylation following the exposure to IR and therefore if it was necessary to examine these organs more closely in future assays. For Western blot analyses the brain and gonad regions were carefully dissected and several specimens were pooled to generate enough protein for extraction to complete Western blot analyses of γH2AvB. The brain and gonad tissue lysates were prepared in a similar manner to whole pupae lysates (using the same lysis buffers and conditions). The upper left panel of Figure 9 (Coomassie stained gel) demonstrated that similar total protein quantities were loaded in all 4 samples (as evidenced by the amount of Coomassie blue staining). In the representative Western blot (upper right-main panel) the γH2AX antibody (human) yielded multiple bands on the Western blot in both brain and gonad samples. Although some of the high molecular weight bands (at >37 kDa) appeared similar in intensity across all samples tested, however there was a clear IR dose-dependent band observed at approximately 15 kDa as expected but also at approximately 75 kDa (for the gonad 240 Gy sample). We believe that the 75 kDa band (in 240 Gy; gonad) represents the primary antibody binding to the γH2AvB protein that existed in a multimer complex, likely to be the histone nucleosome that was not fully dissociated. Importantly there was a very clear increase in γH2AX signal following IR at 240 Gy in both brain and gonad QFly pupae samples, almost none detected at 0 Gy (at approximately 15 kDa). This area of the Western blot membrane was expanded and is shown in the lower panel of Figure 9. Since there was a similar response of γH2AvB to IR in both gonad and brain we felt there was no need to carefully dissect specimens in future studies as the whole organisms would provide a high yield of total protein for Western blot analyses.

 

 

 

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Figure 9. Comparison of two anatomical regions showing γH2AvB response. Upper (left) panel shows the Coomassie stained gel (with molecular weight markers shown on the left side). Total protein loaded was similar for all samples: brain 0 and 240 Gy; gonad 0 and 240 Gy. The region of interest at approximately 15 kDa has been expanded as shown in the lower panel.

 

3.6 Short-term kinetics of H2AvB phosphorylation

Phosphorylated H2AvB (γH2AvB) was detected following exposure of pupae to doses as low as 10 Gy of IR (Figure 10A). The phosphorylation of H2AvB occurred rapidly and could be detected at 5 min post-IR exposure, peaking at approximately 20 min post IR exposure (Figure 10B). There was a gradual decline of γH2AvB over a period of 24 h, however, there was still significant γH2AvB present 24 h post IR exposure, indicating that only a proportion of γH2AvB was dephosphorylated within 24 h. As expected, 60 Gy IR exposure led to a higher level of γH2AvB relative to the pupae exposed to 10 Gy. Alkaline phosphatase treatment of a histone extract from IR-treated (70 Gy, 24 h post IR) pupae abolished γH2AvB detection (Figure 10C), confirming that the antibody detected only the phosphorylated form of H2AvB, at the SQ-motif. Since irradiated samples at other life stages (egg versus larvae) of B. tryoni also elicit a γH2AvB response, an increase in γH2AvB response following IR exposure at 150 Gy, the standard dose used for QFly post-harvest disinfestation was also shown (Figure 10D).

 

 

 

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Figure 10. Short-term kinetics of H2AvB phosphorylation. (A) Total pupae lysates were prepared and γH2AvB responses are shown for 0, 10, and 60 Gy IR at 5 min, 20 min, 2 h, and 24 h following IR exposure. β-actin is shown on the lower panels to demonstrate loading controls (225 μg protein on each lane). (B) The γH2AvB signal from (A) was quantified using ImageJ and data were plotted with the following symbols: 0 Gy (filled circles), 10 Gy (filled squares), and 60 Gy (filled triangles). (C) 24 h post-IR exposed pupae were subjected to the acid precipitation method to extract histones. Treatment of samples with alkaline phosphatase (+) abolished the γH2AvB signal, which remained in non-treated samples (-). The data shown confirmed that the IR-induced H2AvB was in the phosphorylated form and was detected by the primary antibody. (D) Western blot analyses of QFly eggs (73 μg protein loaded; left panel) or larvae right panel (105 μg protein loaded) demonstrating detectable γH2AvB signal in different QFly life stages. β-actin was used as a protein loading control.

3.7 γH2AvB dose-response to previous ionising radiation exposure

The above data indicate a clear phosphorylation-dependent γH2AvB signal following IR exposure compared with non-irradiated samples. To further investigate the effect of IR on QFly pupae at different doses, particularly covering and exceeding the range most often used for SIT and to disinfest produce, pupae were exposed to a wide dose range (up to 400 Gy) and then frozen at -80oC

 

 

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24 h post-IR. Figure 11A shows a representative Western blot demonstrating a dose-dependent increase in the γH2AvB signal. The maximum signal was produced at the highest tested dose of 400 Gy and yielded an approximate 10-fold increase above non-irradiated pupae. γH2AvB signal was detected in QFly pupae at doses as low as 20 Gy, however, in Figure 11A this is not particularly clear since this Western blot was exposed for ECL under conditions that would clearly show the higher end doses (>80 Gy) of the Western blot. To compare the results of 3 separate assays, data were normalized using β-actin as a loading control. Since there were differences between imaging exposure times and therefore the band intensities between separate assays, data were then further corrected to the “maximum” signal (i.e. at 400 Gy) to account for these potential differences in imaging and incubation conditions. This allowed the slope and fit of the lines of γH2AvB responses to be appropriately compared in separate assays as shown in Figure 11B inset. This figure also demonstrates the high linear correlation of γH2AvB with IR dose (r2 > 0.9).

 

Figure 11. γH2AvB dose-response following IR. The intensity of γH2AvB signal in QFly pupae (24 h post IR) is proportional to IR exposure. (A) Western blot showing the mean γH2AvB signal at approximately 15 kDa (upper panel) increases in proportion to the IR dose up to the maximum exposure of 400 Gy tested for this assay. The lower panel shows the β-actin loading controls. (B) ImageJ software was used to quantify the integral of the bands in (A) upper and lower panels. γH2AvB signal from three independent assays (see inset) was corrected for the amount of β-actin loaded and data (as % of maximum) was plotted against IR dose to allow for differences in incubating conditions and imaging exposure times. Data are mean ± SEM.

3.8 γH2AvB – long term response

Interestingly, our data show a very strong γH2AvB signal in QFly pupal lysates from exposures as low as 20 Gy, at least 24 h post-IR (Figure 11). This led us to examine whether the γH2AvB signal was evident at even longer time points post-IR, as this would potentially provide a useful biomarker to demonstrate that samples were previously exposed to IR. Figure 12 demonstrates that the dose effect of IR on γH2AvB signal was clearly observed at 24 h post-IR (for doses of 0, 70 & 240 Gy), however, at five days post–IR the γH2AvB signal in pupal lysates was substantially reduced

 

 

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compared with one day post-IR (the same amount of total protein was loaded in all samples to allow direct comparisons). It should be noted that in some of our earlier Western blot assays we did occasionally observe a very low amount of γH2AvB signal (approximately 15 kDa) after 70 Gy exposure at five days post-IR, when higher amounts of total protein were loaded and when longer ECL exposure times were used. These preliminary observations led us to believe that there was indeed a measureable persistent γH2AvB signal even 5 days post-IR exposure. Figure 12A (lower right panel, labeled “overexposed”) shows a longer development time on the same Western blot membrane and a dose-responsive γH2AvB signal became more evident, albeit not as intense as when analyzed at one day post-IR. This suggests that despite a large decline in phosphorylated γH2AvB levels between one and five days post-IR exposure in QFly pupae, a persistent or residual γH2AvB signal remained.

