Aurélie Docquier

Pierre-Olivier Harmand

Samuel Fritsch

Maia Chanrion

Jean-Marie Darbon

Vincent Cavailles
email: cavailles<vincent.cavailles@inserm.fr

The transcriptional coregulator RIP140 represses E2F1 activity and discriminates breast cancer subtypes Aur lie Docquier

Keywords: Breast Neoplasms, genetics, pathology, Cell Cycle, Cell Line, Tumor, E2F1 Transcription Factor, antagonists & inhibitors, Female, Gene Expression Regulation, Neoplastic, Humans, Neoplasms, Basal Cell, genetics, Nuclear Receptor Co-Repressor 1, physiology, Transcriptional Activation, Transfection breast cancer, cell proliferation, E2F1, RIP140, transcription

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The transcriptional coregulator RIP140 represses E2F1 activity and discriminates breast cancer subtypes.

INTRODUCTION

Cell cycle control is a fundamental process that governs cell proliferation and is frequently altered during tumorigenesis. E2Fs and their heterodimer partners, DPs, are central regulators of cell cycle progression and directly regulate the expression of a broad spectrum of genes involved for instance, in cell cycle regulation, DNA replication and repair, apoptosis, differentiation or development , .

[1 2 ] E2F1, which was discovered as a protein promoting the transition to S phase, was the founding member of the E2F family which comprises eight members in mammals. Among this family, some were initially presented as activator E2Fs (E2F1, 2, and 3) while the " "

other members were mostly known as transcription repressors although this classification now appeared too simplistic ( and reference [START_REF] Degregori | Distinct and Overlapping Roles for E2F Family Members in Transcription, Proliferation and Apoptosis[END_REF] herein). E2F transcriptional activity was shown to be regulated by a large number of coactivators or corepressors including the so-called pocket proteins which form the retinoblastoma (RB) tumor suppressor family (pRB, together with the related proteins p107 and p130) .

[3 ]

RB attenuates E2F action by recruiting transcriptional corepressors such as histone deacetylases (HDACs) to E2F-regulated promoters, thus mediating transcriptional repression of E2F-regulated genes , . RB is a critical component of the cell cycle control machinery [4 5 ] and, as a consequence, its loss or inactivation is a major mechanism by which cancer cells attain a growth advantage during tumorigenesis . [START_REF] Knudsen | Retinoblastoma tumor suppressor: where cancer meets the cell cycle[END_REF] Our laboratory is engaged in the characterization of various transcriptional repressors which regulate another important class of transcription factors, namely nuclear hormone receptors. These receptors such as the estrogen and androgen receptors are also important regulators of cell proliferation and strongly influence the growth of hormone-dependent cancers . These receptors control gene [START_REF] Ko | Targeting steroid hormone receptor pathways in the treatment of hormone dependent cancers[END_REF] expression through the recruitment of a large set of coregulatory proteins which regulate, either positively or negatively, chromatin structure and transcription initiation. Our work is mainly focused on RIP140 (Receptor Interacting Protein of 140 kDa also known as NRIP1), a nuclear protein of 1158 amino acids, initially identified as a transcription cofactor of estrogen receptors and shown to regulate energy homeostasis in metabolic tissues (for a review see ). RIP140 is an atypical coregulator since, despite its recruitment by [START_REF] Augereau | The nuclear receptor transcriptional coregulator RIP140[END_REF] agonist-liganded nuclear receptors, it exhibits a strong transcriptional repressive activity. We and others have deciphered the molecular mechanisms involved in this transrepression and identified four repressive domains within the RIP140 molecule . We also

[9 ] [START_REF] Christian | Characterization of four autonomous repression domains in the corepressor receptor interacting protein 140[END_REF] demonstrated that RIP140 expression was increased upon estrogen or androgen stimulation, in breast and prostate cancer cells, [11 ] [12 ] respectively.

The present study identified RIP140 as a novel transcriptional repressor of E2F1 activity which significantly decreased E2F1-target gene expression both on transfected reporter constructs and on endogenous genes. In human breast cancers, a decrease in RIP140 expression is inversely correlated with E2F1-target gene expression and significantly associated with basal-like tumors which exhibit bad prognosis. Altogether this work identifies RIP140 as a new key actor of the E2F pathway and as a potential new prognostic marker in oncology.

MATERIALS AND METHODS

Plasmids and reagents

The . The 17M5 Glob-Luc construct and plasmids allowing RIP140 expression , , have been described previously. The 13 ] β [9 11 12 ] pEGFP-C2-RIP140 vector was a kind gift of Dr J. Zilliacus . The deletion of the E2F interaction domain in the RIP140 sequence [START_REF] Zilliacus | Regulation of glucocorticoid receptor activity by 14--3-3-dependent intracellular relocalization of the corepressor RIP140[END_REF] (from residues 119 to 199) was done using the QuickChange XL from Stratagene. The pRL-CMVbis plasmid (Ozyme, Saint Quentin Yvelines, France) was used to normalize transfection efficiency.

Cell culture, RNA extraction and quantitative PCR

HeLa, MCF-7 and HEK293T human cancer cell lines were cultured as previously described . Total RNA was extracted from cells

[9 ]
using the TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA (2 g) was subjected to a reverse-transcription step using the μ Superscript II reverse transcriptase (Invitrogen). Real-time quantitative polymerase chain reaction was performed using a SYBR Green approach (LightCycler, Roche Diagnostics, Meylan, France). Primer sequences are available upon request. For each sample, results were corrected for RS9 mRNA levels (reference gene) and were normalized to a calibrator sample.

Transient transfection, luciferase assays and cell cycle analysis

MCF-7 cells were plated in 96-well plates (2.10 cells per well) 24h prior to DNA transfection with Jet-PEI (0.25 g of total DNA).

interaction assay and coimmunoprecipitation In vitro

translation and GST pull-down assays were performed as previously described . For coimmunoprecipitations, expression In vitro [9 ] plasmids for E2F1 or c-myc-tagged RIP140 were transfected in HeLa cells using JetPEI (Ozyme, Saint Quentin Yvelines, France). After cell lysis in 50 mM Tris-HCl, pH8, 0.5 Nonidet P-40 supplemented with protease inhibitors, Transfected RIP140 and E2F1 were % immunoprecipitated with the 9E10 monoclonal antibodies against the c-myc epitope or with the anti-E2F1 antibody (C-20) covalently bound to protein G-Sepharose beads. After incubation at 4 C during 2 h and 5 washes, immunoprecipitated proteins were eluted in °Laemmli sample buffer, resolved by SDS PAGE and detected by western blotting using primary antibodies against E2F or c-myc epitope.

-

Interaction of endogenous proteins (coimmunoprecipitation and ChIP analysis)

For coimmunoprecipitation of endogenous proteins, 700 g of MCF-7 cell nuclear extracts (prepared using the NE-PER kit from μ Thermo Scientific) were incubated with 2 g of anti-E2F1 monoclonal antibody (KH95 sc-251 Santa Cruz) for 3 hours at room μ temperature. Beads coupled to Protein G (Ademtech G0433) were added to the immune complex (2 hrs at room temperature) and after 3 washes with lysis buffer, beads were resuspended in 20 l of lysis buffer and analysed by western blotting using primary antibodies μ specific for E2F1 (KH95 sc-251 Santa Cruz) and RIP140 2656C6a (sc-81370 Santa Cruz). For ChIP analysis, MCF-7 cells (70% confluent) were synchronized using 4mM hydroxyurea (HU) during 24h and the block was released by changing the medium with 10% FCS supplementation for the indicated time. After PBS washing and cross-linking with 3, 7 formaldehyde during 10 min at 37 C, we % ° used the ChampionChIP One-Day Kit (SABiosciences) according to the manufacturer s recommendations and using either the antibody ' KH95 (sc-251 SantaCruz) or 2656C6a (sc-81370 Santa-Cruz) for E2F1 and RIP140 respectively, or no antibody as a control. Q-PCR was then performed using the Power SYBR Green PCR master mix (Applied Biosystems) on an Applied Biosystems 7300 thermal cycler with 2 l of material per point. Primers flanking the E2F site of the cyclin D1 promoter were 5 -GCAGCGGGGCGATTTGCATT-3 and 5

μ ′ ′ ′
-AGCAAAGATCAAAGCCCGGCAGAG-3 . The input DNA fraction corresponded to 1 of the immunoprecipitation.

′ %

Microarray analysis

Microarray data (accession number GSE1992) of the study from Hu et al.

were obtained from the Gene Expression Omnibus [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF] (GEO) database ( ). Expression data from the 170 sample experiment were downloaded as http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi normalised and log2 transformed Cy5/Cy3 ratios, where tumor sample RNA and human universal reference RNA were labeled with Cy5 and Cy3, respectively. Hierarchical pairwise average-linkage clustering of the 170 tumor specimens was performed on the basis of expression of RIP140 and 6 E2F1-target genes using the Cluster and TreeView software with median-centered gene expression values and Pearson correlation as similarity metrics. Results were analyzed for statistical significance using the 2-tailed Student s test. For all ' t analyses, < 0.05 was considered as significant. p

RESULTS

RIP140 interacts with E2F1

Based on published data reporting that nuclear receptor coregulators were involved in the regulation of E2F1 activity , we

[16 -18 ]
hypothesized that RIP140 might also act as a transcriptional modulator of the E2F pathway.

Using GST pull-down assays, we first investigated whether RIP140 was able to interact with E2F1. We therefore performed in vitro pull-down assays with GST-E2F1 and labeled full-length RIP140. As shown in (left panel), data clearly demonstrated in vitro Figure 1B the binding of RIP140 to E2F1. We then tried to delineate the respective binding sites on the two proteins. The use of deletion mutants of E2F1 and RIP140 corresponding to the N-terminal or the C-terminal moiety of the two proteins (fused to GST for E2F1 and in vitro translated for RIP140) suggested the presence of at least two interaction domains on each protein ( right panel). Indeed, the two Figure 1B translated fragments of RIP140 (fragment 1 to 480 and 480 to 1158) were retained by the GST-E2F1 chimaeric proteins in vitro encompassing regions from residues 1 to 123 or 123 to 437.

