J T Silverstein

R L Vallejo

Y Palti

T D Leeds

C E Rexroad

T J Welch

G D Wiens

V Ducrocq

Rainbow trout resistance to bacterial cold-water disease is moderately heritable and is not adversely correlated with growth 1

Keywords: challenge test, disease resistance, genetic correlation, growth, heritability, rainbow trout

The objectives of this study were to estimate the heritabilities for and genetic correlations among resistance to bacterial cold-water disease and growth traits in a population of rainbow trout (Oncorhynchus mykiss). Bacterial cold-water disease, a chronic disease of rainbow trout, is caused by Flavobacterium psychrophilum. This bacterium also causes acute losses in young fish, known as rainbow trout fry syndrome. Selective breeding for increased disease resistance is a promising strategy that has not been widely used in aquaculture. At the same time, improving growth performance is critical for efficient production. At the National Center for Cool and Cold Water Aquaculture, reducing the negative impact of diseases on rainbow trout culture and improving growth performance are primary objectives. In 2005, when fish averaged 2.4 g, 71 full-sib families were challenged with F. psychrophilum and evaluated for 21 d. Overall survival was 29.3% and family rates of survival varied from 1.5 to 72.5%. Heritability of postchallenge survival, an indicator of disease resistance, was estimated to be 0.35 ± 0.09. Body weights at 9 and 12 mo posthatch and growth rate from 9 to 12 mo were evaluated on siblings of the fish in the disease challenge study. Growth traits were moderately heritable, from 0.32 for growth rate to 0.61 for 12-mo BW. Genetic and phenotypic correlations between growth traits and resistance to bacterial cold-water disease were not different from zero. These results suggest that genetic improvement can be made simultaneously for growth and bacterial cold-water disease resistance in rainbow trout by using selective breeding.

INTRODUCTION

Bacterial cold-water disease (BCWD), a chronic disease of rainbow trout (Oncorhynchus mykiss), is common in US rainbow trout aquaculture and is caused by Flavobacterium psychrophilum (Fp). This bacterium also causes acute losses in young fish, known as rainbow trout fry syndrome. Methods for prevention of BCWD are limited, and no licensed vaccine is currently available.

There is evidence of genetic variation for resistance to Fp infection in salmonids. For example, [START_REF] Nagai | Differences in susceptibility to Flavobacterium psychrophilum among three stocks of ayu, Plecoglossus altivelis[END_REF] showed differences in susceptibility to Fp challenges among 3 stocks of ayu (Plecoglossus altivelis), and [START_REF] Henryon | Selective breeding provides an approach to increase resistance of rainbow trout (Onchorhynchus mykiss) to the diseases, enteric redmouth disease, rainbow trout fry syndrome, and viral haemorrhagic septicaemia[END_REF] showed significant additive genetic variation for resistance to Fp challenge in a Danish rainbow trout broodstock. This evidence of genetic variation in resistance to Fp suggests that selective breeding may improve the survival of rainbow trout.

Growth performance is another trait of primary importance to many rainbow trout producers, and selection for growth is generally included in most breeding programs [START_REF] Gjerde | Design of fish breeding programs[END_REF]. Typical harvest BW for rainbow trout in the United States is 400 to 500 g, but recently the demand for larger fish has increased (National Agricultural Statistics Service, 2007) The relationship between growth-to-harvest BW and resistance to Fp in rainbow trout has not been report-ed previously. [START_REF] Johansen | The effects of chronic immune stimulation on muscle growth in rainbow trout[END_REF] speculated that because growth and an active immune system are resource-demanding systems, an unfavorable correlation between disease resistance and growth performance may exist. However, estimates of genetic correlation between growth and general survival have typically been positive, between 0.18 and 0.37 [START_REF] Fjalestad | Genetic improvement of disease resistance in fish: An overview[END_REF]. Correlations between growth and specific disease resistance require empirical testing. Therefore, our objectives were to determine the phenotypic and genetic parameters for resistance to Fp in rainbow trout, and to determine the genetic and phenotypic relationships between resistance to Fp and growth.

MATERIALS AND METHODS

The institutional Animal Care and Use Committee protocol used in this research was NCCCWA Animal Care and Use Committee Protocol Number 029.

Fish

Data were from 71 full-sib families produced in January 2005 from 56 sires and 65 dams from the National Center for Cool and Cold Water Aquaculture (Kearneysville, WV) synthetic population initiated in 2002 and 2003 [START_REF] Silverstein | Genetic variation measured by microsatellites among three strains of domesticated rainbow trout (Oncorhynchus mykiss, Walbaum)[END_REF][START_REF] Johnson | Development and evaluation of a new microsatellite multiplex system for parental allocation and management of rainbow trout broodstocks[END_REF]. The 71 families were composed of 33 paternal half-sib families, 20 maternal half-sib families, and 18 full-sib families, with no maternal or paternal half sibs. The original domesticated strains that contributed to the synthetic population were 1) University of Washington, Donaldson; 2) Kamloops/Puget Sound Steelhead cross; 3) College of Southern Idaho, House Creek; and 4) Ennis NFH Shasta strain. These strains were selected because of their documented domestication. Parents from the original strains were tested and determined to be free of viral hemorrhagic septicemia virus, infectious hematopoetic necrosis virus, infectious pancreatic necrosis virus, infectious salmon anemia virus, and Renibacterium salmoninarum, the causative agent of bacterial kidney disease. Parents of the 2002 and 2003 founding populations were mated in a partly factorial design [START_REF] Berg | A comparison of mating designs for inference on genetic parameters in fish[END_REF]. Approximately onehalf of the 2002 and 2003 families were F 1 crosses between different strains, and one-half were pure strains. Parents for the 2005 population were selected primarily from the 2003 population based on family growth performance, but some 3-yr-old parents from the fastest growing families of the 2002 population were also used. Pedigree data were available beginning with grandparents of fish hatched in 2005.

