Etienne Bezault
email: ebezault@reed.edu

Xavier Rognon

Frederic Clota

Karim Gharbi

Jean-Francois Baroiller

Bernard Chevassus

Analysis of the Meiotic Segregation in Intergeneric Hybrids of Tilapias

Tilapia species exhibit a large ecological diversity and an important propensity to interspecific hybridisation. This has been shown in the wild and used in aquaculture. However, despite its important evolutionary implications, few studies have focused on the analysis of hybrid genomes and their meiotic segregation. Intergeneric hybrids between Oreochromis niloticus and Sarotherodon melanotheron, two species highly differentiated genetically, ecologically, and behaviourally, were produced experimentally. The meiotic segregation of these hybrids was analysed in reciprocal second generation hybrid (F2) and backcross families and compared to the meiosis of both parental species, using a panel of 30 microsatellite markers. Hybrid meioses showed segregation in accordance to Mendelian expectations, independent from sex and the direction of crosses. In addition, we observed a conservation of linkage associations between markers, which suggests a relatively similar genome structure between the two parental species and the apparent lack of postzygotic incompatibility, despite their important divergence. These results provide genomics insights into the relative ease of hybridisation within cichlid species when prezygotic barriers are disrupted. Overall our results support the hypothesis that hybridisation may have played an important role in the evolution and diversification of cichlids.

Introduction

Interspecific hybridisation has been suggested to be an important evolutionary force that generates biological diversity by the recombination of genetic material among divergent lineages [START_REF] Arnold | Natural Hybridization and Evolution[END_REF][START_REF] Rieseberg | Plant speciation[END_REF][START_REF] Mallet | Hybridization as an invasion of the genome[END_REF]. Hybridisation has been shown to facilitate adaptation, the emergence of evolutionary novelty, and the isolation of new species [START_REF] Rieseberg | Major ecological transitions in wild sunflowers facilitated by hybridization[END_REF][START_REF] Baack | A genomic view of introgression and hybrid speciation[END_REF][START_REF] Via | The ecological genetics of speciation[END_REF][START_REF] Mallet | Hybridization, ecological races and the nature of species: empirical evidence for the ease of speciation[END_REF]. The role of introgressive hybridisation has also been shown in case of humanmediated evolution and especially domestication [START_REF] Arnold | Natural hybridization and the evolution of domesticated, pest and disease organisms[END_REF]. For instance, interspecific hybridisation has been widely used for aquaculture purposes in a large variety of species [START_REF] Bartley | The use of interspecific hybrids in aquaculture and fisheries[END_REF].

Convincing evidence suggests that inter-specific hybridisation has played an important role during evolution and diversification of cichlid fish [START_REF] Seehausen | Hybridization and adaptive radiation[END_REF]. Some cichlid adaptive radiations may have been initiated through hybridisation between distantly related lineages, forming a "hybrid swarm," such as the radiations of Lakes Victoria [START_REF] Seehausen | Nuclear markers reveal unexpected genetic variation and a Congolese-Nilotic origin of the Lake Victoria cichlid species flock[END_REF], Malawi [START_REF] Joyce | Repeated colonization and hybridization in Lake Malawi cichlids[END_REF], and Makgadikgadi [START_REF] Joyce | An extant cichlid fish radiation emerged in an extinct Pleistocene lake[END_REF]. Hybridisation can also occur later during the process of radiation between divergent or already diverged species, forming "syngameon", as suggested for some Lake Tanganyika lineages [START_REF] Schelly | Phylogenetic relationships of the lamprologine cichlid genus Lepidiolamprologus (Teleostei: Perciformes) based on mitochondrial and nuclear sequences, suggesting introgressive hybridization[END_REF][START_REF] Rüber | Population structure in two sympatric species of the Lake Tanganyika cichlid tribe Eretmodini: evidence for introgression[END_REF][START_REF] Salzburger | Speciation via introgressive hybridization in East African cichlids?[END_REF]. The vast majority of the studies conducted so far on cichlid speciation and the potential influence of hybridisation have focused on the East African Great Lakes radiations, especially the Haplochromines. However, the Tilapiines sensu lato [START_REF] Klett | What, if anything, is a Tilapia? Mitochondrial ND2 phylogeny of tilapiines and the evolution of parental care systems in the African cichlid fishes[END_REF][START_REF] Schwarzer | The root of the East African cichlid radiations[END_REF] appear to be an extremely interesting group to study the evolutionary role of hybridisation. Several cases of introgressive hybridisation have been recorded in the wild, under natural conditions, either during the process of adaptive radiation, as in Cameroonian crater lake [START_REF] Schliewen | Reticulate sympatric speciation in Cameroonian crater lake cichlids[END_REF], or during paleoenvironmental fluctuations [START_REF] Rognon | Large extent of mitochondrial DNA transfer from Oreochromis aureus to O. niloticus in West Africa[END_REF]. Several cases have also been reported following anthropogenic perturbations, either between sympatric species after important habitat modifications such as dam building [START_REF] Pouyaud | Génétique des populations de tilapias d'intérêt aquacole en Afrique de l'Ouest[END_REF] or between allopatric species after introduction of species outside their natural distribution area [START_REF] Daget | Hybridation introgressive entre deux espèces de Sarotherodon (Pisces, Cichlidae) dans un lac de Madagascar[END_REF][START_REF] Gregg | Introgressive hybridization of tilapias in Zimbabwe[END_REF]. Furthermore, numerous interspecific hybrids have been produced for aquaculture [START_REF] Wohlfarth | The unexploited potential of tilapia hybrids in aquaculture[END_REF], mainly among Oreochromis species [START_REF] Bartley | The use of interspecific hybrids in aquaculture and fisheries[END_REF][START_REF] Eknath | Use and exchange of genetic resources of Nile tilapia (Oreochromis niloticus)[END_REF][START_REF] Wohlfarth | The heredity of sex determination in tilapias[END_REF]. The production of tilapia hybrids has two main purposes: the production of monosex populations by crossing between species with opposite sex determination system [START_REF] Hulata | Progeny-testing selection of tilapia broodstocks producing all-male hybrid progenies-Preliminary results[END_REF][START_REF] Mair | Sex determination in the genus Oreochromis-2. Sex reversal, hybridisation, gynogenesis and triploidy in O. aureus Steindachner[END_REF][START_REF] Baroiller | Environment and sex determination in farmed fish[END_REF][START_REF] Cnaani | Genetics of sex determination in tilapiine species[END_REF][START_REF] Baroiller | Tilapia sex determination: where temperature and genetics meet[END_REF] and the improvement of phenotypic traits, such as body colour, growth, or tolerance to environmental conditions [START_REF] Wohlfarth | The unexploited potential of tilapia hybrids in aquaculture[END_REF]. However, it is only for the latter that introgressive hybridisation is performed, with the production of breeding population of hybrid origin, aiming at being propagated and selected throughout successive reproductive events. This has been conducted mostly crossing pairs of closely related species (e.g., Oreochromis niloticus and O. aureus, [START_REF] Hulata | Evaluation of Oreochromis niloticus × O. aureus hybrid progeny of different geographical isolates, reared under varying management regimes[END_REF]), but also using more complex crossing schemes involving multiple species (e.g., Red Florida stain, between Oreochromis urolepis hornorum, O. mossambicus, O. niloticus, and O. aureus) [START_REF] Brummett | Development of biochemical dichotomous keys for identification of American populations of Oreochromis aureus, O. mossambicus, O. niloticus, O. urolepis hornorum and red tilapia[END_REF].