 

 

 

Figure 12. γH2AvB signal after 5 days. γH2AvB signal in QFly pupae was reduced at five days post-IR. (A) Western blot showing a dose-dependent increase in γH2AvB signal 1 day after IR exposure (0, 70 and 240 Gy). However, at five days post IR, the γH2AvB response was not easily visible in this representative assay until the Western blot membrane was allowed to develop with a longer imaging time (“overexposed”) as shown in (B). 100 µg protein was loaded in all lanes.

 

To further examine whether we could detect γH2AvB signal at least 5 days after IR exposure (at the standard dose used for SIT), the effect of 70 Gy IR on γH2AvB signal using whole QFly pupal lysates 1 day and 5 days post-IR was investigated. The γH2AvB response was quantified by Western blot as shown in Figure 13A (left “pupal lysate” panels, lanes 1 and 2) demonstrating a significant γH2AvB signal at approximately 15 kDa. β-actin and cytochrome c oxidase subunit II were used as loading controls and confirmed that equivalent amounts of protein had been loaded for each treatment. To confirm the specific association of the γH2AvB signal with cell nuclei and to improve the γH2AvB signal nuclear proteins were isolated by an acid precipitation method as described previously (Shechter, et al. 2007). When 15 µg total nuclear protein extract was examined by Western blot analysis (shown in lane 5 and 6 of Figure 13A, labeled “histone extract”) the γH2AvB signal following 70 Gy IR clearly yielded a higher signal than that of the equivalent amount of protein from the whole “pupal lysate” when either 15 µg or 150 µg protein was loaded (Figure 13A).

 

 

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This enrichment of nuclear γH2AvB protein observed was also associated with a higher γH2AvB signal at 0 Gy. Nevertheless, the IR response of γH2AvB signal was clearly distinguishable from background levels and several fold more intense at 70 Gy compared with 0 Gy. The absence of any detectable signal coming from β-actin (cytoplasm) and cytochrome-C oxidase subunit II (a mitochondrial protein) in the histone extract (Figure 13A, lanes 5 and 6) demonstrates that the histone extract was relatively free from these latter proteins as expected, and confirmed that the nuclear extract method employed did not result in significant cytoplasmic or mitochondrial contamination, whilst significantly enriching the histone fraction. Therefore, it appears that the nuclear histone extraction method offers a convenient way to partially purify and concentrate low levels of persistent IR-induced γH2AvB signal from QFly.

Since our objective was to detect any long-term persistent γH2AvB signal in irradiated QFly pupae the histone extract method was subsequently used to concentrate the γH2AvB signal as outlined earlier. Figure 13B shows a representative Western blot experiment using whole lysate from QFly pupae (120 μg protein) and nuclear extracts (6 μg protein), five days post-IR. Under the same duration of exposure times using ECL, Figure 13B left panels (lane 1 and 2) show no apparent γH2AvB signal response to 70 Gy IR using 120 µg total protein loaded, compared to a strong signal using the histone extract with only 6 µg total nuclear protein loaded (i.e. 20 times less protein, compare lanes 2 and 4 of Figure 13B). The IR-induced signal (70 Gy) was clearly evident and significantly higher than the background (0 Gy) signal. Since QFly are able to survive and withstand relatively high doses of IR, it was hypothesised that adult QFly specimens produced from irradiated pupae would contain persistent γH2AvB (as observed recently with minipig skin samples after receiving a dose of 50 Gy IR (Ahmed, et al. 2012)). Figure 13C demonstrates that persistent IR-responsive γH2AvB signal was observed in adult QFly at 17 days post-IR, in nuclear extract samples. Although later time points were not investigated, this may be a convenient method to identify samples previously exposed to IR as pupae and therefore, may have application for SIT.

To address whether individual pupae show variation in their γH2AvB response following IR exposure, the total lysate and histone extraction techniques were scaled down in order to examine γH2AvB responses of individual pupae. Figure 13D demonstrates that when replicate individual pupae were lysed and used for Western blot analyses, there was some variation of the γH2AvB produced in response to IR as expected. Overall, all pupae from the 0 Gy group (individual pupae lysates were loaded in lanes 1-6, Figure 13D) had significantly less γH2AvB signal compared with individual pupae exposed to 70 Gy IR (24 h post IR), as shown in Figure 13D, lanes 7-12. The γH2AvB signal was quantified using ImageJ and results are shown on the right panel of Figure 13D, with 70 Gy (n=6) significantly higher (P<0.001) than 0 Gy (n=6). Furthermore, the histone extraction method was successfully scaled down in a similar manner so that individual pupae could be subjected to the nuclear extraction method to increase the γH2AvB signal per total protein tested. Pupae exposed to 70 Gy had a significantly higher amount of γH2AvB signal (P<0.001) in the individual histone preparations as demonstrated by the Western blot from the single pupae replicates when compared with 0 Gy (Figure 13E).

 

 

 

 

 

 

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Figure 13. γH2AvB response following 70 Gy exposure at different time post IR. (A) Left panel show γH2AvB response from whole pupal lysates (150 µg protein loaded; 0 vs 70 Gy; lanes 1 and 2). γH2AvB was not observed in the same Western blot membrane when the sample was diluted 10-fold to 15 µg protein (lanes 3 and 4). However, when 15 µg total protein from the histone extract was loaded, the 70 Gy sample (lane 6) showed an intense signal exceeding that observed from the total pupal lysates at 70 Gy (lane 2). The absence of cytoplasmic proteins (as observed in lanes 5 and 6, lower panels) including β-actin and cytochrome C oxidase subunit II confirmed the relative purity of the histone extract. (B) γH2AvB signal in QFly pupae was reduced at 5 days post-IR as confirmed by analyses of total pupal lysates (lanes 1 and 2). However, significant γH2AvB signal was observed in the histone extract from QFly pupae five days post-IR (70 Gy; lanes 3 and 4). (C) Histone fraction showing significant γH2AvB signal 17 days post-IR (70 Gy) compared with 0 Gy. (D) Variability of the γH2AvB response in individual pupae is shown for 0 Gy (n=6; lanes 1-6) or 70 Gy (n=6; lanes 7-12) in the upper panel. The lower panel shows the β-actin loading controls. (E) Variability of the γH2AvB response in histone extracts from individual pupae that were exposed to 0 Gy (n=6; lanes 1-6) or 70 Gy (n=6; lanes 7-12). For both (D) and (E) all samples shown were run on the same Western blot to allow direct comparison. Bar charts to the right of (D) and (E) represent the mean ± SEM of the band intensities (integral) as determined by ImageJ analyses. Lower panels in (B), (C) and (E) are loading controls showing that the Coomassie-stained gels have equivalent amount of protein. ***P<0.001.

 

 

 

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3.9 Visual scoring of IR-induced γH2AvB signal in isolated nuclei

Microscope slides were prepared containing isolated nuclei from either 1st instar larvae 2 days post IR or pupae 24 h post IR as described in methods. Figure 14A shows representative images of nuclei (blue) with γH2AvB foci (red), with the scoring criteria recorded above each image. Figure 14B is a summary of the results from scoring approximately 100 γH2AvB foci at each IR dose; 0, 90, 240 and 400 Gy. Some of the data from the table in Figure 14B are further plotted and shown in Figure 14C, which demonstrate a dose-dependent decrease in the proportion of nuclei with zero foci scored or a dose-dependent increase in the frequency of nuclei containing “groups of foci” following IR exposure (Figure 14D). In another assay, to score the frequency of γH2AvB foci nuclei were classified as small, medium or large (obtained from 2 days post IR of 1st instar larvae) when comparing 0 Gy (n=790) and 90 Gy (n=609). Figure 15A shows representative images of small, medium and large nuclei, with approximately 30-35% of nuclei being classified as “large”, 60-75% as “medium” whilst the remainder were classified as “small” (Figure 15B). When “large” nuclei were scored only, those that contained >1 γH2AvB foci per nucleus (Figure 15C) showed a frequency of approximately 17% (mean of duplicates) from the 90 Gy samples compared with the 0 Gy samples where no nuclei were scored with >1 γH2AvB foci per nucleus (Figure 15D). Although these scoring methods demonstrated a dose dependent increase in the number and size of γH2AvB foci following IR exposure, it was a laborious procedure and remains somewhat subjective due to the difficulty in scoring quantitatively.