Eggs were fertilized over a 5-wk period between January 7, 2005, andFebruary 8, 2005. Each full-sib group was reared in a separate incubator compartment. The hatch timing of the families was synchronized by manipulating the incubation water temperature. All egg lots hatched within a 4-d period between March 2, 2005, and March 6, 2005. After hatch, 600 randomly chosen siblings from each full-sib family were moved to separate 200-L family tanks and reared on a flow-through spring water system. Approximately 66 d posthatch, three 25-fish groups were weighed from each family to provide an average fish BW for each family. The average (SD) over all families was 2.4 (0.3) g. One week later, between 40 and 80 fish were removed from each family tank and moved into the challenge facility (see challenge details below). Fish remaining in the family tanks were periodically culled randomly to maintain biomass densities below 50 kg/m 3 . At 5 mo posthatch, when fish averaged 47.1 (9.9) g, between 10 and 50 fish from each full-sib family were tagged with passive integrated transponder tags (Avid, Norco, CA), and all families were combined into five 1.3-m circular tanks, each with a volume of approximately 1,000 L. Fish were divided into more tanks as they grew, to maintain biomass densities between 30 and 70 kg/m 3 . Fish were fed most of their daily ration with Arvotec (Huutokoski, Finland) automatic feeders, and once each day, the fish were fed by hand to apparent satiation.

Growth Evaluation

Growth performance measures were individual BW at 9 and 12 mo posthatch (9BW and 12BW, respectively) and growth rate, evaluated as the thermal growth coefficient (TGC) between 9 and 12 mo posthatch. The TGC provides a measure of fish growth rate on a linear scale for the typical production phase of most fish species [START_REF] Iwama | A simple growth model for salmonids in hatcheries[END_REF][START_REF] Jobling | The thermal growth coefficient (TGC) model of fish growth: A cautionary note[END_REF][START_REF] Lankford | Associations between plasma growth hormone, insulin-like growth factor-I, and cortisol with stress responsiveness and growth performance in a selective breeding program for rainbow trout[END_REF], and is calculated in relation to degree days (T × t), where T is water temperature in Celsius and t is time in days. Calculation of TGC is as follows:

TGC = [( 3 √W f -3 √W i )/(T × t)] × 1,000,
where W f is the final BW and W i is the initial BW.

Challenge Protocol

The Fp challenge was carried out in the National Center for Cool and Cold Water Aquaculture challenge facility, where 120 eight-liter tanks were available. Forty full-sib fish were allocated to a single 8-L family tank (i.e., fish from different families were not commingled). From 42 of the 71 families, a replicate group of 40 fish was allocated; thus, 113 tanks were used. The fish were allowed 1 wk to acclimate before challenge, and were fed daily by hand to apparent satiation. Fish were challenged with a virulent strain of Fp CSF-259-93 [START_REF] Crump | Antigenic characterization of the fish pathogen Flavobacterium psychrophilum[END_REF][START_REF] Maclean | The structure of the lipopolysaccharide Oantigen produced by Flavobacterium psychrophilum (259-93)[END_REF] at approximately 80 d posthatch. The bacterial culture was prepared as described by [START_REF] Lafrentz | Characterization of serum and mucosal antibody responses and relative percent survival in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization and challenge with Flavobacterium psychrophilum[END_REF], and 8.8 × 10 6 cfu/ fish was administered to each fish by intraperitoneal injection. The water temperature during challenge was 13.0 ± 0.5°C. Fish that died during the 21-d period after challenge were recorded for day of mortality and examined for clinical signs of disease. We assumed that the longer a fish survived, the greater was its resistance to Fp infection [START_REF] Henryon | Selective breeding provides an approach to increase resistance of rainbow trout (Onchorhynchus mykiss) to the diseases, enteric redmouth disease, rainbow trout fry syndrome, and viral haemorrhagic septicaemia[END_REF]. Fish that did not die during the 21-d challenge were recorded as censored observations.

Survival Analysis

Time to event data (e.g., survival data) arise in several applied fields, and events (e.g., death) are generally considered "failures." A common feature of survival data is the occurrence of censored observations. Censored data arise for individuals that do not have a failure event during the predefined observation period, or for individuals arbitrarily withdrawn from the study before completion of the predefined observation period. Thus, the only knowledge from a censored record is that the individual survived for the entirety of its observation period. Survival data can be right, left, or interval censored [START_REF] Klein | Survival Analysis: Techniques for Censored and Truncated Data[END_REF]. Observations available on the failure times of n individuals are assumed to be independent. Analysis of survival data that contain censored records requires statistical methods that 1) incorporate event and censored data jointly to assess the dependence of failure time on explanatory variables, and 2) appropriately specify the underlying, or baseline, hazard function [START_REF] Kalbfleisch | The Statistical Analysis of Failure and Time Data[END_REF]. A classical approach to analyze censored and uncensored data jointly is to use survival analysis models, such as proportional hazards models. These can be either parametric (e.g., assuming a Weibull baseline hazard) or semiparametric, with an arbitrary hazard baseline [START_REF] Cox | Regression models and life-tables[END_REF].

However, proportional hazard models often assume that failure times are expressed on a continuous time scale, with no ties between ordered failure times [START_REF] Cox | Regression models and life-tables[END_REF][START_REF] Kalbfleisch | The Statistical Analysis of Failure and Time Data[END_REF]. When survival after challenging experimental animals with a diseasecausing pathogen is evaluated for relatively short periods of time (e.g., 21 d) and is measured with limited precision (i.e., observations are made infrequently), this assumption is violated. In such cases, the time scale is discrete with few classes. For these discrete data, survival analysis using standard proportional hazard models is a priori incorrect.

In this study, we had many ties between ordered failure times, because the survival data were collected for 21 d after exposure to the disease-causing pathogen and observations were made only once daily. Furthermore, the plot of ln(-ln[S(t)]) against ln(t), where S(t) is the Kaplan-Meier survivor function and t is time, did not produce a straight line (data not shown), which indicated that the Weibull distribution was not a good fit for the survival data [START_REF] Kalbfleisch | The Statistical Analysis of Failure and Time Data[END_REF]. A more convenient strategy was to use the grouped data model of [START_REF] Prentice | Regression analysis of grouped survival data with application to breast cancer data[END_REF]. This discrete proportional hazards model is a fully parametric one for which the baseline hazard is estimated at every discrete time point and for which the definition of hazard is modified to take into account the discrete time scale without violating the proportional hazard model [START_REF] Ducrocq | Extension of survival analysis to discrete measures of longevity[END_REF].

The survival times of Fp-challenged fish were described with a grouped data model, for which the hazard function h(τ i ;x j ) within the interval τ i = (t i-1 ,t i ) of a particular animal j, characterized by a vector of explanatory x j variables, can be written as

h h i j i j ( ; ) ( )exp , ' t t x x = { } 0 b
where h 0 (τ i ) is the discrete baseline hazard function and β is a vector of regression coefficients of the explanatory variables on survival times. An extension of the previous model is obtained by adding random effects [START_REF] Ducrocq | A Bayesian analysis of mixed survival models[END_REF], and the resulting mixed model (also known as a frailty model) can be written as

h h i j j i j j ( ; , ) ( )exp , ' ' t t x z x z u = + } { 0 b
where u is a vector of random variables and z j is an incidence vector.