In order to further map the respective binding sites on RIP140, we used deletion mutants of RIP140 fused to GST. Data shown in (left panel) indicated that the N-terminal region of RIP140 encompassing residues 27 to 439 exhibited the strongest binding of Figure 1C translated full-length E2F1. No binding at all was observed with the central region of RIP140 whereas a faint interaction was in vitro detectable upon long exposure with the C-terminal region (residues 683 to 1158). This E2F1 binding region was clearly confirmed when we used the translated fragment of E2F1 corresponding to residues 1 to 123 ( , left panel). in vitro Figure 1C Moreover, using a series of deletion mutants in the N-and C-terminal moieties of RIP140, we showed that the minimal N-terminal E2F1 interaction domain (EID1) encompassed a region of 80 amino acids spanning from residues 119 to 199 whereas EID2 was mapped to a region spanning from residues 916 to 1158 ( and , right panel). Finally, an E2F1 mutant lacking the Figure 1A Figure 1C transactivation domain which encompasses residues 380 437 was significantly less efficient (more than 7-fold decrease) than wild-type -E2F1. This region corresponds to the pRB interaction domain which is strongly impaired by the Y411H mutation . Very interestingly, [19 ] this Y411H E2F1 mutant was also less efficient to interact with RIP140 ( ), suggesting that the C-terminal RIP140 binding site Figure 1D on E2F1 might overlap that of pRB.

To demonstrate that this interaction between RIP140 and E2F1 also occurred in intact cells, we then set up co-immunoprecipitation experiments. As illustrated in (top panel), RIP140 was found associated with immunoprecipitated E2F1 and the reverse Figure 2A experiment confirmed the interaction between the two proteins (bottom panel). All controls performed with the sole expression of one partner, or with the use of beads alone confirmed the specificity of the interactions. In order to emphasize these results, we then analyzed the association between endogenous proteins in MCF7 breast cancer cells. Coimmunoprecipitation experiments demonstrated that E2F1 was able to specifically pull-down endogenous RIP140 (

). Finally, chromatin immunoprecipitation (ChIP) analysis was Figure 2B performed in MCF-7 cells on E2F binding sites in the cyclin D1 promoter which is transcriptionally repressed by E2F1

. Data [START_REF] Watanabe | Inhibition of cyclin D1 kinase activity is associated with E2F-mediated inhibition of cyclin D1 promoter activity through E2F and Sp1[END_REF] indicated a concomitant increase in E2F1 and RIP140 recruitment upon G1/S transition ( ). Altogether, these results illustrated Figure 2C the interaction between endogenous RIP140 and E2F1, and binding of RIP140 on an E2F1 target promoter, thus strengthening the data presented in . Figure 1

RIP140 inhibits E2F1 transactivation

We next tested the ability of RIP140 to control E2F1 transactivation in transient transfection experiments using expression vectors for both E2F1 and DP1, together with different luciferase reporter vectors known to be regulated by E2Fs. We first used an artificial reporter RIP140 controls E2F1 activity Clin Cancer Res . Author manuscript Page / 4 12 plasmid containing three copies of the E2F binding site upstream the thymidine kinase promoter (E2F -TK-Luc). As shown in

(left panel), when transfected in MCF-7 breast cancer cells, we observed a significant inhibition of E2F1 activity upon overexpression of RIP140. Similar inhibition of E2F1 transactivation by RIP140 (ranging from 4-to 8-fold) was obtained on two other reporter constructs corresponding to the natural cyclin E or ARF promoters ( , middle and right panels, respectively) and, as shown in , Figure 3A Figure 3B the cyclin E promoter was dose-dependently repressed by RIP140. Moreover, the same repressive effect of RIP140 on E2F1 was observed upon transient transfection of other human cancer cell lines such as HeLa or HEK293T (data not shown) and was not restricted to E2F1.

Indeed, we found that the two other activator E2Fs E2F2 and E2F3 not only interacted with RIP140 but were similarly i.e. in vitro inhibited upon RIP140 overexpression (see supplementary Figure S2).

In order to further characterize the mechanism of this inhibitory effect, we used plasmids allowing the expression of Gal4-E2F1 chimeric proteins fusing the DNA-binding domain of the Gal4 yeast transcription factor to the COOH-terminal moiety of E2F1 (amino acids 123 to 437 or 380 to 437 which both contain the transactivation domain). In these conditions, we obtained a similar dose-dependent transcriptional repression upon RIP140 overexpression ( ), suggesting that this effect was not due to a regulation of the binding Figure 3C of E2F1 to DNA or to its heterodimerization partners (DP proteins). Finally, we compared, the effect of RIP140 to that of the pocket protein pRB, and found that, in our experimental conditions, E2F1 activity on the cyclin E promoter was repressed to comparable levels although the residual transactivation by E2F1 (compared to basal level) was higher with RIP140 than with pRB ( left panel). On Figure 3D the Gal-E2F1(380 437) chimaeric protein, the effect of RIP140 was slightly less efficient than that of pRB probably reflecting the loss of the N-terminal binding site for RIP140 (see above). Altogether, these results demonstrated that RIP140 was able to interact with E2F1 and to repress its transcriptional activity.

RIP140 decreases cell cycle progression and regulates E2F-target gene expression

Having demonstrated that RIP140 was a novel repressor of E2F1 activity, we sought to directly address whether this regulation might impact cell proliferation. We first examined the effect of RIP140 overexpression on cell cycle distribution in various human cancer cell lines. HeLa cells were transfected with a GFP-RIP140 expression vector and two populations overexpressing (GFP-RIP ) or not (GFP-RIP + ) RIP140 were separated and analyzed for cell cycle distribution by FACS. i.e. MCF-7 breast cancer cells and HEK293T transformed embryonic kidney cells ( right panel), thus suggesting that this effect of Figure 4A RIP140 could represent a general feature. Controls corresponding to cells transfected with GFP alone and sorted as performed for GFP-RIP cells (data not shown) indicated that the effect was indeed due to the overexpression of RIP140.

+

Since the data shown in suggested that the NH2-terminal EID1 (residues 119 to 199) was the strongest binding site for Figure 1 in translated full-length E2F1, we generated the corresponding RIP140 mutant ( EID1 deleted from amino acids 119 to 199) as a fusion vitro Δ with GFP. In transient transfection experiments, we found that this EID1 mutant was significantly less efficient (p<0.01) to repress Δ E2F1/DP1 transactivation on the cyclin E promoter (

). This effect was specific of E2F transactivation since wild-type RIP140 Figure 4B and the EID1 deletion mutant inhibited estrogen receptor transcriptional activity to the same extend (data not shown). We also analyzed Δ the effect of the EID1 mutant on cell cycle ( ). We found that its overexpression in MCF7cells led to an increase in the Δ

proportion of cells in G1 (as compared to GFP alone) and this increase was significantly lower (p<0.01) than that obtained with the wildtype GFP-RIP40 vector. These results thus indicated that deletion of the main E2F1 interacting domain significantly impaired RIP140 ability to repress E2F transactivation and to block cell cycle progression.

In an attempt to explain the growth effect associated with RIP140 expression, we then analyzed by quantitative RT PCR, the steady-state levels of various mRNAs known to be transcribed from E2F-target genes . As shown in , we first noticed that in [START_REF] Degregori | The genetics of the E2F family of transcription factors: shared functions and unique roles[END_REF] Figure 4D cells overexpressing the GFP-RIP140 expression vector, the mRNA levels of CCNE, CCNB2, CDC2 and CDC6 were strongly decreased as compared to cells which did not overexpressed GFPRIP140. The negative regulation was however not general for all E2F-traget genes since for instance, the DHFR mRNA levels were not significantly regulated ( ). Altogether, these results indicated that RIP140 Figure 4C controls cell cycle progression and regulates endogenous E2F-target gene expression.

Inverse correlation between RIP140 and E2F1-target genes in human breast cancers

To determine the biological relevance of E2F control by RIP140 and to validate the regulation by RIP140 of some of the E2F1-target genes in human cancers, we analyzed the expression of RIP140 and E2F1-target genes on a tumor microarray data set representing 170 breast cancer patients . We first selected among 16 known E2F1-target genes shown to be related to tumor proliferation, those [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF] presenting the most pronounced differential expression in tumors with low levels of RIP140 tumors with high levels of RIP140 (the two vs groups including tumors with log2 RIP140 expression values lower and upper than 0, respectively). As shown in , six genes ( Figure 5A and

) which presented the most important and significant differences in expression ratio CCNE1, MYBL2, BIRC5, E2F1, CCNB2 CDC6

between tumors with low and high RIP140 mRNA levels were selected for clustering analysis. It should be noted that three of these genes and were found regulated by RIP140 in MCF-7 cells ( ). i.e. CCNE1, CDC6 CCNB2 Figure 4C The gene expression signature comprising RIP140 and the 6 selected E2F1-target genes was used to cluster the 170-point data set and is displayed as a condition tree in . This map clearly showed two regions of gene coregulation, low levels of RIP140 mRNA Figure 5B being associated with high levels of the E2F1-target genes whereas in tumors expressing high levels of RIP140 mRNA, the E2F1-target genes were underexpressed. As shown in , the mean expression levels of the six E2F1-target genes were significantly lower (p Figure 5C = 1.5E-20) in the group of tumors with high levels of RIP140. By contrast, although RIP140 is a known estrogen-target gene, the median expression of ESR1 was not significantly higher in tumors expressing high low levels of RIP140 mRNA (respectively 0.41 and 0.29, vs p>0.05). Together, these data showing that RIP140-deficiency is inversely correlated with a signature of E2F1-target genes in human breast cancer, thus strongly strengthen our results. in vitro Low RIP140 mRNA levels are associated with basal-like human breast cancers " "

Human breast tumors are diverse in their natural history and their responsiveness to treatments. Variations in transcriptional programs account for much of the biological and clinical heterogeneity of breast cancers. Using hierarchical clustering of gene expression patterns, human breast tumors have been classified into five distinct subtypes arising from at least two distinct cell types (basal and luminal epithelial cells) and associated with significant differences in clinical outcome . Using the tumor microarray data set from Hu [START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF] et al. [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF] , we investigated how RIP140 could discriminate between these molecular subtypes and whether the regulation of E2F1-target genes by ] RIP140 could be observed in these distinct subclasses. We anticipated that it could be the case since a proliferation gene cluster including E2F1-target genes has been shown to be differentially expressed through the breast tumor subtypes and reported as a hallmark of a [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF] series of prognosis molecular signatures , ;

. The 170 tumors were ranked according to RIP140 gene expression and divided [22 23 ] [24 ] into two equal groups of 85 tumors with low and high RIP140 expression levels, respectively. As expected, the E2F1-target metagene expression almost mirrored the RIP140 mRNA levels in these two tumor groups (

). Interestingly, as shown in , the Figure 6A Figure 6B group of tumors with the lowest levels of RIP140 gene expression (left panel) included 87.5 of the tumors identified as basal-like tumors % (black box) and only 17.9 of those diagnosed as luminal-like tumors (white box). By contrast, the group with high levels of RIP140 % mRNA (right panel) contained 82.1 of the luminal-subtype tumors and only 12.5 of the basal-subtype tumors. It should be noted that % % 75.2 of the tumors included in this high RIP140 expression group belonged to the luminal subtype while only 5.9 were classified as

% %
basal-like tumors. In agreement with the fact that the low and high RIP140 expression groups exhibited opposite contents of luminal-and basal-like tumors, we found that basal-like tumors express low RIP140 and high E2F1-target metagene levels, respectively. By contrast, those identified as luminal-like tumors expressed high RIP140 and low E2F1-target metagene levels, respectively ( ). Indeed, the Figure 6C RIP140 gene expression was 3.1-fold higher in luminal-than in basal-like tumors while the E2F1-target metagene level was 2.1-fold higher in basal-than in luminal-like tumors. Using the same data, we also analyzed the partition of luminal-like and basal-like tumors according to the expression of the gene or those of the E2F co-activators NCOA3, NCOA6, CBP and PCAF. Interestingly, variations RB of RIP140 expression were the most powerful to discriminate between luminal-and basal-like tumors in this cohort (Figure S1).