 

Figure 14. Visual scoring of the frequency of γH2AvB foci in pupae 24 h post IR. (A) Representative images of nuclei stained with DAPI (blue). γH2AvB foci are seen as red spots within the nuclei and were scored as follows; either no foci, 1 focus, >1 foci or groups of foci. Approximately 100 nuclei were scored for each IR dose (0, 90, 240 or 400 Gy) and is summarized in (B) whilst the proportion of nuclei with no γH2AvB foci is shown in (C) or proportion of nuclei with groups of γH2AvB foci is shown in (D).

 

 

 

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Figure 15. Visual scoring of the frequency of γH2AvB foci in 1st instar larvae 2 days post IR. (A) Representative fluorescence microscopy images of nuclei stained with DAPI (blue). Nuclei were classified in 3 broad categories as large, medium or small. (B) The frequency of large, medium and small nuclei was similar for 0 and 90 Gy IR exposure. (C) A representative fluorescence microscopy image showing three γH2AvB foci (green spots indicated with arrows) in a “large” nucleus. There was no difference between the number of foci scored in small and medium sized nuclei, however when only large nuclei were scored, approximately 17% (mean of duplicates) of large nuclei yielded >1 foci per nucleus following 90 Gy IR exposure, compared with 0% at 0 Gy exposure as shown in (D).

 

 

3.10 Persitent, long-term γH2AvB signal in adult Q-flies

Whilst processing adult Q-flies for analyses we noted that there appeared to be bleaching (to white) of the scutellum of Q-flies that had been previously exposed to IR as pupae. An example of this bleaching effect following 70 Gy IR (as pupae) is shown in Figure 16. Since this observation may be variable and difficult to quantify, we did not pursue this, however it was worth noting. Additionally, a random sample of 10 adult Q-flies were chosen and weighed. The total weight for the Q-flies appeared to decrease with increasing IR doses as shown in Table 3.

 

 

 

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Figure 16. Bleaching of the scutellum of adult Q-flies following 70 Gy IR. In a random sampling of adult Q-flies exposed to 70 Gy IR as pupae, it was noted that the scutellum appeared bleached (white) compared with controls (not exposed to IR).

 

   

 

 

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Table 3. Total weight of 10 randomly selected adult Q-flies; 0, 70, 160 Gy

Sample type  Weight (g) 

17 days adult QFly (0 Gy; no radiation)  0.1645 

17 days post‐IR adult QFly (70 Gy)  0.1549 

17 days post‐IR adult QFly (160 Gy)  0.1458 

 

 

To further validate the long-term (17 days) post IR γH2AvB response (as shown in the Western blot in Figure 13C), we employed immunofluorescence methods using nuclear extracts in combination with LSC. Representative LSC images of adult QFly nuclei stained with DAPI (blue) and demonstrating the γH2AvB signal are shown in Figure 17A–C. To determine whether long-term persistent γH2AvB signal could be observed at low and high doses, QFly pupae were exposed to 0, 20 or 240 Gy and then allowed to emerge as adults. The γH2AvB signal (green) was observed within nuclei 17 days post-IR, in doses as low as 20 Gy. Figure 17D shows that the mean (± SEM) integral fluorescence (from LSC) was significantly increased (P<0.001) following 20 Gy IR (n = 3078 nuclei) or 240 Gy IR (n = 3571 nuclei) compared with 0 Gy IR (n = 2656 nuclei). Figure 17E demonstrates that both 20 and 240 Gy IR exposure resulted in a significantly higher percentage of nuclei containing a γH2AvB signal compared with 0 Gy (control). The fluorescence integral of those nuclei with a positive γH2AvB signal identified from Figure 17E were quantified and reported in Figure 17F (as mean ± SEM). Figure 17F demonstrates that the γH2AvB signal (integral) was also significantly elevated in adult QFly nuclei 17 days post IR at the low dose of 20 Gy (P<0.01) as well as the higher dose of 240 Gy (P<0.05). The area of the γH2AvB signal in nuclei was examined as shown in Figure 17G. Although the area of γH2AvB signal appeared to be dose-dependent at 20 and 240 Gy, this increase was not statistically significant. The overall findings illustrated in Figure 17 further confirmed that γH2AvB signals persisted in emergent adult Q-flies for at least 17 days post IR (irradiated as pupae) and that quantitative scoring could be achieved using an automated fluorescence laser scanning cytometer.

 

 

 

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Figure 17. Quantification of γH2AvB signal in isolated nuclei by laser scanning cytometry. Representative laser scanning cytometry images of QFly nuclei showing (A) DAPI only (blue), (B) γH2AvB signal only (green) and (C) “merged” images which show the DAPI and γH2AvB signal overlaid. (D) Mean ± SEM of the integral fluorescence per nucleus of all nuclei examined including nuclei that lacked any measurable γH2AvB signal; n = 2656, 3078 and 3571 nuclei for 0, 20 and 240 Gy samples, respectively. (E) The percentage of nuclei examined that contain a measurable γH2AvB signal above background, increased significantly from approximately 7% in 0 Gy samples to 9.3% in 20 Gy samples (P<0.01) and to 23.7% of nuclei in 240 Gy samples (P<0.0001 by chi-squared test). To further examine if there was a greater γH2AvB signal in the 20 and 240 Gy samples compared with 0 Gy samples, only those nuclei with a measurable γH2AvB signal were analyzed and the results are reported in (F) as the mean integral (± SEM). Finally, the mean contoured areas of the total γH2AvB signal per nucleus are shown in (G). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

 

 

 

 

 

 

 

 

 

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3.11 Development of an ELISA to quantify γH2AvB signal

In order to make a simplified test for IR-induced DNA damage as measured by phosphorylation of H2AvB in QFly, we proposed that an Enzyme-Linked Immunosorbent Assay (ELISA) could be developed. Initially we prepared an indirect ELISA which involved attaching the (sample) proteins to the surface of positively charged 96-well plates by treatment with poly-lysine. A schematic is shown in Figure 18. Figure 19A shows samples in a 96-well plate with increasing concentrations of total protein from a nuclear extract. Figure 19B is a representative result from a single assay whilst Figure 19C shows the result with increasing concentration of total protein (from A). Although there was an increase in the level of γH2AvB signal at all protein concentrations tested in the 400 Gy sample compared with the 0 Gy sample, it appeared there was an approximate 2-fold increase at 400 Gy compared with 0 Gy which was substantially lower than that observed with Western blot assays (approximately 10-20 fold).

 

 

Figure 18. Concept of the γH2AvB Indirect ELISA assay.

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 19. Indirect ELISA of histone extracts. γH2AvB was quantified by indirect ELISA in pupae (histone extracts) 24 h post IR. (A) Example of a region of a 96-well plate showing the coloured reagents after the reaction occurred. (B) Representative result from a single assay using 100 μg/ml total protein comparing 0 vs 400 Gy. (C) Representative results from a single assay at various protein concentrations (0 vs 400 Gy).