Model Selection. Preliminary analyses to assess the significance of the random effect of challenge tank nested within family and the fixed effect of average family BW (covariate) on the response variable days to death (DTD) were performed by using REML and likelihood ratio tests implemented in the R statistical package from the Bioconductor Software Project [START_REF] Gentleman | Bioconductor: Open software development for computational biology and bioinformatics[END_REF]. To perform these analyses, we developed an R script by using the statistical methods outlined by [START_REF] Pinheiro | Mixed-Effects Models in S and S-PLUS. 1st ed[END_REF]; this R script is available online (http://www.ars.usda.gov/Research/ docs.htm?docid=8033). Both effects were significant (P < 0.05) in the preliminary analyses and were therefore included in the survival analysis models.

Frailty Models. Bayesian analysis of the frailty models developed by [START_REF] Ducrocq | A Bayesian analysis of mixed survival models[END_REF] can be applied to the grouped data model [START_REF] Prentice | Regression analysis of grouped survival data with application to breast cancer data[END_REF] for discrete data [START_REF] Ducrocq | Extension of survival analysis to discrete measures of longevity[END_REF], and this method was implemented in the Survival Kit Version 3.12 program [START_REF] Ducrocq | The Survival Kit-A FORTRAN package for the analysis of survival data[END_REF] by using the following discrete failure time frailty models.

Animal model: •

h h a i j j k i j j k ( ; , , ) ( )exp , ' ' ' t t x z z x z z r 1 2 0 1 2 = + + } { b
where b j is a vector for the fixed animal BW covariate, dom tank or family effects, and x j , z 1j , and z 2k are incidence matrices.

a

Sire-dam model:

• h h i j j k i j j k ( ; , , ) ( )exp , ' ' ' t t x z z x z s z r 1 2 0 1 2 = + + } { b
where b j is a vector for the fixed animal BW covariate, s is the vector of random sire-dam effects, r is the vector of random tank or family effects, and x j , z 1j , and z 2k are incidence matrices.

Animal s animal 2 ( ) and sire-dam s sire dam -( )

2
variance components and EBV were estimated by using the Bayesian approach outlined by [START_REF] Ducrocq | A Bayesian analysis of mixed survival models[END_REF]. The reliability (R) for survival EBV when using the sire-dam model was estimated as

R S EP sire dam = - ( ) 1 4 2 2 s - ,
where SEP 2 is the squared SE of EBV.

Heritability of Survival.

The effective heritability of survival was estimated from both models by using expressions modified from [START_REF] Yazdi | Heritability, reliability of genetic evaluations and response to selection in proportional hazards models[END_REF].

Animal model: •

h animal animal 2 2 1 4 2 1 = + s s ( ) ,
Sire-dam model:

• h fam s ire dam 2 2 2 2 1 = + ( ) s s / .
where fam is for full-sib families.

Growth Analysis

For growth data, the MTDFREML software developed by [START_REF] Boldman | A manual for use of MTDFREML. A set of programs to obtain estimates of variances and covariances[END_REF] was used to analyze the data, using animal and sire-dam models for an observation vector y:

y = Xβ + Z 1 a + Z 2 u + e,
where β is a vector of fixed effects, a is a vector of random animal or sire-dam genetic effects, u is a vector of random effects of family tank before tagging individuals and pooling families into larger tanks, and X, Z 1 , and Z 2 are design matrices. Fixed effects included rearing tank after tagging and sex. Using these models, we obtained estimates of genetic (V animal and V sire-dam ), common environment (V familytank ), residual (V R ), and phenotypic variances (

V A + V familytank + V R ).

Genetic and Phenotypic Correlations

The EBV were derived from animal and sire-dam models for survival, 9BW, 12BW, and TGC. Genetic correlations among growth traits were estimated from 2-trait animal models in MTDFREML. Genetic correlations between growth traits and survival were estimated by using 2 approaches. First, family mean EBV were calculated from the animal model EBV, and Pearson correlations between family mean EBV were calculated. Correlations were weighted by the variance of the growth data attributable to differences in the number of individuals sampled per family for growth traits. Second, Pearson correlations between parent EBV from sire-dam models were calculated. Phenotypic correlations among growth traits were estimated from MTD-FREML. Phenotypic correlations between growth and DTD were estimated as Pearson correlations between family mean growth and DTD phenotypic data.

RESULTS

In this experiment, 4,656 fish were challenged and 1,350 survived until the termination of the study (i.e., 71% of the fish challenged with Fp died during the 21-d challenge), with a mean DTD of 8.1 d. Mortality was low through d 4 (1.6%•d -1 ), and then increased to more than 5%•d -1 through d 9, resulting in more than 54% mortality by d 9 (Figure 1). One major peak of mortality was centered at d 6, with a smaller secondary increase at d 13 to 14. At d 18, mortality declined to less than 1%•d -1 .

There was substantial variation among full-sib families for mortality rates, which ranged from 27.5 to 98.5% (Figure 2). Survival of the Fp challenge had a clear additive genetic component; survival heritability estimates were 0.35 ± 0.09 and 0.43 ± 0.03 from the animal and sire-dam models, respectively. Variance attributable to tank within families accounted for only 1.6% of the phenotypic variance (data not shown). Body weight at the time of challenge had a significant effect on survival. Resistance to BCWD increased with increasing BW; each 1-g increase in BW conferred a 0.7 ± 0.2-d increase in DTD (P < 0.001).

Heritability estimates from animal models for 9BW, 12BW, and TGC were 0.59, 0.61, and 0.32, respectively (Table 1). The common environmental effect (i.e., family tank before tagging) accounted for 5 and 2% of variation in 9BW and 12BW,respectively. Genetic correlations between survival and 9BW, 12BW, and TGC were ≤0.10 (absolute value) and were not different from 0 when estimated by using family mean EBV from the animal models (Table 2). Similarly, these genetic correlations were ≤0.09 (absolute value) when estimated by using parental EBV from the sire-dam models (data not shown). Phenotypic correlations between survival and growth traits were weak and were not different from 0 (Table 2). Genetic and phenotypic correlations between 9BW and 12BW were large (≥0.86), whereas correlations of 9BW and 12BW with TGC were ≤0.69 (Table 3). Correlations between 12BW and TGC were considerably stronger than correlations between 9BW and TGC.

