Mahmoud Alhosin

Abdurazzag Abusnina

Mayada Achour

Tanveer Sharif

Christian Muller

Jean Peluso

Thierry Chataigneau

Claire Lugnier

Valérie B Schini-Kerth

Christian Bronner

Guy Fuhrmann
email: guy.fuhrmann@unistra.fr

Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator

Keywords: Apoptosis, caspase, thymoquinone, tumor suppressor protein p73, UHRF1

Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A c c e p t e d M a n u s c r i p t

Introduction

The p73 gene produces a protein homolog to p53 with similar functions [START_REF] Kaghad | Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers[END_REF]. p73 regulates the transcription of several p53 target genes, including the apoptosisregulating gene PUMA, a Bax transactivator, and the cell cycle regulatory genes p21 Waf1/Cip1 [START_REF] Levrero | The p53/p63/p73 family of transcription factors: overlapping and distinct functions[END_REF] and p16 INK4A [START_REF] Boominathan | Some facts and thoughts: p73 as a tumor suppressor gene in the network of tumor suppressors[END_REF]. Transcriptional activation of these genes leads to the induction of cell-cycle arrest and/or apoptosis [START_REF] Levrero | The p53/p63/p73 family of transcription factors: overlapping and distinct functions[END_REF]. It is therefore expected that many p53-responsive genes could also be targets of p73, especially those genes which respond to DNA damage and could initiate cell cycle arrest and/or apoptosis. This could explain that cells lacking functional p53 have the ability to undergo apoptosis through a p53independent pathway when p73 is expressed [START_REF] Jost | p73 is a human p53-related protein that can induce apoptosis[END_REF]. Apart its p53-mimetic activity, p73 is also a major component of specific signaling cascades, like the caspase-independent cell death (CICD) [START_REF] Kitagawa | Caspase-independent mitotic death (CIMD)[END_REF].

Under physiological conditions, the basal expression of the p73 gene is kept extremely low and is only up-regulated in response to cellular stress [START_REF] Kaghad | Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers[END_REF]. This explains that, in spite of extensive searches, mutations of the p73 gene are rarely detected in primary tumors [START_REF] Ikawa | p53 family genes: structural comparison, expression and mutation[END_REF], but aberrant hypermethylation of the p73 promoter region and subsequent inactivation of the p73 gene have been reported in acute lymphoblastic leukemia (ALL) [START_REF] Corn | Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5' CpG island methylation[END_REF]. The consequences of this hypermethylation and its maintenance are not clearly understood but they likely target the expression of specific genes involved in the DNA damage response. We hypothesized that UHRF1 (Ubiquitin-like, containing PHD and RING Finger domains, 1) could be a major effector of the p73 deregulation, since this nuclear protein is known to be over-expressed in numerous cancer cell lines and tissues [8][9][START_REF] Bronner | The UHRF family: oncogenes that are drugable targets for cancer therapy in the near future?[END_REF][START_REF] Unoki | UHRF1 is a novel molecular marker for diagnosis and the prognosis of bladder cancer[END_REF].

A c c e p t e d M a n u s c r i p t 4 Several studies have shown that UHRF1 participates in the control of cell proliferation and cell cycle transition from G1 to S, by regulating the expression of several genes, including RB1 and p16 INK4A [START_REF] Jeanblanc | The retinoblastoma gene and its product are targeted by ICBP90: a key mechanism in the G1/S transition during the cell cycle[END_REF][START_REF] Achour | The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression[END_REF]. This suggests that pathological overexpression of UHRF1, by repressing permanently the expression of specific tumor suppressor genes, could induce disorders in the G1/S progression and consequently promote tumor development [START_REF] Bronner | The UHRF family: oncogenes that are drugable targets for cancer therapy in the near future?[END_REF]. In agreement with our hypothesis, it has been shown that the activation of different cell cycle checkpoints during DNA damage-induced apoptosis leads to a down-regulation of UHRF1 [9]; such deregulation has been described to be dependent on the p53/p21 WAF1/CIP1 pathway [START_REF] Arima | Downregulation of nuclear protein ICBP90 by p53/p21Cip1Waf1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition[END_REF]. It should be noted that reduction of UHRF1 expression solely can suppress proliferation and induce apoptosis of cancer cells whose p53 is inactivated [START_REF] Abbady | ICBP90 expression is downregulated in apoptosis-induced Jurkat cells[END_REF]. This suggests that UHRF1 functions as a component in the DNA damage response pathways and that it plays a role in the maintenance of genomic stability. In this point of view, accumulating evidences have shown that UHRF1 acts as a multi-modular protein involved in the maintenance of the chromatin status and its propagation during cell division. Indeed, UHRF1 binds to methylated DNA and recruits DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) through the SRA (SET and RING finger Associated) domain [START_REF] Achour | The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression[END_REF][START_REF] Unoki | ICBP90, an E2F-1 target, recruits HDAC1 and binds to methyl-CpG through its SRA domain[END_REF][START_REF] Citterio | Np95 is a histone-binding protein endowed with ubiquitin ligase activity[END_REF][START_REF] Bostick | UHRF1 plays a role in maintaining DNA methylation in mammalian cells[END_REF][START_REF] Sharif | The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA[END_REF]. These protein-protein interactions precede S phase entry and could be required for cell cycle progression [START_REF] Bronner | The UHRF family: oncogenes that are drugable targets for cancer therapy in the near future?[END_REF][START_REF] Milutinovic | Epigenomic stress response. Knockdown of DNA methyltransferase 1 triggers an intra-S-phase arrest of DNA replication and induction of stress response genes[END_REF]. UHRF1 therefore, ensures the crosstalk between DNA methylation and histone modifications, promoting the maintenance of the epigenetic code and its transmission from a mother cell to the descent cells [START_REF] Unoki | Drug discovery targeting epigenetic codes: the great potential of UHRF1, which links DNA methylation and histone modifications, as a drug target in cancers and toxoplasmosis[END_REF]. For these reasons it is suspected that a down-regulation of UHRF1 in response to DNA damage has dramatic consequences on the cell viability.

A c c e p t e d M a n u s c r i p t 5 A number of studies have shown that thymoquinone (TQ) is a potent cytotoxic and genotoxic drug over a broad range of human cancer cells [START_REF] Gali-Muhtasib | Thymoquinone: a promising anti-cancer drug from natural sources[END_REF]. It has been suggested that TQ, as a DNA damaging agent, is a potential reactive oxygen species (ROS) producer which exerts its anti-cancer effects by inhibiting cell growth, arresting cell cycle progression and inducing subsequently apoptosis [START_REF] Worthen | The in vitro anti-tumor activity of some crude and purified components of blackseed, Nigella sativa L[END_REF][START_REF] Shoieb | In vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone[END_REF][START_REF] Gali-Muhtasib | Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes[END_REF][START_REF] Gali-Muhtasib | Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism[END_REF][START_REF] El-Mahdy | Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells[END_REF][START_REF] Roepke | Lack of p53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells[END_REF][START_REF] Gali-Muhtasib | Thymoquinone triggers inactivation of the stress response pathway sensor CHEK1 and contributes to apoptosis in colorectal cancer cells[END_REF]. p53-dependent [START_REF] Gali-Muhtasib | Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism[END_REF] and p53-independent pathways [START_REF] Worthen | The in vitro anti-tumor activity of some crude and purified components of blackseed, Nigella sativa L[END_REF][START_REF] El-Mahdy | Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells[END_REF] have been evidenced to explain the cellular actions of TQ. In HCT-116 colorectal cancer cells, TQ-induced apoptosis involves an upregulation of both p53 and p21 WAF1/CIP1 expressions, concomitantly with a downregulation of the expression of the anti-apoptotic protein Bcl-2 [START_REF] Gali-Muhtasib | Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism[END_REF]. In p53-null myeloblastic leukemia HL-60 cells [START_REF] El-Mahdy | Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells[END_REF] and in p53-null osteosarcoma MG63 cells [START_REF] Roepke | Lack of p53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells[END_REF], the anti-cancer activities of TQ involve alterations of the Bax/Bcl2 ratio and caspase activations, but the precise mechanisms remain unknown.

The aim of the present study is to determine in the p53-deficient Jurkat cell line [START_REF] Cheng | Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines[END_REF] whether an activation of the p73 gene, via a TQ-induced DNA damage, could target the anti-apoptotic UHRF1 gene with subsequent cell cycle arrest and apoptosis.

Our results show that TQ produces intracellular ROS, promotes a DNA damage-related cell cycle arrest and triggers apoptosis through the activation of a p73-dependent mitochondrial and cell cycle signaling pathway, followed by a down-regulation of UHRF1. We hypothesize that this p73-dependent down-regulation of UHRF1 prevents epigenetic code replication and thus hinders the "cancer signature" to be inherited by the daughter cancer cells.

Material and methods

A c c e p t e d

M a n u s c r i p t 6

Cell Culture, Treatment and Transfection

The human leukemic T-cell line Jurkat (clone E6-1) was cultured as previously described [START_REF] Achour | The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression[END_REF]. A 100 mM solution of TQ (Sigma-Aldrich, St. Louis, MO, USA) was prepared in 100% DMSO (DiMethylSulfOxide; Millipore S.A.S., Molsheim, France) and appropriate working concentrations were prepared with the cell culture medium; the final concentration of DMSO was of 0.1% in both control and treated conditions.

Transient transfections of p73 siRNA duplex (sc-36167; Santa Cruz Biotechnologies,

Santa

Cruz, CA, USA), UHRF1 siRNA duplex (5' -GGUCAAUGAGUACGUCGAUdTdT-3'; [START_REF] Achour | The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression[END_REF]) or scramble siRNA duplex (5'-GGACUCUCGGAUUGUAAGAdTdT-3'; [START_REF] Achour | The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression[END_REF]) were performed with lipofectamine 2000 (Invitrogen, Eugene, OR, USA), following the manufacturer's recommendations.

Transient transfections with pSG5 or pSG5-UHRF1 plasmid were performed as previously described [START_REF] Hopfner | ICBP90, a novel human CCAAT binding protein, involved in the regulation of topoisomerase II expression[END_REF]. Experiments using a specific inhibitor of caspase-3 (Z-DEVD-FMK) were carried out according to the manufacturer's instructions (Millipore S.A.S.).

Cell proliferation, viability and apoptosis assays

Cells were seeded on 6-multiwell plates at a density of 2x10 CellQuest software (BD Biosciences) was used for the analysis of the data. At least 10,000 events were recorded, iteratively increased if possible, and represented as dot plots.