Theoretically, while the evolutionary potential of a hybrid lineage is ultimately dependent on its ability to successfully occupy a peak of the local adaptive landscape through the advantage conferred by its original combination of phenotypic traits, hybrid propagation initially requires the maintenance of a stable gene pool through generations. Most of theoretical and empirical studies are focussing on the adaptive role of hybridisation (see review by Stelkens and Seehausen [START_REF] Stelkens | Genetic distance between species predicts novel trait expression in their hybrids[END_REF]); however very few studies have investigated the transmission of hybrid genomes across generations.

Various hybrid meiotic mechanisms deviating from classical diploid Mendelian segregation and inheritance have been reported, especially in fish [START_REF] Chevassus | Modification du phénotype sexuel et du mode de reproduction chez les poissons Salmonidés : inversion sexuelle hormonale, gynogenèse, hybridation interspécifique et polyploïdisation[END_REF][START_REF] Chevassus | Hybridization in fish[END_REF]. Such mechanisms may be classified in four main categories: (1) hybridisation can induce variable ploidy levels by the production of nonreduced (diploid) gametes; this mechanism is likely to lead to the isolation of polyploidy lineages as, for example, in Barbus species [START_REF] Chenuil | A test of the hypothesis of an autopolyploid vs. allopolyploid origin for a tetraploid lineage: application to the genus Barbus (Cyprinidae)[END_REF]; (2) hybridisation can induce clonal gynogenetic reproduction by the suppression of syngamy (i.e., absence of fusion and elimination of male pronucleus) and the mitotic or meiotic restoration of diploidy, such as observed in Poecilia formosa [START_REF] Hubes | Apparent parthenogenesis in nature, in a form of fish of hybrid origin[END_REF]; (3) hybridogenesis can be achieved by selective meiosis, in which the genome of one of the parents (generally the paternal one) is preferentially eliminated from the gametes leading to the next generation, as described in hybrid females of Poeciliopsis [START_REF] Schultz | Hybridization, unisexuality and polyploidy in the teleost Poeciliopsis (Poeciliidae)[END_REF]; (4) modification of recombination within the hybrid genomes, caused by the slight structural divergences between the associated genomes, might affect the homogeneity of the hybrid gene pool, leading, for example, to the maintenance of a mosaic hybrid genome and a fitness deficit [START_REF] Butlin | Recombination and speciation[END_REF][START_REF] Woram | A genetic linkage map for Arctic char (Salvelinus alpinus): evidence for higher recombination rates and segregation distortion in hybrid versus pure strain mapping parents[END_REF][START_REF] Rogers | Linkage maps of the dwarf and normal lake whitefish (Coregonus clupeaformis) species complex and their hybrids reveal the genetic architecture of population divergence[END_REF][START_REF] Rogers | Combining the analyses of introgressive hybridisation and linkage mapping to investigate the genetic architecture of population divergence in the lake whitefish (Coregonus clupeaformis, Mitchill)[END_REF].

To investigate hybrid meiotic segregation in cichlids, we previously produced an intergeneric hybrid between two highly divergent species using in vitro fertilisation: the Nile tilapia, Oreochromis niloticus, and the black-chinned tilapia, Sarotherodon melanotheron [START_REF] Baroiller | Production of 2 reciprocal intergeneric hybrids between Oreochromis niloticus and Sarotherodon melanotheron[END_REF]. All hybrids were found to be viable and fertile for at least three generations [START_REF] Baroiller | Production of 2 reciprocal intergeneric hybrids between Oreochromis niloticus and Sarotherodon melanotheron[END_REF]. The divergence between the two species is estimated between 6.4 Myrs and 21.4 Myrs using the age of radiation of Oreochromis and Oreochromines as an approximation [START_REF] Schwarzer | The root of the East African cichlid radiations[END_REF]. Additionally, these two species show high levels of differentiation in morphology, ecology, behaviour, and physiology. O. niloticus is a maternal mouthbrooder, fast growing and freshwater stenotopic species, whereas S. melanotheron is a paternal mouthbrooder, slow growing, brackish water eurytopic species. Selecting two highly differentiated parental species for the experimental hybridisation potentially increases the possibility of generating original trait association in hybrids compared to the parental species (including transgressive characters) but also increases the likelihood for reproductive incompatibility or unusual meiotic mechanisms, compared to hybridisation between closely related or sister species. Thus, these inter-generic hybrids represent an original and well-adapted model to study the association and segregation of parental genes within cichlid hybrid genomes.

Microsatellite markers provide valuable tools for a wide range of genetic investigations, including species comparison using PCR primers developed in one species and crossamplified in closely related taxa [START_REF] Estoup | Microsatellites and minisatellites for molecular ecology: theoretical and empirical considerations[END_REF][START_REF] Jarne | Microsatellites, from molecules to populations and back[END_REF]. Here we took advantage of the availability of a large number of markers cloned in O. niloticus [START_REF] Lee | Microsatellite DNA markers for genetic mapping in Oreochromis niloticus[END_REF] positioned onto the tilapia genetic maps [START_REF] Kocher | A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus)[END_REF][START_REF] Agresti | Breeding new strains of tilapia: development of an artificial center of origin and linkage map based on AFLP and microsatellite loci[END_REF][START_REF] Mcconnell | Microsatellite marker based genetic linkage maps of Oreochromis aureus and O. niloticus (Cichlidae): extensive linkage group segment homologies revealed[END_REF][START_REF] Lee | A second-generation genetic linkage map of tilapia (Oreochromis spp.)[END_REF] and their high rate of cross-species amplification among tilapias [START_REF] Bezault | Microsatellites cross-species amplification across some African cichlids[END_REF], to study the mechanism of hybrid meiotic segregation in experimental hybrids between O. niloticus and S. melanotheron. We tested (1) whether hybrid meiotic segregations follow diploid Mendelian inheritance and (2) whether this model of meiotic segregation allows the maintenance of a stable hybrid gene pool across generations.

Material and Methods

Biological Material.

The meiosis of first generation hybrids (F1) between the 2 parental species O. niloticus (On) and S. melanotheron (Sm) was studied for both reciprocal hybrid crosses (♀ On × ♂ Sm called G1 and ♀ Sm × ♂ On called G 1) and both sexes. Analysis was conducted on backcross (BC) and second-generation hybrid (F2) families. While F2 crosses allow studying hybrid meiosis for both parents in each family, BC progeny allowing the study of only one hybrid meiosis per family can be more informative with respect to the parental origin of alleles, and further allow the analysis of a parental meiosis, which provide an internal pure-species control segregation.

Four backcross families performed on O. niloticus (due to its better known reproduction biology and greater ease to be manipulated) were analysed to study the meiotic segregation of both reciprocal hybrid types and both sexes (♀ G1, ♂ G1, ♀ G 1 & ♂ G 1), and the pure-species segregation of O. niloticus in both sexes (♀ On & ♂ On) (Table 1). In addition, two different hybrid F2 families (♀ G 1 × ♂ G 1 and ♀ G1 × ♂ G 1) were analysed to look at the segregations and allele associations within a full hybrid genome (Table 1). Finally, an independent pure S. melanotheron family (♀ Sm & ♂ Sm; P-Sm) was studied to provide information on the meiotic segregation of the other parental species (Table 1).

For each family, 50 randomly sampled individuals were analysed.

Microsatellites Markers.