DISCUSSION

The potential to improve a population of rainbow trout for resistance to BCWD through selective breeding depends first on there being an additive genetic component to variation in resistance. Furthermore, the value of using selective breeding for this trait is conditioned by how selection for BCWD resistance may affect other traits. In this study, we found a moderate heritability for resistance to BCWD, measured as DTD, and no significant correlation with growth to market sizes. These findings are encouraging because they suggest that selection to improve BCWD resistance will not be antagonistic to improved growth; thus, both traits can be improved simultaneously.

Additive genetic variation for resistance to specific pathogens has been reported for many animals, including fish [START_REF] Chevassus | Genetics of resistance to disease in fishes[END_REF][START_REF] Van Muiswinkel | The influence of environmental and genetic factors on the disease resistance of fish[END_REF][START_REF] Bishop | Genetics of disease resistance in sheep and goats[END_REF]. In particular, [START_REF] Henryon | Selective breeding provides an approach to increase resistance of rainbow trout (Onchorhynchus mykiss) to the diseases, enteric redmouth disease, rainbow trout fry syndrome, and viral haemorrhagic septicaemia[END_REF] also found resistance to Fp to be heritable. Nevertheless, their estimates were considerably less, at 0.07 compared with our current estimate of 0.35. In both studies, intraperitoneal injection was the route of pathogen delivery. Differences in fish size, strain of the pathogen, and other environmental differences could be responsible for the difference in estimates. Additionally, any heritability estimate is particular to a population and environment, and the response to this challenge may have had a stronger genetic basis than the challenge conducted by [START_REF] Henryon | Selective breeding provides an approach to increase resistance of rainbow trout (Onchorhynchus mykiss) to the diseases, enteric redmouth disease, rainbow trout fry syndrome, and viral haemorrhagic septicaemia[END_REF]. The difference between heritability estimates from the animal and sire-dam models (0.35 and 0.43, respectively) provides an idea of the magnitude of nonadditive genetic effects on the inheritance of BCWD resistance, although the data structure was not ideal to interpret the differences in variances.

The effect of BW at the time of challenge on resistance to BCWD was positive in this study, and a similar conclusion was reached by [START_REF] Henryon | Selective breeding provides an approach to increase resistance of rainbow trout (Onchorhynchus mykiss) to the diseases, enteric redmouth disease, rainbow trout fry syndrome, and viral haemorrhagic septicaemia[END_REF]; however, the BW data from the 2 studies did not measure exactly the same thing. In the current study, mean BW of each full-sib family 1 wk before challenge was used as a covariate in the statistical model. In the study by [START_REF] Henryon | Selective breeding provides an approach to increase resistance of rainbow trout (Onchorhynchus mykiss) to the diseases, enteric redmouth disease, rainbow trout fry syndrome, and viral haemorrhagic septicaemia[END_REF], each fish was weighed when it was removed from the tank after death or at the end of the experiment, and these data were included as a covariate. In both cases, fish BW was positively related to resistance, suggesting that greater BW at pathogen exposure confers some advantage.

In our mating design, some of the families were paternal half sibs, some were maternal half sibs, and some did not have half sibs. This design required us to estimate family breeding values as mean of the EBV of the animals to estimate genetic correlations between postchallenge survival and growth traits. However, we also used the sire-dam model to estimate breeding values for growth traits by using MTDFREML and to estimate postchallenge survival by using Survival Kit, which enabled us to estimate reliabilities of the parental EBV. The reliabilities of the sire-dam EBV averaged 0.73, 0.73, and 0.60 for 9BW, 12BW, and TGC, respectively, and 0.88 for survival (data not shown). These large accuracies suggest that our estimates of 0 for genetic correlations between growth and survival are good approximations, and support our results when using EBV from the animal models.

Genetic correlations between resistance to BCWD and BW at later ages (i.e., 9 and 12 mo posthatch) and growth rate have not been reported previously. The nonsignificant correlations found here are promising for selective breeding, because selection for improvement in one trait should not have antagonistic effects on the other. A favorable genetic correlation between Fp resistance and growth would be desirable, because selection for either trait would result in improvement in the other.

Table 1. Sample sizes (n), means and their SD (g), and heritabilities and their SE for growth traits A weak and nonsignificant correlation between resistance to BCWD and growth means that to improve both traits would require selection pressure on each trait. The method expected to give the most rapid improvement in merit is simultaneous selection by using an index incorporating economic weights and heritabilities for each trait, and genetic and phenotypic correlations between the traits [START_REF] Falconer | Introduction to Quantitative Genetics[END_REF].

Favorable genetic correlations between general survival and growth were previously observed in salmon and trout. Estimates of correlation between general survival and fingerling growth were 0.23 to 0.37 (reviewed in [START_REF] Gjedrem | Genetic improvement of cold-water fish species[END_REF]; however, correlations between growth and resistance to specific diseases are less consistent.

The relationship between growth and disease resistance is not consistent among different species or among different diseases. Work in the shrimp (Penaeus vannamei) has shown a favorable genetic correlation (~0.4) between general survival and growth (Gitterle et al., 2005a); however, an unfavorable correlation (between -0.55 and -0.64) was found between growth and resistance to white spot syndrome virus (Gitterle et al., 2005b). [START_REF] Gjedrem | Genetic variation in susceptibility of Atlantic salmon to furunculosis[END_REF] found a correlation of 0.3 between vibriosis and growth among matched groups of fingerling Atlantic salmon (Salmo salar), whereas we found a correlation near 0 for 2.4-g rainbow trout challenged with Fp and compared with their growth rate to market size.

The heritabilities of 9BW and 12BW and growth rate between 9 and 12 mo were moderate to high and were somewhat greater than previous estimates for rainbow trout at similar ages. Other estimates of BW heritability were between 0.48 and 0.55 [START_REF] Elvingson | Genetic and environmental components of variation in body traits of rainbow trout (Oncorhynchus mykiss) in relation to age[END_REF] and between 0.15 and 0.38 [START_REF] Kause | Feed efficiency of rainbow trout can be improved through selection: Different genetic potential on alternative diets[END_REF]. The differences in growth rates among families and the moderate to high heritabilities suggest considerable potential for growth improvement in this population.