Cell cycle phase distribution analysis and quantitation of hypodiploid sub-G0/G1 cell population

Cells were plated in 80 cm 2 culture flasks at a density of 1.5x10 5 cells/ml, grown for 24h and exposed to TQ at different concentrations for different times. Cells were then prepared as previously described [START_REF] Sharif | Selective proapoptotic activity of polyphenols from red wine on teratocarcinoma cell, a model of cancer stem-like cell[END_REF]. Cellular DNA content was assessed by flow cytometry in either a Guava EasyCyte Plus HP system (Guava Technologies, Hayward, CA, USA) or a BD FACSCalibur system (BD Biosciences).

Assessment of DNA fragmentation pattern

A c c e p t e d M a n u s c r i p t 8 Genomic DNA was prepared according to the manufacturer's instructions (Qiagen, Courtaboeuf, France), separated by electrophoresis on a 1% agarose gel and visualized under UV light with ethidium bromide.

Analysis of the production of ROS metabolites

Treated and untreated cells, seeded at an initial density of 2x10 6 cells/well in 6-well plates, were stained for 30min at room temperature with a 1mol/l dihydroethidium (DHE; Sigma Aldrich) solution and then subjected to flow cytometric analyses (BD FACSCalibur, BD Biosciences). 10,000 events were recorded per experiment.

Mitochondrial membrane potential measurement

Cells grown as described above, were incubated for 15 min at 37°C in PBS supplemented with 40 nM of [3,3' -Dihexyloxacarbocyanine iodide] -DiOC6; Sigma Aldrich-and 1 g/ml PI, followed by FACS analysis (BD FACSCalibur, Becton Dickinson). At least, 5,000 cells were analyzed for each sample.

Western blot analysis

Proteins from cell lysates were extracted, separated on 10-15% SDS-polyacrylamide gels and transferred to membranes as previously described [START_REF] Achour | The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression[END_REF][START_REF] Fuhrmann | Repression of Oct-4 during embryonic cell differentiation correlates with the appearance of TRIF, a transiently induced DNAbinding factor[END_REF]. Immunoblotting was performed by using either a mouse monoclonal anti-p73 antibody (BD Biosciences Pharmingen), a rabbit polyclonal anti-p16 antibody (Proteogenix, Oberhausbergen, according to the manufacturer's instructions. Membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Signals were visualized as previously described [START_REF] Achour | The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression[END_REF] and subjected to optical densitometric (OD) quantification by using NIH's Image J software.

Statistical analysis

Data were presented in a bar graph form, expressed as means ± S.E.M. from at least three independent experiments and statistically subjected to the one-way ANOVA test.

Significance levels were defined in accordance with the standard notation.

Results

TQ inhibits cell growth and induces cell cycle arrest of Jurkat cells

The Jurkat cell line was used to identify and characterize the molecular mechanisms induced by TQ. We first analyzed the effects of TQ on the growth parameters of this cell line. Cell proliferation (Fig. 1A) and cell viability (Fig. 1B) following TQ treatment, were decreased in a concentration-dependent manner. In our experimental Because cell growth is a result of the progression of the cells through the different phases of the cell cycle, we next determined the effects of TQ on the cell cycle distribution (Fig. 1C). Slight modifications were already detectable at 10 M of TQ and a significant accumulation of the cell population in G0/G1 phase was observed for higher concentrations. It appears therefore that TQ is able to inhibit the growth of Jurkat cells by promoting cell cycle arrest at the G0/G1 phase.

TQ induces apoptosis in Jurkat cells

We next investigated whether TQ could induce apoptosis in Jurkat cells. As shown in Fig. 2A, increasing concentrations of TQ are associated with increasing number of apoptotic cells. TQ began to trigger apoptosis at 10 μM while at 30 μM, a major proportion of cells was concerned. As expected, the calculated half-maximal effect of TQ on apoptosis was 24.7 + 0.3 μM (24h treatment). For 48h of treatment with TQ, this value was 23.2 + 0.3 μM (results not illustrated). Hence, these data were consistent with those obtained from cell proliferation assays. As a next step, cell cycle phase distribution analysis was focused on the detection of specific G0/G1 apoptotic cells; as shown in Fig. 2B, increasing concentrations of TQ led to increasing number of hypodiploid sub-G0/G1 cells. In agreement with these data, the intensity of the genomic 2C). Finally all these results demonstrate the occurrence of a p53independent DNA damage-related apoptosis in the p53-mutated Jurkat cells, when exposed to TQ.

TQ induces the generation of ROS and the breakdown of m in Jurkat cells

We suspected that TQ, like many quinone compounds (e.g. denbinobin), can trigger apoptosis by generating ROS [START_REF] Sánchez-Duffhues | Denbinobin inhibits nuclear factor-kappaB and induces apoptosis via reactive oxygen species generation in human leukemic cells[END_REF], that in turn should induce mitochondrial membrane disruption. We therefore determined by flow cytometry the levels of the DNA intercalating fluorescent marker ethidium which is produced after intracellular oxidation of DHE. As shown in Fig. 3A, cells exposed to increasing concentrations of TQ exhibited enhanced accumulation of intracellular ROS. When compared to untreated cells, a 77% increase of the fluorescence emitted by the ROS-induced oxidation of DHE was detected in cells treated with 100 M of TQ (Fig. 3A, Fig. 3B). In parallel, the effects of TQ on the mitochondrial membrane potential status were investigated by determining the uptake rate of DiOC6, a mitochondrial specific and voltage-dependent fluorescent dye. Fig. 4 shows that the number of cells, emitting high fluorescence levels, decreases when 20 M or higher concentrations of TQ were used, indicating a dramatic drop of m. These results suggest that the DNA-damaging agent TQ induces apoptosis by producing ROS metabolites and triggering mitochondrial membrane potential loss in the p53-mutated Jurkat cells. ). This up-regulation, observed in a concentration-dependent manner, concerned both  and  isoforms of p73 and was correlated with increasing expression levels of the tumor suppressor protein p16 INK4A (Fig. 5A). Interestingly, p73 re-expression was accompanied by a transient over-expression of caspase-3 cleaved subunits, only observed in cells exposed to 10 μM of TQ. At higher concentrations of TQ however, caspase-3, became undetectable in their active conformation (Fig. 5A).

p73 is up-regulated and UHRF1 is down-regulated in TQ-treated Jurkat cells

There are several lines of evidence that UHRF1 deregulation may impair the control of G1/S transition during cell cycle checkpoint activation [8, 9, 14]. Since such activation is known to occur during the DNA damage-related apoptosis of TQ [START_REF] Worthen | The in vitro anti-tumor activity of some crude and purified components of blackseed, Nigella sativa L[END_REF][START_REF] Shoieb | In vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone[END_REF][START_REF] Gali-Muhtasib | Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes[END_REF][START_REF] Gali-Muhtasib | Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism[END_REF],

we analyzed the expression levels of UHRF1 and its partners DNMT1 and HDAC1 in TQ-treated Jurkat cells. As shown in Fig. 5A, treatment of Jurkat cells with TQ (up to 10 M) resulted in a faint decrease in the expression levels of UHRF1 and DNMT1. In contrast, the expression levels of HDAC1 appeared to increase. However at higher concentrations of TQ, all the three proteins became undetectable. Taken together, these results also observed after 48h of TQ treatment (Fig. 5B), indicate that TQ-induced apoptosis in Jurkat cells is associated with an activation of the cell cycle checkpoint regulator p73 and a down-regulation of the UHRF1/DNMT1/HDAC1 complex.

A c c e p t e d M a n u s c r i p t 13

TQ rapidly induces apoptosis, cell cycle arrest and deregulation of p73 and UHRF1 expressions in Jurkat cells

In order to determine more precisely the chronology of the cellular and molecular events induced by TQ, we analyzed the time-course effects of TQ on Jurkat cells, at a concentration corresponding to its half-maximal activity (25 M). As shown in Fig. 6A and 6B, a notable number of cells in apoptosis appeared within 3h of treatment, suggesting that the cell cycle progression is rapidly slowing down after TQ exposition.

Accordingly, significant accumulation of cells in G0/G1 phase could be evidenced after 6h of treatment (Fig. 6C). Interestingly, increased levels of p73 could be detected within 3h of TQ exposition (Fig. 6D); this up-regulation is accompanied with a progressive down-regulation of UHRF1, in association with the appearance of a lower molecular weight form (Fig. 6D). Moreover the up-regulation of p73 was also correlated with a marked over-expression of caspase-3 cleaved subunits. Thus, for the concentrations of TQ above 10M that showed an absence of cleaved caspase-3 after 24h or 48h (Figs. 5

A & 5B), there was an increased expression of the activated caspase-3 in the early hours of treatment. These results suggest that TQ-induced apoptosis is linked to a rapid deregulation of p73 and UHRF1 expressions in Jurkat cells.

Knockdown of p73 counteracts TQ-induced UHRF1 down-regulation, cell cycle arrest and apoptosis in Jurkat cells

To determine whether p73 could act in Jurkat cells as a main regulator of the apoptotic signaling pathway activated by TQ, we attempted an acute depletion of p73 by proteins could be evidenced (Fig. 7B). The knockdown of p73 also allowed reactivation of cell cycle progression since the percentage of the cell populations in G0/G1 and S phase is significantly inversed when compared to that of TQ-treated cells transfected with scramble siRNA (Fig. 7C). Consistently, a significant decrease in the number of apoptotic cells could be observed (Fig. 7D). These data clearly demonstrated that UHRF1 is down-stream of p73 in the TQ-induced apoptotic pathway. In order to address unambiguously whether UHRF1 is targeted by p73 and is involved in the apoptotic process, we further determined the direct relationships between a p73 deregulation, modulations of UHRF1 expression and apoptosis in Jurkat cells, in the absence of TQ. As shown in Fig. 8A, siRNA-induced decrease of the basal expression levels of p73 upregulated UHRF1 expression. Moreover UHRF1 siRNA which induced the decrease of the basal expression levels of UHRF1 (Fig. 8B) significantly increased the number of apoptotic cells when compared to the controls (Fig. 8C), showing that UHRF1 exhibits anti-apoptotic properties. Altogether these results indicate that TQ triggers apoptosis in Jurkat cells through a DNA damage response involving the activation of the tumor suppressor protein p73, which is able to induce a downregulation of the expression of the anti-apoptotic protein UHRF1.