In an attempt to obtain an improved signal-to-background a sandwich ELISA was developed (Figure 20). In a sandwich ELISA assay the “capture” antibody which is tethered to the charged surface is often used to pull down the protein(s) of interest out of a complex mixture, whilst non-bound proteins are washed away. Then another antibody that is specific for a different region of the protein is commonly used to enhance specificity, and finally, a secondary “labelled” antibody is used for detection. In our sandwich ELISA, we used an antibody for capture that was developed using the N-terminal sequence AGGKAGKDSGKAKAKA which is a conserved histone variant H2A.Z, which would capture all histone H2A proteins regardless of level of phosphorylation. Figure 21 shows a representative Western blot which confirmed that the antibody binds a (histone) protein that is not affected by IR dose.

 

 

 

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Figure 20. Concept of the γH2AvB Sandwich ELISA assay.

 

 

 

Figure 21. Anti H2A.Z antibody binds to histone H2A proteins independent of previous IR exposure. Since the Anti H2A.Z antibody binds to all H2A proteins regardless of level of phosphorylation, it was then used as a “capture” antibody in sandwich ELISA assays.

 

 

 

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Following capture of the H2A proteins (including γH2AvB) with the H2A.Z antibody, the specific probing antibody directed to Bactrocera tryoni (QFly) C-terminal protein sequence QDPQRKNTVILS*QGY was used followed by a HRP-labeleld secondary antibody (as shown in the schematic, Figure 20). Preliminary investigations with ELISA demonstrated that total pupae lysates (from 24 h post IR pupae) yielded no differences between γH2AvB levels when comparing 0 vs 400 Gy (not shown), however when using a Western blot the expected increase in signal following IR exposure was clearly evident (See Figure 22). It is speculated that the total lysate did not work in terms of the ELISA assay detecting an increase in γH2AvB signal, since the histones including the H2AvB protein existed in a tightly bound multimer (octamer) complex which could not be captured by the H2A.Z antibody due to steric hindrance. In Western blots, this same protein complex becomes fully dissociated into its constituent components due to the denaturing conditions used for Western blots. Thus the antibody was free to bind to the monomeric γH2AvB in Western blots. Alternatively, when a nuclear extract was prepared comparing 0 vs 400 Gy at several total protein concentrations in the sandwich ELISA, an increase in the γH2AvB signal was observed (Figure 23) with the 400 Gy samples. It was likely that the monomeric H2AvB dissociated from the multimeric histone complex during the preparation of the histones since there were several acid precipitation steps which would normally result in protein dentauration/unfolding.

 

 

Figure 22. Representative Western blot showing a result using QFly specific γH2AvB antibody.

 

 

 

 

 

 

 

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Figure 23. Sandwich ELISA of histone extracts. γH2AvB was quantified by sandwich ELISA in pupae (histone extracts) 24 h post IR. Representative results from a single assay (in triplicate) 0 - 100 μg/ml total protein comparing 0 vs 400 Gy.

 

 

3.12 Preliminary detection of γH2AvB homlog in Mediterranean fruit fly (MedFly)

We undertook to make a preliminary assessment of whether the same biomarker could be detected in irradiated MedFly pupae. To this end we obtained pupae irradiated at three doses (40, 80 and 160 Gy) and assessed them with the Drosophila γH2AX antibody using the Western blot technique similarly to that conducted for QFly. This proved successful, with the biomarker being detected in samples from each dose (Figure 24A). As for QFly, -actin was used as a housekeeping protein and the ratio of biomarker:actin was calculated and shown to increase with increasing dose. 

 

 

 

 

 

 

 

 

 

 

Sandwich ELISA Q‐fly nuclear extract

g/ml protein

Absorban

ce at 492 nm

25 50 75 100

‐0.1

0.0

0.1

0.2

0.3

0.4

0.5

0 Gy

400 Gy

 

 

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Figure 24. Preliminary detection of γH2AvB homolog in Mediterranean fruit fly. (A) Western blot: 16kDa H2AvB homolog was detected in whole pupal lystes of Mediterranean fruit fly (Medfly) irradiated at 40, 60 and 160 Gy, using Drosophila H2AX primary antibody (with -actin primary antibody used as a houekeeping control). 70ug protein extract from each cohort was run on PAGE gel and transferred to nitrocellulose membrane. From left to right 1: Medfly pupae exposed to 40 Gy irradiation; 2: Medfly pupae exposed to 80 Gy irradiation; 3: Medfly pupae exposed to 160 Gy irradiation; 4: two-colour protein ladder (BioRad). (B) Quantitative analysis of Western blot data. The ratio of the band intensities of the H2AvB homolog versus the loading control (-Actin) were graphed for the three tested irradiation doses for Medfly pupae. Data represents Mean ± SEM, technical duplicates (n=2), each dose representative of total protein from 10 pupae.

 

 

 

 

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4 Discussion

4.1 Summary

Phosphorylation of the C-terminal tail of H2AX proteins in nucleosomes located in the vicinity of DNA DSBs is one of the earliest responses to IR-induced DNA damage (Rogakou, et al. 1998; Olive, Banath. 2004). A γH2AX homolog has not been reported previously in tephritid fruit flies, including the commercially important QFly (B. tryoni), although the expression of a H2AX variant (H2AvD) has been reported in the vinegar fly D. melanogaster (Madigan, et al. 2002). In this study the QFly pupae exposed to IR were shown to have an elevated level of phosphorylated H2A protein (termed γH2AvB). Consistent with reports for other species (Rogakou, et al. 1999), irradiated QFly pupae showed a strong γH2AvB signal of approximately 15 kDa when examined using Western blot. The γH2AvB sequence was identified using 454 sequencing and found to be identical to G. morsitans. The identity and partial sequence of the IR-induced, phosphorylated histone was also confirmed by LC-ESI-MS/MS (data not shown, mass spectrometry was carried out by the Adelaide Proteomics Centre, University of Adelaide, SA, Australia). A linear dose-response of γH2AvB up to the maximum IR dose tested (400 Gy) was observed in QFly pupae 24 h post IR. However, at 5–17 days post IR, the γH2AvB signal had declined significantly when analysed in whole pupal lysates. In contrast, the persistent (5 days post-IR and beyond) γH2AvB response remained dose-responsive and was easily measurable by either Western blot or immunofluorescence methods such as LSC when in enriched histone extracts. The dose-dependent response with doses used for SIT (70 Gy) and disinfestation of fruit (up to 400 Gy), shows that γH2AvB may be useful as a marker of previous IR exposure in assays that support these commercially important applications.

4.2 Identification of γH2AvB

γH2AX is highly conserved across a wide taxonomic range of organisms (Redon, et al. 2002; Friesner, et al. 2005) and is a well-characterized histone protein that is known to be responsive to IR-induced DNA DSBs (Huang, et al. 2004; Olive, Banath. 2004; Roch-Lefevre, et al. 2010). We identified the sequence of a H2AX homolog protein in the QFly, B. tryoni (termed H2AvB; GenBank Accession #KC161252). It was found that H2AvB is approximately 96% similar to the vinegar fly D. melanogaster H2AvD, approximately 54.8% similar to human H2AX and interestingly, identical to the human disease vector G. morsitans (which is also the subject of SIT (Mutika, et al. 2013)). Our preliminary experiments demonstrated that an antibody designed to the human C-terminal tail sequence of γH2AX, KKAATQA[PSer]QEY, showed similar IR-induced γH2AvB signal compared with the antibody used for detection of D. melanogaster γH2Av as used in this study, which revealed a protein of approximately 15 kDa. The C-terminal amino acid sequence of human histone H2AX consists of ASQEY whereas for D. melanogaster the equivalent sequence is LSQAY. Although the C-terminal sequence for B. tryoni is slightly different from both human and Drososphila, it therefore appears that the antibody recognition site is likely to be mostly targeted towards recognizing the SQ phosphorylation motif, which is conserved across species. Indeed, others have used antibodies based on the human sequence of phosphorylated H2AX and found that it cross-reacts with histone H2A (phosphorylated) variants from many diverse taxa, including plants (Rogakou, et al. 1999; Friesner, et al. 2005). Therefore, it was not surprising in this study that the H2AvD antibody (based on the Drosophila sequence) yielded a single intense band on Western blots (following IR) corresponding to phosphorylated H2AvB in the B. tryoni samples.