In summary, we demonstrated that rainbow trout survival after Fp injection challenge was a moderately heritable trait in our broodstock population. These results have favorable implications for selective breeding for increased disease resistance and have provided an important experimental resource for investigation into the mechanistic basis of immunity to this pathogen. In 2 additional studies, we demonstrated a positive corre-lation between family survival and average spleen size [START_REF] Hadidi | Spleen size predicts resistance of rainbow trout to Flavobacterium psychrophilum challenge[END_REF]. In addition, we reported an association between survival and 2 microsatellites (OMM3089 and OMM1189) that map near the major histocompatibility IB region [START_REF] Johnson | Suggestive association of major histocompatibility IB genetic markers with resistance to bacterial cold water disease in rainbow trout (Oncorhynchus mykiss)[END_REF]. Thus, the fish crosses identified in this study are likely to continue to provide an important experimental resource. However, it is important to recognize that several caveats remain. All our work has used a single bacterial clone isolated from F. psychrophilum strain CSF 259-93 and all challenge work has used injection challenge. Further laboratory-based experiments and field trials are underway to determine the relevance of these findings to performance under farm conditions.

  is the vector of random animal genetic effects with the assumption the numerator relationship matrix, r is the vector of ran-Silverstein et al.

Figure 1 .

 1 Figure 1. Daily mortality over time. Each bar represents the number of mortalities for the days after challenge. In 2005, when fish averaged 2.4 g, 71 full-sib families were challenged with Flavobacterium psychrophilum, and survival was evaluated for 21 d.

Figure 2 .

 2 Figure 2. Percentage mortality of the groups challenged. Replicated families are represented by the average of the 2 replicates. In 2005, when fish averaged 2.4 g, 71 full-sib families were challenged with Flavobacterium psychrophilum, and survival was evaluated for 21 d.

Table 2 .

 2 Approximate genetic (r g ) and phenotypic (r p ) correlations between postchallenge survival and growth traits 1

	Trait	r g	r p
	9BW 2	-0.09 (-0.31 to 0.15)	0.01 (-0.08 to 0.09)
	12BW 2	-0.10 (-0.33 to 0.13)	-0.06 (-0.07 to 0.05)
	TGC 3	0.09 (-0.15 to 0.31)	-0.13 (-0.39 to 0.22)

1

Point estimates and range enclosing the 95% confidence limits (in parentheses) are given.

2

9BW and 12BW = BW at 9 and 12 mo posthatch, respectively.

3 TGC = thermal growth coefficient between 9 and 12 mo posthatch [units are grams divided by degree days (T × t), where T is water temperature in Celsius and t is time in days].

Table 3 .

 3 Genetic (below diagonal) and phenotypic (above diagonal) correlations among BW and thermal growth coefficient (TGC) traits TGC = thermal growth coefficient between 9 and 12 mo posthatch [units are grams divided by degree days (T × t), where T is water temperature in Celsius and t is time in days].

	Item	9-mo BW	12-mo BW	TGC
	9BW 1		0.86	0.15
	12BW 1	0.93 ± 0.02		0.62
	TGC 2	0.39 ± 0.14	0.69 ± 0.08
	1 9BW and 12BW = BW at 9 and 12 mo posthatch, respectively.

2

at INRA Institut National de la Recherche Agronomique on October 17, 2013 www.journalofanimalscience.org Downloaded from at INRA Institut National de la Recherche Agronomique on October 17, 2013 www.journalofanimalscience.org Downloaded from at INRA Institut National de la Recherche Agronomique on October 17, 2013 www.journalofanimalscience.org Downloaded from at INRA Institut National de la Recherche Agronomique on October 17, 2013 www.journalofanimalscience.org Downloaded from at INRA Institut National de la Recherche Agronomique on October 17, 2013 www.journalofanimalscience.org Downloaded from at INRA Institut National de la Recherche Agronomique on October 17, 2013 www.journalofanimalscience.org Downloaded from at INRA Institut National de la Recherche Agronomique on October 17, 2013 www.journalofanimalscience.org Downloaded from at INRA Institut National de la Recherche Agronomique on October 17, 2013 www.journalofanimalscience.org Downloaded from at INRA Institut National de la Recherche Agronomique on October 17, 2013 www.journalofanimalscience.org Downloaded from

The authors thank Eric Squires, Jim Everson, Jon Leasor, Josh Kretzer, David Payne, Chris Campbell, and Jennifer Harper (USDA, ARS, National Center for Cool and Cold Water Aquaculture, Kearneysville, WV) for technical assistance; Scott LaPatra (Clear Springs Foods, Buhl, ID) for the Flavobacterium psychrophilum strain used to challenge the fish; and Maureen Purcell (Western Fisheries Research Center, Biological Resources Discipline, US Geological Survey, Seattle, WA) for critical review of this

manuscript. 2 Present address: 5601 Sunnyside Ave, Rm 4-2106,

Beltsville, MD 20705. 3 Corresponding author: Yniv.Palti@ars.usda.gov Received May 6, 2008. Accepted November 13, 2008.


G Manolescu

L Favaro

F Knappe

G Girard

B Poumellec
email: bertrand.poumellec@universite-paris-saclay.fr.

H Renner

J E Bourrée

E Brinkmeyer

H Poumellec

A L Renner

Structural transformations induced by direct UV writing in MDECR-PECVD silica layer exhibiting negative index change

Keywords: 71, 55, Jv

Structural transformations induced by direct UV writing in MDECR

Introduction

We were successful in achieving, using 244 nm CW laser for direct UV writing, negative index change in H, Ge co-doped silica layer elaborated by Matrix Distributed Electron Cyclotron Resonance (MDECR-PECVD), an improvement of PECVD method [START_REF] Daineka | High density plasma enhanced chemical vapor deposition of optical thin films[END_REF] . The amplitude of the index change is of the order of -10 -2 at a UV beam speed as large as 1 mm/s. We have therefore a material that can be potentially used for new application achievements because it exhibits strong negative index changes [START_REF] Billi | presented at the Advances in Optical Materials[END_REF][START_REF] Knappe | presented at the Bragg Gratings Photosensitivity Poling[END_REF] .