UHRF1 down-regulation in TQ-treated Jurkat cells leads to apoptosis via a caspase-dependent mechanism

A c c e p t e d M a n u s c r i p t 15

We suspected that TQ, at a concentration corresponding to its half-maximal effects, could activate a p73-dependent caspase-dependent pathway which could then trigger the down-regulation of UHRF1 and subsequently apoptosis. We therefore examined the effects of a caspase-3 specific inhibitor on TQ activity in Jurkat cells. Indeed, Z-DEVD-FMK was able to counteract the decrease of UHRF1 expression (Fig. 9A) and the apoptotic process (Fig. 9B) induced by 25 M of TQ. Moreover, an acute overexpression of UHRF1 after TQ treatment can partially rescue the cells from apoptosis, thus mimicking the effects of a caspase-3 inhibitor (Fig. 9C). The results suggest that the TQ-activation of the p73-dependent mitochondrial caspase-dependent pathway induces UHRF1 down-regulation which explains, at least in part, the observed apoptosis.

Discussion

It has been reported that the pro-apoptotic activity of TQ on cancer cells occurs in p53 wild-type cells through an up-regulation of both the tumor suppressor protein p53 and the cyclin dependent kinase inhibitor p21 Waf1/Cip1 which in turn induces G1 cell cycle arrest and apoptosis [START_REF] Gali-Muhtasib | Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism[END_REF]. In p53-null cells however, the molecular mechanisms leading to TQ-induced mitochondrial reactivity are poorly documented.

The present study demonstrates that TQ triggers apoptosis in the p53-deficient Jurkat cell line through the production of ROS and the activation of the cell cycle checkpoint regulator p73. Accordingly it has been shown that ROS production can be an efficient activator of p73 expression [START_REF] Singh | Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway[END_REF]. On the other hand, different point mutations in the p53 gene have been evidenced in Jurkat cells, mostly at the C-terminal A c c e p t e d M a n u s c r i p t 16 basic domain [START_REF] Cheng | Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines[END_REF], which is required for the activation of the p21 Waf1/Cip1 gene, for cell cycle arrest and for apoptosis [START_REF] Zhu | Definition of the p53 functional domains necessary for inducing apoptosis[END_REF]. Thus, the p53-dependent pathway is not involved in the effects of the ROS producer TQ on Jurkat cells.

Similarly to p53, the structural and functional homolog p73 can promote cell cycle arrest and apoptosis when over-expressed [START_REF] Jost | p73 is a human p53-related protein that can induce apoptosis[END_REF]. Moreover, it has been reported that cellular stress signals can induce endogenous expression of p73 in p53 null or mutant cells, engaging a p53-independent apoptotic pathway [START_REF] Zaika | Oncogenes induce and activate endogenous p73 protein[END_REF][START_REF] Willis | p73 can suppress the proliferation of cells that express mutant p53[END_REF]. Accordingly p73 seems to act as a cellular gatekeeper by preventing the proliferation of TQ-exposed Jurkat cells;

obviously the sharp re-expression of p73 that we observed in response to TQ, triggers G0/G1 cell cycle arrest and apoptosis. This cell reactivity is likely a down-stream effect of p73 since the knockdown of p73 in TQ-treated cells restored cell cycle progression and proliferation. It has been observed that early increased expression of p73 in response to cell stress is a consequence of an accumulation at the protein level [START_REF] Irwin | Chemosensitivity linked to p73 function[END_REF]. It could therefore be hypothesized that the proteasome-mediated proteolytic degradation of p73 is rapidly blocked after TQ-treatment, leading to a stabilization of the protein.

To determine whether the cell cycle checkpoint regulator p73 possesses an operative activity, we analyzed the expression of some of its down-stream effectors in TQ-treated Jurkat cells. Interestingly, a transient TQ concentration-dependent upregulation of caspase 3 cleaved subunits has been shown. First of all, this suggests that TQ exerts its apoptotic activity through caspase-dependent and caspase-independent pathways. p73, like p53, is known to promote the activation of caspases [START_REF] Levrero | The p53/p63/p73 family of transcription factors: overlapping and distinct functions[END_REF]. However, only p73 is involved in a caspase-independent cell death (CICD), which protects cells from aneuploidy by inducing their death when chromosome missegregation occurs [START_REF] Kitagawa | Caspase-independent mitotic death (CIMD)[END_REF].

Interestingly it has been observed that the switch from a caspase-dependent to a

A c c e p t e d M a n u s c r i p t caspase-independent pathway allows the progression from a specific cell death state to another [START_REF] Rebbaa | Caspase inhibition switches doxorubicin-induced apoptosis to senescence[END_REF]. Jurkat cells treated with high concentrations of TQ likely switch to a particular cell death program which involves the p73-dependent CICD pathway.

We have previously shown in HCT116 cells, a colon cancer cell line, that the activation of the p53-dependent p21 Waf1/Cip1 pathway is able to down-regulate UHRF1 [START_REF] Arima | Downregulation of nuclear protein ICBP90 by p53/p21Cip1Waf1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition[END_REF]. Here we show in Jurkat cells, the most abundant known source of UHRF1 ( [START_REF] Hopfner | ICBP90, a novel human CCAAT binding protein, involved in the regulation of topoisomerase II expression[END_REF]; personal laboratory observations), that a p73-dependent pathway also regulates UHRF1.

Indeed, TQ induces the activation of the cell cycle checkpoint regulator p73, which in turn represses UHRF1 expression in the p53 mutant Jurkat cells. The knockdown of p73 in either untreated or TQ-treated cells modulates UHRF1 expression, indicating that UHRF1 is a down-stream effector of p73 in this cell type. Our data also show that TQ down-regulates DNMT1, which is not surprising considering that DNMT1 is a privileged partner of UHRF1 [START_REF] Achour | The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression[END_REF]. Otherwise the variations of HDAC1 expression levels after TQ treatment, at least during the first 24h, remain to be elucidated.

Interfering effects between direct and indirect actions of TQ could however be invoked.

We have previously observed that UHRF1 reduction after activation of the p53dependent pathway is consecutive of both a transcriptional suppression and a protein degradation enhancement [START_REF] Arima | Downregulation of nuclear protein ICBP90 by p53/p21Cip1Waf1-dependent DNA-damage checkpoint signals contributes to cell cycle arrest at G1/S transition[END_REF]; this suggests that the p73-dependent down-regulation of UHRF1 we evidenced, likely results from the same processes. Accordingly the lower molecular weight form we observed within few hours after TQ-treatment could be related to increased UHRF1 degradation after caspase activation. explains this correlation remains unknown [START_REF] Bronner | The UHRF family: oncogenes that are drugable targets for cancer therapy in the near future?[END_REF]. In view of the important role that the epigenetic integrator UHRF1 plays in the maintenance of the chromatin status, its down-regulation should necessarily lead to the loss of genome integrity and cell death.

Accordingly, we observed that UHRF1 knockdown in untreated cells, as well as an acute overexpression of UHRF1 in TQ-treated cells, have direct incidences of the cell apoptosis rate. UHRF1 appears therefore to have in our experimental settings antiapoptotic properties, in agreement with previous observations [START_REF] Bronner | The UHRF family: oncogenes that are drugable targets for cancer therapy in the near future?[END_REF][START_REF] Abbady | ICBP90 expression is downregulated in apoptosis-induced Jurkat cells[END_REF][START_REF] Unoki | Drug discovery targeting epigenetic codes: the great potential of UHRF1, which links DNA methylation and histone modifications, as a drug target in cancers and toxoplasmosis[END_REF]. One interesting idea therefore is that preventing the epigenetic code to be replicated after UHRF1 deregulation leads to the activation of an apoptotic pathway. But this hypothesis needs further investigations.

In conclusion, this is the first report which shows that a natural compound induces apoptosis by acting on the epigenetic integrator UHRF1. By using a p53 mutant cell line, we have shown that TQ produces ROS metabolites and acts through a p73dependent mitochondrial pathway which targets UHRF1 and likely DNMT1. This pathway could involve either a caspase-dependent or caspase-independent activation (see graphical abstract). However the role of CICD on UHRF1 expression and activity remains unclear and its study is currently under investigation. Our data also highlight a new property of TQ which could be used to prevent the epigenetic code to be transmitted from a mother cell to the daughter cells.

A c c e p t e d M a n u s c r i p t 7 counting

 7 using the trypan blue exclusion method (Invitrogen). The viability rate was obtained by dividing the number of trypan blue-negative cells by the total number of cells. Cell apoptosis rate was assessed by flow cytometer (BD FACSCalibur system, BD Biosciences, San Diego, CA, USA) using the Annexin V-FITC/propidium iodide (PI) apoptosis assay (BD Biosciences), following the manufacturer's recommendations.

A c c e p t e d M a n u s c r i p t 9 France

 9 ), a rabbit polyclonal anti-cleaved caspase-3 antibody (Cell Signaling Technology, Danvers, MA, USA), a mouse monoclonal anti-UHRF1 antibody[START_REF] Hopfner | ICBP90, a novel human CCAAT binding protein, involved in the regulation of topoisomerase II expression[END_REF], a mouse monoclonal anti-DNMT1 antibody (Stressgen Biotechnologies, Victoria, BC Canada), a rabbit polyclonal anti-HDAC1 antibody (USBiological, Swampscott, MA, USA), or a mouse monoclonal anti-beta actin antibody (Abcam, Paris, France),

A c c e p t e d M a n u s c r i p t 10 conditions

 10 , calculated concentrations of TQ-induced half-maximal effects on cell proliferation and viability were respectively of 24.2 + 0.3 M and 24.3 + 0.2 M for 24h of treatment. When treated with TQ for 48h, these values were 23.3 + 0.2 M and 23.1 + 0.4 M respectively (data not illustrated). These results indicate that Jurkat cells respond to TQ within 24h and at the previously published concentrations[START_REF] Shoieb | In vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone[END_REF][START_REF] Gali-Muhtasib | Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes[END_REF][START_REF] Gali-Muhtasib | Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism[END_REF][START_REF] El-Mahdy | Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells[END_REF][START_REF] Roepke | Lack of p53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells[END_REF].

A c c e p t e d M a n u s c r i p t 11 DNA

 11 smears of the TQ-treated Jurkat cells increased in a concentration-dependent manner (Fig.

A c c e p t e d M a n u s c r i p t 12 In

 12 order to define the nature of the cell cycle checkpoint signaling pathway which could be activated in response to the TQ-induced DNA damage, we first examined the expression status of p73. In the absence of TQ, the expression of p73 in Jurkat cells was faintly detectable; however when concentrations of TQ reached 10 M, a sharp increase in the expression of p73 was observed after either 24h or 48h of treatment (Figs. 5A & 5B

A c c e p t e d M a n u s c r i p t 14 transfecting

 14 TQ-treated cells with specific siRNA against p73. As shown in Fig.7A, p73 knockdown restored UHRF1 expression at levels similar to those observed for untreated cells; accordingly a normalization of the expression level ratio of the two

  Indeed a specific inhibitor of caspase-3 can recover UHRF1 expression and rescue cells from apoptosis induced by TQ. Interestingly it has been shown that repression of UHRF1 expression after treatment with different inducers of cell cycle checkpoints regulators is associated A c c e p t e d M a n u s c r i p t 18 with cell cycle arrest in G0/G1 and cell proliferation inhibition [9]. Although accumulating data have highlighted a correlation between the reduction of UHRF1 expression and cell cycle arrest or apoptosis, the molecular mechanisms which clearly

A c c e p t e d M a n u s c r i p t 20 [ 8 ]Fig. 1 .