Microsatellites markers were selected from published markers isolated in O. niloticus [START_REF] Lee | Microsatellite DNA markers for genetic mapping in Oreochromis niloticus[END_REF] and successfully amplified in both O. niloticus and S. melanotheron [START_REF] Bezault | Microsatellites cross-species amplification across some African cichlids[END_REF]. Markers were selected based on cross amplification efficiency, polymorphism among and within O. niloticus and S. melanotheron, and their position on the genetic map of tilapias [START_REF] Kocher | A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus)[END_REF][START_REF] Agresti | Breeding new strains of tilapia: development of an artificial center of origin and linkage map based on AFLP and microsatellite loci[END_REF][START_REF] Mcconnell | Microsatellite marker based genetic linkage maps of Oreochromis aureus and O. niloticus (Cichlidae): extensive linkage group segment homologies revealed[END_REF][START_REF] Lee | A second-generation genetic linkage map of tilapia (Oreochromis spp.)[END_REF]. The analysis of the meiotic segregation was conducted using a total of 30 microsatellite markers distributed across the tilapia genome, allowing to compare the segregation of both punctual genomic locations, represented by single independent loci, as well as larger genomic segments represented by two to four linked loci. Overall, the 30 selected markers represented 15 of the 24 linkage groups (LGs) defined in O. niloticus genetic map, 8 of each were represented by more than one marker. Two unmapped loci were also included in the analysis (Table 2).

Genotyping of Microsatellites.

Genomic DNA was extracted from fin clips stored in ethanol using phenol-chloroform protocol [START_REF]Marqueurs microsatellites: isolement à l'aide de sondes nonradioactives, caractérisation et mise au point[END_REF]. Genotypes were obtained by PCR amplification using radioactively (P 33 ) labelled primers and 6% acrylamide gel electrophoresis [START_REF]Marqueurs microsatellites: isolement à l'aide de sondes nonradioactives, caractérisation et mise au point[END_REF][START_REF] Estoup | Parentage assignment using microsatellites in turbot (Scophthalmus maximus) and rainbow trout (Oncorhynchus mykiss) hatchery populations[END_REF]. For each microsatellite marker, optimal amplification conditions, namely annealing temperature and MgCl 2 concentration for coamplification of alleles from heterospecific origin, were obtained from a cross-species amplification study in over 15 species of African cichlids, including both parental target species [START_REF] Bezault | Microsatellites cross-species amplification across some African cichlids[END_REF]. Specific PCR conditions for each locus are indicated in Table 2.

All parents used to produce the experimental progeny were genotyped first at all selected loci to identify informative markers for each family. Each family was genotyped for all informative loci in the entire set of optimised markers (n = 30), whereas the F2 hybrid progeny was only genotyped across the restricted set of independent markers (n = 14) (Table 2; details in supplementary Table S1 available online at doi:10.1155/2012/817562).

Statistical Analysis

2.4.1. Genetic Diversity. The test of amplification efficiency and the estimates of genetic diversity within and across the two-target parental species have been conducted over the set of individuals analysed during cross-species amplification study and the set of pure parents and grandparents of the seven experimental progenies included in the analysis of meiotic segregation [START_REF] Bezault | Microsatellites cross-species amplification across some African cichlids[END_REF]. For each locus, the number of observed alleles was recorded for each parental species, as well as the number of shared alleles between them. The presence of null alleles in the studied loci was estimated across all breeder individuals, based on the repetitive occurrence of a nonamplification result in parents, and/or the significant departure from Mendelian inheritance associated with a pattern characteristic of the segregation of at least one nonamplified allele (i.e., based on the overall pattern of alleles segregation in the entire progeny). For each species, the proportion of polymorphic loci (P < 0.95) and the mean allele number per loci were calculated. The number of shared alleles, between O. niloticus and S. melanotheron, as percent of total number of observed alleles, was calculated per locus and across loci. For the purpose of this study, the existence of shared alleles between parental species and/or their low allelic diversity can potentially lead to cases where marker segregation is not fully informative (e.g., when both parents are heterozygous for the same alleles). These cases were identified based on the genotyping of all parent individuals, prior to the progeny genotyping.

Analysis of Meiotic

Segregation. The analysis of the meiotic segregation was conducted for each individual parent and family, as well as across all pure and hybrid segregations, considering either each locus independently or collectively. In order to accurately account for false positives due to multiple testing, a sequential Bonferroni correction was applied [START_REF] Rice | Anylizing tables of statistical tests[END_REF], considering the multiple tests performed either within a given progeny/breeder-segregation across independent loci or for a given locus or pair of loci across the independent tested segregations/families. Accordance of the observed segregations to Mendelian expectations was tested using a Chi 2 test to detect possible [START_REF] Lee | Microsatellite DNA markers for genetic mapping in Oreochromis niloticus[END_REF][START_REF] Kocher | A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus)[END_REF][START_REF] Lee | A second-generation genetic linkage map of tilapia (Oreochromis spp.)[END_REF], optimised PCR conditions established from cross-species amplification study of microsatellites across 15 African cichlid species (labeled primer " * ", annealing temperature and Magnesium concentration (mM)-Bezault et al., [START_REF] Bezault | Microsatellites cross-species amplification across some African cichlids[END_REF]); the size range and diversity of alleles within each parental species as well as the number of shared alleles between them and the presence of null allele (N) have been estimated from the set of individuals used for cross-priming analysis and the parents of all families studied here; the set of independent loci analysed in all backcross and F2 hybrid families are indicated in bold.

Loci

GenBank distortion of segregation. Sequential Bonferroni correction of P-value for multiple tests was performed across loci separately within each cross. The balance of global meiotic transmission of both parental alleles was tested within each hybrid segregation using a Chi 2 test across all loci within each breeder segregation. Homogeneity of reciprocal backcrosses was tested by comparison of genotypic distributions between families for each locus using Chi 2 tests, with global tests combining these results across loci for each families comparison using Fisher's method [START_REF] Sokal | Biometry, the Principles and Practice of Statistics in Biological Research[END_REF]. Comparisons were carried out between progeny from the same hybrid type (G1 or G 1), or the same hybrid way (♀ or ♂), and overall cases. Pairwise linkage analysis was performed using LinkMFex [START_REF] Danzmann | LINKMFEX: Linkage analysis package for outcrossed mapping families with male or female exchange of the mapping parent[END_REF] using an LOD score of 3 as threshold for significance. Linkage associations with a 2 ≤ LOD Score < 3 were considered as suggestive. Recombination rates were compared using a Chi 2 test.

Results

Microsatellite Diversity.

A high percentage of the markers were polymorphic in both species (Table 2), with a slightly lower diversity in S. melanotheron (77%) than in O. niloticus (97%). A similar pattern was observed for the mean allelic diversity per locus and species (2.8 and 4.3, resp.). Null alleles were detected for a total of 7 markers, with a higher frequency S1).

Families BC-A BC-B BC-C BC-D F2-A F2-B Breeders ♀ G1 ♂ G1 ♀ G 1 ♂ G 1 ♀ G 1 ♂ G 1 ♀ G1 ♂ G 1 # l o c i 2

Analysis of Meiotic Segregation.

A total of 4 out of 232 segregations (1.7%) showed significant evidence of segregation distortion (P < 0.05 after correction for multiple testing). Three significant tests were observed in hybrid genomes (2 in ♂ G 1, UNH-008 & UNH-216, and 1 in ♀ G1, UNH-197) and a single significant test in pure species (♀ S. melanotheron, UNH-135) (details in Supplementary Table S1).

Out of the 8 hybrid parents analysed, equal transmission of alleles from both parental and species origin was observed in the vast majority of the cases (n = 7), representing at least one meiotic segregation of each hybrid cross (G1 and G 1) and sex (Table 3). The hypothesis of equal transmission of parental species alleles was rejected in a single hybrid male (BC-D family), which globally undertransmitted its paternal alleles of O. niloticus origin (Table 3). This was consistent with the results from the locus-level analysis for this individual, which revealed evidence of unequal allele transmission for 2 significant loci from 3 different LGs after Bonferroni correction, always in the direction of undertransmission of O. niloticus paternal alleles (details in Supplementary Table S1).