 

 

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4.3 Kinetics and persistence of γH2AvB signal

Many studies have analyzed the kinetics of phosphorylation and dephosphorylation of H2AX, with IR shown to induce maximal amounts of γH2AX in cells at times often less than 10 min after exposure to IR followed by a decline in γH2AX signal over a period of hours (Rogakou, et al. 1998; Madigan, et al. 2002; Olive, Banath. 2004; Roch-Lefevre, et al. 2010). Previous reports using Drosophila S2 tissue culture cells have suggested that the phosphorylation of H2Av increases within minutes following IR exposure, but then declines significantly after several hours (Madigan, et al. 2002). The rapid loss of the phosphorylated H2Av was likely due to regulated dephosphorylation of H2Av and was similar to that reported for radiation-induced phosphorylation/dephosphorylation kinetics in mammals (Rogakou, et al. 1998). Indeed, γH2AX quantification assays have been proposed as the basis of protocols for biological dosimetry following IR events (Roch-Lefevre, et al. 2010). Although the absolute number of phosphorylated γH2AX molecules declines over a period of hours and days post-IR, a recent study in mice showed a dose-dependent response of γH2AX foci in nuclei up to 7 days after exposure to IR (Bhogal, et al. 2010). The residual γH2AX foci at 24-72 h post-IR are believed to represent misrepaired DNA DSBs, unrepaired DNA with ongoing genomic instability, S-phase cells or apoptotic cells (Liu, et al. 2008). In Drosophila S2 cultured cells, the percentage of phosphorylated H2AX variant (H2Av) was shown to have reduced almost to non-irradiated levels within 3 h after the initial IR dose (Madigan, et al. 2002). Similarly, in cultured human microvascular endothelial cells exposed to 2–16 Gy IR, a transient increase in γH2AX signal was observed to peak at 1 h post IR and return to background levels 24 h post IR (Kataoka, et al. 2006). In this study, the γH2AvB response observed in whole tissue displayed kinetics that were less transient than that of cultured cells and persisted at measurable levels for at least 17 days, although the signal was considerably reduced even 1-5 days post IR. It should be noted that doses used in human studies are generally much less than applied here, as the doses used for SIT and disinfestation of insects are well beyond what can be tolerated by humans. Thus, the persistence of the phosphorylated protein may be related to the higher IR-doses were tested. The basis for the relatively high IR-tolerance of insects is not clear, however, it is conceivable that it may be partly related to the persistence of the phosphorylated histone. A recent study that used Göttingen minipig skin biopsies found that radiation induced γH2AX foci (50 Gy) were observed in approximately 60% of cells 4 h after IR. The number of γH2AX foci was found to be significantly less after 70 days following IR exposure; however, there remained a significantly higher number of γH2AX foci per epidermal keratinocyte compared with controls (Ahmed, et al. 2012). In our study there was a strong positive linear correlation (r2>0.9) in γH2AX signal over a dose range of 0–400 Gy, corresponding to a 20-fold increase in signal above the background (non-irradiated) level. It is therefore likely that high IR doses are necessary to observe the long–term persistent γH2AX or γH2AvB signals. Indeed, after 17 days post IR (240 Gy) we found that approximately 25% of nuclei had a measurable γH2AvB signal as determined by LSC. Although LSC detected a small amount of measurable background signal in 0 Gy (control) QFly adults in approximately 7% of nuclei, no 0 Gy γH2AvB signal was observed by Western blotting (Figure 13C). Therefore, it appeared LSC may prove to be a more sensitive method to detect and quantify γH2AvB signal in nuclei that are persistent many days after exposure to the IR event. Bonner et al. (Rogakou, et al. 1999) previously suggested there is potentially a low level of γH2AX in non-irradiated cells. Our study is in agreement with Bonnet et al (see discussion below) that additionally confirmed the necessity for the phosphorylation of putative Ser137 within the SQ motif of γH2AvB to allow detection by our primary antibody, through abolishing the signal via treatment of the histone extract with alkaline phosphatase.

At five days post IR exposure, an IR-induced γH2AvB signal in whole pupal lysates was observed via Western blotting (depending on amount of protein loaded on gels and imaging exposure times). Therefore, the nucleosome (histone) extraction procedure was used and this resulted in a substantial

 

 

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enrichment of the γH2AvB signal compared with the use of the whole pupal lysates. In the non-irradiated whole pupal lysate we did not detect any γH2AvB. However, in the non-irradiated histone fraction, we observed a basal γH2AvB signal in the non-irradiated 5 day samples. However, at 17 days (QFly adults) we did not observe a γH2AvB signal in the 0 Gy samples, possibly indicating that “basal” level of γH2AvB is life-stage specific and is dependent on the level of cellular differentiation.

4.4 Fluorescence microscopy and laser scanning cytometry

Visual scoring of γH2AvB signals (foci) in individual nuclei was possible; however this procedure was was laborious and subject to “interpretation” and therefore remains subjective. If a simple scoring procedure was applied as done in this study, i.e. count the number of nuclei that contain >1 γH2AvB foci or groups of foci, then the visual scoring protocol may be useful for higher throughput determination of previous IR exposure. Indeed, for 1st instar larvae 2 days post IR we were able to demonstrate there were zero nuclei scored that contained >1 γH2AvB foci per nucleus in the 0 Gy control samples as compared with 90 Gy samples, of which 17% of nuclei had >1 γH2AvB foci per nucleus. For longer times post IR exposure we developed an “automated” scoring procedure was developed using LSC for successful automated quantitation of IR-induced γH2AvB signal in adult QFly nuclei 17 days post-IR as pupae. Our LSC results support data obtained by Western blot analyses and also provide a visualisation of the signal. The iCyte® software allows for automated scoring and quantitation of nuclei and events within them, and therefore LSC could be useful for future studies to investigate additional parameters associated with IR induction of γH2AvB (e.g. γH2AvB signal related to cell cycle phases) at a tissue-specific level. Additionally, LSC could be used to simultaneously detect γH2AvB signal with a dependant DNA repair mechanism protein such as ATM or other markers such as caspases (for apoptosis), to yield more information on cell-cycle dynamics.

4.5 Development of an ELISA for γH2AvB

Since Enzyme-Linked Immunosorbent Assays (ELISA) method utilises the specificity of antibodies coupled to an easily-assayed enzyme, it was envisioned that developing an ELISA assay may prove inexpensive and rapid. In the latter part of this project both indirect and sandwich ELISA assays were developed for measurement of IR-induced γH2AvB with the antibodies that were used successfully for Western blotting. Interestingly, it was not possible to routinely detect γH2AvB in total lysates (e.g. from 24 h post IR pupae) by the ELISA method, yet in Western blots the γH2AvB signal was always observed even at low doses such as 10 Gy. It was therefore speculated that total lysates contain the γH2AvB in a non-denatured form which is likely to be inaccessible to the antibody and thus prevents the antibody binding to the γH2AvB proteins in the nucleosome/histone complex. However in Western blot procedures the histone complex becomes dissociated into its constituent monomer histone proteins due to the denaturing conditions used and the γH2AvB proteins are then able to freely bind the γH2AvB antibody. This concept is further supported by the observation that nuclear extracts containing (denatured) histone proteins were easily detectable in ELISA assays using IR exposed samples. In these samples it is likely that the processing procedures, which included acid denaturation steps, induced dissociation of the histone complexes into their constituent monomers which were then available to freely bind with the ELISA antibodies. From the preliminary work done with ELISA assays it appeared there is potential to optimise the ELISA conditions in order for a higher throughput assay to be developed to measure whether QFly samples had indeed experienced previous IR exposure. Western blotting may be

 

 

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prove to be a more sensitive method but the dynamic range for Western blots may be a limiting factor for quantifying the γH2AvB response accurately.