 2081 Figure captions

Fig. 2 .A c c e p t e d M a n u s c r i p t 25 GenomicFig. 3 .

 2253 Fig. 2. Concentration-dependent apoptosis induced by TQ in Jurkat cells. Cells were

Fig. 4 .

 4 Fig. 4. Concentration-dependent m disruption induced by TQ in Jurkat cells. Cells

Fig. 5 .

 5 Fig. 5. Effects of TQ on UHRF1, DNMT1, HDAC1, p73, p16 and cleaved caspase-3

A c c e p t e d M a n u s c r i p tFig. 6 .

 6 Fig. 6. Time-course of the effects of TQ on the number of apoptotic cells, cell cycle

Fig. 7 .A c c e p t e d M a n u s c r i p t 27 0

 727 Fig. 7. Effects of p73 knockdown on TQ activity in Jurkat cells. Untransfected cells (2;

Fig. 8 .

 8 Fig. 8. Direct relationships between p73 knockdown, UHRF1 deregulation and apoptosis in Jurkat cells. Cells were seeded at a density of 2 x  10 5 cells/ml and grown

Fig. 9 .

 9 Fig. 9. Rescue effects after treatment with a caspase-3 inhibitor or an acute

  Fig. 1.

Figure 3 Page 33 of 38

 338 Figure 3

  Fig. 4.

Figure 4 Page

 4 Figure 4

Figure 6 Page

 6 Figure 6

Figure 8 A

 8 Figure 8

Figure 9

 9 Figure 9

Acknowledgments


Massimo Blandino
email: massimo.blandino@unito.it

Amedeo Reyneri

Francesca Vanara

Michelangelo Pascale

Miriam Haidukowski

Claudio Campagna

Managing fumonisin contamination in maize kernels through the timing of insecticide application against European corn borer

Keywords: Mycotoxins -fumonisins, Mycotoxins -fusarium Food Types: Animal feed, Cereals and grain maize, European corn borer, insecticide treatment, pyrethroids, Fusarium, 24 fumonisins, mycotoxins

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction 2

Fusarium verticillioides (Sacc.) Niremberg is the most common toxigenic fungus in maize 3 in temperate areas which causes ear rot disease and fumonisin accumulation in kernels 4 (Logrieco et al., 2002;Munkvold et al., 2003b). Fumonisins are receiving increasing 5 attention by the scientific community since fumonisin B 1 (FB 1 ) and B 2 (FB 2 ) are the most 6 common mycotoxins found in maize kernels throughout the world (Doko and Visconti, 7 1994; Placinta et al. 1999;Soriano and Dragacci, 2004). These toxins have been shown to 8 cause a number of health problems in livestock and laboratory animals (Marasas et al., 9 2000) and have been associated with an increased incidence of human esophageal 10 cancer in some parts of the world (Yoshizawa et al., 1994;Marasas, 1995). Therefore, 11 admissible maximum levels for fumonisins B 1 and B 2 have recently been set by the 12 European Commission in maize and maize-based foods (EC, 2007).

13 14
Maize is consumed either directly or indirectly when processed into food and feed 15 products. In food production, maize is generally dry milled to produce flours and grits 16 which can be processed for snacks, breakfast cereals, cooked or extruded products 17 (Serna-Saldivar et al., 2001). Since interesting new uses of dry milling foodstuffs can be 18 found in gluten-free products intended for people with gluten intolerance (celiac disease) 19 and for foods used for infants (baby food), the demand for maize for food processing has 20 increased in the last few years.

22

Since fumonisin contamination starts in the field, maize grain safety can be improved 23 through agronomic prevention strategies, which are able to reduce Fusarium infection and 24 fumonisin synthesis (Munkvold, 2003a). The occurrence of fumonisins is mainly related to ear injuries caused by Lepidoptera borers (Avantaggiato et al., 2002;Papst et al., 2005).

1
In temperate maize areas, the insect most frequently associated with F. verticillioides and 2 fumonisin synthesis is Ostrinia nubilalis Hübner (European corn borer -ECB) (Mason et 3 al., 1996;Dowd, 2003). The larvae of this insect tunnel into the stalks and ears of the 4 plant, causing a yield reduction and loss of grain quality (Godfrey et al., 1991). Two 5 generations of ECB larvae usually occur per year in North Italy: the first generation attacks 6 plants during the mid to late vegetative stages and the second generation attacks during 7 the reproductive stages (from early milk stage to maturity). The second generation larvae, 8 above all, plays an important role in the epidemiology of F. verticillioides in maize (Sobek 9 and Munkvold, 1999) and the insect damage of ears can increase the fumonisin 10 contamination of kernels (Logrieco et al., 2003). ECB feeding activity is crucial in maize 11 grain fumonisin contamination: damaged ears can suffer fumonisin contamination at a 40 12 times higher rate than healthy ones (Alma et al., 2005). Several studies have established 13 that the control of ECB clearly affects fumonisin levels in maize kernel at harvest; this has 14 been demonstrated through the use of methods such as insecticide treatment (Blandino et 15 al, 2008a;Folcher et al., 2009), biological control with parasitoids (Dowd, 2000) and 16 genetic control involving GMO Bt technology (Munkvold et al., 1999;Papst et al., 2005). 17 Aerial applications of spray formulations of Bt appear less interesting since Bt toxins show 18 a low persistence and is susceptible to a rapid degradation by sunlight (Tamez Guerra et 19 al., 1996). Despite the vast amount of literature available on the biology and biological 20 control of O. nubilalis, the use of chemical insecticides is still the main method for ECB 21 management, where Bt maize cultivation is not permitted (Saladini et al., 2008). Insecticide (Blandino et al., 2008a). On the other hand, insecticide treatments are not always able to 1 guarantee maize production with contamination levels of fumonisins below the thresholds 2 (Saladini et al., 2008). Since fumonisin contamination is positively related to the number of 3 tunnels in ears (Alma et al., 2005), a lower grain contamination could be guaranteed by 4 increasing the efficacy of the insecticide treatment, to obtain a major reduction of ECB 5 larval injury in the ears. Increasing the number of insecticide applications is not 6 practicable, because it is not economically favourable and could have a high impact on 7 non-target biota, in particular on the natural enemies of ECB and could lead to a high 8 development of other pests (Ayyappath et al., 1996). The timing of the insecticide 9 application is an important technical aspect that needs to be better understood in order to 10 improve ECB and fumonisin control, since a correct application is crucial to reach the 11 larvae during the few days between the time they hatch and when they enter into the stalk 12 and ear (Mason et al., 1996). 1 for each year and site.

12 13

The applied insecticide was pyrethroid lambda-cyhalothrin (Karate® Zeon, formulation: 2009), was used to describe the development of ECB and fungal ear rot incidence. In the derivatives were analyzed by reversed-phase HPLC with fluorescence detection (λ ex. =335

10 logistic model applied, Y = 1*[1*A -1 + (B 0 *B 1 X )] -1 ,
13 nm, λ em. =440 nm). The limit of detection (LOD) of the analytical method was 20 ng g -1 for 14 FB 1 and FB 2 .

15 16

Statistical analysis 17

The relationship between the ECB and fungal ear rot incidence and severity and the The normal distribution and homogeneity of variances were verified for the data collected The two years were subject to different meteorological trends, as far as both rainfall and 12 temperature (expressed as growing degree days, GDDs) from flowering to harvesting are 13 concerned (Table 2). The year 2007 had more rainfall in May and June, but less in 14 September, compared to the previous year. In this last year, the GDDs from January to 15 May were higher than those observed in 2006, while in 2007 the GDDs were lower from 16

June to July and from September to October. In both years, site A had more rainfall than 17 site B, while the two sites had similar GDDs. and peaked about 20 days later (Table 1). In the first year, the captures at flight peak were The increase in ECB incidence and severity during the crop maturation for some 3 representative treatments (NT, T1, T3 and T6) for each site and year is reported in figures 4 1 and 2, respectively. The parameters, coefficients of determination (R 2 ) and level of 5 significance (P) for the logistic and linear models shown in figures 1 and 2 and those of the 6 other insecticide application timings not shown, are reported in tables 3 and 4.

7

In 2006, the first ear injuries caused by ECB larvae in the untreated plots appeared at 8 GDD=1009 and GDD=1108, in site A and site B, respectively, while in 2007, the first ears

9
showing damage were recorded at GDD=1141 and GDD=1334, respectively. In both sites,

10
the ECB incidence increased rapidly between GDD 1200 and 1500 in 2006 and between

11

GDD 1300 and 1700 in 2007 (Fig. 1). However, figure 2 shows that ECB severity 12 increased linearly over the whole the crop maturation period until the physiological maturity 13 stage.

15

In 2006, the maximum level of ECB incidence in the untreated control (NT) was reached at 16 GDD=1510 (96%) and at GDD=1569 (93%), in sites A and B, respectively. In this year, 17 treatment T3 showed the lowest increase in ECB incidence and severity, while the T1 and 18 T6 insecticide applications resulted in a similar ear damage development to the untreated 19 control. The maximum level of ECB incidence in the T3 treatment was 34% and 38% in 20 sites A and B, respectively, and was reached at approximately GDD=1700 for both sites.

21

In 2007, the maximum level of ECB incidence in the untreated control was reached at 22 GDD=1706 (45%) and at GDD=1810 (75%), in site A and site B, respectively. In this year, At harvest, significant differences (P<0.001) between the treatments were observed for 4 ECB incidence and severity in both years and sites (Table 5). In 2006, treatments T3 and respectively. In both sites, the fungal ear rot incidence increased rapidly between GDD 6 1400 and 1600 in 2006 and between GDD 1500 and 1700 in 2007 (Fig. 3). However,

7
fungal ear rot severity increased linearly during maize ripening until the physiological 8 maturity stage (Fig. 4). As observed for ECB, the insecticide application reduced the 9 impact and delayed the appearance of ear rot symptoms compared to the untreated 10 control, with an effect which was proportional to the efficacy of the ECB control. In both 11 years and sites, treatment T3 resulted in the lowest increase in fungal ear rot incidence 12 and severity, followed by treatment T1, while the T6 insecticide applications resulted in a 13 fungal ear rot development that was similar to the untreated control. At harvest, significant differences (P<0.001) between the treatments were observed for 16 ECB incidence and severity in both years and sites (Table 5). In 2006, treatments T3 and 17 T4 in site A and treatments T2 to T5 in site B showed a significantly lower fungal ear rot As far as ECB is concerned, the T3 treatment showed the lowest fungal ear rot incidence 2 and severity at harvest, with the exception of the experiment conduced in 2007 in site B,

3
where the highest efficacy to control ear rot was observed for treatment T4. The reduction in fumonisin level can be expressed by a parameter, efficacy (E), defined by Furthermore, an early insecticide treatment, applied at the end of maize flowering without 9 adult captures, showed significantly lower fungal ear rot symptoms and fumonisin 10 contamination than a treatment applied after the flight peak. Thus, the higher efficacy of 11 early pyrethroid application is probably due to the control of larvae which initiate feeding 12 earlier during the maize ripening stages and which have a greater potential to cause a 13 higher Fusarium development and fumonisin contamination than those hatched at the end 14 of adult flight, which initiate feeding on harder and drier kernels (Mason et al., 1996).