When we considered allelic distributions observed among backcross progeny, according to hybrid type and/or sex, only the comparison between hybrid males exhibited significant heterogeneity (P < 0.01) (Table 4). However, the comparison between the 2 types of hybrids, G1 versus G 1, and globally among the 4 different sex and hybrid types did not reveal any evidence of deviation from homogeneity.

Genetic linkage was detected in 16 cases (Table 5), of which 14 cases were expected from previous publications [START_REF] Kocher | A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus)[END_REF][START_REF] Agresti | Breeding new strains of tilapia: development of an artificial center of origin and linkage map based on AFLP and microsatellite loci[END_REF][START_REF] Mcconnell | Microsatellite marker based genetic linkage maps of Oreochromis aureus and O. niloticus (Cichlidae): extensive linkage group segment homologies revealed[END_REF][START_REF] Lee | A second-generation genetic linkage map of tilapia (Oreochromis spp.)[END_REF], but 2 being unexpected among these studies. The , but only in one case for each of these pairs. One unexpected linkage (UNH-008 & UNH-124) was significant in 6 parents. The other unexpected association (UNH-106 & UNH-207) was only found in one parent out of 3. Finally one unmapped marker, UNH-117, was assigned to the LG 5 with a significant linkage in 2 parents. The occurrence of these linkage associations was checked comparatively between the 2 pure and the hybrid genomes. All but one was significant in S. melanotheron parents, and every linkage significantly established in O. niloticus showed significant linkage in hybrid parents. Recombination rates were compared using a Chi 2 test between parents of opposite sex and/or genetic types in pure and hybrid individuals (see details in Supplementary Table S3). Out of 16 pairs of linked loci (i.e., for which a significant linkage has been detected in at least one parent), heterogeneity of recombination rate in at least one comparison was observed for 2 pairs of loci (i.e., UNH-125 & UNH-138 (LG16) and UNH-131 and UNH-135 (LG3)). These 2 cases where homogeneity of recombination rate was rejected were detected in comparisons involving pure-species parents. In both cases, evidence of heterogeneity was observed within species (in O. niloticus comparisons, between sexes and among males, resp.) as well as between species (i.e., between O. niloticus and S. melanotheron). No evidence of significant heterogeneity was observed among hybrid meioses.

Discussion

We have characterized meiotic segregation in experimental intergeneric hybrids between two highly differentiated Oreochromine cichlid species, O. niloticus and S. melanotheron, from which no natural hybrid has ever been reported, due to ecological and reproductive behaviour divergence, even if sympatric in some basins of West Africa [START_REF]Faunafri-World Wide Web electronic[END_REF][START_REF] Trewavas | Tilapiine Fishes of the Genera Sarotherodon, Oreochromis and Danakilia[END_REF][START_REF] Philippart | Ecology and distribution of tilapias[END_REF]. Our analysis permitted to track the origin and transmission of alleles across 17 independently mapped anchors distributed over the tilapia genome, including 8 genomic segments represented by 2 to 4 linked loci to survey variation of recombination between hybrids, parental species, and/or sexes. All hybrid parents showed systematic exclusion of the alleles from each of the two parental species (i.e., hybrid parents transmitted either the O. niloticus allele or the S. melanotheron allele but never both or none). The strict disjunction of both O. niloticus and S. melanotheron alleles at each locus confirms locus homology between these two species and indicates the diploid state of these hybrids, at both the sequence and genome organisation level. The balanced segregations of loci lead to Mendelian transmission of both parental species alleles to the next generation for both reciprocal ways of crosses (G1 & G 1). A stable hybrid gene pool was maintained through meiosis, although segregation distortions were observed in very few cases (1.7%). As we observed no preferential allele elimination, neither in terms of parental species nor cross-direction (maternal/paternal origin), the observed departures from expected Mendelian inheritance may be due to local meiosis irregularities (i.e., male G 1 hybrid from BC-D), rather than a general effect of hybridisation.

The vast majority of linkage associations between markers expected in O. niloticus based on existing genetic maps [START_REF] Kocher | A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus)[END_REF][START_REF] Agresti | Breeding new strains of tilapia: development of an artificial center of origin and linkage map based on AFLP and microsatellite loci[END_REF][START_REF] Mcconnell | Microsatellite marker based genetic linkage maps of Oreochromis aureus and O. niloticus (Cichlidae): extensive linkage group segment homologies revealed[END_REF][START_REF] Lee | A second-generation genetic linkage map of tilapia (Oreochromis spp.)[END_REF] were confirmed in this study. Moreover these associations appeared well conserved in S. melanotheron and hybrids. This is consistent with a high level of synteny conservation between tilapia species, as previously observed between the closely related O. niloticus and O. aureus [START_REF] Mcconnell | Microsatellite marker based genetic linkage maps of Oreochromis aureus and O. niloticus (Cichlidae): extensive linkage group segment homologies revealed[END_REF] and further extends this observation to distantly related species among Oreochromines. Recombination rates appear very similar between sexes as well as among hybrids and pure species. We did not observe a contraction of the genetic map as may be expected in hybrids because of reduced recombination due to structural genomic differences between parental species [START_REF] Mcconnell | Microsatellite marker based genetic linkage maps of Oreochromis aureus and O. niloticus (Cichlidae): extensive linkage group segment homologies revealed[END_REF]. Taken together our results allow us to reject the hypothesis of meiotic irregularities or specific meiotic processes (i.e., nonhomoploid biparental transmission) in the hybrid between O. niloticus and S. melanotheron.

Our study also allowed us to demonstrate Mendelian transmission during the F1 hybrid meiosis, independent of the sex and/or direction of crosses (G1 or G 1), leading to the maintenance of a stable and balanced hybrid gene pool through generations. These results, especially the strict disjunction of parental alleles at each locus and the conservation of linkage groups without genetic map contraction, suggest a close genomic structure between both parental species and their hybrids [START_REF] Mcconnell | Microsatellite marker based genetic linkage maps of Oreochromis aureus and O. niloticus (Cichlidae): extensive linkage group segment homologies revealed[END_REF][START_REF] Lee | A second-generation genetic linkage map of tilapia (Oreochromis spp.)[END_REF]. The analysis of phenotypic traits segregating in hybrids is particularly interesting in this context, especially considering the numerous divergent traits between the two parental species, such as morphology, reproductive behaviour, and physiological adaptive capacity. Physiological traits, for example, growth and salinity tolerance, show intermediate phenotypes in F1 hybrids [START_REF] Baroiller | Production of 2 reciprocal intergeneric hybrids between Oreochromis niloticus and Sarotherodon melanotheron[END_REF][START_REF] Lemarié | A simple test to estimate the salinity resistance of fish with specific application to O. niloticus and S. melanotheron[END_REF]. In experimental conditions, the fertility of hybrids has been established up to the F2 and viability up to F3 (for both hybrid and backcross progeny-unpublished data). Fertility problems have been previously reported in interspecific fish hybrids (see review by Bartley et al. [9]), not only at the initial hybrid stage (i.e., sterility in F1 generally due to ploidy perturbations) but also in subsequent hybrid generations, as in the case of hybrid models where high fitness is observed in early hybrids (F1) followed by a strong fitness breakdown in subsequent generations (F2+). Such examples were observed in tilapia during a complex four-species crossing project between three Oreochromis and one Sarotherodon [START_REF] Agresti | Breeding new strains of tilapia: development of an artificial center of origin and linkage map based on AFLP and microsatellite loci[END_REF], where the viable and fertile two-way F1 hybrids led to four-way F2 hybrids unable to produce viable progeny [START_REF] Hulata | Fertility problems in the second generation of a four-species tilapia cross[END_REF]. Such rapid introgression of four genomes in only two generations might have exacerbated instability of the resulting gene pool leading to fertility problems. This phenomenon would have been avoided or strongly reduced in case of a two-way hybridisation and/or increasing the number of generations to mix the genomes, especially if it involved an introgressive (backcross) scheme and/or closely related parental species. In any case, the existence of a late fitness disruption in hybrids tends to suggest the absence of major genomic reorganisation between Oreochromines species, in favour of small genomic rearrangement(s), for example, microinversions and insertions, structural variations and/or divergent gene evolution. These elements can all be potentially responsible for the establishment of postzygotic isolation resulting from the increase of Dobzhansky-Muller genetic incompatibilities [START_REF] Dobzhansky | Studies on hybrid sterility. II. Localization of sterility factors in Drosophila pseudoobscura hybrids[END_REF]. Over time, both premating and postmating incompatibilities are expected to accumulate between divergent lineages, with a relative rate depending on different parameters, including the geographical mode of speciation, the existence of sexual dimorphisms, and/or strong sexual selection, overall yielding to the "speciation clock" [START_REF] Coyne | Speciation[END_REF].