Altogether, these data suggested that, in breast cancers, low RIP140 mRNA expression was associated with high E2F1-target gene levels and basal-like tumors.

DISCUSSION

RIP140 was initially identified as a transcriptional repressor of ligand-activated nuclear hormone receptor, involved in the control of ovarian functions and metabolic pathways (for a review, see

). In the present study, we identified RIP140 as a novel transcriptional [START_REF] Christian | Metabolic regulation by the nuclear receptor corepressor RIP140[END_REF] repressor of E2F1 and as a new important regulator of cell proliferation. Based on both protein-protein interaction assays, in vitro coimmunoprecipitation and ChIP experiments, our data clearly demonstrate that RIP140 is an E2F1 partner. Transcriptional repression of E2F1 activity by RIP140 was observed in transient transfections on various E2F-target promoters and strong inverse correlations between RIP140 and E2F-target genes were noted upon overexpression of RIP140 in breast cancer cells. More interestingly, the same observation ( low levels of RIP140 associated with high expression of E2F1 targets) was made using data obtained on a set of 170 human breast i.e. cancer samples.

Other nuclear receptor cofactors were previously reported as E2F regulators. However, most of these studies dealt with transcriptional activators such as CBP , PCAF or more recently ASC-2/NCOA6 or AIB1/NCOA3 . RIP140, acting as a negative [START_REF] Morris | Regulation of E2F transcription by cyclin E-Cdk2 kinase mediated through p300/CBP co-activators[END_REF] [16 ] [START_REF] Kong | Interaction and functional cooperation of the cancer-amplified transcriptional coactivator activating signal cointegrator-2 and E2F-1 in cell proliferation[END_REF] [18 ]

regulator of E2F1, thus appears to act similarly to the well-known pocket proteins although no structural similarities could be detected [START_REF] Du | Retinoblastoma family genes[END_REF] between RIP140 and pRB, p107 or p130. It has been shown that pocket proteins exhibit some specificity towards E2F family members.

Indeed, pRB targets preferentially the activator E2Fs (E2F1, 2 and 3), whereas p107 and p130 are involved in the regulation of E2F4 and

E2F5

. We are currently trying to determine whether RIP140 exhibits a similar specificity amongst E2F transcription factors and our [START_REF] Iaquinta | Life and death decisions by the E2F transcription factors[END_REF] preliminary results indicate that RIP140 is not specific for E2F1, E2F2 and E2F3 and also interacts with repressor E2Fs (Figure S2).

Very interestingly, our data also identified RIP140 as a new regulator of cell proliferation with a significant impact on cell cycle progression since transient overexpression of RIP140 significantly decreased the number of cells in S phase. The function of RIP140 in cell proliferation was supported by the observation that DNA synthesis (assessed by incorporation of 5-ethynyl-2 -deoxyuridine) was ′ significantly increased in tissues from RIP140 knock-out mice as compared to wild-type littermates (M. Lapierre, unpublished data).

[27 ]

Moreover, in addition to their roles on cell cycle and cell proliferation, E2F1 and/or RB are also key players in the control of major biological processes such as apoptosis or differentiation and deregulation of this pathway is of major relevance in tumorigenesis , .

[1 3 ] Experiments are therefore currently in progress in our laboratory to evaluate the relevance of RIP140 on the control of these parameters and more globally to determine whether RIP140 loss has a direct impact on cancer formation. Based on the data published on pocket proteins, this might not be obvious since, although mice are prone to develop pituitary and thyroid tumors , no increase in RB / -+ [START_REF] Jacks | Effects of an Rb mutation in the mouse[END_REF] tumor formation upon p107 or p130 gene invalidation was observed, except in an background . In addition, several studies RB / -+ [3 ][29 ] have reported that deregulation of the E2F signaling pathway could be linked to antiestrogen resistance in breast cancers . It will be [START_REF] Bosco | The retinoblastoma tumor suppressor modifies the therapeutic response of breast cancer[END_REF] therefore very important to investigate whether RIP140, through its effects on E2F signaling, could be involved in resistance of mammary tumors to antiestrogen therapy. markers for predicting relapse in breast cancer. Interestingly, when we analyzed published transcriptomic data obtained on human breast tumors , we clearly demonstrated that RIP140 mRNA levels discriminate between the different cancer subtypes classified as basal-or [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF] luminal-like based on the basis of molecular profiling. Our results indicate that low RIP140 mRNA levels correlate with basal-like tumors whereas those expressing high levels of RIP140 mRNA are mostly luminal-like (see and S1). Figure 6 These data therefore suggest that RIP140 may help to improve molecular signatures used to classify breast cancers. However, further work is needed to assess the association of RIP140 expression with clinical outcome and to determine the relative contribution of this gene as compared to the different markers previously identified.

Ackowledgements:

Grant support: This work was supported by the Institut National de la Sant et de la Recherche M dicale , the University of Montpellier I, Footnotes: The 170 samples from (A) Differential statistical analyses between 2 groups of tumor specimens defined by RIP140 expression values. the Hu study were separated into two groups according to their RIP140 expression levels. Fifty-five (low RIP140 expression) and et al [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF] 115 (high RIP140 expression) tumors exhibited log 2-transformed RIP140 expression values lower and upper than 0, respectively. The median expression of each of 16 E2F1-target genes was determined within each of the 2 groups and the ratios of the respective median values were calculated.

. Six (B) Hierarchical clustering of the 170 samples on the basis of expression of RIP140 and E2F1-target genes E2F1-target genes with expression ratios 1.45 between high and low RIP140 expression tumor groups were selected (boxed in panel A) and were ranked according to RIP140 gene expression and divided into two equal groups (85 tumors) exhibiting low and high RIP140 [START_REF] Hu | The molecular portraits of breast tumors are conserved across microarray platforms[END_REF] expression, respectively. Expression values for RIP140 and E2F1-target metagene (defined as the mean expression of the 6 E2F1-target genes selected in ) are expressed as means SE and -values (Student s test) are indicated.

  E2F1 and DP1 expression vectors were given by Dr C. Sardet (IGMM, Montpellier, France), the E2F(3)TK-Luc and cyclin E-Luc reporter plasmids by Dr L. Fajas (IRCM, Montpellier) and the pGL2-ARF-Luc construct ( 735 to 75) by Dr S-Y Shieh (Taipei, Taiwan) -+ [

4 μ

 4 Luciferase (firefly and renilla) values from transient transfection were measured and all data were expressed as mean SD. For cell [9 ] ± cycle analysis, cells were transfected with the RIP-GFP expression plasmid and the two populations (RIP-GFP and RIP-GFP ) were -+ separated. Cell cycle was analyzed with a FACs-Vatange flow cytometer (Becton-Dickinson, San Jose, CA) after propidium iodide labeling. The CellQuest and ModFit softwares were used to analyze data.

- 1 (

 1 As illustrated in (left panel), 48 hr after transfection, GFP-RIP cells (which overexpress RIP140) showed a strong and Figure4A+ significant decrease in the S-phase cell population (from 22.1 to 10.1 ) with a concomitant increase in the number of G -phase cells % % from 65.7 to 86.3 ). Interestingly, a similar decrease in the fraction of S-phase cells was observed in two other human cell lines % %

  du Cancer , the Ligue Nationale contre le Cancer , the Association pour la Recherche sur le Cancer and the Fondation Lejeune . P.-O. H. and S. F. were recipients of fellowships from the Ligue Nationale contre le Cancer . We are grateful to Dr Claude Sardet for plasmids, to Sandrine Bonnet for technical assistance and to Drs St phan Jalaguier and Patrick Augereau for critical reading of the é manuscript.

Figure 1 Figure 3

 13 Figure 1 RIP140 binds to E2F1 The figure shows the four repressive domains (RD) and the two-E2F1 interaction (A) Schematic representation of the RIP140 molecule. domains (EID) mapped on RIP140. GST pull-down assays were carried out as (B and C) interaction between RIP140 and E2F1. In vitro described in using GST or GST-E2F1 with S-labelled wild-type RIP140 (B) or GST-RIP140 mutant proteins to retain Material and Methods 35 S-labelled wild-type E2F1 (C) Inputs represent 25 of the material used in the assays.GST pull-down assays carried out with the 35

Figure 4 .

 4 Figure 4 RIP140 controls cell cycle progression . Cells were transfected by pEGFP-C2-RIP140 plasmid and analyzed by flow cytometry (A) Effect of RIP140 over-expression on cell cycle as described in Left panel shows an experiment performed in HeLa cells with the profiles of relative iodide Materials and Methods. fluorescence versus the number of cells for each population (GFP-RIP GFP-RIP ). In the right panel, the breakdown of the population + vs according to cell cycle phase is indicated for each condition (mean of 3 independent experiments). (B and C) Deletion of the N-terminal (B). MCF-7 cells were transiently transfected with E2F1 and DP1 together with the cyclinE-Luc and expression E2F1 interaction domain. vectors for wild-type RIP140 or the EID1 mutant. (C) Analysis of the proportion of MCF-7 cells in G1 after transfection of expression Δ vectors for wild-type or EID1 mutated RIP140. Statistical analysis were performed using the Mann-Whitney test ( p< 0.01).Δ **

Figure 5

 5 Figure 5Inverse correlation between RIP140 and E2F1-target gene expression in breast cancerThe 170 samples from (A) Differential statistical analyses between 2 groups of tumor specimens defined by RIP140 expression values. the Hu study were separated into two groups according to their RIP140 expression levels. Fifty-five (low RIP140 expression) and et al

≥

  used for cluster analysis of the 170 tumor specimens. (C) Boxplot analyses of the expression values for RIP140 and E2F1-target. The expression value of the E2F1-target metagene was defined in each tumor metagene into the 2 groups defined by RIP140 expression as the mean expression of the 6 E2F1-target genes selected in (A). RIP140 and metagene expression values were visualised for the low and high RIP140 tumor groups using boxplots.