15 Moreover, moulds brought by the first larvae take advantage of a less compact and more 16 easily colonizable substrate (Yates and Jaworski, 2000). In the present experiments, the insecticides were applied on early planted maize. It was 10 demonstrated in previous works that later sowing times caused a higher incidence of ECB 11 larvae damage (Anderson et al., 2003) and this is one of the main causes of a higher 12 fumonisin contaminations (Blandino et al., 2009b).

13

The information on the correct application of insecticides against ECB obtained from this 14 research, should be introduced into a integrated field programme to enhance the 15 prevention of fumonisin in maize destined for the food chain (Blandino et al., 2009a). The fitted lines are logistic curves fitted to the accumulated GDD calculated from 1 st of January. See table 3 9 for details of the fitted models. 10 The linear lines are fitted to the accumulated GDD calculated from 1 st of January. See table 4 for details of 9 the fitted models. 10 The fitted lines are logistic curves fitted to the accumulated GDD calculated from 1 st of January. See table 3 9 for details of the fitted models. The linear lines are fitted to the accumulated GDD calculated from 1 st of January. See

Z Logistic model Y = 1*[1*u -1 + (B 0 *B 1 X )]
-1

; Y represents ECB or fungal ear rot incidence, A is the observed 6 asymptotic maximum incidence level, B 0 is related to initial disease, B 1 is the damage progress rate and X is 7 the accumulated GDD calculated from the 1 st of January. The equations were fitted to the mean value of each 8 treatment. The parameters were calculated by SPSS non-linear regression. 9

5

The equations were fitted to the mean value of each treatment. The growing degree-days were calculated 6 from the 1 st of January. The parameters were calculated by SPSS linear regression. 7

22

  treatment against second generation larvae of ECB plays an increasing role in maize crop 23 practices and several insecticides, mainly synthetic pyrethroids, are currently labelled for 24 ECB control in maize and generally applied after the first generation flight peak of adults 25

  this study was to investigate the effect of the timing of the insecticide 15 application against the second generation larvae of ECB with pyrethroid lambda-16 cyhalothrin on insect damage, fungal ear symptoms and fumonisin B 1 and B 2 17 contamination in maize cultivated in NW Italy. sowing date and insecticide application on fumonisin contamination in maize 22 kernels was studied in 2006 and 2007 in 2 sites in NW Italy: site A (Vignale, 45° 29' N, 8° 23 37' E; altitude 160 m., in a sandy-loamy textured soil) and site B (Vigone, 44° 51' N, 7° 30' 24 E; altitude 256 m, in a sandy-medium textured soil). timings (T1-T7) were compared to an untreated control (T0), 2 in each site and in each year. The insecticide treatments were applied at approximately 7-3 d intervals, starting from maize flowering [Growth stage (GS) 65, Weber and Bleiholder, 4 1990; Lancashire et al., 1991], with wet silks (T1).

  activity was monitored by means of a cone trap placed outside the 7 experimental plots, baited with sex pheromone (E:Z=97:3) to attract males and with 8 phenylacetaldehyde (PAA) for females. The sex pheromones and PAA dispenser were 9 replaced each 15 and 30 d, respectively. Adults were removed from the trap and counted 10 every 1-2 d. The sowing, silking and harvest dates, the ECB flight peak and the insecticide 11 application dates are reported in table

1437

  capsule suspension, Syngenta Crop Protection, Italy) at 0.019 kg AI ha -1 . Treatments were 15 carried out with self-propelled sprayers (Agri JS 826, Kertitox ® ), with a hydraulically 16 adjustable working height (0.70 -3.50 m) in order to spray the maize crop after flowering. 17 Twenty fan nozzles on the air-assisted boom applied a spray volume of 400 l ha -1 at a 18 pressure of 200 kPa; the operation speed was 10 km h -1 . Air-assisted spraying uses 19 relatively large volumes of low-pressure air, which is generated by a fan, to direct the 20 spray onto the crop. 21 22 Studies were carried out each year on the commercial dent corn hybrid Pioneer 3235 23 (FAO 600; 130 d). The previous crop was maize each year. The treatments were assigned 24 to experimental units using a completely randomized block design with 3 replicates. Each 25 20 rows, separated by two untreated buffer rows on either side; the plot 1 length was 40 m. 2 During the crop maturation, 30 ears were visually evaluated in each plot for the ECB 4 damage incidence and severity and fungal ear rot symptoms every 10 days, starting from 5 the milk stage (GS 73), until the physiological maturity stage (GS 89). 6 At the end of maturity, 100 ears (included ears used for the evaluation of ECB and fungal 8 ear rot incidence and severity at harvest) were collected by hand in each plot and shelled 9 using an electric sheller. The ears were collected at a grain moisture content of between 10 23-27%. The kernels in each plot were mixed thoroughly to obtain a random distribution 11 and 5 kg samples were taken to analyze the fumonisin (FB 1 and FB 2 ) content. incidence was calculated as the percentage of ears per plot with kernel 15 injuries or apical and basal tunnels in the cob due to larvae activity. The ECB damage 16 severity was calculated as the percentage of kernels per ear with injuries due to larvae 17 activity. A scale of 1 to 7 was used in which each numerical value corresponds to a 18 percentage interval of surfaces exhibiting visible kernel damage due to larvae activity 19 according to the following schedule: 1 = no injuries, 2 = 1-5%, 3 = 6-10%; 4 = 11-20 %, 5 = 20 21-35%, 6 = 35-60%, 7 > 60% (Blandino et al., 2008a).21The fungal ear rot incidence was calculated as the percentage of ears per plot with 22 symptoms, while the fungal ear rot severity was calculated as the percentage of kernels 23 per ear with symptoms. A scale of 1 to 7 was used in which each numerical value 24 corresponds to a percentage interval of surfaces exhibiting visible symptoms of the 25 26-50%, 6 = 51-75%, 7 > 75%(Blandino et al. 2008a). The ECB damage 2 severity and ear rot severity scores were converted to percentages of ears exhibiting 3 symptoms and each score was replaced with the mid-point of the interval.

4 5

 4 The data recorded from the visual evaluation of the ears during the crop maturation were 6 used to describe the increase in ECB and fungal ear rot symptoms for the insecticide 7 application timings. A logistic model, based on accumulated growing degree days (GDD) 8 according to an analogous approach set up by Trnka et al. (2007) and Maiorano et al.9

  18accumulated GDD was studied through nonlinear and linear regression in order to obtain 19 parameters for each insecticide application timing. 20 21

22

  at harvest, by performing the Kolmogorov-Smirnov normality test and the Levene test,23respectively. An analysis of variance (ANOVA) was utilized to compare ECB damage and 24 fungal ear rot incidence and severity and fumonisin B 1 + B 2 content, using a completely 25 . Each experiment was analyzed separately since there were 1 significant interactions of the treatment with year and site. Multiple comparison tests were 2 performed according to the Student-Newman-Keuls test on the treatment means. The 3 SPSS Version 13.0 for Windows statistical package, (SPSS Inc., Chicago), was used for 4 the statistical analysis. The incidence and the severity values of ECB and fungal ear were 5 previously transformed using y'=arcsin√x*180/π, as percentage data derived from 6 counting. The concentration of fumonisins was transformed using the equation y'=ln(x+1)

  , the flight activity of the first-generation moths started at the beginning of July

  21

22

  higher compared to those observed in the second year, while site B showed more 23 captures than site A. On the basis of flight monitoring information, the T5 treatment was 24 applied in each site and year at approximately the ECB flight peak, while the last treatment 25 was performed about 12-15 days after the ECB flight peak. 1 2

  23treatment T3 reached the maximum level of ECB incidence at approximately GDD=1761 24 (16%) and GDD=1875 (43%), in site A and site B, respectively. Treatments T1 and T4 25 final ECB incidence and severity, which was reached earlier than in 1 treatment T3.

5

  T4 in site A and the T2 to T5 treatments in site B showed a significantly lower ECB 6 incidence and severity than the untreated control (NT). In 2007, in site A, a significantly 7 lower ECB incidence and severity was shown compared to NT for the timing of the 8 insecticide application from 7 days after maize flowering (T2) to approximately 7 days after 9 the adult flight peak (T6), while in site B, only the last time of application, applied at 10 approximately 15 days after the adult flight peak (T7), was not significantly different from 11 the untreated control. In both sites, in 2006, and in site A in 2007, the T3 treatment 12 showed the lowest ECB incidence and severity, while both these parameters increased 13 moving to an earlier or later treatment. In 2007, in site B, the highest reduction of ECB 14 damage on ears was observed for treatment T4.

  18incidence than the untreated control (NT), while a significantly lower ear rot severity was 19 also observed for treatments T1 and T2 in site A, and T1 and T6 in site B. In 2007, in site 20 A, a significantly lower fungal ear rot incidence was shown compared to NT for all the 21 insecticide application timings, while ear rot severity was significantly lower than NT in the 22 T2 to T6 treatments. In site B, with the exception of treatments T6 and T7 for ear rot 23 incidence, all the insecticide application timings significantly reduced the fungal ear rot 24 incidence and severity compared to the untreated control.