As cichlids are known to exhibit sexual selection, typically associated with male breeding colour polymorphism [START_REF] Maan | Intraspecific sexual selection on a speciation trait, male coloration, in the Lake Victoria cichlid Pundamilia nyererei[END_REF], theory predicts a faster loss of premating than postmating compatibility. In the species used in this study, pre-mating isolation is total, most notably due to the divergence of reproductive behaviour between the two parental species (i.e., O. niloticus is a maternal mouthbrooder whereas S. melanotheron is a paternal mouth-brooder, which explains the need for in vitro fertilisation for the production of F1 hybrids between these species). Post-mating isolation also appears to be relatively weak and probably only due to drift in the absence of reinforcement mechanisms between the two parental species. This process might be different depending International Journal of Evolutionary Biology on the cichlid lineage considered, based on their rate of transition between reproductive systems. For instance, while Oreochromines exhibit maternal, paternal, and biparental mouth-brooding systems, the most species-rich lineage, Haplochromines, exhibits exclusively maternal mouthbrooding reproductive systems [START_REF] Keenleyside | Parental care[END_REF][START_REF] Barlow | Mating systems among cichlid fishes[END_REF]. The absence of pre-mating barrier based on reproductive behaviour among species would either be compensated by other component(s) of pre-mating isolation system, such as sexual selection mediated by male colour polymorphism, or represent an overall lower level of premating isolation, which would be prone to generate an increased rate of postzygotic accumulation (i.e., through reinforcement process).

Two recent studies have investigated the dynamics of hybridisation in Haplochromine cichlids [START_REF] Stelkens | Phenotypic novelty in experimental hybrids is predicted by the genetic distance between species of cichlid fish[END_REF]. While phenotypic novelty does increase with the genetic distance between parental hybridizing lineages [START_REF] Stelkens | Phenotypic novelty in experimental hybrids is predicted by the genetic distance between species of cichlid fish[END_REF], the accumulation of reproductive incompatibilities is also building up with divergence time [START_REF] Stelkens | The accumulation of reproductive incompatibilities in African cichlid fish[END_REF]. This later study demonstrates that, along the axis of species divergence, pre-mating isolation is built up fast initially (i.e., due to mate choice and sexual selection) without increasing much later (e.g., due to the highly conserved courtship behaviour and the absence of change in reproductive system across the entire lineage), whereas postmating incompatibilities only start accumulating at relatively later stages of divergence. Such a pattern may have led to "evolutionary viable" hybridisation between lineages diverging for a very long time (i.e., estimation of the hybrid unviability after 4.4-18.4 Myrs, depending on the molecular clock calibration used [START_REF] Stelkens | The accumulation of reproductive incompatibilities in African cichlid fish[END_REF]). Considering the latest estimates of divergence time between the radiation of the entire Oreochromines tribe (12.8-21.4 Myrs) and the radiation of the genus Oreochromis (6.4-9.7 Myrs) [START_REF] Schwarzer | The root of the East African cichlid radiations[END_REF], the experimental hybrids between O. niloticus and S. melanotheron stand at the later end of the continuum tested in Haplochromines. Our results then appear consistent with the findings drawn from hybridisation in Haplochromines, while adding an independent estimate from another major cichlid tribe. Additionally, the present study extends the previous results by demonstrating the maintenance of normal meiotic mechanisms in hybrids between highly divergent species, long after the completion of pre-mating isolation (i.e., once postmating isolation would be expected to have already started accumulating).

The important role of interspecific hybridisation during the evolution of cichlids, especially the processes of adaptation and diversification, has been extensively discussed [START_REF] Seehausen | Hybridization and adaptive radiation[END_REF]. Hybridisation is thought to occur when populations are subjected to important environmental changes, such as anthropogenic perturbations [START_REF] Daget | Hybridation introgressive entre deux espèces de Sarotherodon (Pisces, Cichlidae) dans un lac de Madagascar[END_REF][START_REF] Gregg | Introgressive hybridization of tilapias in Zimbabwe[END_REF][START_REF] Trewavas | Tilapiine Fishes of the Genera Sarotherodon, Oreochromis and Danakilia[END_REF][START_REF] Seehausen | Cichlid fish diversity threatened by eutrophication that curbs sexual selection[END_REF] and hydroclimatic changes [START_REF] Rognon | Large extent of mitochondrial DNA transfer from Oreochromis aureus to O. niloticus in West Africa[END_REF], and when populations invade a new environment [START_REF] Seehausen | Hybridization and adaptive radiation[END_REF], enabling these populations to undergo rapid adaptive response and even radiation. Evidence suggests that many of the largest cichlid lake radiations may have been initiated and fuelled through hybridisation between distantly related lineages [START_REF] Seehausen | Nuclear markers reveal unexpected genetic variation and a Congolese-Nilotic origin of the Lake Victoria cichlid species flock[END_REF][START_REF] Joyce | Repeated colonization and hybridization in Lake Malawi cichlids[END_REF][START_REF] Joyce | An extant cichlid fish radiation emerged in an extinct Pleistocene lake[END_REF], which invaded the new empty environment and started interbreeding to form a hybrid swarm, predisposing the colonizing lineages to diversify. One of the crucial issues in the hypothesis of hybrid swarm origin of adaptive radiations is to identify the mechanism by which hybrid lineage(s) can be initiated and propagated. Demonstrating that hybridisation between highly distantly related Oreochromines species can lead to meiotic processes following diploid Mendelian segregation and the maintenance of a stable and recombining hybrid gene pool across generations appears to strongly support this hypothesis. Overall, this study provides functional insights into hybridisation in cichlids, when prezygotic barriers are disrupted, despite important divergence time between lineages, and therefore supports the idea that interspecific hybridisation has the potential to play an important role in the evolution and diversification of cichlids.

Table 1 :

 1 Description of the experimental families analysed, including the type of cross, the genetic origin of breeders (i.e., pure O. niloticus) (On) or S. melanotheron (Sm), and reciprocal 1st generation hybrid G1 (♀ On × ♂ Sm) and G 1 (♀ Sm × ♂ On) and the number of studied individuals.