Figure 6

 6 Figure 6 RIP140 expression discriminates luminal and basal breast cancer subtypes The 170 breast tumors of the study from Hu (A) E2F1-target metagene expression in the low and high RIP140 expression tumor groups. et al.were ranked according to RIP140 gene expression and divided into two equal groups (85 tumors) exhibiting low and high RIP140

.

  For the two groups of tumors defined in (A) and expressing low and high RIP140 expression levels and molecular breast tumor subtypes mRNA (left and right panel, respectively), luminal-like (white), basal-like (black), ERBB2 (left-hatched), normal-like (grey) and + unclassified (right-hatched) tumors are indicated. The statistical significance was assessed using a Chi2 analysis ( 3.9E-13). p = (C) RIP140 . The expression values of RIP140 and E2F1-target and E2F1-target metagene expression levels in luminal-and basal-like tumors metagene in the basal-(n 40) and luminal-like (n 78) tumors of the cohort are expressed as means SE and p-values are indicated.

[START_REF] Carascossa | Receptor-interacting protein 140 is a repressor of the androgen receptor activity[END_REF]

[START_REF] Carascossa | Receptor-interacting protein 140 is a repressor of the androgen receptor activity[END_REF]

[START_REF] Carascossa | Receptor-interacting protein 140 is a repressor of the androgen receptor activity[END_REF]

[START_REF] Carascossa | Receptor-interacting protein 140 is a repressor of the androgen receptor activity[END_REF]

Mol Cell Biol . 2004 ; 24 : 5157 -71 19 . Shan B , Durfee T , Lee WH . Disruption of RB/E2F-1 interaction by single point mutations in E2F-1 enhances S-phase entry and apoptosis . Proc Natl Acad Sci U S A .

[START_REF] Carascossa | Receptor-interacting protein 140 is a repressor of the androgen receptor activity[END_REF]

[START_REF] Carascossa | Receptor-interacting protein 140 is a repressor of the androgen receptor activity[END_REF]

[START_REF] Carascossa | Receptor-interacting protein 140 is a repressor of the androgen receptor activity[END_REF]

[START_REF] Carascossa | Receptor-interacting protein 140 is a repressor of the androgen receptor activity[END_REF]

CONFLICT OF INTEREST

The authors declare no conflict of interest. [START_REF] Augereau | The nuclear receptor transcriptional coregulator RIP140[END_REF] 12

Figure 2

Interaction between RIP140 and E2F1 in intact cells HeLa cells were either transfected with pCDNA3-E2F1 (lanes 4 6) or (A) Co-immunoprecipitation of RIP140 and E2F1.

-pEF-c-mycRIP140 (lanes 7 9) alone or co-transfected with pCDNA3-E2F1 and pEF-c-mycRIP140 (lanes 10 12). Immunoprecipitations were -done using the anti-E2F1 polyclonal antibody (IP E2F1) or the anti-c-myc monoclonal antibody (IP RIP140) and Western blots were performed with the same antibodies as indicated. For each immunoprecipitation (IP), inputs and non-specific retention on beads alone are shown.

Nuclear extracts from MCF-7 human breast cancer cells were (B) Coimmunoprecipitation of endogenous proteins. immunoprecipitated using the anti-E2F1 polyclonal antibody C20 (sc-193 Santa-Cruz) or an irrelevant antibody (sc-510 anti-Gal4 DBD antibody Santa-Cruz). Western blots were performed as indicated with the same E2F1 antibody and with the anti-RIP140 monoclonal antibody (sc-81370 Santa Cruz). Input representing 10 of the amount used for immunoprecipitation is also shown.


Dr Bernard Vercambre
email: bernard.vercambre@cirad.fr

Biology and population dynamics of the potential sugarcane Fiji disease vector Perkinsiella saccharicida (Homoptera: Delphacidae) in Réunion: evidence of its loss of biotic potential

Keywords: Perkinsiella saccharicida, biology, population dynamics, sugarcane, biological control, Tytthus spp., Réunion

There is a risk that irrigation schemes that have been developed to enhance sugarcane, Saccharum spp, cropping on the island of Réunion could markedly broaden the distribution range in which outbreaks of the insect Persinkiella saccharicida Kirkaldy (Homoptera: Delphacidae), a sugarcane Fiji virus disease vector, have already occurred. This trend was confirmed by the results of laboratory studies and monthly surveys carried out in six sugarcane fields distributed throughout the island. A complex of efficient entomophagous organisms were also observed, including Tytthus spp. (Hemiptera: Miridae), and they were found to be capable of reducing P. saccharicida populations. These latter planthoppers also seemed to have lost part of their biotic potential as compared to those of the same species in its endemic area abroad. It is thus recommended that pest species be carefully monitored in cropping areas where large-scale irrigation schemes have been developed.

INTRODUCTION

The sugarcane commodity channel is a major economic entity on the island of Réunion. This crop is presently grown on a total area of 25 900 ha, or 60% of the usable arable land. The current increased productivity of this sugar-and fuel-oriented subsector has led to an increase in the sugarcane cropping area. French public authorities, with EU-funding support, have thus set up a large-scale irrigation scheme that is geared towards developing dry areas on the island. This spectacular project aims to transfer excess water from the eastern side of the island, under high rainfall conditions, to the western side where water is in short supply [START_REF] Payet | Aménagement hydroagricoles de la zone ouest. Panorama Agricole et Sucrier[END_REF]. This water diversion project-whereby water will be tapped from sites with high rainfall (Mafate, Salazie) and transited via some 30 km of tunnels through relatively inaccessible, rugged mountain areas-is aimed at making about 9 000 additional hectares of land suitable for sugarcane cropping (Fig. 1). In 1998, 1 500 ha was already being irrigated and the project will be under way until 2010, so a further 7 500 ha will also gradually be developed.

However, in terms of crop pests, this newly irrigated area has risks, especially of promoting the spread of certain piercing-sucking insects such as Perkinsiella saccharicida Kirkaldy (Homoptera: Delphacidae), which belongs to the species complex that was reviewed by [START_REF] Fennah | New species and new records of Perkinsiella (Hemiptera: Delphacidae) from Papua New Guinea[END_REF]. This potential Fiji disease (FDV) vector is present in Réunion. FDV was first identified in 1890 in Fiji but is endemic to Papua New Guinea [START_REF] Kerr | Southern Sugar Saga[END_REF]. The pathogen belongs to the Rheoviridae family and is ultimately capable of killing the host plant [START_REF] Rott | A guide to sugarcane diseases[END_REF]. FDV has been detected in a third of all countries colonised by P. saccharicida, including Madagascar where the insect arrived in the eastern coastal region in the early 1950s. The fact that a susceptible sugarcane variety (M134-32) was planted on over 80% of the sugarcane cropping area apparently triggered this epidemic [START_REF] Sigwalt R | Note sur l'insecte vecteur de la maladie de Fidji : Perkinsiella saccharicida Kirk[END_REF]. In Australia, this also occurred after cv POJ 2878 (1930)(1931)(1932)(1933)(1934)(1935)(1936)(1937)(1938)(1939)(1940) and then cv NCo 310 were sown in the Bundaberg region in [1969][1970][1971][1972][1973][1974][1975][1976][1977]. Although FDV symptoms have not been detected in Madagascar since 1971, the conclusions of an FAO mission report state that it is not possible to definitively conclude that the disease has been eradicated from this region [START_REF] Egan | Constat d'éradication de la maladie de Fidji. Madagascar. Rapport de mission[END_REF].

In Réunion, in the late 1980s, professional in sugar industry stakeholders are already worried about the potential impact of this type of disease since 80% of the cropping area has been replanted with cv R 570. This variety is high yielding but, according to the findings of tests carried out by the Australian Bureau of Sugar Experiment Stations, is highly susceptible to FDV. As no sure data are available on Fiji disease patterns in Madagascar-a country that is not far from Réunion and with which there is substantial trade-professional Réunion sugar industry stakeholders asked the French Agricultural Research Centre for International Development (CIRAD; Centre de coopération Internationale en Recherche Agronomique pour le Développement, France) to conduct a study to assess the potential benefits of preventive biological control treatments. Although this piercing-sucking insect is not yet infected, there is still a risk, especially since the virus may also be transmitted via sugarcane cuttings.

Here we present the results of a study which was carried out in 1989 and 1990 on the biology and population dynamics of P. saccharicida under the conditions that prevail in Réunion. No previous studies have focused on this insect on the island. Despite the age of these data, they will still provide a starting point for future studies conducted to check whether large-scale irrigation development schemes could change the disease-vector status of P. saccharicida and to assess the risk of FDV developing on the island.

MATERIAL AND METHODS

The study was carried out at six sugarcane cropping sites in Réunion (Fig. 1). Sugarcane is grown at elevations ranging from 0 to 600 m around the entire island. The mean monthly temperatures are shown in Fig. 2. Data collected at the measuring station closest to the study site was corrected to account for the difference in elevation between this and the six other study sites (± 0.75°C per 100 m elevation, correlation coefficient > 0.90, [START_REF] Chopart | Relations entre l'altitude et la température mensuelle de l'air dans l'ouest de la Réunion[END_REF]. The eastern and western sides of the island differed markedly in terms of rainfall levels, while the northern and southern sides were transitional (Fig. 1, Table 1).

Experimental studies on the biology of the insect

Experimental studies were carried out on the biology of P. saccharicida in order to gain insight into its population patterns in the natural environment and to be able to compare the results with those obtained in other parts of the world where this pest prevails. These studies were conducted in climate-controlled rooms (25 ± 1°C, 60-70% r.h.; L16:D8).