  (P<0.001) of the insecticide application timing on fumonisin occurrence 7 in maize kernels was observed in both years (Table5). Only treatment T6 in 2007 in site B 8 and treatment T7 in 2007 and in 2006 in site B, were not significantly different from the 9 untreated control. 10 11 As for ECB and fungal ear rot, the T3 treatment showed the lowest fumonisin 12 contamination in each experiment. Contamination of these mycotoxins increased when the 13 treatment was moved to earlier or later dates. Treatment T4 only had a significantly higher 14 fumonisin contamination than T3 in 2007 in site A, while treatment T2 was not significantly 15 different from the previous two insecticide application timings in either year in site B. 16 For the other application timings, the insecticide application at maize flowering (T1) 17 resulted in a significantly lower contamination than the treatment applied at approximately 18 7 days after the ECB flight peak (T6) in 2006 in site A and in 2007 in site B.19 20

5(

  The results of these experiments confirm the important link between fumonisin 6 contamination, fungal development and ECB activity in the damage of maize ears (Sobek 7 andMunkvold, 1999; Dowd, 2003). ECB larvae are vectors of Fusarium spp.; they cause 8 entry wounds and carry fungal inoculum from the plant surface to the ears. It has been 9 demonstrated that in temperate areas F. verticillioides is favoured by ECB larvae feeding 10 more than other Fusarium species(Lew et al., 1991; Munkvold et al., 1999). The 11 development of F. verticillioides on damaged area of kernels caused by second generation 12 ECB is the predominant cause of fumonisin contamination in natural conditions in maize 13 fields in North Italy (Blandino et al., 2008b). 14 When the insects were controlled by chemical applications, the fumonisin amounts clearly 15 decreased within the harvested kernels. The collected data also underline the importance 16 of timing of insecticide application in order to improve the effectiveness of the fumonisin 17 contamination control. Under natural conditions, with relatively high ECB levels, the 18 treatments applied 7-10 days before the ECB adult flight peak (T3), in correspondence to 19 the constant increase in adult captures, showed the best efficacy to control the insect 20 damage on ears and the lowest fungal ear rot development and fumonisin contamination.21At this insecticide application timing, fumonisins decreased within the maize grains with an 22 efficacy of more than 90%. This study underlines how a single insecticide treatment with 23 pyrethroids, when properly applied, is able to provide a reduction in ear mould and 24 fumonisin that is almost comparable with those observed in several experiment with Bt 25 ). Since pyrethroids are only effective when the ECB is in its larval status and has2 not yet penetrated the stalk or ear, and they have approximately 15-d residual activity3Rinkleff et al., 1995), the correct treatment window for their application ranges between 4 the first captures of 1 st generation adults and the flight peak. If the treatment is delayed 5 until after the adult flight peak, larvae from the eggs deposited early in the egg-laying 6 period enter the plant and are not controlled effectively by the insecticide, causing ear 7 injuries are responsible for fumonisin contamination.

  8

  fields cultivated in areas with a high ECB pressure, the control of second 19 generation larvae of this insect by an insecticide application is essential to reduce 20 fumonisin levels in kernels intended for human consumption.21In order to obtain a lower yield reduction, associated whit a lower fumonisin contamination, 22 as a consequence of the development of ECB injuries on maize ears, the optimum timing 23 of pyrethroid insecticides is comprised between the beginning of a consistent adult flight 24 activity and the flight peak.

  calculated as the percentage of ears with ECB damage, based on 3 replications of 30 7 ears each. 8

1

  Figure 2.

  calculated as the mean number of tunnels per ear, based on 3 replications of 30 ears 7 each. 8

1

  Figure 3.

  incidence was calculated as the percentage of ears with symptoms, based on 3 replications of 7 30 ears each. 8

  10

1

  Figure 4.

  severity was calculated as the mean percentage of kernels with symptoms per ear, based on 7 3 replications of 30 ears each. 8

U 8 X6

 8 ECB incidence was calculated as the percentage of plants with ECB damage, based on 3 replications of 30 5 plants each. 6V ECB severity was calculated as the percentage of ears with ECB damage, based on 3 replications of 30 ears 7 each. Fungal ear rot incidence was calculated as the percentage of ears with symptoms, based on 3 replications of 9 30 ears each. 10 Y Fungal ear rot severity was calculated as the mean percentage of kernels with symptoms per ear, based on 11 3 replications of 30 ears each. 12 Means followed by different letters are significantly different (the level of significance is shown in the table). 1The ECB and fungal ear rot incidence and severity means reported are values transformed using 2 y'=arcsin√x*180/π, as percentage data derived from counting. The fumonisin contamination means reported 3 are transformed [ T; y'= ln (x + 1)] and not transformed (N) values. 4Page 34 of 34 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

  Y represents ECB or fungal ear rot

	1
	2	cloramfenicol peptone (DCPA) and incubated at 20 °C. The Fusarium colonies were
	3	identified after 7 to 10 days through colony and conidial morphology, as reported by
	4	Nelson et al. (1983).
	5
	6	Fumonisin analyses
	7 8	F A 5 kg representative sample of grain from each plot was freeze-dried and milled. A o r representative sub-sample of the milled material was analyzed for fumonisins according to
	9 10	the AOAC Official Method N.995.15 (AOAC, 2000). Briefly, maize samples (50 g) were P e extracted with 100 mL of methanol-water (3:1, v/v) by blending for 3 min. 10 mL of the
	11 12 11 12	incidence, A is the asymptotic maximum incidence level observed, B 0 is related to the filtered extracts were cleaned-up using strong anion exchange (SAX) cartridges and e r derivatized with an o-phthaldialdehyde (OPA)/2-mercaptoethanol solution. Fumonisin-OPA
	13	initial disease, B 1 is the rate of damage and symptom progress and X is the accumulated R
	14 15 16 17	e GDD. Both B 0 and B 1 are estimated parameters in the logistic model. ECB and fungal ear rot severity were instead modelled as a linear function of the accumulated GDD. GDD accumulation was calculated from the 1st of January (Mason et al., 1996) by summing the v i e average of the daily maximum (T max ) and minimum (T min ) temperatures. A base w
	18 19 20	temperature (T base ) and a temperature cut-off (T cut ) were defined and set equal to 10°C and 30°C, respectively (Got and Rodolphe, 1989). If T min and/or T max were lower than T base , T base was considered. If T min and/or T max were greater than T cut , T cut was considered. O n l y
	21	Fungal isolation and identification were performed through a morphological analysis to
	22	verify which Fusarium species were involved to the greatest extent occurrence of
	23	symptoms. The evaluation of Fusarium infections was carried out using 100 kernels
	24	randomly sampled from the untreated control at dough stage (GS 85). The kernels were
	25	surface disinfested for 3 min in a 2% solution of sodium hypochlorite, then rinsed 3 times

15 16 Fungal ear rot incidence and severity

		Food Additives and Contaminants
	1	different treatments is reported in figures 3 and 4, respectively. The parameters,
	2	coefficients of determination (R 2 ) and levels of significance (P) for the logistic and linear
	3	models are reported in tables 3 and 4. In 2006, the first mold symptoms in the untreated
	4	plots appeared at GDD=1261 and GDD=1395, in sites A and B, respectively, while in
	5	2007, the first ears showing fungal ear rot were recorded at GDD=1301 and GDD=1430,
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y

17

The most frequently isolated fungal species in the kernel samples taken at the dough 18 stage (GS 85) in each year was F. verticillioides. The mean percentage of kernels infected 19 by this species in the untreated control (T0) was on average 65% and 37% in the years 20 2006 and 2007, respectively (data not shown). Other Fusarium species, such us F. 21 graminearum, F. subglutinans, F. culmorum, F. oxysporum, were found each year, but 22 only on some isolated samples.

23 24 The increase in fungal ear rot incidence and severity during crop maturation for the 25 Page 12 of 34 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

  In the present study, the monitoring of insect injuries during maize ripening clearly showed 2 that the chemical treatment not only reduced the ECB severity, but also delayed the

	1 1
	2	Since, according to the data collected, the optimum insecticide application timing begins
	3 3	after the trap catches indicated a constant adult ECB activity, the monitoring of adult flight
	4 4	appearance of ECB damage and shifted the insect activities to a later ear development activity with either pheromone or light traps could not give a timely prediction to efficiently
	5 5	stage. As Calvin et al. (1988) reported, the impact of ECB feeding declines from the drive the management of chemical applications in a certain area. ECB development and
	6 6	beginning of the reproductive period to physiological maturity. At present, the pyrethroid activity risk assessment models, based on GDD accumulation, could be used as
	7 7 8	F o r treatment applied after the first generation ECB flight peak is the normal practice of farmers in North Italy, since they usually spray insecticide when adult catches decline, F instruments to help farmers estimate the correct timing of chemical applications during the o r growing season (Got et al., 1997).
	9	P P
		e e
		e r e r
		R R
		e e
		v i e v i e
	17	w w
	18 19 20 21 18 19 20 21	The higher effect caused by larvae that arrive at the beginning of the maize ripening stages on ear moulds and fumonisin content could also be supported by data reported by Munkvold et al (1999). Since the Fusarium ear rot and fumonisin concentration were O n l y this pest and fumonisin contamination. Moreover, determining the best distribution timing for each insecticide class could lead to a differentiation of the products in function of the O n l spray timing, in order to obtain high efficacy treatments throughout the entire chemical y
	22 22	higher in manually infested ECB treatments, the authors suggested that this effect was ECB control period. The possible interaction between application timing and type of
		related to the timing of the manual infestation, which occurred at an early reproductive insecticide used still needs further investigation, in order to find the best timing of
	25 25
		18

23

stage, thus allowing the maximum time for fumonisin accumulation before harvest 24

(Munkvold et al;1999)

.

8

after the peak has been reached. In our experiments, the efficacy to reduce fumonisin 9 contamination observed with a pyrethroid application at approximately 7 days after the 10 ECB flight peak was between 26 and 54%, confirming values observed in previous studies 11

(Alma et al., 2005; Saladini et al., 2008; Blandino et al., 2008a)

. Considering the higher 12 efficacy on reducing fumonisin content observed in this research with earlier applications, 13 it can be stated that the timing of the chemical treatment needs to be reconsidered. 14 15 Rinkleff et al. (1995) reported that application timing may be particularly flexible for 16 pyrethroids which have good control and residual activity on neonates. On the other hand, 17 insecticides with higher contact residual toxicity could play a major role in the control of 23 application for each insecticide and to obtain a larger window for useful chemical 24 treatments at a farm scale.