	Families	Type of crossing	Female	Breeders	Male	No. Ind.
	BC-A	Backcross	Hybrid Gl		O. niloticus	50
	BC-B	Backcross	O. niloticus		Hybrid Gl	50
	BC-C	Backcross	Hybrid G l		O. niloticus	50
	BC-D	Backcross	O. niloticus		Hybrid G l	50
	F2-A	Hybrid F2	Hybrid G l		H y b r i d G l	50
	F2-B	Hybrid F2	Hybrid Gl		Hybrid G l	50
	P-Sm	Pure Cross	S. melanotheron		S. melanotheron	50

Table 2 :

 2 Microsatellite loci analysed with indications of GenBank accession number, repeat structure, and linkage group according to O. niloticus

Table 3 :

 3 Test of balanced meiotic transmission of alleles from both parental species (On for O. niloticus and Sm for S. melanotheron) for each of the 8 hybrid F1 breeders (G1 (♀ On × ♂ Sm) and G 1 (♀ Sm × ♂ On)); Chi 2 values and associate P-values are given; significant (i.e., biased transmission) tests (a = 0.05) when applying sequential Bonferroni correction (n = independent tests) are indicated in bold.

Table 4 :

 4 Comparisons of allelic distributions observed among the 4 backcross progeny; the number of loci implicated, the Fisher's test value and the associated P-values are given; significant heterogeneity (a = 0.05) is indicated in bold (see Table1for detail about hybrid types).

	Comparisons	# Loci	Fisher's test	P
	Global	25	65.51	0.069
	Hybrids G1	26	44.91	0.747
	Hybrids G 1	20	54.46	0.063
	Hybrids ♀	24	49.08	0.429
	Hybrids ♂	22	68.8	0.010
	♂ G1/♀ G 1	23	42.82	0.606
	♀ G1/♂ G 1	21	55.93	0.074
	large majority of expected linkage associations (73%) were
	confirmed, including the 2 unexpected cases (i.e., UNH-008
	& UNH-146, and UNH-154 & UNH-207, with LOD > 3 in 5
	and 7 segregations, resp.). Three expected associations were
	not observed: UNH-008 & UNH-103 (LG 17) showed no
	cosegregation, whereas UNH-102 & UNH-138 (LG 16) and
	UNH-130 & UNH-197 (LG 23) showed a suggestive linkage
	(LOD > 2)

Table 5 :

 5 Comparison of linkage associations expected from tilapia genetic maps and observed within the 7 studied families; linkage group (LG) is taken from the previously published

	BC-A BC-B BC-C BC-D P-Sm	♀ G1 ♂ O. nil. ♀ O. nil. ♂ G1 ♀ G 1 ♂ O. nil. ♀ O. nil. ♂ G 1 ♀ S. mel. ♂ S. mel.	Rec LOD Rec LOD Rec LOD Rec LOD Rec LOD Rec LOD Rec LOD Rec LOD Rec LOD Rec LOD	0.42 0.28 0.42 0.28 0.42 0.28 0.46 0.07 0.35 0.9	0.22 3.61 0.10 7.74 0.08 8.73 0.20 3.97 0.09 7.95 0.06 9.58	0.04 11.4 0.08 9.00 0.02 12.92 0.00 15.05 0.00 14.45	0.36 0.86 0.24 3.08 0.22 3.61 0.3 1.79 0.32 1.44 0.36 0.86 0.28 2.18 0.26 2.61	0.41 0.3 0.46 0.07 0.36 0.86 0.4 0.44 0.38 0.63 0.28 2.18 0.38 0.63	0.44 0.16 0.42 0.28 0.24 3.08	0.02 12.05 0.04 10.56 0.23 3.04 0.1 7.99 0.10 7.99	0.00 15.05 0.02 12.92 0.02 12.92 0.02 12.92 0.04 11.4	NT 0.00 15.5 0.02 12.34	0.12 7.08 0.2 4.19 7.08 0.24 3.08 0.12 6.84 0.18 4.6 0.12	0.18 4.82 0.06 9.85 0.10 7.74 0.09 7.95 9.85 0.10 7.74 0.06	0.
	Families	Locus A LG Locus B LG References	UNH-008 17 UNH-103 17 A, D	UNH-008 17 UNH-124 4	UNH-008 17 UNH-146 4 B versus A, D	UNH-102 16 UNH-125 16 A, C	UNH-102 16 UNH-138 16 A, C	UNH-106 3 UNH-207 6	UNH-115 3 UNH-131 3 A, D	UNH-115 3 UNH-135 3 A, D	UNH-117 UNH-149 5	UNH-123 12 UNH-189 12 A, C, D	UNH-124 4 UNH-146 4 C, D	UNH-125 16 UNH-138 16 A, B, C, D

genetic maps (A:

[START_REF] Kocher | A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus)[END_REF]

; B:

[START_REF] Mcconnell | Microsatellite marker based genetic linkage maps of Oreochromis aureus and O. niloticus (Cichlidae): extensive linkage group segment homologies revealed[END_REF]

; C:

[START_REF] Agresti | Breeding new strains of tilapia: development of an artificial center of origin and linkage map based on AFLP and microsatellite loci[END_REF]

; D:

[START_REF] Lee | A second-generation genetic linkage map of tilapia (Oreochromis spp.)[END_REF]

); the recombination rate (Rec) is given with the associated LOD score; significant linkages (LOD > 3) are represented in bold, while suggestive linkages (LOD > 2) are underlined; see Table

1

for detail about hybrid types.

02 12.92 0.08 9.00 0.00 15.05 0.12 7.08 0.06 10.12 0.08 9.00 0.00 13.85 0.04 10.27 0.24 3.08

	0.48 0.02	0.44 0.16	0.02 12.05 0.04 11.12	0.00 14.75 0.04 11.12	0.08 9.00 0.13 6.60 0.08 8.47
				0.02 12.92
	0.28 2.18		0.06 10.12 0.02 12.05 0.21 3.58	11.4 0.02 12.92 0.02 12.92 0.04	0.20 4.19
	0.46 0.07	0.33 1.31 0.02 12.63	0.08 9.00 0.04 11.4	0.02 12.92	0.23 3.42
	A, C	A, C	A, D	A versus D	A, D
	UNH-130 23 UNH-197 23	UNH-130 23 UNH-216 23	UNH-131 3 UNH-135 3	UNH-154 6 UNH-207 6	UNH-197 23 UNH-216 23

Acknowledgments

The authors would like to thank Stephane Mauger and Rene Guyomard for laboratory assistance; Martial Derivaz, Pierre Morisens, and Jerome Lazard for their support in the scientific program of intergeneric hybridisation of tilapias; Irene Keller, Ole Seehausen, Martine Maan, and Matthias Stoeck for stimulating discussions, as well as the two anonymous referees and the editor, R. Craig Albertson, for their valuable comments on the paper. This study was supported by grants from the CIRAD and INRA (France).


Vincent Cottin
email: vincent.cottin@chu-lyon.fr

Jean-François Cordier

Athol U Wells

Centrilobular emphysema combined with pulmonary fibrosis results in improved survival: a response

Better survival in combined pulmonary fibrosis and emphysema than in lone pulmonary fibrosis: bias or reality? A response to Centrilobular emphysema combined with pulmonary fibrosis results in improved survival by Todd et al., Fibrogenesis & Tissue Repair 2011, 4:6.