Life cycle studies Adult insects that had been reared under routine conditions [START_REF] Taniguchi | Effects of Fiji disease-resistant sugarcane (Saccharum sp.) on the biology of the sugarcane delphacid[END_REF] were deposited on sugarcane leaves and left there for 2 h (Fig. 6, A.). Leaves on which eggs had been detected were placed in plexiglass boxes (20 x 10 x 2 cm 3 ) with green moistened blotting paper on the bottom. This technique ensured high humidity in the boxes and facilitated the detection of nymphs after hatching. After 8 days, the eggs were monitored twice daily. Forty eggs were monitored overall. Each nymph was subsequently transferred to a cylindrical plastic box (4 cm dia., 1 cm height) with moistened blotting paper on the bottom and a screened opening to ensure air circulation. The nymphs were fed sugarcane leaves. They were monitored four times daily so as to determine exactly when moulting occurred. At each moult, exuviae were examined and removed from the boxes.

Fertility studies

After emergence, pairs of adults were placed in containers made with the bottoms of plastic water bottles (8 cm dia., 20 cm height). They were sealed with muslin and the bottoms were coated with moistened sterile sand in which a sugarcane leaf was inserted. The leaf was replaced daily throughout the adult life cycle and checked under a magnifying glass for the presence of egg clusters. After a female had laid its first egg, the length of the pre-oviposition period was noted. Then the number of clusters and eggs per cluster were recorded until the female's death so as to determine its fertility rate. Ten pairs of insects were monitored in triplicate under the same rearing conditions.

Study on the effects of temperature on egg development As field populations were found to decline during the cold season, a specific study was carried out to determine the egg incubation period according to temperature. Twenty egg clusters (reared eggs) were studied overall. The four treatments were as follows: i/ usual rearing conditions (25°C, L16:D8); ii/ 25°C, L11:D13); iii/ 20°C, L16:D8); and iv/ natural conditions: mean 22°C (min. 16.5°C; max. 28°C, L11:D13).

Studies on the population dynamics of P. saccharicida and associated organisms

The population dynamics of P. saccharicida and its antagonists were studied in two phases, i.e. a preliminary scouting operation and a monthly survey. The population dynamics criteria used in this study were the number of P. saccharicida adults and egg clutches. Visible adults were counted on 4 x 10 adjacent sugarcane stalks growing within a 1 ha area per plot. Eggs were counted on leaf 5 (from the top of the stem) on 10 groups of three adjacent sugarcane stems. This latter monitoring operation was facilitated by the fact that an incision made by P. saccharicida in a sugarcane leaf rib during oviposition provided a port of entry for the fungus Glomerella tucumenensis (Speg.) Arx & E. Müller and the egg clusters were often surrounded by a reddish halo (Fig. 6, B and C). Fifty egg clusters per plot were selected on leaves 7 and 8 so as to quantify the efficacy of antagonistic organisms in controlling this pest on the basis of the aspect of the destroyed eggs (Table 5).

The leaf ribs were incubated in plastic bottles containing a small amount of water so that the newly laid eggs would have time to develop signs of viability or parasitism, and they were dissected 5-6 days after harvest. The focus was especially on detecting potential entomopathogens. Fifty nymphs and adults captured with a D-Vac were squashed, stained with Loeffler's methylene blue, while 50 others were fixed in Zenker-formol fixative, embedded in paraffin, cut into 7 μm thick slices and stained according to the Mann-Dominici method.

Statistical treatment

For the laboratory studies, we calculated the means and standard deviations when the population distributions were normal. Wilcoxon (adult life cycle) and Kruskal-Wallis (life cycle according to temperature) tests were used to highlight significance differences. For counts in sugarcane fields, we adopted the sequential sampling plan developed by [START_REF] Allsopp | Sampling Distribution and Sequential Sampling Plans for Perkinsiella saccharicida Kirkaldy (Homoptera: Delphacidae) and Tytthus spp. (Hemiptera: Miridae) on Sugarcane[END_REF] based on Iwao's regression indices and relationships between the variance and mean according to Taylor's power law. The accuracy levels were 10 and 25% according to the mean observed densities.

RESULTS

Experimental studies on the biology of P. saccharicida

Life cycle

The complete life cycle from egg to emergence was around 43.7 days at 25°C. The sex ratio was balanced. The mean egg incubation period was 15 days. Table 2 gives the lengths of the five nymphal instar periods. The high losses recorded for the first instar were due to the fact that some young, fragile and hard to detect nymphs were crushed. The 4 th to 5 th instar moult may have been hampered by the low humidity conditions. Clutches contained one to seven eggs (mean 3.7 eggs; N = 106 clusters) (Table 3).Despite the high variability in the adult lifespan (females: 5-61 days; males: 3-24 days), the mean lifespan of females was significantly higher than that of males (23.5 vs 11.3 days; P=0.02 according to the Wilcoxon test, SAS Institute Inc., 2004. SAS On lineDoc® 9.1.1.3 Cary, NC: SAS Institute Inc., NPAR1WAY procedure). Acute mortality periods were detected for both sexes (5-6 days and then 11-12 days). Half of the female population was affected by these critical periods, whereas the other half lived for at least 30 days.

Female fertility

The preoviposition period was determined for 27 out of the 35 females tested, considering the early mortalities recorded (5-6 days). This period ranged from 5 to 14 days (mean 9.6 ± 2 days). For isolated pairs, if the male died before 6 days old, no viable eggs were laid. The number of eggs laid per female was 14-145 (mean 74.7). The usual variation coefficients could not be used in this latter case. The graphs show a discontinuous series of peaks.

Influence of the photoperiod and temperature on eggs The photoperiod had no impact on the incubation period. However, P. saccharicida was sensitive to temperature changes: the development period was 2.6-fold longer when the temperature declined by 5°C (Table 4). The low percentage of hatching at 20°C was mainly due to cold-induced leaf rib contraction which led to crushing of the eggs. Despite this problem, the differences in incubation period were significantly different at the three tested temperatures (P=0.0001 according to the Kruskal-Wallis test, SAS Institute Inc., 2004. SAS On lineDoc® 9.1.1.3 Cary, NC: SAS Institute Inc., NPAR1WAY procedure).

Survey of population variations

P. saccharicida population dynamics

The fact that the P. saccharicida population dynamics trends were similar at all monitoring sites on the island is interesting, as clearly noted at the Savanna site (Fig. 3). The reference scale for all sites was based on the maximum values obtained at a single site (Savanna) where P. saccharicida showed marked population variations (up to 300 egg clusters and 3.8 adults per stem), with a very uniform series of transitions between egg laying periods and adult emergence. At Savanna, the emergence of the main 1989-1990 generation was detected as early as November 1989, after the visible disappearance of the previous generation noted during the preliminary scouting. The population decline began in April, even though the conditions were still suitable from three standpoints: climate, insect biology and vegetative stage of the sugarcane plants. The same overall patterns were noted at the other sites but to a less marked extent. An imbalance between the number of observed egg clusters and the resulting adult populations was noted, although we found that on average an egg cluster could give rise more than three offspring.

The preliminary scouting operation revealed that P. saccharicida was present in sugarcane fields throughout the island, even at elevations as high as 600 m, but the following survey confirmed that the numbers were not high enough, even at Savanna, to directly cause any crop damage. Generally, two winged forms of adults were noted: macropterous individuals with normal wings, and brachypterous-type koeliopterous individuals with wings that extended to the end of the abdomen and were tipped with a tegument that was midway between macroand brachypterous forms. As previously reported in Mauritius [START_REF] Williams | The sugarcane delphacid and their natural enemies in Mauritius[END_REF], koeliopterous males were found in lower numbers (1.9%) than koeliopterous females (20.9%) (N = 769), whereas only females have been reported (unknown levels) in South Africa [START_REF] Harris R | Perkinsiella saccharicida Kirkaldy (Homoptera: Delphacidae) an insect pest of sugarcane in Southern Africa[END_REF]. The mean number of eggs per cluster was 3.5. The monitored upper leaf surfaces (leaves 5-8 from the top) had been the site of 84.3% of clusters (N = 362). The sex ratio was balanced, even though we noted variations of between 40 and 60%, in favour of either sex. P. saccharicida was always associated with another delphacid species, i.e. Dicranotropis muiri Kirkaldy. The biology of this latter species is close to that of the studied P. saccharicida, but these species may be distinguished on the basis of several differences: D. muiri adults are smaller and yellowish in colour, they roost on the under side of leaves, contrary to P. saccharicida, which roosts on the upper side, egg clusters are covered with a transparent veil and there are one or two eggs per oviposition, which are also smaller (-20 %) [START_REF] Williams | The sugarcane delphacid and their natural enemies in Mauritius[END_REF].

Antagonistic organisms

List of inventoried organisms Six main entomophagous organisms that attack P. saccharicida, i.e. egg parasites or predators, was identified (Table 5). The nymphs were preyed upon by a Dryinidae hymenopteran species (Pseudogonatopoides mauritianus Williams). So-called 'stylopisation' may be noted, whereby a larval 'sac' protrudes from the abdomen between tergites 5 and 6, but this phenomenon was only observed in a few adult males (0.5%). Note that Anagrus optabilis (Perkins) and Aprostocetus pallidipes (Perkins) are common parasites of P. saccharicida and D. muiri. A scant number of dead D. muiri individuals were collected-this death was due to the development of a Strepsipteran Elenchus Curtis sp. Complex parasitism behaviour: first an egg parasite, then becomes a predator when the larva hatched from a parasited egg and subsequently eats the other eggs in the cluster (Fig. 6, plate E.) Larva parasitisation Several spider species were found during the D-Vac sampling, but they were not identified. The predatorial status of certain spider species that weave a web at the leaf-stem interface where P. saccharicida is often found was confirmed.

No fungal diseases were noted. Moreover, screenings for intracellular infectious agents such as rickettsia and viruses were negative and no cytoplasm modifications were detected. Nuclei of all monitored individuals had not been modified and seemed normal. However, a symbiotic yeast was detected in the haemolymph. Relatively few of these symbionts were detected in some individuals as compared to others.

Population dynamics of the main entomophagous organisms

The dissections regularly revealed cumulated variations in the antagonism of P. saccharicida due to the three entomophagous genera at the different sites (Fig. 4). Tytthus mundulus (Breddin) was identified throughout the island. Tytthus parviceps (Reuter) was only detected in the Savanna area, but at high densities. This site showed clear trends with respect to entomophagous population dynamics. A low percentage of egg infestation was noted from December to February (Fig. 5). This period occurred 1-2 months prior to the increase in adult populations noted in the field, i.e. the length of one generation. The percentage of eggs destroyed by mirids gradually increased from March to over 80% in May. Tytthus spp. were dominant at this time, whereas the egg parasitism rate was found to have dropped (Fig. 5). At all sites, the viable egg frequency was high between November-December and February, decreasing gradually as a result of the combined action of all antagonists, which had even more of an impact than the thermal factor. The mortality rates of the different antagonists were identical, but predatorial bugs were by far the most efficient antagonist. A. pallidipes was found in very low numbers, as it had never parasitised more than 10% of the egg clusters as the Delphacidae population density increased (Fig. 6,E). A. optabilis was the most efficient, having parasitised up to 40% of the eggs. Its population numbers increased until February-April, and subsequently declined to the benefit of Tytthus spp.