4.6 Detection of γH2AvB homolog in Mediterranean fruit fly (Medfly)

Because we knew that antibody made against Drosophila melanogaster was capable of detecting γH2AvB of QFly and that this histone protein is highly conserved amongst flies, we decided to undertake a preliminary experiment to assess whether the same protein could be detected in Medfly, using the same method. As expected, this was able to be achieved (Figure 24A). The signal from protein extracts from whole irradiated Medfly pupae was dose dependent and increased with increasing irradiation dosage. Unfortunately, we did not have access to non-irradiated samples for comparison against non-irradiated controls, but nonetheless, the protein was detectable and there was a 2.7 fold increase in H2AvB homolog signal when Medfly were irradiated at 160 Gy compared to those receiving 40 Gy (Figure 24B). This result may have applicability for SIT programs utilising Medly, such as those conducted in South Australia.

This result, although preliminary demonstrates the potential applicability of the biomarker to other pest flies. Given the high level of conservation of the biomarker across a range of organisms (including mammals, plants and invertebrates) it is likely that the biomarker could be adapted to assess irradiation damage of all insect pests. Additionally, it may be possible to use the biomarker for the produce itself although it is not known how it would respond to irradiation in plant parts that have been removed from the parent plant.

4.7 Layperson’s Summary

We have been successful in identifying a protein in QFly (called H2AvB), which is altered (to produce H2AvB) due to breaks in the flies’ DNA caused by irradiation. The alteration of this protein allows it to become active and bind to the broken DNA as part of the repair process. We showed that the higher the dose of irradiation, the higher the amount of H2AvB that is produced in the flies; thus H2AvB is a biomarker of irradiation exposure in QFly. This was tested over irradiation doses of 0 Gy (not irradiated) to 400 Gy, which covers the standard irradiation doses used for post-harvest disinfestation (150 Gy) and doses used to produce sterile flies for Sterile Insect Technique eradication programs (~70Gy). Furthermore, the biomarker was detectable for a significant period after irradiation treatment (up to 17 days).

We were able to detect the H2AvB using standard methods and have developed an ELISA test for its detection; this is routine technology for detection of many specific proteins in biological samples (e.g. commercial virus testing). To our knowledge this is the only test capable of retrospectively determining if an organism has been previously irradiated. A key advantage of our approach is that the biomarker is present in all insects and so the test should be adaptable to most pests of growing and stored produce. Our findings provide a key technological capability required to facilitate broad uptake of irradiation for commercial post-harvest disinfestation applications and to confidently assess imported produce or to confirm treatment of exported produce, should live pests be found. This addresses the potential market failure associated with loss of key disinfestation chemicals fenthion and dimethoate, which is the primary reason the project was undertaken. The next steps involve incorporation of the test into commercial and quarantine facilities, and to broaden the range of pests it can detect.

 

 

 

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Technology Transfer

This was a proof-of-concept largely focused on molecular science, including searching for a biomarker of irradiation exposure in Queensland fruit fly, and validating this biomarker under controlled conditions. It was envisioned that if successful, further R&D would be required to provide commercial adoption of the technology. Thus, findings have been disseminated largely to the scientific community as per the list below. It should be noted that the biomarker data have been peer reviewed (see Siddiqui 2013); publication in Mutagenesis is important in demonstrating that the work is grounded in science and that industry is undertaking best practice. There has been a range of informal discussions with HAL and relevant industry (e.g. Steritech) and government representatives (from Australia and New Zealand), in terms of adopting the technology. These discussions have led to submission of an Expression of Interest for further funding with that aim. Additionally, CSIRO has established a Biosecurity Flagship, which would support further research in this area. Mohammad Sabbir Siddiqui, Erika Filomeni, Maxime François, Samuel R. Collins, Tamara

Cooper, Richard V. Glatz, Phillip W. Taylor, Michael Fenech and Wayne R. Leifert (2013). Exposure of insect cells to ionising radiation in vivo induces persistent phosphorylation of a H2AX homolog (H2AvB). Mutagenesis (in press).

Siddiqui, S., Filomeni, E., Francois, M., Collins, S.R., Glatz, R.V., Taylor, P.W., Fenech, M. & Leifert, W.R. (2012). H2AX is a biomarker of ionizing radiation induced DNA damage in the fruit fly, Bactrocera tryoni. ComBio 2012, Adelaide, 23-27 September.

Glatz, R.V. (2009). SARDI Entomology Biosecurity Collaborations. Presented to Federal Plant Health Committee meeting, Adelaide, November 12th.

 

 

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Recommendations

Our work has identified γH2AvB as a potential biomarker and biodosimeter indicative of previous irradiation exposure in Queensland fruit fly and potentially many other insects. We have incorporated this biomarker into methods such as ELISA and Western blotting, each of which could be integrated into commercial disinfestation and quarantine facilities. There are four broad areas that should be addressed through future funding.

1. Improving understanding of the biomarker’s response. Future studies that focus on γH2AvB as a potential biomarker of IR-induced DNA damage should extend the time course following irradiation exposure and use tissue section immunohistochemistry or immunofluorescence techniques that will allow identification of tissue-specificity of γH2AvB signals in QFly. Furthermore understanding the kinetics of γH2AvB phosphorylation/dephosphorylation in different life stages of QFly would also be of benefit. As the biomarker is associated with DNA-damage, its response to other environmental challenges (such as toxins) also needs to be assessed.

2. Broadening the pests targeted by the irradiation test. While QFly was used as a model to identify a candidate biomarker, the biomarker occurs in most organisms (all insects) where it is involved in similar DNA-repair mechanisms. Therefore, the opportunity exists to adapt the test to a suite of insects of market access and biosecurity concern. This would increase the disinfestation options for a broad cross section of horticultural producers. It is also possible that the biomarker may be useful for testing the produce itself.

3. Incorporation of the test into commercial irradiation facilities and/or quarantine facilities. The availability of the irradiation test means that the test can be used to assess the marker under commercial irradiation procedures and use it in a commercial context to gather baseline treatment data. Applications could include providing a stronger certification “brand” and/or confirming exported produce was treated if pests are found after export. Additionally, incorporation into quarantine facilities would allow Australia to confidently assess prior irradiation of imported produce (currently we rely only on certification from the place of origin).

4. Promotion of the availability and benefits of irradiation to industry and the public. While the regulatory and technological framework exists for broad commercial uptake of irradiation for produce disinfestation, many producers may be unaware of its applicability/advantages for their particular product. Similarly, the public needs to be informed of the broad use of irradiation as a sterilization technique, its advantages for food disinfestation, and its high level of safety in terms of food quality, particularly in comparison to the chemical options being withdrawn due to health concerns.