We read with great interest the somewhat thought-provoking article by Todd et al. [START_REF] Todd | Centrilobular emphysema combined with pulmonary fibrosis results in improved survival[END_REF], which addressed the prognosis of patients with combined pulmonary fibrosis and emphysema (CPFE). The authors found that survival in CPFE was better than that of patients with idiopathic pulmonary fibrosis (IPF) without emphysema or with minimally extensive emphysema ('lone fibrosis'), in striking contrast to previous results [START_REF] Cottin | Groupe d'Etude et de Recherche sur les Maladies "Orphelines" Pulmonaires (GERM"O"P): Combined pulmonary fibrosis and emphysema: a distinct underrecognised entity[END_REF][START_REF] Mejia | Idiopathic pulmonary fibrosis and emphysema: decreased survival associated with severe pulmonary arterial hypertension[END_REF][START_REF] Antoniou | Idiopathic pulmonary fibrosis: outcome in relation to smoking status[END_REF]. From these counterintuitive results, it is argued that tobacco-smokinginduced pulmonary inflammation may be protective against the progression of fibrosis. However, in reaching this conclusion, we feel the authors have failed to sufficiently consider a number of potential biases that are evident from the stated data.

Firstly, amongst biopsied patients, fibrotic non-specific interstitial pneumonia (NSIP) was more prevalent in association with CPFE (6 of 20, 30%) than in the remaining groups (7 of 53, 13%). If these proportions are extrapolated to the whole cohort, including nonbiopsied cases, the prevalence of fibrotic NSIP was significantly higher in the CPFE cases. Furthermore, this imbalance may have been amplified by the inclusion of non-biopsied cases: it is well recognized that NSIP when combined with emphysema may mimic honeycombing and can, therefore, be confused with genuine IPF [START_REF] Akira | Usual interstitial pneumonia and nonspecific interstitial pneumonia with and without concurrent emphysema: thin-section CT findings[END_REF]. A bias towards NSIP would also explain the unexpectedly higher female rate in the CPFE group.

Secondly, the way in which transplantation was handled in the survival analysis is very open to question. It has been usual to censor at date of transplantation, and not to handle a transplant event as a death. The authors point out that the rate in transplantation did not differ between groups. However, as at least a third of 'deaths' (n = 34) were transplant episodes, it is likely that this way of handling transplant events as deaths distorted the Kaplan-Meier curves.

Thirdly, potential lead-time bias was a significant concern in this study. For example, patients with CPFE were more frequently ever-smokers than their counterparts with lone fibrosis, and conceivably were more likely to suffer from chronic bronchitis and thus to seek medical advice or to draw medical attention. A better outcome in patients with CPFE compared with patients with lone fibrosis may therefore reflect earlier identification of disease.

Lastly and most importantly, patients with CPFE (pulmonary fibrosis with significant emphysema) and those with lone fibrosis had comparable carbon monoxide diffusion capacity (DLco) in the study by Todd et al. [START_REF] Todd | Centrilobular emphysema combined with pulmonary fibrosis results in improved survival[END_REF]. As DLco reflects the combined impact of emphysema and fibrosis [START_REF] Kiakouama | Conditions associated with severe carbon monoxide diffusion coefficient reduction[END_REF], it follows that patients with more extensive emphysema must have had less extensive fibrosis, to end up with the same average level of DLco. In other words, based on DLco levels, patients with CPFE must have had significantly less extensive fibrosis than the lone fibrosis group.

In essence, Todd et al. report better survival, using a highly controversial outcome analysis, in a patient sub-group (CPFE) with a higher prevalence of fibrotic NSIP and much less extensive fibrosis. These confounding factors would seem to adequately account for the observed group differences. Thus, whilst Todd et al. should be commended for addressing the question of the relative prognosis of CPFE versus IPF, the biases discussed here make it difficult to reach clear conclusions. Such studies stimulate research by suggesting unreliable yet intriguing conclusions. Adequately conducted studies with adjustment for severity, and extent of fibrosis, and control of confounding factors are now needed to address the unresolved issue of whether CPFE and lone IPF may differ in outcome.

Authors' contributions VC conceived the study and drafted the manuscript; AW designed the study and contributed to draft the manuscript; JFC designed the study and contributed to draft the manuscript; all authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.


Marie-Noëlle Rosso
email: rosso@sophia.inra.fr

Paulo Vieira

Janice De Almeida-Engler

Philippe Castagnone-Sereno

Danchin Eg

Hussey Rs

Proteins secreted by root-knot nematodes accumulate in the extracellular compartment during root infection

Keywords: plant pathogen, effector, immunocytochemistry, root-knot nematode, secretion, plant apoplasm

Root-knot nematodes are biotrophic parasites that invade the root apex of host plants and migrate towards the vascular cylinder where they induce the differentiation of root cells into hypertrophied multinucleated giant cells. Giant cells are part of the permanent feeding site

required for nematode development into the adult stage. To date, a repertoire of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection has been identified. However, the precise role of these candidate effectors during root invasion or during giant cell induction and maintenance remains largely unknown. Primarily, the identification of the destination of nematode effectors within plant cell compartment(s) is crucial to decipher their actual functions. We analysed the fine localization in root tissues of five nematode effectors throughout the migratory and sedentary phases of parasitism using an adapted immunocytochemical method that preserves host and pathogen tissues. We showed that secretion of effectors from the amphids or the oesophageal glands is tightly regulated during the course of infection. The analysed effectors accumulated in the root tissues along the nematode migratory path and along the cell wall of giant cells, showing the apoplasm as an important destination compartment for these effectors during migration and feeding cell formation.

TEXT

Nematodes secrete effectors within plant tissues to promote infection

During plant infection, pathogens secrete effectors that promote disease by manipulating plant cell functions or plant defenses. Similarly, root-knot nematodes (RKN), genus Meloidogyne, secrete proteins within the plant tissues to assist parasite establishment. RKN are biotrophic pathogens that display a highly specialized feeding relationship with their hosts. Infective juveniles penetrate the root apex and migrate intercellularly to the vascular cylinder. There, they settle and select a few cells that will differentiate into hypertrophied and multinucleated giant cells in response to secreted nematode effectors. Six to eight giant cells surrounded by dividing vascular parenchyma cells form the gall, or feeding site, that provides the nutrients required for completion of the nematode life cycle and offspring production. Understanding the role of nematode effectors is a crucial issue to decipher the molecular events leading to the establishment of the parasite, the differentiation of the giant cells and the manipulation of plant defenses throughout interaction.

The oesophageal glands are three cells connected to the oesophagus that produce proteins secreted during infection through a protrusible stylet at the buccal orifice. The stylet is compared to the type three secretion system (TTSS) of bacteria and functions as a syringe to inject secretions in the plant tissues and to retrieve nutrients from the cytoplasm of feeding cells. [START_REF] Williamson | Nematode pathogenesis and resistance in plants[END_REF][START_REF] Sobczak | Ultrastructure of feeding plugs and feeding tubes formed by Heterodera schachtii[END_REF] The amphids, two chemosensory organs located on the nematode head and open to the exterior via a prominent pore, are important secretory organs involved in the perception of chemotactic environment stimuli. 3 To date, approximately hundred proteins potentially secreted by RKN have been identified (for a review see ref. 4). Only few RKN proteins have been shown to be secreted within host tissues during parasitism but the end localization of these secretions is still unknown. [START_REF] Doyle | Cloning and characterisation of an esophageal-gland specific pectate lyase from the root-knot nematode Meloidogyne javanica[END_REF][START_REF] Curtis | Identification and in situ and in vitro characterization of secreted proteins produced by plant-parasitic nematodes[END_REF][START_REF] Lopez De Mendoza | Immunolocalisation in planta of secretions from parasitic stages of Meloidogyne incognita and M. hispanica[END_REF][START_REF] Doyle | Meloidogyne javanica chorismate mutase 1 alters plant cell development[END_REF] One single study provided a clear demonstration for secretion of a RKN protein (the calreticulin Mi-CRT) in the feeding site and showed abundant protein accumulation along the cell wall of giant cells in the vicinity of the stylet tip. [START_REF] Jaubert | In planta secretion of a calreticulin by migratory and sedentary stages of root-knot nematode[END_REF] In order to get a broader view on the secretory activity of the nematode during infection, we used sera directed against five different candidate effectors from the RKN M. incognita and analyzed the localization of the secreted proteins throughout infection.