The starting idea was to use an elaboration procedure that allows including hydrogen in the layer at a level of a few mol % i.e. larger than it can be achieved by high pressure loading for getting a large positive index change, or even larger one without any post-deposition treatment. But, finally, we obtained not positive index change but strong negative one. As for such an index change amplitude, a volume change is expected, we have measured the surface topography (Figure 1 a). We have found two domains according to UV light power P and writing speed v when the UV absorbing layer is coated with a H:SiO 2 layer (Figure 1 c). In the first domain, the effect increasing with the laser power and 1/v, in the second the effect intensity decreases with 1/v.

2

The amplitude measured can be as large as 200 nm but we did not know the associated microscopic change. To precise the mechanisms, we measured UV absorption since the first step of the process is obviously an absorption in the UV range by one or several photons. Then, as the most labile species in our material is the OH containing species, we measured the OH absorption in the IR range. Lastly, as the magnitude of the index change is quite large (almost 1%), we have searched for a change of structure by Raman spectroscopy.

2.

Experimental details Direct UV written areas (squares of 0.4 mm×0.4 mm) at different power (from 3 to 57 mW) and velocities (from 30 to 1000 µm/s) in a planar waveguide sample have been analyzed by IR absorption and Raman microprobe. For UV absorption, a smaller number of squares of larger dimensions (1x1 mm 2 ) have been written. The sample consists of a 4.25±0.25 µm thick layer of H:Ge:SiO 2 silica-on-Suprasil deposited by MDECR-PECVD 1 covered by a 13.5±1.5 µm thick cladding layer of MDECR H:SiO 2 . Direct UV writing was performed using a CW frequency doubled Ar-ion laser (λ at 244 nm) which was focused by an optical lens to obtain a 1/e 2 beam diameter of about 4.µm. UV absorption spectra were measured with a Varian Cary spectrophotometer between 190 and 300 nm with two collimated beams for a proper localization of the beams into the irradiated areas. IR absorption spectra were measured in the range 2000-4000 cm -1 using a Fourier transform IR spectrometer Nicolet and an infrared microscope (Continuum), both purged with dry air. Spectra were obtained using a Globar light source, KBr beam splitter and a MCT (HgCdTe) detector. They were acquired averaging 64 scans and the spectral resolution was consistently chosen (4 cm -1 ). The analyzed spot had a diameter of 100 µm. Raman spectra were measured at atmospheric conditions using a T 64000 Jobin Yvon confocal microRaman spectrometer, which was equipped with a liquid nitrogen-cooled CCD detector. The Argon laser beam at 514 nm wavelength was focused in the depth of the sample (50× objective) and the Raman scattering signal was collected and focused back to the detector. The confocal aperture was opened to 200 µm. The full spectra preliminarily calibrated with a silica sample, covered a wavenumber ranging from 200 to 4000 cm -1 . The accumulation time was 200 s. The recorded spectra were treated firstly by removing the baseline in order to eliminate the offset recorded in the spectra, i.e. by extracting the minimum intensity at an energy where no vibration is expected. Spectra were normalised by dividing each of them by their value measured at 440 cm -1 .

Results

UV absorption

UV absorption The UV absorption intensity is quite strong for this kind of sample. It is currently around 6000 cm -1 at 200 nm i.e. the absorption length is of the order of 2 µm and is located in the H:Ge:SiO 2 layer. The UV absorption in the cladding has been measured negligible. Under UV irradiation, UV absorption decreases rapidly and proportionally over the UV range by 50%, even at laser power as low as 10 mW (see Figure 2 a). This observation does not depend much on the writing speed. Beyond 4 mW, the absorption decrease saturates. This saturation is reached for lower writing speed at lower power. The effect appears to be stronger for large power and also large speed as it was observed for topography. These results are collected in Figure 2 b and seems coincide approximately with topography observations (see Figure 1 b) i.e. the domain extension are similar. The OH, H 2 O content is similar either in the pure silica layer and in the Ge doped layer i.e. giving rise to an absorption of 250 cm -1 . Changes of IR absorption spectra after irradiation are plotted in the Figure 3 a. The main change is located around 3200-3300 cm -1 for laser power larger than 15 mW (H 2 O absorption), it is a decrease. Below this power, and for large speed, the change is a small increase (maximum 0.02 in optical density). The effect of laser power and writing speed on the absorption at 3200 cm -1 are presented in the Figure 3 b. The change remains small, even at large power, the change is smaller than 10% on the initial H 2 O content. The Raman spectrum does not change after irradiation with low laser power whatever the writing speed. It seems to be also unchanged under large power and low speed. For large power and large speed, drastic changes are detected around 1500 cm -1 and 2800 cm -1 (see Figure 4 a). The change appearing at 2800 cm -1 seems to be roughly proportional to the one around 1500 cm -1 . The energy shift of these features does not change when excitation line is changed indicating that they are Raman scattering not fluorescence. The variation of their intensity with laser power and writing velocity is shown in Figure 4 b. From that one, we can define three domains, although two domains (I and II) looks similar without exhibiting change on the Raman spectrum but we think that they exist compared to the other optical properties. As a matter of fact, for 100 µm/s, the two domains are separated by a change on the Raman spectrum. The group at 1500 cm -1 is composed by two lines: one at 1338 and another one at 1598 cm -1 . The group at 2800 cm -1 is composed by three lines: 2667, 2910, 3200 cm -1 . Other structures are detected at 2208 cm -1 disappearing under irradiation, and two at 3570 and 3670 cm -1 exhibiting a constant intensity. The bands at 490 nm and 606 cm -1 called defect bands are also decreasing after irradiation.

IR absorption spectrum

Raman spectrum

Discussion

Change in UV absorption

The first step in the photosensitivity mechanisms is clearly the absorption in the H:Ge:SiO 2 since the absorption intensity is very strong (several 1000 cm -1 ) in this layer. The spectral diffusion of the excitation leads to strong bleaching of the whole UV spectrum at least between 190 and 300 nm but it saturates rapidly. The change of UV spectrum could have been related to a bleaching of SiE' centers absorbing at 195 nm and known as related to UV induced positive index change but using ESR we will show in another paper that this is not the case, actually.

Change in IR absorption spectrum in the range of OH linkages

The OH and H 2 O contents in this kind of material are almost the same. They reach each of them 2 mol % either in the core or in the cladding material. Nevertheless, the change is small (a few % of the total absorption) showing a slight increase of H 2 O for low power and an increase of OH and a decrease of H 2 O for larger power.