 

 

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Acknowledgements

We thank various others for their valued input into this project. Q-flies were generously provided from the stock used by New South Wales Department of Primary Industries for Sterile Insect Technique releases. Dr Tamara Cooper’s expertise in preparing QFly mRNA as well as transcriptomic data analysis is gratefully acknowledged. Dr Erika Filomeni contributed to this project as part of a research training program from the University of Pisa, Faculty of Mathematics, Physics and Natural Sciences. We thank Dr Maxime François for his expertise in laser scanning cytometry. We thank Mrs Jannatul Tuli for helping to develop the ELISA assay. The authors thank the staff at The Adelaide Proteomics Centre (School of Molecular & Biomedical Science, University of Adelaide) for discussions and analysis of QFly protein samples.

 

 

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References

Abramoff, M.D., Magalhaes, P.J., Ram, S.J., 2004. Image Processing with ImageJ. Biophotonics International 11 (7), 36-42.

Ahmed, E.A., Agay, D., Schrock, G., Drouet, M., Meineke, V., Scherthan, H., 2012. Persistent DNA Damage After High Dose in Vivo Gamma Exposure of Minipig Skin. PLoS One 7 (6), e39521.

Anderson, L., Henderson, C., Adachi, Y., 2001. Phosphorylation and Rapid Relocalization of 53BP1 to Nuclear Foci upon DNA Damage. Mol. Cell. Biol. 21 (5), 1719-1729.

Ant, T., Koukidou, M., Rempoulakis, P., Gong, H.F., Economopoulos, A., Vontas, J., Alphey, L., 2012. Control of the Olive Fruit Fly using Genetics-Enhanced Sterile Insect Technique. BMC Biol. 10 51-7007-10-51.

Bhogal, N., Kaspler, P., Jalali, F., Hyrien, O., Chen, R., Hill, R.P., Bristow, R.G., 2010. Late Residual Gamma-H2AX Foci in Murine Skin are Dose Responsive and Predict Radiosensitivity in Vivo. Radiat. Res. 173 (1), 1-9.

Burma, S., Chen, B.P., Murphy, M., Kurimasa, A., Chen, D.J., 2001. ATM Phosphorylates Histone H2AX in Response to DNA Double-Strand Breaks. J. Biol. Chem. 276 (45), 42462-42467.

Chehab, N.H., Malikzay, A., Appel, M., Halazonetis, T.D., 2000. Chk2/hCds1 Functions as a DNA Damage Checkpoint in G(1) by Stabilizing p53. Genes Dev. 14 (3), 278-288.

Christmann, M., Tomicic, M.T., Roos, W.P., Kaina, B., 2003. Mechanisms of Human DNA Repair: An Update. Toxicology 193 (1-2), 3-34.

Collins, S.R., Weldon, C.W., Banos, C., Taylor, P.W., 2009. Optimizing Irradiation Dose for Sterility Induction and Quality of Bactrocera Tryoni. J. Econ. Entomol. 102 (5), 1791-1800.

Deem, A.K., Li, X., Tyler, J.K., 2012. Epigenetic Regulation of Genomic Integrity. Chromosoma 121 (2), 131-151.

Downs, J.A., Lowndes, N.F., Jackson, S.P., 2000. A Role for Saccharomyces Cerevisiae Histone H2A in DNA Repair. Nature 408 (6815), 1001-1004.

Fernandez-Capetillo, O., Lee, A., Nussenzweig, M., Nussenzweig, A., 2004. H2AX: The Histone Guardian of the Genome. DNA Repair (Amst) 3 (8-9), 959-967.

Follett, P.A., Phillips, T.W., Armstrong, J.W., Moy, J.H., 2011. Generic Phytosanitary Radiation Treatment for Tephritid Fruit Flies Provides Quarantine Security for Bactrocera Latifrons (Diptera: Tephritidae). J. Econ. Entomol. 104 (5), 1509-1513.

Follett, P.A., Armstrong, J.W., 2004. Revised Irradiation Doses to Control Melon Fly, Mediterranean Fruit Fly, and Oriental Fruit Fly (Diptera: Tephritidae) and a Generic Dose for Tephritid Fruit Flies. J. Econ. Entomol. 97 (4), 1254-1262.

Friesner, J.D., Liu, B., Culligan, K., Britt, A.B., 2005. Ionizing Radiation-Dependent Gamma-H2AX Focus Formation Requires Ataxia Telangiectasia Mutated and Ataxia Telangiectasia Mutated and Rad3-Related. Mol. Biol. Cell 16 (5), 2566-2576.

Furuta, T., Takemura, H., Liao, Z.Y., Aune, G.J., Redon, C., Sedelnikova, O.A., Pilch, D.R., Rogakou, E.P., Celeste, A., Chen, H.T., Nussenzweig, A., Aladjem, M.I., Bonner, W.M., Pommier, Y., 2003. Phosphorylation of Histone H2AX and Activation of Mre11, Rad50, and Nbs1 in Response to Replication-Dependent DNA Double-Strand Breaks Induced by Mammalian DNA Topoisomerase I Cleavage Complexes. J. Biol. Chem. 278 (22), 20303-20312.

Goll, M.G., Bestor, T.H., 2002. Histone Modification and Replacement in Chromatin Activation. Genes Dev. 16 (14), 1739-1742.

Gotz, S., Garcia-Gomez, J.M., Terol, J., Williams, T.D., Nagaraj, S.H., Nueda, M.J., Robles, M., Talon, M., Dopazo, J., Conesa, A., 2008. High-Throughput Functional Annotation and Data Mining with the Blast2GO Suite. Nucleic Acids Res. 36 (10), 3420-3435.

 

 

51

51

Huang, X., Halicka, H.D., Darzynkiewicz, Z., 2004. Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage (DNA Double-Strand Breaks). Curr. Protoc. Cytom Chapter 7 Unit 7.27.

Huyen, Y., Zgheib, O., Ditullio, R.A.,Jr, Gorgoulis, V.G., Zacharatos, P., Petty, T.J., Sheston, E.A., Mellert, H.S., Stavridi, E.S., Halazonetis, T.D., 2004. Methylated Lysine 79 of Histone H3 Targets 53BP1 to DNA Double-Strand Breaks. Nature 432 (7015), 406-411.

IAEA-TECDOC-1427, 2004. Irradiation as a Phytosanitary Treatment of Food and Agricultural Commodities. Proceedings of a Final Research Coordination Meeting Organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture 2002. International Atomic Energy Agency (IAEA) 181pp.

Kataoka, Y., Bindokas, V.P., Duggan, R.C., Murley, J.S., Grdina, D.J., 2006. Flow Cytometric Analysis of Phosphorylated Histone H2AX Following Exposure to Ionizing Radiation in Human Microvascular Endothelial Cells. J. Radiat. Res. 47 (3-4), 245-257.

Kinner, A., Wu, W., Staudt, C., Iliakis, G., 2008. Gamma-H2AX in Recognition and Signaling of DNA Double-Strand Breaks in the Context of Chromatin. Nucleic Acids Res. 36 (17), 5678-5694.

Kumano, N., Haraguchi, D., Kohama, T., 2008. Effect of Irradiation on Mating Ability in the Male Sweetpotato Weevil (Coleoptera: Curculionidae). J. Econ. Entomol. 101 (4), 1198-1203.

Lane, D.P., 1992. Cancer. p53, Guardian of the Genome. Nature 358 (6381), 15-16. Lang, J., Smetana, O., Sanchez-Calderon, L., Lincker, F., Genestier, J., Schmit, A.C., Houlne, G.,

Chaboute, M.E., 2012. Plant gammaH2AX Foci are Required for Proper DNA DSB Repair Responses and Colocalize with E2F Factors. New Phytol. 194 (2), 353-363.

Liu, S.K., Olive, P.L., Bristow, R.G., 2008. Biomarkers for DNA DSB Inhibitors and Radiotherapy Clinical Trials. Cancer Metastasis Rev. 27 (3), 445-458.