Nematode proteins are secreted within the apoplasm along the path of migrating nematodes

In order to reach the vascular tissue where they establish their feeding site, nematodes need to penetrate and migrate along the root facing physical and molecular barriers imposed by the host.

Genome sequencing has revealed in M. incognita an extraordinary diverse repertoire of cell wall degrading and modifying enzymes. [START_REF] Abad | Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita[END_REF] We explored the localization of ß-1,4-endoglucanases and expansins during RKN infection using a serum specific to the cellulose binding module (CBM2) appended to these enzymes. In addition, we analysed the localization of a new pectate lyase from M. incognita (Mi-PEL3). Both sera showed the secretion of the enzymes through the stylet along the nematode head and in the apoplasm of root cells during migration, suggesting a role in plant cell wall loosening for root invasion (Fig. 1 A,B). [START_REF] Vieira | The plant apoplasm is an important recipient compartment for nematode secreted proteins[END_REF] Similarly, we observed abundant and continuous secretion through the stylet of an aspartyl protease-like protein in the apoplasm at the forefront of migrating juveniles. The potential role of this new aspartyl protease-like protein as an effector was further supported by its phylogenetic position in a cluster clearly distinct from all other eukaryotes, including other nematodes, suggesting that it could have evolved independently towards a specific function related to plant parasitism. Enzymatic assays will be necessary to further unravel its role in infection.

A Meloidogyne candidate avirulence protein (MAP-1) produced in the amphids of pre-parasitic juveniles was identified in avirulent nematode lines that trigger Mi-1 resistance in tomato. [START_REF] Semblat | Molecular cloning of a cDNA encoding an amphid-secreted putative avirulence protein from the root-knot nematode Meloidogyne incognita[END_REF] At the time of its discovery, map-1 did not show any significant similarity in databases. [START_REF] Semblat | Molecular cloning of a cDNA encoding an amphid-secreted putative avirulence protein from the root-knot nematode Meloidogyne incognita[END_REF] However, based on the whole-genome sequence of the nematode 10 , a wide phylogenetic analysis recently positioned M. incognita map-1 homologues into a cluster of genes encoding expansin-like proteins, [START_REF] Danchin | Multiple lateral gene transfers and duplications have promoted plant parasitism ability in nematode[END_REF] thus confirming individual homologies found with related genes from other nematode species. [START_REF] Hanse | Mining the secretome of the root-knot nematode Meloidogyne chitwoodi for candidate parasitism genes[END_REF][START_REF] Haegeman | The role of pseudo-endoglucanases in the evolution of nematode cell wall-modifying proteins[END_REF] Our immunolocalization study showed secretion of MAP-1 from the amphids of migrating juveniles, supporting a role of the protein during the early steps of infection. Whether this role is related to cell wall loosening is still to be determined. The formation of the nematode feeding site is determined by the molecular dialogue between the nematode and its susceptible plant host. Our localization studies have demonstrated that two proteins, one produced by the amphids (MAP-1) and one secreted through the stylet (6D4), accumulate at the plant cell wall during giant cell formation and maintenance (Fig. 1 C,D), suggesting that the intercellular space may be an important interface during nematode feeding site induction and maintenance. Our finding here suggests that MAP-1 is not only secreted in planta by migrating juveniles but could have an additional role during the initiation of the feeding site. The protein 6D4 was identified by screening a panel of antibodies that reacted with the secretory oesophageal glands of the nematode. [START_REF] Davis | Novel immunization procedures used to develop monoclonal antibodies that bind to specific structures in Meloidogyne spp[END_REF] Unfortunately, this protein could not be purified and its sequence is still unknown. Interestingly, we only observed residual secretion of CBM2-bearing enzymes and pectate lyase by sedentary nematodes, suggesting that these proteins have no role in the cell wall expansion and thickening associated with giant cell formation. Rather, the plant cell wall degrading enzymes that are induced during infection [START_REF] Jammes | Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsis[END_REF][START_REF] Barcala | Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells[END_REF] could be responsible for the cell wall modifications occurring in the already hypertrophied giant cells.

Nematode proteins are secreted during parasitism within the nematode-feeding site

Discussion

Recent yeast two hybrid screens have identified cytoplasmic proteins as potential plant targets for nematode secreted proteins (for a review see ref. 4). However, our in planta observations of five different RKN proteins secreted from three distinct secretory organs of the nematode, i.e., amphids, subventral and dorsal glands, reveal that the plant apoplasm constitutes an important compartment where these proteins are delivered during distinct phases of parasitism. Although we cannot exclude that the tiny amount of secreted proteins or epitope modifications might prevent the detection of the proteins into the plant cells, we clearly demonstrate that these proteins accumulate in the plant apoplasm during migratory and sedentary phases of infection. Flourishing data on oomycete and fungus effectors demonstrate that the extracellular space is the first destination compartment for secreted effectors that are subsequently translocated into the cytoplasm of recipient plant cells. [START_REF] Dodds | Effectors of biotrophic fungi and oomycetes: pathogenicity factors and triggers of host resistance[END_REF] No such translocation system has been evidenced so far for nematodes and such a hypothesis would be conflicting with the stylet acting as a syringe able to directly inject nematode effectors within plant cells.

However, a role for nematode effectors in the apoplasm of feeding cells has recently been proposed by Wang et al. [START_REF] Wang | Dual roles for the variable domain in protein trafficking and host-specific recognition of Heterodera glycines CLE effector protein[END_REF] who have shown the ability of a CLE-like peptide secreted from the cyst nematode Heterodera glycines to traffic outside plant cells from the cytoplasm. Once in the apoplasm, the nematode CLE-like peptides act as ligand mimics of plant CLE peptides and interact with receptors at the plasma membrane. [START_REF] Replogle | Nematode CLE signaling in Arabidopsis requires CLAVATA2 and CORYNE[END_REF] Except for plant cell wall loosening during infection, no function has been described so far for RKN effectors in the apoplasm of plant cells. Yet, the observed accumulation of nematode secreted proteins in the apoplasm seems significant enough to be perceived by the plant. Indeed, the plant plasma membrane is a sensor for pathogen-associated molecular patterns (PAMPs) and effector proteins that are perceived by plant pattern-recognition receptors or resistance proteins (for review see refs [START_REF] Jones | The plant immune system[END_REF][START_REF] Segonzac | Activation of plant pattern-recognition receptors by bacteria[END_REF] ).

As a consequence, accumulation of RKN secreted proteins in the apoplasm could affect the interplay between nematode effectors and plant responses.

Figure 1 .

 1 Figure 1. Immunodetection of Meloidogyne incognita secreted proteins during

  Comment citer ce document : Rosso, M.-N. (Auteur de correspondance), Vieira, P. C., De Almeida-Engler, J., Castagnone-Sereno, P. (2011). Proteins secreted by root-knot nematodes accumulate in the extracellular compartment during root infection. Plant Signaling and Behavior, 6 (8), 1-3. DOI : 10.4161/psb.6.8.16290 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in : Plant signaling & behavior (2011), Vol. 6, N°.8, p. 1-3, DOI: 10.4161/psb.6.8.16290 Journal homepage: http://www.landesbioscience.com/journals/psb/ 6