Change in Raman spectrum

The sharp bands at 490 cm -1 and at 606 cm -1 called defect bands generally, which are specific of the pure silica Raman spectra are due to intermediate range order in the form of highly regular rings of bonds. They have been attributed to the uncoupled symmetric oxygen breathing motion in the puckered four-and planar three-membered rings respectively [START_REF] Galeener | Vibrational decoupling of rings in amorphous solids[END_REF] . This attribution has been confirmed recently by DFT vibrational frequency calculation [START_REF] Pasquarello | Identification of Raman defect lines as signatures of ring structures in vitreous silica[END_REF] . We observe, that irradiation of sample induce a decrease of the intensity of the defect bands, interpreted as a reduction of the four-and three-membered ring units 6 . These changes vary with increasing fluence. This is consistent with a decrease of the glass density. In the spectral region between 1000 cm -1 and 1500 cm -1 drastic changes are induced by irradiation. The dominant bands located at 1338 cm -1 have been assigned to the symmetric vibration of silanone =Si=O 7 having two oxygen atoms in the back bonds [START_REF] Chabal | Silanone (Si=O) on Si(100): intermediate for initial silicon oxidation[END_REF] . The band at 1598 cm -1 could be comparable to the one mentioned by Skuja et al. [START_REF] Skuja | Quantitative analysis of the concentration of interstitial O 2 molecules in SiO 2 glass using luminescence and Raman spectrometry[END_REF] about interstitial oxygen, but here it is Raman line and not luminescence. It is possible that it is a symmetric vibration of another kind of silanone bonds weakened by a hydrogen bonds. The band at around 2208 cm -1 has been previously detected in the Raman spectra of H 2 -bearing silica glass [START_REF] Skuja | Quantitative analysis of the concentration of interstitial O 2 molecules in SiO 2 glass using luminescence and Raman spectrometry[END_REF] and was attributed to Si or Ge hydrides stretching vibrations. It disappears under irradiation. The intensity of the bands observed in the spectral region between 2500 cm -1 and 3250 cm -1 increase almost proportionally to the previous lines around 1000-1500 cm -1 . Composed by three bands (2667, 2910, 3200 cm -1 ), the two first bands could be assigned to vibration of unusual H containing species (numerical investigations are still in progress). The last band contains a contribution from H 2 O symmetric stretching vibration mode [START_REF] Davis | Quantitative infrared spectroscopic measurement of hydroxyl concentrations in silica glasses[END_REF] . Vibrations of Ge-OH and Si-OH groups are located at around 3570 cm -1 and 3670 cm -1 , respectively. Their constancy is consistent with the ones in IR absorption spectrum.

5.

Conclusion: Photosensitivity mechanisms The index change calculated by Kramers Kronig relation from UV absorption changes using Gaussian fitting reaches -7 10 -4 . The index change from IR absorption change reaches +10 -4 . So, UV is the first step of the mechanisms in any case but not the origin of refractive index change. IR absorption changes are also not responsible of the refractive index change. Topography results, instead of a densification, normally observed for usual germanium-doped silica materials and associated to a UV induced positive index change, show an expansion after UV laser irradiation. This phenomenon is at the base of the measured negative index change, as the same amplitude of the index change is roughly obtained by Lorentz-Lorenz relation and elastic calculations. Furthermore, we can distinguish, by topographic analysis, two different regions in the P-v domain, revealing distinct phenomena or processes contributing to the material expansion: a light induced expansion and an effect of laser heating. Raman spectra after UV irradiation for laser power from 4 to 60 mW and writing velocity of 30 to 1000 µm/s shows changes at 1330, 1590, 2660 and 2920 cm -1 . Those ones are related to structural transformations involving a three fold co-ordinated oxygen i.e. a silanone 7 . We demonstrate that Ge acts as a light absorber due to the strong UV absorption, leading to a heating effect that can propagate also into neighboring pure H:SiO 2 layer. As a matter of fact, irradiation of this layer without the neighbouring H:Ge:SiOH layer has no effect but the same Raman spectrum can be obtained in this material simply by heating at 600°C, 1/2 h. The Raman observations according to P, v leads to define regions in this sample that almost coincide with the ones obtained in sample that does not have a cladding 2 . There are the followings.

Region I (low laser power, whatever the beam velocity): we observe a reorganization of H 2 O population (different hydrogen bonds). On UV absorption, a large change is detected (the absorbing defect is bleached), half of the absorption disappears. On the contrary, no difference is seen on Raman scattering because, the structure change detected in IR and UV absorption represent a small contribution. Such transformation has also a small impact on the specific volume change because the glass connectivity does not change and thus the topography change is small.

Region III (large laser power, large beam velocity): The Raman spectrum shows several kinds of silanone. They can be produced by the following kind of reaction: SiOSi → Si=O + Si. This reaction exhibits an energy of 3.1 eV as calculated by quantum chemical software Gaussian using small cluster. The corresponding change in polarisability is smaller than 2%. The change of index arises mainly of the change of volume. IR absorption shows that water molecules are consumpted and OH groups are produced in the course of irradiation. We think about transformation of H 2 O into OH: by SiOSi + H 2 O → 2SiOH. UV saturate (no further transformation of the UV absorbing defects), topography change shows an expansion because connectivity decreases as indicated by the silanone formation. Here, heating effect on hydrated silica glass is the driving force. GeH 2 groups also disappear and the glass is on the way to decompose by forming Si or Ge nanocrystals. We do not see nanocrystals on Raman spectrum when the sample has a cladding like the sample used in this paper but we have detected the signature at 300 cm -1 of Ge nanocrystals when the cladding is absent.

Region II (large laser power, small beam velocity): Raman spectrum is restored, silanone linkages are erased. Here, the heating effect is so large that the remelting and either the return of the glass to its original state (when there is a cladding) or GeO and O 2 outgassing occur similarly to the case of Nishii et al. [START_REF] Nishii | Origin of enormous photon-induced volume expansion of GeO 2 -SiO 2 thin glass films[END_REF] for which there was no cladding. These authors found in Ge:SiO 2 layer obtained by sputtering, a very large negative index decrease of 0.045 with an expansion of the glass of 18% but the formation of porous glass as mentioned by the authors are not detected here.

6.

Figure 1 :

 1 Figure 1: Topography results obtained on MDECR H:Ge:SiO 2 coated layer after 244 nm-CW-through-microscope irradiation. (a) example of UV induced surface swelling, (b) the domains in the (P, 1/v) plane based on the results.