P ara s it is m c o nt ro l pa t t e rns in

DISCUSSION

This survey clearly highlighted the densities of P. saccharicida populations under the diverse range of conditions that prevail in Réunion, along with its annual population dynamics and natural control mechanisms, while also generating some data on biological criteria, some of which were especially heterogeneous (adult lifespan, egg number/female). This diversity could not be solely explained by the rearing conditions, and we put forward the hypothesis that several groups of individuals were present. In sugarcane fields, the mean recorded densities were likely representative, considering the sampling intensity. It is understood that, in visual monitoring surveys, all adult insects present will not be detected and nymphs populations that are relatively hidden under the leaf sheaths will be overlooked. Moreover, some egg clusters may be partially located on the under side of leaves [START_REF] Laverde | Nota de Investigatión. Perkinsiella saccharicida : el saltahojas hawaiano[END_REF].

The number of adults per stem was the most reliable criterion for comparing our results with previously published data. Only the Savanna site (Site A, maximum adults per stem = 3.8 in March 1990) was in compliance with the accuracy levels (P = 0.10) defined in the statistical studies of [START_REF] Allsopp | Sampling Distribution and Sequential Sampling Plans for Perkinsiella saccharicida Kirkaldy (Homoptera: Delphacidae) and Tytthus spp. (Hemiptera: Miridae) on Sugarcane[END_REF] for adult insect counts (at least 3.5 adults per stem in sequential counts comparable to those conducted in our study). This original feature showed that the insect seemed to have a preference for a mean temperature of 25°C and especially high humidity conditions maintained by steady irrigation, thus confirming the results obtained in Madagascar [START_REF] Sigwalt R | Note sur l'insecte vecteur de la maladie de Fidji : Perkinsiella saccharicida Kirk[END_REF]. At the Gol site (site B) and La Vigne site (site E), the accuracy level was only P = 0.25 for this type of sampling. [START_REF] Sosa | Perkinsiella saccharicida Kirkaldy, a vector disease found in the United States[END_REF] published differences ranging from 9 to 36 adults per stem in Florida, while [START_REF] Ayquipa | Evaluation of a heavy infestation with Perkinsiella saccharicida Kirk. (Homoptera: Delphacidae) on sugarcane in Cooperativa Casa Grande, Trujillo[END_REF] and [START_REF] Laverde | Nota de Investigatión. Perkinsiella saccharicida : el saltahojas hawaiano[END_REF] reported similar densities in South America (7-34 according to the former author and 25 adults per stem and up to 40 for the latter). In Hawaii, according to [START_REF] Egan | Monitoring the Fiji epidemic in sugarcane in Bundaberg, Australia. Plant virus epidemiology[END_REF], this number could reach 300, which means there would be substantial crop loss due to direct sap sucking prior to the introduction of predatorial bugs in 1920. [START_REF] Bull R | Population studies on the sugarcane leaf-hopper (Perkinsiella saccharicida Kirk.) in the Bundaberg district[END_REF] indicated 60 adults per stem in Australia in February 1974, with the P. saccharicida migration-triggering threshold being 20 adults.

The biological performances of P. saccharicida could also be compared to those recorded in other countries. The incubation time and life cycle are in agreement with previous findings [START_REF] Williams | Handbook of The Insects and Other Invertebrates of Hawaiian Sugar Cane Fields. Experiment Station of the Hawaiian Sugar Planters[END_REF][START_REF] Williams | The sugarcane delphacid and their natural enemies in Mauritius[END_REF][START_REF] Sigwalt R | Note sur l'insecte vecteur de la maladie de Fidji : Perkinsiella saccharicida Kirk[END_REF][START_REF] Bull R | A study of the sugar cane leafhopper Perkinsiella saccharicida Kirk (Homoptera: Delphacidae) in the Bundaberg District of South-Eastern Queensland[END_REF]Yepes et al.,1988;[START_REF] Laverde | Nota de Investigatión. Perkinsiella saccharicida : el saltahojas hawaiano[END_REF][START_REF] Fernandez | Notas biologicas de Tytthus parviceps Reuter (Hemiptera: Miridae)[END_REF][START_REF] Mendoza Mora | Management of Perkinsiella saccharicida in sugarcane in Ecuador[END_REF]. Amongst the other possible criteria, the number of eggs per cluster is the easiest and surest to monitor (Fig. 6. D). It was found to be 3.5 in Réunion, Mauritius and Madagascar, and ranged from 4.6 to 5.6 [START_REF] Bull R | A study of the sugar cane leafhopper Perkinsiella saccharicida Kirk (Homoptera: Delphacidae) in the Bundaberg District of South-Eastern Queensland[END_REF], with egg clusters consisting of 1 to 21 eggs in Australia [START_REF] Bull R | Population studies on the sugarcane leaf-hopper (Perkinsiella saccharicida Kirk.) in the Bundaberg district[END_REF], or a reduction of about 25-50% in Réunion. In Florida, the mean is 4.5, with egg clusters of 1-12 eggs [START_REF] Sosa | Perkinsiella saccharicida Kirkaldy, a vector disease found in the United States[END_REF], whereas means of 4.1 were obtained in Colombia [START_REF] Laverde | Nota de Investigatión. Perkinsiella saccharicida : el saltahojas hawaiano[END_REF][START_REF] Laverde | Nota de Investigatión. Perkinsiella saccharicida : el saltahojas hawaiano[END_REF]4.3 in South Africa (Harris, 1970). The mean lifespan of female adults and their fertility were also lower in Réunion than previously recorded in Australia: 23 days and 75 eggs per female in Réunion, for 1-2 months and 300 eggs in Australia. The rearing conditions could be partially responsible for this difference, but the Australian data were also obtained with laboratoryreared insects, so other factors must also have been involved. Our results were in line with those obtained by Frappa (1955) in Madagascar. He observed the same level of early adult mortality, the same thresholds (up to 12 days) and low egg cluster levels. His life cycle results, which were obtained at a mean temperature of 26-27°C, were comparable to ours. Frappa (1955) also noted that P. saccharicida was sensitive to temperature variations. The similar results obtained on the biology of P. saccharicida in the two islands, whose sugarcane history is partly linked, provides additional proof of the negative evolution of this insect, even if it was not possible to pursue the research in Reunion due to the financial specifications and time limit of the project. The other previously published results come from reviews or are not accurate enough to determine the actual variability in the biological features considered, probably in association with the statistical differences noted in our study. They are correlated with the capacity of piercing-sucking insects to host many primary symbiotic organisms that are essential for their survival, along with secondary organisms that have a less fundamental but sometimes a considerable impact [START_REF] Oliver | Facultative bacterial symbionts in aphids confer resistance to parasitic wasps[END_REF][START_REF] Weeks | Incidence of a new sex-ratio-distorting endosymbiotic bacterium among arthropods[END_REF]. Variability in the number of symbiotic yeasts detected per planthopper sampled in Réunion could be explained by the loss of symbionts during fixation and subsequent treatments affecting the analysed samples, or could reflect a fluctuation associated with causes intrinsic to the insects.

As there is natural variability in the insect-symbiont association, it would be interesting to determine if there is a correlation between this variation and the differences in life cycle and fertility noted in the biological study.

All of these conclusions suggest that P. saccharicida lacks part of its biotic potential in Réunion and Madagascar. Only a few individuals of this species must therefore have been initially introduced, or this loss could have been due to genetic depletion or loss of symbionts which could not be rectified in this island environment. These are broad ranging issues for which few results have been obtained and would thus warrant further research.

Our results on the annual population dynamics of P. saccharicida, its growth during the hot and humid seasons, and its decline under the influence of less suitable abiotic conditions were in agreement with most previous findings [START_REF] Harris R | Perkinsiella saccharicida Kirkaldy (Homoptera: Delphacidae) an insect pest of sugarcane in Southern Africa[END_REF][START_REF] Bull R | Population studies on the sugarcane leaf-hopper (Perkinsiella saccharicida Kirk.) in the Bundaberg district[END_REF][START_REF] Sosa | Seasonal abundance and temperature sensitivity of sugarcane Delphacid (Homoptera: Delphacidae)[END_REF]. [START_REF] Bull R | A study of the sugar cane leafhopper Perkinsiella saccharicida Kirk (Homoptera: Delphacidae) in the Bundaberg District of South-Eastern Queensland[END_REF][START_REF] Bull R | Population studies on the sugarcane leaf-hopper (Perkinsiella saccharicida Kirk.) in the Bundaberg district[END_REF] proposed that there are four generations per year, with the adults disappearing almost completely during the southern winter (June-August), and the nymphs being concentrated at the base of sugarcane stubble in autumn (April-May), taking shelter under dry leaves. The factors underlying this evolution are the change to cooler climatic conditions and the gradual harvesting of sugarcane crops between June and December. In addition to the temperature and humidity, a third less recognized factor is therefore the diversity of crop vegetative stages available, so when the insect arrives in a crop field it is able to quickly select the most suitable host plant for its development. Note, however, that [START_REF] Williams | The sugarcane delphacid and their natural enemies in Mauritius[END_REF] found that in Mauritius P. saccharicida was more readily visible during the southern winter, whereas in Hawaii this higher visibility was found to be associated with the increase in the percentage of more prolific brachypterous females and with the higher susceptibility of associated entomophagous organisms to the declining temperature and high winds [START_REF] Williams | Handbook of The Insects and Other Invertebrates of Hawaiian Sugar Cane Fields. Experiment Station of the Hawaiian Sugar Planters[END_REF]).

The regional differences noted in the insect's population dynamics in Réunion should also be considered with respect to the abiotic conditions. For an insect that is so susceptible to temperature changes, the mean monthly differences of 1.5-2.5°C between Savanna (site A) and La Vigne (site E), or 2.1-3°C between Savanna and Beaufonds (site D) must have had an impact on its population density. The regularity of the humidity conditions could be a further influential factor, since the most abundant populations were located in dry areas that were regularly and abundantly irrigated (Savanna), as compared to areas that were also highly humid, but to a more variable extent because of the temporary and irregular rainy season (Beaufonds, La Vigne) (Table 1).