  Mancini for their expert technical assistance. The funds for this research were provided by Crambidae) on fumonisin contamination in maize kernels. Crop Prot. 27:1432-1436. Got B, Piry S, Migeon A, Labatte JM. 1997. Comparison of different models for predicting Papst C, Utz HF, Melchinger AE, Eder J, Magg T, Klein D, Bohn M. 2005. Mycotoxins 1 produced by Fusarium spp. in isogenic Bt vs. non Bt maize hybrids under European Placinta CM, D'Mello JPF, Macdonald AMC. 1999. A review of worldwide contamination of 4 cereal grains and animal feed with Fusarium mycotoxins. Anim Feed Sci Tech.Effect of the timing of the insecticide application on the ECB incidence evolution during

	1 1	2009. Comparative activity of agrochemical treatments on mycotoxin levels with Figure 1.
	2 2 2	sowing date and insecticide application against European corn borer (Lepidoptera: model (FUMAgrain) on fumonisin synthesis by Fusarium verticillioides in maize grain
	3 3 3	Prot. 28: 302-308. in Italy. Crop Prot. 28:243-256. corn borer pressure. Agron J. 97:219-224.
	4 4 4	Blandino M, Tamietti G, Vanara F, Visentin A. 2008b. Role and importance of different EC 2007. Commission Regulation (EC) No 1126/2007 of 28 September 2007 amending Marasas WFO, Miller JD, Visconti A. 2000. Fumonisin B 1 . Environ Health Criter. 219:1-
	5 5 5 5	infection pathways on the final Fusarium infection and fumonisin contamination on Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in 150.
	6 6 7 6 6 7 8 6 7 8	F o r F o r maize. In: Proceedings of 9th International Congress of Plant Pathology, Torino, August 24-29, Journal of Plant Pathology 90 (2, Supplement), 317. Blandino M, Reyneri A, Colombari G, Pietri A. 2009a. Comparison of integrated field F o r foodstuffs as regards Fusarium toxins in maize and maize products. Official Journal of the European Union, L255, p. 14-17. F o r Marasas WFO. 1995 Fumonisins: their implications for human and animal health. Nat Toxins 3:193-198. Masoero F, Meschini M, Rossi F, Grandini A, Pietri A. 1999. Nutritive value, mycotoxin F o r 78:21-37. Rinkleff JH, Hutchison WD, Campbell CD, Bolin PC, Bartels DW. 1995. Insecticide toxicity in European Corn Borer (Lepidoptera: Pyralidae): ovicidal activity and residual F o r F o r
	9	P P P P contamination and in vitro rumen fermentation of normal and genetically modified corn P mortality to neonates. J Econ Entomol. 88(2): 246-253. P P
		e e e e (Cry1A(B)) grown in northern Italy. Maydica 44:205-209. e e e
		e r e r e r e r e r e r e r
		R R R R R R R
		e e e e e e e
		v i e v i e v i e v i e v i e v i e v i e
		w w w w w w w
	17 18 17 18	weeds. Ann Appl Biol. 119:561-601. maize kernels. J Econ Entomol. 92(3):503-509.	O n l O n l O n l O n l O n l O n l O n l
	18 19 19 19	y y y Logrieco A, Mulè G, Moretti A, Bottalico A. 2002. Toxigenic Fusarium species and y y Soriano JM, Dragacci S. 2004. Occurrence of fumonisins in foods. Food Res Int. 37(10): y y
	19 20 20 20 20	Tetranychidae) following insecticide applications in corn. Environ Entomol. 25(5):933-Econ Entomol. 93:1669-1679. mycotoxins associated with maize ear rot in Europe. Eur J Plant Path. 108:597-609. 985-1000.
	20 21 21 21	937. ears. Eur J Plant Path. 109:705-713. Dowd PF. 2003. Insect management to facilitate preharvest mycotoxin management. J Logrieco A, Bottalico A, Mulè G, Moretti A, Perrone G. 2003. Epidemiology of toxigenic Tamez Guerra P, McGuire MR, Roldan HM, Wong LJG, Shasha BS & Vega, FE. 1996.
	22 22	Bakan B, Melcion D, Richard-Molard D, Cahagneir B. 2002. Fungal growth and Fusarium Toxicol Toxin Rev. 22 (2):327-350. fungi and their associated mycotoxins for some Mediterranean crops. Eur J Plant Sprayable granule formulation for Bacillus thuringiensis. J Econ Entomol. 89:1424-
	23 23 23 23	Path. 109:645-667. 1430.
	25 23
			21 1

7

Syngenta Crop Protection S.p.A. 8 References 9 Alma A, Lessio F, Reyneri A, Blandino M. 2005. Relationships between Ostrinia nubilalis 10 Hübner (Lepidoptera Crambidae), crop technique and mycotoxin contamination of 11 corn kernel in Northwestern Italy. Int J Pest Manag. 51(3):165-173. 12 Anderson PL, Weiss MJ, Hellmich RL, Hoffmann MP, Wright MG. 2003. Millet preference, 13 effects of planting date on infestation, and adult and larval use of proso millet by 14 Ostrinia nubilalis (Lepidoptera: Crambidae). J Econ Entomol. 96(2):361-369. 15 Avantaggiato G, Quaranta F, Desiderio E, Visconti A. 2002. Fumonisins contamination of 16 maize hybrids visibly damaged by Sesamia. J Sci Food Agric. 83:13-18.

17

Ayyappath R, Witkowski JF, Higley LG. 1996. Population changes of spider mites (Acari: 21 mycotoxin content in isogenic traditional maize and genetically modified maize grown 22 in France and Spain. J Agric Food Chem. 50:728-731.

2 8 programmes for the reduction of fumonisin contamination in maize kernels. Field 9 Crop Res.111:284-289. 10 Blandino M, Reyneri A, Vanara F. 2009b. Effect of sowing time on toxigenic fungal 11 infection and mycotoxin contamination of maize kernels. J Phytopathol. 157:7-14. 12 Calvin DD, Knapp MC, Xingquan K, Poston FL, Welch SM. 1988. Influence of European 13 Corn borer (Lepidoptera: Pyralidae) feeding on various stages of field corn in 14 Kansas. J Econ Entomol. 81(4):1204-1208. 15 Doko MB, Visconti A. 1994. Occurrence of fumonisins B 1 and B 2 in corn and corn-based 16 human foodstuffs in Italy. Food Addit Contam. 11:433-439.

17

Dowd PF. 2000. Indirect reduction of ear molds and associated mycotoxins in Bacillus 18 thuringiensis corn under controlled and open field conditions: utility and limitation. J 22 Folcher L, Jarry M, Weissenberger A, Gérault F, Eychenne N, Delos M, Regnault-Roger C. 1 regard to corn borers and Fusarium mycoflora in maize (Zea mays L.) fields. Crop 7 8 development time of the European corn borer (Lepidoptera, Pyralidae). Environ 9 Entomol. 26:46-60. 10 Godfrey LD, Holtzer TO, Spomer SM, Norman JM. 1991. European corn borer 11 (Lepidoptera: Pyralidae) tunneling and drought stress: effects on corn yield. J Econ 12 Entomol. 84(6):1850-1860. 13 Got B., Rodolphe F. 1989. Temperature-dependent model for European Corn borer 14 (Lepidoptera: Pyralidae) development. Environ Entomol. 18(1):85-93. 15 Lancashire PD, Bleiholder H, Longelüddcke P, Stauss R, Van Den Boom T, Weber E, 16 Witzenberger A. 1991. An uniform decimal code for growth stages of crops and 10 Mason CE, Rice ME, Calvin DD, Van Duyn JW, Showers WB, Hutchison WD, Witkowski 11 JF, Higgins RA, Onstad DW, Dively GP. 1996. European corn borer ecology and 12 management. North Central Regional Extension Publication 327. Iowa State University, 13 Ames, Iowa, 57 pp. 14 Munkvold GP, Hellmich, RL, Rice LG. 1999. Comparison of fumonisin concentrations in 15 kernels of transgenic Bt maize hybrids and nontransgenic hybrids. Plant Dis. 83:130-16 138. 17 Munkvold GP. 2003a. Cultural and genetic approaches to managing mycotoxins in maize. 18 Annu Rev Phytopathol. 41:99-116. 19 Munkvold G.P. 2003b. Epidemiology of Fusarium diseases and their mycotoxins in maize 21 Nelson PE, Toussoun TA, Marasas WFO. 1983. Fusarium species: an illustrated manual 22 for identification. Pennsylvania State University, University Park.

3 9 Saladini M, Blandino M, Reyneri A, Alma. 2008. The impact of insecticide treatments on 10 Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and their influence on the 11 mycotoxin contamination of maize kernels. Pest Manag Sci. 64:1170-1178. 12 Serna-Saldivar SO, Gomez MH, Rooney LW. 2001. Specialty corns, 2nd edition. Boca 13 Raton (Florida): A.R. Hallauer. Chapter 11, Food uses of regular and specialty corns 14 and their dry-milled fraction; p. 303-337. 15 Sobek EA, Munkvold GP. 1999. European Corn Borer (Lepidoptera: Pyralidae) Larvae as 16 vectors of Fusarium moniliforme, causing kernel rot and symptomless infection of 1 2 maize maturation.

table 4

 4 Main trial information and date of insecticide application in the field experiments conducted

	for details of

1

Table 2 .

 2 Total rainfall and accumulated growing degree days (GDD) from January to October 2006-

	2
	2007 in the research sites.

1

Table 3 .

 3 Parameters (u, B 0 and B 1 ) Z , coefficients of determination (R 2 ) and level of significance (P) 2 for the logistic model fitted to progress curves of ECB and fungal ear rot incidence on