Madigan, J.P., Chotkowski, H.L., Glaser, R.L., 2002. DNA Double-Strand Break-Induced Phosphorylation of Drosophila Histone Variant H2Av Helps Prevent Radiation-Induced Apoptosis. Nucleic Acids Res. 30 (17), 3698-3705.

Mastrangelo, T., Chaudhury, M.F., Skoda, S.R., Welch, J.B., Sagel, A., Walder, J.M., 2012. Feasibility of using a Caribbean Screwworm for SIT Campaigns in Brazil. J. Med. Entomol. 49 (6), 1495-1501.

Matsuoka, S., Rotman, G., Ogawa, A., Shiloh, Y., Tamai, K., Elledge, S.J., 2000. Ataxia Telangiectasia-Mutated Phosphorylates Chk2 in Vivo and in Vitro. Proc. Natl. Acad. Sci. U. S. A. 97 (19), 10389-10394.

Matsuoka, S., Huang, M., Elledge, S.J., 1998. Linkage of ATM to Cell Cycle Regulation by the Chk2 Protein Kinase. Science 282 (5395), 1893-1897.

Mendez-Acuna, L., Di Tomaso, M.V., Palitti, F., Martinez-Lopez, W., 2010. Histone Post-Translational Modifications in DNA Damage Response. Cytogenet. Genome Res. 128 (1-3), 28-36.

Mutika, G.N., Kabore, I., Seck, M.T., Sall, B., Bouyer, J., Parker, A.G., Vreysen, M.J.B., 2013. Mating Performance of Glossina Palpalis Gambiensis Strains from Burkina Faso, Mali, and Senegal. Entomol. Exp. Appl. 146 (1), 177-185.

Oliva, C.F., Jacquet, M., Gilles, J., Lemperiere, G., Maquart, P.O., Quilici, S., Schooneman, F., Vreysen, M.J., Boyer, S., 2012. The Sterile Insect Technique for Controlling Populations of Aedes Albopictus (Diptera: Culicidae) on Reunion Island: Mating Vigour of Sterilized Males. PLoS One 7 (11), e49414.

Olive, P.L., Banath, J.P., 2004. Phosphorylation of Histone H2AX as a Measure of Radiosensitivity. Int. J. Radiat. Oncol. Biol. Phys. 58 (2), 331-335.

Pandita, T.K., Richardson, C., 2009. Chromatin Remodeling Finds its Place in the DNA Double-Strand Break Response. Nucleic Acids Res. 37 (5), 1363-1377.

Park, E.J., Chan, D.W., Park, J.H., Oettinger, M.A., Kwon, J., 2003. DNA-PK is Activated by Nucleosomes and Phosphorylates H2AX within the Nucleosomes in an Acetylation-Dependent Manner. Nucleic Acids Res. 31 (23), 6819-6827.

 

 

52

52

Paull, T.T., Rogakou, E.P., Yamazaki, V., Kirchgessner, C.U., Gellert, M., Bonner, W.M., 2000. A Critical Role for Histone H2AX in Recruitment of Repair Factors to Nuclear Foci After DNA Damage. Curr. Biol. 10 (15), 886-895.

Prakash, S., Sung, P., Prakash, L., 1993. DNA Repair Genes and Proteins of Saccharomyces Cerevisiae. Annu. Rev. Genet. 27 33-70.

Redon, C., Pilch, D., Rogakou, E., Sedelnikova, O., Newrock, K., Bonner, W., 2002. Histone H2A Variants H2AX and H2AZ. Curr. Opin. Genet. Dev. 12 (2), 162-169.

Riches, L.C., Lynch, A.M., Gooderham, N.J., 2008. Early Events in the Mammalian Response to DNA Double-Strand Breaks. Mutagenesis 23 (5), 331-339.

Roch-Lefevre, S., Mandina, T., Voisin, P., Gaetan, G., Mesa, J.E., Valente, M., Bonnesoeur, P., Garcia, O., Voisin, P., Roy, L., 2010. Quantification of Gamma-H2AX Foci in Human Lymphocytes: A Method for Biological Dosimetry After Ionizing Radiation Exposure. Radiat. Res. 174 (2), 185-194.

Rogakou, E.P., Boon, C., Redon, C., Bonner, W.M., 1999. Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo. J. Cell Biol. 146 (5), 905-916.

Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S., Bonner, W.M., 1998. DNA Double-Stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139. J. Biol. Chem. 273 (10), 5858-5868.

Rouse, J., Jackson, S.P., 2002. Interfaces between the Detection, Signaling, and Repair of DNA Damage. Science 297 (5581), 547-551.

Sancar, A., Lindsey-Boltz, L.A., Unsal-Kacmaz, K., Linn, S., 2004. Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints. Annu. Rev. Biochem. 73 39-85.

Savic, V., Yin, B., Maas, N.L., Bredemeyer, A.L., Carpenter, A.C., Helmink, B.A., Yang-Iott, K.S., Sleckman, B.P., Bassing, C.H., 2009. Formation of Dynamic Gamma-H2AX Domains Along Broken DNA Strands is Distinctly Regulated by ATM and MDC1 and Dependent upon H2AX Densities in Chromatin. Mol. Cell 34 (3), 298-310.

Sedelnikova, O.A., Rogakou, E.P., Panyutin, I.G., Bonner, W.M., 2002. Quantitative Detection of (125)IdU-Induced DNA Double-Strand Breaks with Gamma-H2AX Antibody. Radiat. Res. 158 (4), 486-492.

Shechter, D., Dormann, H.L., Allis, C.D., Hake, S.B., 2007. Extraction, Purification and Analysis of Histones. Nat. Protoc. 2 (6), 1445-1457.

Song, Y.H., 2005. Drosophila Melanogaster: A Model for the Study of DNA Damage Checkpoint Response. Mol. Cells 19 (2), 167-179.

Soopaya, R., Stringer, L.D., Woods, B., Stephens, A.E., Butler, R.C., Lacey, I., Kaur, A., Suckling, D.M., 2011. Radiation Biology and Inherited Sterility of Light Brown Apple Moth (Lepidoptera: Tortricidae): Developing a Sterile Insect Release Program. J. Econ. Entomol. 104 (6), 1999-2008.

Steller, H., 2008. Regulation of Apoptosis in Drosophila. Cell Death Differ. 15 (7), 1132-1138. Stiff, T., O'Driscoll, M., Rief, N., Iwabuchi, K., Lobrich, M., Jeggo, P.A., 2004. ATM and DNA-PK

Function Redundantly to Phosphorylate H2AX After Exposure to Ionizing Radiation. Cancer Res. 64 (7), 2390-2396.

Sun, N.K., Sun, C.L., Lin, C.H., Pai, L.M., Chao, C.C., 2010. Damaged DNA-Binding Protein 2 (DDB2) Protects Against UV Irradiation in Human Cells and Drosophila. J. Biomed. Sci. 17 27.

van Gent, D.C., van der Burg, M., 2007. Non-Homologous End-Joining, a Sticky Affair. Oncogene 26 (56), 7731-7740.

Zhao, H., Albino, A.P., Jorgensen, E., Traganos, F., Darzynkiewicz, Z., 2009a. DNA Damage Response Induced by Tobacco Smoke in Normal Human Bronchial Epithelial and A549 Pulmonary Adenocarcinoma Cells Assessed by Laser Scanning Cytometry. Cytometry A. 75 (10), 840-847.

 

 

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Zhao, H., Traganos, F., Darzynkiewicz, Z., 2009b. Kinetics of the UV-Induced DNA Damage Response in Relation to Cell Cycle Phase. Correlation with DNA Replication. Cytometry A. 77 (3), 285-293.