The direct effects of the environmental conditions were boosted by the additional impact of a complex of predators and parasitoids. It is likely that the increase in Tytthus spp. populations was responsible for the decreased egg parasitism noted. Indeed, bugs will attack P. saccharicida eggs regardless of whether they are infested with parasites or not. The high efficacy of bugs could also be partly explained by the fact that it has a faster life cycle, i.e. 26 days as compared to 40 days for the delphacid [START_REF] Fernandez | Notas biologicas de Tytthus parviceps Reuter (Hemiptera: Miridae)[END_REF]. This diversity of antagonistic organisms present is surprising since no introductions had been officially recorded. The two most common parasitoids (n.b. because of its lifestyle, the impact of Aprostocetus was likely underestimated) could have been naturally introduced upon the arrival of living sugarcane rootstock that had been shipped from other countries during varietal exchanges, which mainly began in the late 18 th century, but it is less likely that this occurred with respect to the two mirid species. This was already noted by the natural absence of bugs on the two islands located close to Réunion (Madagascar, Mauritius) [START_REF] Sigwalt R | Note sur l'insecte vecteur de la maladie de Fidji : Perkinsiella saccharicida Kirk[END_REF][START_REF] Williams | The sugarcane delphacid and their natural enemies in Mauritius[END_REF]. It is more likely that they were introduced in Réunion during Mauritian biological control operations targeting these species in 1956-1957 [START_REF] Greathead | A review of biological control in the Ethiopian region[END_REF]. However, few reports are available on populations inducing severe crop damage simply due to sap sucking, as occurred in Hawaii, prior to the introduction of predatorial bugs [START_REF] Egan | Monitoring the Fiji epidemic in sugarcane in Bundaberg, Australia. Plant virus epidemiology[END_REF] or in Ecuador recently [START_REF] Mendoza Mora | Management of Perkinsiella saccharicida in sugarcane in Ecuador[END_REF]. During our active research, we observed no signs of diseases in the insect, whereas studies have been carried out in South America to assess the efficacy of two entomopathgenic fungi (Metarhizium sp. and Beauveria sp.) [START_REF] Rico | Evaluation and identification of pathogens of Perkinsiella saccharicida (Hom: Delphacidae) in sugarcane[END_REF][START_REF] Badilla | Control of the leaf-hopper Perkinsiella saccharicida with the fungi Beauveria bassiana and Metarhizium anisopliae[END_REF]. In Madagascar, [START_REF] Giannotti | Observation d'un microorganisme d'un type inhabituel chez un insecte homoptère[END_REF] detected microorganisms resembling mycoplasms of relatively unknown origin and role.

The findings of this study and the situation described indicated that a complex of insects antagonistic to P. saccharicida is present and is efficiently fulfilling its antagonistic roles, even though we observed inevitable competition between egg parasites and egg predators. As part of the Reunion water diversion project, this uneven balance should to be closely monitored to check for possible changes over time [START_REF] Ameixa | Agricultural policy-induced landscape changes: effect on carabid abundance and their biocontrol potential[END_REF].

The temperate tropical climatic conditions did not seem to be optimal for this stenothermal insect, which is susceptible to even minor decreases in temperature. Its biological capacities also seem to have been reduced following its introduction in this island environment, which took place long ago. As another favourable factor, Francki et al. (1986) showed that P. saccharicida was a poor virus vector. These authors deposited adults on Fiji disease infected sugarcane plants and noted that only 15% of the 537 planthoppers tested were infected and only 6% were able to transmit the virus to healthy plants.

The Savanna area could eventually be closely monitored for early detection of Fiji disease infestation, since three factors at this site may be conducive to the development of this disease: it has the highest temperatures on the island, humidity is constant because of the irrigation system, and the sugarcane crop is present at all of its development stages.

It would also be essential to ensure that only cuttings from quarantine glasshouses are imported, especially if they are coming from a country where the Fiji disease virus is present. Madagascar is the closest country in this respect.

Finally, the sugarcane cropping area should be managed so as to avoid situations where one variety prevails too markedly over the others, unless this is unavoidable because of one variety's overriding economic potential. For the highest yielding variety R 570, which is still grown on over 70% of the total sugarcane cropping area, the two parents have two varieties in their ancestry (cv POJ 2878 and R445) which have been ranked at level 7 on an Australian scale of Fiji disease susceptibility. Hence, the situation must be closely monitored while ensuring that all of the above mentioned precautionary measures be taken. Finally, it would be important to assess the impact of the introduction of irrigation on the western part of the island on the population dynamics of all other insects present, such as the scale insect Aulacaspis tegalensis Zehntner (Homoptera: Diaspididae), another piercing-sucking sugarcane pest that could have a negative impact on crop yields.

ACKNOWLEGEMENTS

These studies were jointly funded by CIRAD-CORDET, Convention 89-151. The data were collected by a CIRAD entomology team (A. Gatignon, L. Lenoir, R. Manikom, J.M. Bègue and L. Cerveau). G. Morel, Professor at the Université de St Denis de la Réunion, provided valuable advice for the microbiology studies. The climatic data were supplied by M. Mézino. The statistical analyses were performed by E. Gozé. C. Fovet-Rabot, M. Duportal, M.J. Linarès and A. Boyer were involved in the editing and layout. I thank M. Vaissayre for his judicious comments. The author is very grateful to all of these people for their kind assistance.

Fig. 1 .

 1 Fig. 1. The sugarcane cropping area and the six P. saccharicida study sites on the island of Réunion.

  Fig. 3. Regional population dynamics of P. saccharicida (Réunion, 1989-1990).Statistics adults/stem: density >3.5, P=0,1 ; density <3.5, P=0,25[START_REF] Allsopp | Sampling Distribution and Sequential Sampling Plans for Perkinsiella saccharicida Kirkaldy (Homoptera: Delphacidae) and Tytthus spp. (Hemiptera: Miridae) on Sugarcane[END_REF]

  Fig. 4. Regional population dynamics of the main entomophagous organisms that attack P. saccharicida (Réunion, 1989-1990).

Fig. 5 .

 5 Fig. 5. Population dynamics of the main entomophagous organisms that attack P. saccharicida showing that the predators (Tytthus spp.) ultimately prevailed over the egg parasitoids (Anagrus sp. and Aprostocetus sp.) (Réunion, Savanna, 1989-1990).

Figure

  Figure 6. P. saccharicida plates.

Table 1 . Rainfall levels (mm) in the study areas recorded during the P. saccharicida survey (Réunion, 1989-1990).

 1

	1989/1990

Fig. 2. Mean monthly temperature patterns recorded during the P. saccharicida survey at the study sites (Réunion, 1989-1990).

Table 2 . Egg incubation times and periods of the five P. saccharicida instars studied under laboratory conditions (25°C, Réunion, 1990).

 2

	Life stages	egg	instar 1	instar 2	instar 3	instar 4	instar 5
	Mean time (in hours)	360±24	88±13	98±8	126±24	136±30	240
	N° individual tested	40	22	18	14	12	4

Table 3 . Reared P. saccharicida egg number distributions (Réunion, 1989-1990).

 3

	Egg number per cluster	1	2	3	4	5	6	7	Total
	Frequency	3	4	27	34	24	11	3	106
	% total	2.8	3.8	25.5	32.1	22.6	10.4	2.8	100

Table 4 . Incubation times of P. saccharicida eggs according to temperature (Réunion, July-August 1990).

 4

	Treatment	Hatching onset	Hatching end	Mean Time Hatching
		(days)	(days)		(%)
	1. (25°C, L.D 16:D8)	13	18	15.5±1.5	100
	2. (25°C, L.D 11:D13)	13	18	15,6±1.6	100
	3. (20°C, L.D 16:D8)	34	-	36.5±(2.3 )	30
	4. (≈22°C, L.D 11:D13)	23	31	26.3±2.1	90
	incomplete data

Table 5 . The six entomophagous organisms detected on P. saccharicida in Réunion (1989-1990) (identifications: G. Delvare, Laboratoire de Faunistique, CIRAD, Montpellier, France).

 5

	Species	Order and family	Type of action
	Tytthus mundulus (Breddin)	Heteroptera, Miridae	Empties eggs by sucking
	Tytthus parviceps (Reuter)	Heteroptera, Miridae	Empties eggs by sucking
	Anagrus optabilis (Perkins	Hymenoptera, Mymaridae	Egg parasitisation
	Aprostocetus pallidipes Perkins	Hymenoptera, Eulophidaae
	Pseudogonatopoides mauritianus	Hymenoptera, Dryinidae

Delphacidae) in Réunion: evidence of its loss of biotic potential -B. Vercambre, 2010, CIRAD 11


El Lazrak
email: lazrak@mirecourt.inra.fr

Ghali

Marc Benoît
email: benoit@mirecourt.inra.fr

Jean-François Mari
email: jfmari@loria.fr

Time-Space Dependencies in Land-Use Successions at Agricultural Landscape Scales

Keywords: HMM2, data mining, temporal cliques

The agricultural landscape can be seen as an assemblage of farm territories. The way farmers organize these territories is a time AND spatial process. Understanding how a land-use succession (LUS) in a parcel depends on LUS of the neighbouring parcels is a milestone to understand the time-spatial organization of the landscape mosaic. In this work, we analyse these time-space dependencies at agricultural landscape scales. We have performed a data mining process based on hidden Markov models (HMM) to identify spatial clusters of similar distributions of LUS in 2 neighbouring parcels, furthermore called cliques. We applied this data mining process to a land-use data set covering the period from 1996 to 2007 of a 350 km² agricultural landscape located within the Niort Plain (France). To take into account the irregular neighbour system of the parcel mosaic, we used a variable depth Hilbert-Peano scan of the area covering the landscape. Through illustrative examples of two contrasted spatial stochastic clusters, we show that considering temporal cliques gives valuable information on the neighbour system in terms of attraction between LUS.