	3
	4	accumulated growing degree-days.
						ECB incidence				Fungal ear rot incidence
		Year	Site	Treatment	A	b 0	b 1	R	2	P	A	b 0	b 1	R	2	P
		2006	A	NT	95.7	2.4*10 8	0.982	0.855	0.008	78.9	1.1*1012 0.9771	0.672	0.046
				T1	75.7	8.5*10 7	0.983	0.768	0.022	53.4	1.6*10 11 0.9794	0.674	0.045
				T2	59.0	3.8*10 10	0.980	0.754	0.025	36.8	5.7*10 10 0.9807	0.703	0.037
				T3	34.5	8.7*10 6	0.985	0.671	0.046	18.9	1.1*10 10 0.9822	0.586	0.076
				T4	57.9	2.7*10 9	0.983	0.724	0.032	52.3	2.4*10 8	0.9834	0.679	0.086
				T5	83.4	5.6*10 7	0.983	0.743	0.027	82.3	8.3*10 12 0.9768	0.865	0.022
				T6	86.8	6.4*10 7	0.982	0.742	0.027	77.8	4.7*1013 0.9746	0.862	0.023
				T7	95.7	2.4*10 8	0.982	0.855	0.008	71.2	4.5*1011 0.9786	0.729	0.066
			B	NT	93.4	1.4*10 10	0.980	0.880	0.006	52.3	5.8*10 10	0.980	0.967	0.003
				T1	75.7	7.4*10 9	0.980	0.896	0.004	29	1.1*10 12	0.979	0.760	0.045
				T2	42.3	7.7*10 10	0.979	0.960	0.001	16.8	1.6*10 11	0.981	0.758	0.048
				T3	37.8	1.4*10 10	0.980	0.938	0.001	12.3	3.3*10 9	0.983	0.967	0.003
				T4	33.4	4.5*10 9	0.981	0.948	0.001	15.7	1.9*10 9	0.983	0.897	0.015
				T5	55.6	4.1*10 9	0.980	0.812	0.014	37.9	4.8*10 9	0.982	0.867	0.021
				T6	75.6	4.1*10 10	0.978	0.926	0.002	40.1	2.4*10 10	0.981	0.969	0.002
				T7	86.7	8.8*10 11 0.9761	0.903	0.004	54.5	6.0*10 10 0.9791	0.980	0.001
		2007	A	NT	45.1	3.8*10 2	0.993	0.819	0.013	62.2	1.7*10 13	0.978	0.830	0.012
				T1	26.0	2.8*10 3	0.992	0.865	0.007	50.8	1.5*10 14	0.977	0.883	0.005
				T2	23.3	6.1*10 2	0.994	0.759	0.024	35.3	2.2*10 18	0.972	0.869	0.007
				T3	17.3	3.2*10 1	0.995	0.858	0.008	38.0	7.8*10 12	0.979	0.750	0.026
				T4	23.9	6.7*10 1	0.994	0.871	0.007	39.1	7.6*10 16	0.974	0.729	0.031
				T5	31.0	1.7*10 1	0.995	0.886	0.005	42.7	6.7*10 5	0.989	0.845	0.010
				T6	35.2	1.5*10 1	0.995	0.817	0.013	45.0	3.2*10 13	0.978	0.827	0.012
				T7	46.0	6.7*10 3	0.991	0.808	0.015	47.2	1.6*10 14	0.977	0.880	0.006
			B	NT	76.0	5.0*10 6	0.987	0.984	0.001	57.0	9.8*10 7	0.986	0.900	0.014
				T1	56.2	1.3*10 6	0.988	0.838	0.029	24.2	2.4*10 7	0.988	0.962	0.003
				T2	43.1	1.1*10 7	0.987	0.994	<0.001	24.3	5.3*10 6	0.989	0.897	0.014
				T3	44.0	5.2*10 6	0.988	0.899	0.014	18.1	1.2*10 23	0.968	0.874	0.020
				T4	25.9	3.8*10 4	0.991	0.890	0.016	14.9	5.0*10 15	0.977	0.783	0.046
				T5	31.2	4.3*10 3	0.992	0.903	0.013	22.4	1.6*10 15	0.977	0.789	0.045
				T6	43.0	2.7*10 5	0.990	0.882	0.018	52.0	1.4*10 17	0.975	0.796	0.049
	5			T7	55.8	1.2*10 7	0.987	0.963	0.003	55.3	2.9*10 17	0.974	0.805	0.046

1

Table 4 .

 4 Slopes, intercepts and coefficients of determination (R 2 ) and level of significance (P) of the 2 relationship between ECB and fungal ear rot severity (%) and accumulated growing degree-

	3
	4	days.
						ECB severity				Fungal ear rot severity
		Year	Site	Treatment	Slope	Intercept	R	2	P	Slope	Intercept	R	2	P
		2006	A	NT	0.016	-16.73	0.984	0.001	0.008	-9.31	0.869	0.021
				T1	0.008	-7.610	0.936	0.007	0.005	-5.93	0.971	0.003
				T2	0.005	-5.57	0.757	0.045	0.002	-1.92	0.694	0.046
				T3	0.002	-2.10	0.813	0.031	0.001	-1.12	0.897	0.019
				T4	0.004	-4.10	0.981	0.001	0.003	-3.25	0.727	0.031
				T5	0.010	-9.38	0.977	0.002	0.006	-6.47	0.965	0.003
				T6	0.011	-10.42	0.844	0.027	0.008	-8.89	0.890	0.019
				T7	0.013	-12.29	0.887	0.017	0.007	-6.77	0.867	0.022
			B	NT	0.024	-26.48	0.858	0.024	0.004	-5.26	0.899	0.026
				T1	0.012	-13.12	0.846	0.019	0.002	-2.73	0.791	0.043
				T2	0.006	-7.62	0.874	0.020	0.001	-1.03	0.751	0.047
				T3	0.005	-5.48	0.846	0.027	0.001	-0.92	0.734	0.064
				T4	0.004	-4.60	0.962	0.003	0.001	-1.61	0.679	0.039
				T5	0.008	-8.68	0.930	0.008	0.003	-3.29	0.908	0.012
				T6	0.015	-17.57	0.909	0.012	0.003	-3.73	0.7913	0.046
				T7	0.016	-18.27	0.983	0.001	0.004	-4.44	0.958	0.004
		2007	A	NT	0.005	-6.65	0.962	<0.001	0.014	-19.16	0.957	0.001
				T1	0.003	-3.26	0.928	0.002	0.009	-11.47	0.954	0.001
				T2	0.002	-2.05	0.717	0.044	0.006	-7.82	0.807	0.015
				T3	0.001	-1.33	0.928	0.002	0.006	-9.09	0.933	0.007
				T4	0.002	-1.54	0.916	0.003	0.007	-8.85	0.761	0.023
				T5	0.002	-1.81	0.991	<0.001	0.007	-8.47	0.784	0.019
				T6	0.003	-3.57	0.918	<0.001	0.009	-9.45	0.946	0.001
				T7	0.004	-4.47	0.965	<0.001	0.009	-11.81	0.905	0.003
			B	NT	0.015	-19.34	0.967	0.003	0.008	-12.16	0.948	0.006
				T1	0.011	-14.81	0.934	0.007	0.004	-5.40	0.931	0.008
				T2	0.008	-11.19	0.914	0.011	0.003	-4.00	0.905	0.013
				T3	0.007	-9.29	0.936	0.007	0.002	-2.41	0.944	0.006
				T4	0.004	-4.58	0.920	0.010	0.001	-1.65	0.855	0.025
				T5	0.004	-5.50	0.883	0.018	0.002	-2.95	0.994	<0.001
				T6	0.011	-15.05	0.841	0.028	0.006	-7.81	0.883	0.018
				T7	0.011	-14.00	0.814	0.036	0.008	-10.94	0.946	0.005

1

Table 5 .

 5 Effect of the timing of the insecticide application on ECB incidence and severity, fungal ear 2 rot incidence and severity at harvest and fumonisin (sum of B 1 +B 2 ) concentration.

	3
	Year	Site		ECB incidence U	ECB severity V	Fungal ear rot	Fungal ear rot severity Y	Fumonisin B 1 + B 2
				(%)	(%)	(%)	(%)	T	N (µg kg -1 )
	2006	A	NT	72.0 a	21.7 a	62.9 a	12.5 a	9.1 a	9330
			T1	63.9 a	18.1 ab	51.0 abc	7.3 bc	7.9 c	2830
			T2	59.2 a	16.7 abc	46.0 abc	6.4 bc	7.7 c	2235
			T3	41.1 b	11.6 c	30.2 c	4.7 c	7.1 d	1170
			T4	42.1 b	13.4 bc	38.2 bc	6.4 bc	6.9 d	970
			T5	63.7 a	16.2 abc	55.3 ab	8.5 abc	8.0 bc	3090
			T6	71.5 a	18.9 ab	62.5 a	9.0 abc	8.4 b	4420
			T7	71.8 a	19.6 ab	61.8 a	9.9 ab	8.6 b	5513
			P (F)	< 0.001	0.001			< 0.001
			SEM Z	2.7	2.7	0.8	0.6	0.2
		B	NT	85.0 a	23.2 a	53.8 a	10.5 a	8.1 a	3150
			T1	66.4 a	16.0 abcd	45.1 abc	7.1 bc	6.9 c	950
			T2	46.5 b	11.7 cd	35.3 bc	5.0 cd	6.0 d	400
			T3	42.3 b	10.1 d	27.3 c	3.7 d	4.8 d	120
			T4	46.0 b	10.4 d	28.1 c	4.9 cd	5.5 d	265
			T5	44.6 b	12.6 cbd	35.2 bc	6.5 bcd	7.1 c	1200
			T6	72.1 a	18.4 abc	48.0 ab	7.2 bc	7.3 bc	1435
			T7	74.8 a	20.1 ab	50.0 ab	8.8 ab	7.7 ab	2130
			P (F)	< 0.001	< 0.001	0.001	< 0.001	< 0.001
			SEM Z	3.5	2.3	1.1	0.5	0.2
	2007	A	NT	42.2 a	14.2 a	50.1 a	13.2 a	8.8 a	6970
			T1	39.3 a	13.6 a	42.1 b	10.8 ab	7.9 b	2790
			T2	24.4 bc	9.1 b	28.3 cd	6.0 c	7.3 c	1530
			T3	18.3 c	4.5 d	12.6 e	2.3 d	6.3 d	540
			T4	18.4 bc	5.2 cd	22.4 d	5.5 c	7.0 c	1160
			T5	28.0 bc	7.0 bc	36.6 bc	9.1 b	7.3 c	1480
			T6	28.4 b	8.4 b	34.8 bc	9.3 b	8.1 b	3380
			T7	40.6 a	13.0 a	42.2 b	12.7 a	8.9 a	7830
			P (F)	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
			SEM Z	2.0	2.4	0.8	0.8	0.2
		B	NT	61.7 a	19.9 a	60.2 a	14.6 a	6.8 a	940
			T1	45.2 c	15.5 b	45.7 b	11.4 bc	6.0 b	400
			T2	42.4 c	15.3 b	42.4 bc	9.5 cd	3.7 c	40
			T3	43.6 c	11.7 c	37.6 c	6.6 e	3.7 c	40
			T4	24.6 d	9.4 c	26.3 d	6.0 e	4.2 c	70
			T5	43.7 c	14.5 b	40.4 bc	8.7 d	5.7 b	290
			T6	54.2 b	14.5 b	55.3 a	11.9 b	6.6 a	700
			T7	60.2 a	18.2 a	55.6 a	12.8 b	6.6 a	755
			P (F)	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
			SEM Z	2.4	2.2	0.7	0.6	0.3

1

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Date of first ECB adult capture July 4 July 6 July 3 July 7 Date of ECB flight peak July 24 July 25 July 23 July 27 Trapping count at ECB flight peak

Acknowledgements

5

The authors would like to thank Fabio Carnaroglio, Alessandro Peila and Mattia Ciro

Year

Site A Site B Site A Site B T1 June 30 June 27 June 26 June 26 T2 July 6 July 5 July 3 July 3 T3 July 13 July 12 July 10 July 10 T4 July 20 July 19 July 16 July 17 T5 July 27 July 26 July 22 July 26 T6 Aug. 3 Aug. 1 July 30 July 31 T7 Aug. 9 Aug. 8 Aug. 7 Aug. 8