Introduction

In agricultural landscapes, land-uses are heterogeneously distributed among different agricultural parcels designed by farmers. At a first glance, the landscape spatial organization and its temporal evolution seem both random. Nevertheless, they reveal the presence of logical processes and driving forces related to the soil, climate, cropping system, and economical pressure. The mosaic of parcels together with their soil-occupancies (OCS) can be seen as a noisy picture generated by these different processes. The understanding of how the temporal succession of a parcel influences the neighbouring parcels is a milestone in the data mining process that aims at extracting knowledge from this mosaic. Furthermore, this piece of knowledge is helpful to simulate coherent agricultural landscapes (Le [START_REF] Ber | Neutral modelling of agricultural landscapes by tessellation methodsapplication for gene flow simulation[END_REF]. Recent studies (Le [START_REF] Ber | Studying Crop Sequences With CarrotAge, a HMM-Based Data Mining Software[END_REF][START_REF] Castellazzi | A systematic representation of crop rotations[END_REF] have shown that the ordered sequences of OCS in each field can be adequately modelled by a Markov process. The OCS at time t depends upon the former OCS at previous times:

1  t , 2  t
, ... . The Markov model or the hidden Markov model (HMM) are able to capture a limited amount of the temporal variability and allow the specification of land-use successions (LUS) in term of which the agricultural landscapes can be described in a more simple way [START_REF] Lazrak | Landscape regularity modelling for environmental challenges in agriculture[END_REF]. Similarly, in the spatial domain, the stochastic modelling of situated observations such as OCS or LUS by means of Markov fields is an elegant way to cluster a landscape into homogeneous patches described by probabilistic distributions of the situated observations. In this work, we process at the same level the temporal and spatial information given by the parcels and their OCS and consider a pair of OCS in 2 neighbouring parcels at time slots tfurthermore called a temporal cliquerather than a single OCS as the basic temporal and spatial information. The stochastic modelling of the temporal cliques allows a spatial and temporal clustering of the landscape and gives valuable informations on the time and spatial dependencies between OCS. Our objective is to develop a generic data mining process, based on HMM and temporal cliques, in order to highlight these time-space dependencies at agricultural landscape scales.

The land-use database

The case study area is a 350 km² agricultural landscape located within the Niort Plain in Poitou-Charentes region, France. This agricultural landscape has been surveyed for more than 12 years (1996 -2007). Every year, two land-use surveys (in April and June) allow to monitor both early harvested and late planted crops. These surveys are stored in a GIS geodatabase, in a vector format. An analysis based on the average frequency of land-uses over the 12-year study period reveals 47 land-uses. These land-uses have been grouped with the help of agricultural experts in 10 categories (table 1) following an approach based on the similarity of crop management.

Table 1. Composition and average frequencies of adopted land-use categories [START_REF] Lazrak | Landscape regularity modelling for environmental challenges in agriculture[END_REF] Land

The agricultural landscape mosaic

The agricultural landscape can be seen as an assemblage of polygons of variable size the parcelswhere each parcel holds a given OCS.

A parcel can be bounded by a road, a path or a limit of a neighbouring parcel. The parcel boundaries can change every year. To take account of this change, each year, the surveyors update the edgesthe boundariesof parcels in the GIS geodatabase. This led to the definition of the elementary parcel as the result of the spatial union of previous parcel edges (figure 1). There are about 20,000 elementary parcels in the study area over the 1996 -2007 period. Each elementary parcel holds one succession of OCS during the study period.

The corpus of land-use data is sampled using a regular grid and is represented in a matrix in which the rows represent the land-uses year by year and the lines, the different grid locations.

Cliques and temporal cliques

Two elementary agricultural parcels represented by 2 polygons are neighbouring if they have at least an edge in common. A clique is a set of parcels in which two unspecified parcels are neighbour. In the mosaic of polygons, the neighbouring relationshipcalled the neighbour systemis irregular. The parcels have a variable number of neighbours in different geographical directions as opposite to digital images where a site has a fixed number of neighbours. In this paper, we consider simple cliques made of 2 neighbouring parcels represented by the 2 centroids of the parcels. Experimental preliminary results show that the OCS distribution in the cliques is isotropic: the direction defined by the 2 centroids does not carry any information. This scanning introduces a spatial warping and a surface normalization in the parcel mosaic. Large parcels are less sampled, whereas no site agglomeration occurs when the curve crosses the parcel boundaries. The longer is the boundary between two polygons, the more frequent is the clique. Of course, the parcels having singular shape cannot be represented with one centroid and some cliques are situated into the same elementary parcel (figure 2). As a matter of fact, the problem of visiting only once the edges or the vertexes of a graph is known to be NP (non polynomial) hard: there is no algorithm running in a reasonable time to solve it [START_REF] Rubin | A search procedure for hamilton paths and circuits[END_REF]. Our irregular spatial sampling is a crude way to avoid this issue.

The occupancies of a site and its neighbour at time t define the temporal clique. At each site l s in the variable depth Hilbert-Peano scan, we have defined a feature vector

t l
o with the OCS held in the ) , ( 1 l l s s  cliques:

)) , ( ), , ((

1 1 1 1      t l t l t l t l t l s s s s o , 1 0    T t , L l   1 (1)
where t l s is the OCS at time t and index l in the variable depth fractal curve. t is a time index running over the study period, and l the spatial index in the L -Length scanning curve. At time t , a landscape is then represented by a ) 1 (  L -Length sequence of overlapping temporal cliques. We consider also 1  T representations to cover the T year length study period due to the overlap artefact. The cliques inside the same elementary parcel result from the variable depth Hilbert-Peano scan. They are not interesting in the present study. To partially deal with this artefact, feature vectors

t l o verifying ) ( 1 t l t l s s   and ) ( 1 1 1     t l t l s s
are removed from the resulted distributions.

The feature vector

The time-space Markovian modelling framework

The way a farmer organizes his territory is a time and spatial process. This time-space dependency becomes more complex at agricultural landscape scales when the agricultural mosaic is built under many farmer's logics. To analyze these dependencies, we rely on 2 assumptions:

1. the OCS of a given field depends upon the OCS of the neighbouring fields (the MRF assumption), and 2. the OCS of a given field in a given year depends also upon the OCS of recent previous years (the Markov chain assumption). We have modelled the spatial structure of the landscape by a MRF whose sites are random variables of temporal cliques. Like in our previous works [START_REF] Ber | Temporal and Spatial Data Mining with Second-Order Hidden Markov Models[END_REF][START_REF] Lazrak | Landscape regularity modelling for environmental challenges in agriculture[END_REF], the MRF has been approximated by a HMM2. This HMM2 has been trained by the EM algorithm on the 1  T temporal representations of the landscape.

The time-space clustering

The stochastic modelling and clustering exhibits patches characterized by distributions of temporal cliques.

Discussion

We have proposed a new representation of agricultural landscapes based on temporal cliques of parcels. To cope with the irregular neighbour system between the parcels, we have specified a variable depth fractal curve that introduces a surface normalization factor and visits the parcels according to their neighbourhood. The sampling becomes irregular and enhances the neighbourhood effects. Considering temporal cliques rather than single OCS gives a valuable information about the neighbour system between OCS and LUS. This shows the different degree of attraction between LUS in this area and therefore describes the landscape through patches. Compared to our previous work [START_REF] Lazrak | Landscape regularity modelling for environmental challenges in agriculture[END_REF], the stochastic modelling of the parcel mosaic based on temporal cliques clusters a landscape into agricultural districts that reveal the LUS and the LUS attraction. We put forward the hypothesis that these agricultural districts capture the temporal and spatial variability and can describe, in a simpler way, the agricultural landscapes to achieve a better understanding of the underlying logical processes.

Figure 1 .

 1 Figure 1. An example of parcel boundary evolution over three successive years. The union of parcel boundaries during this period leads to the definition of seven elementary parcelsFollowing Benmiloud and Pieczynski[START_REF] Benmiloud | Estimation des paramètres dans les chaînes de Markov cachés et segmentation d'images[END_REF][START_REF] Pieczynski | Markov models in image processing[END_REF], we have approximated the Markov field by scanning the 2-D landscape representation with a Hilbert-Peano curve (figure2). The Markov field is then represented by a Markov chain. To take into account the irregular neighbour system, we have first regularly sampled the area covering the landscape (eg.1 point every 20 m), next have introduced an Hilbert-Peano scanning and finally, have adjusted the fractal depth to the elementary parcel size. The figure2illustrates this concept. The sites lying in the same elementary parcel are agglomerated into one point as far they draw the fractal motif. Two successive sites in the L -Length fractal curve ) , ( 1 l l s s  ,

Figure 2 .

 2 Figure 2. Variable depth Hilbert-Peano scan to take into account the parcel size. Two successive merging in the bottom left parcel yield to the agglomeration of 16 points

-use category Land-use Cumul. frequency

	Wheat	Wheat, bearded wheat, cereal	0.337
	Sunflower	Sunflower, reygrass followed by sunflower	0.476
	Rapeseed	Rapeseed	0.600
	Urban	Built area, peri-village, road	0.696
	Grassland	Grassland of various types, alfalfa, ...	0.774
	Maize	Maize, ryegrass followed by maize	0.850
	Forest	Forest or hedge, wasteland	0.884
	Winter barley	Winter barley	0.918
	Ryegrass	Ryegrass, ryegrass followed by ryegrass	0.942
	Pea	Pea	0.964
		Spring barley, grape vine, clover, field bean,
	Others	ryegrass, cereal-legume mixture, garden/market	1.000
		gardening, ...

Table 2 .

 2 Table 2 is a simple example involving the patches tagged as Urban by the stochastic clustering. We can see that the Grassland and Urban categories are stable in the time and have a mutual strong attraction. Less frequent is the neighbourhood occupied by crop successions involving Wheat, Rapeseed and Sunflower. Temporal cliques in the patches tagged as Urban by the stochastic clustering. Items are listed in decreasing order of frequency.

		• The analysis of rows t S and	S	t		1	shows the time dependencies at the site
		level whereas the analysis of rows	t N and	N	t		1	shows the same time
		dependencies at the neighbour level;
		• similarly, the analysis of rows		S and	N shows the attraction between
												t	t
		OCS;
		• furthermore, the joint analysis permits to quantify the attraction between
		LUS.
	S	t	N	t	S	t		1				t		1

Acknowledgments

This work was supported by the ANR-ADD-COPT project, the API-ECOGER project and the ANR-BiodivAgrim project. We thank the CNRS team in Chizé for their data records obtained from their "Niort Plain data base".

The table 3 is an other example that represents the most frequent items of temporal cliques in the patches holding crop successions including Sunflower, Wheat, and Rapeseed. This table shows clearly that, in theses patches, the OCS located nearby a parcel will be held soon in this parcel. Most likely, this time-space relationship is dictated by the type of crop rotations practiced in this cluster. In fact, a previous data mining study [START_REF] Lazrak | Landscape regularity modelling for environmental challenges in agriculture[END_REF] on the same land-use data base allowed to discover that the main rotations involving Sunflower, Wheat, and Rapeseed in the study area are the quadrennial rotation: (Sunflower-Wheat-Rapeseed-Wheat), and the biennial rotations: (Sunflower-Wheat) and (Rapeseed-Wheat). Furthermore, this spatial cluster describes an open-field agricultural area because the temporal cliques involving either Forest or Grassland in the neighbourhood are not represented.