Figure 2 :

 2 Figure 2: UV absorption change before and after irradiation. (a) the optical density change at 200 nm according to laser power and laser velocity. (b) the domains in the (P, 1/v) plane based o these results.

Figure 3 :

 3 Figure 3: Infrared absorption of direct UV written areas (a) absorption change in the OH/H 2 O absorption range for three couples of power and beam velocity. The inset shows the absorption spectrum before and after irradiation for P=40 mW and v=100 µm/s (b) intensity change at 3200 cm -1 in the H 2 O absorption range at large powers (>15 mW) with velocities from 30 to 1000 µm/s. Lines, here, are just guides for the eye.

Figure 4 :

 4 Figure4: Raman scattering in directly UV written areas (a) normalised spectra until 4000 cm -1 for power level from 32 to 57 mW with writing velocity of 300 µm/s. (b) plot of Raman intensity at 1338 cm -1 for laser power from 0 to 57 mW and writing velocity from 30 to 1000 µm/s. (c) scheme of the different domains based on these results.

Acknowledgments

We would like to acknowledge the European Commission for the financial support, which allowed this work, in the frame of the R&D PLATON project (IST-2002-38168) and of the ODUPE Research Training Network (RTN-CT-2000-00045).

7.


Hélène Roumes

Ludovic Leloup

Elise Dargelos

Jean-Jacques Brustis

Laetitia Daury
email: daury@enscbp.fr

Patrick Cottin

Calpains: Markers of tumor aggressiveness?

Keywords:

Rhabdomyosarcoma (RMS) are soft-tissue sarcoma commonly encountered in childhood. RMS cells can acquire invasive behavior and form metastases. The metastatic dissemination implicates many proteases among which are μ-calpain and m-calpain.

Study of calpain expression and activity underline the deregulation of calpain activity in RMS.

Analysis of kinetic characteristics of RMS cells, compared to human myoblasts LHCN-M2 cells,

shows an important migration velocity in RMS cells. One of the major results of this study is the positive linear correlation between calpain activity and migration velocity presenting calpains as a marker of tumor aggressiveness. The RMS cytoskeleton is disorganized. Specifying the role of μand m-calpain using antisense oligonucleotides led to show that both calpains up-regulate αand β-actin in ARMS cells. Moreover, the invasive behavior of these cells is higher than that of LHCN-M2 cells. However, it is similar to that of non-treated LHCN-M2 cells, when calpains are inhibited.

In summary, calpains may be involved in the anarchic adhesion, migration and invasion of RMS.

The direct relationship between calpain activity and migration velocities or invasive behavior indicates that calpains could be considered as markers of tumor aggressiveness and as potential targets for limiting development of RMS tumor as well as their metastatic behavior.

Rhabdomyosarcoma cells

Migration Invasion

Cytoskeleton

Alveolar and embryonal rhabdomyosarcoma (ARMS and ERMS respectively) are soft-tissue sarcoma commonly encountered in childhood and adolescence. These rhabdomyosarcoma (RMS) arise from immature cells which fate is to form striated skeletal muscle. These cells express, similarly to muscle cells, some muscle specific proteins such as myogenic factors (MRF), desmin, and myogenin [START_REF] Dias | Myogenic regulatory protein (MyoD1) expression in childhood solid tumors: diagnostic utility in rhabdomyosarcoma[END_REF][START_REF] Parham | Immunohistochemical study of childhood rhabdomyosarcomas and related neoplasms. Results of an intergroup rhabdomyosarcoma study project[END_REF]. ARMS are characterized by two chromosomal translocations: t(2;13)(q35;q14) or t(1;13)(p36;q14) [START_REF] Fredericks | The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcoma is a more potent transcriptional activator than PAX3[END_REF]. ERMS histological type is associated with a loss of heterozygosis at the 11p15 locus [START_REF] Scrable | Chromosomal localization of the human rhabdomyosarcoma locus by mitotic recombination mapping[END_REF]. RMS can acquire invasive behavior and can form metastases in lung, bone, marrow, and lymphatic nodes. The development of such metastases is associated with poor prognosis [START_REF] Dagher | Rhabdomyosarcoma: an overview[END_REF]. The comprehension of mechanisms that regulate cancer cells migration and invasion may be a key for the development of new therapies for limiting metastases. Among the many proteases implicated in cell motility, the calpains play an essential role in regulating migration and invasion phenomena, both involved in metastases development [START_REF] Mamoune | Calpain-2 as a target for limiting prostate cancer invasion[END_REF][START_REF] Xu | Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu-and m-calpain in association with increased secretion, cell migration, and invasion[END_REF][START_REF] Xu | Suppression of cancer cell migration and invasion by protein phosphatase 2A through dephosphorylation of mu-and m-calpains[END_REF]. Targeting calpains may present a novel approach toward restraining metastasis and development of RMS cancers. Calpains are calcium-dependent cysteine proteases which best-characterized members are μ-calpain and m-calpain (i.e. CAPN1 and CAPN2 respectively), known as ubiquitous heterodimeric enzymes consisting of a large 80-kDa catalytic subunit and a smaller regulatory subunit (about 28 kDa, css1 or css2). Css1 is ubiquitous while css2 is tissue-specific [START_REF] Schád | A novel human small subunit of calpains[END_REF].

Calpain activity is Ca 2+ -dependent. Among the different factors regulating calpain activity, the critical factor is its specific endogenous inhibitor, calpastatin [START_REF] Wendt | Interaction of calpastatin with calpain: a review[END_REF]. Calpastatin is a ubiquitous protein regulated by several factors [START_REF] Raynaud | Four promoters direct expression of the calpastatin gene[END_REF] that requires Ca 2+ to bind and inhibits calpains [START_REF] Otsuka | Purification of the Ca 2+ -dependent proteinase inhibitor from bovine cardiac muscle and its interaction with the millimolar Ca 2+ -dependent proteinase[END_REF].

The present study focuses on the role of both μand m-calpain in the metastatic characteristics of the two cancer cell lines, ARMS and ERMS, assuming the role of calpains in cell adhesion and migration (that are limited steps of invasion and metastasis).

To reach that goal, we first characterized the calpain system in RMS before studying the role of the calpains in RMS cell adhesion, on RMS morphology and cytoskeleton organization, as well as in RMS migration characteristics and invasive behavior. Non-tumor human myoblasts LHCN-M2 [START_REF] Zhu | Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies[END_REF] were chosen as non-metastatic control cells.

The main result is that calpains play a crucial role in adhesion, high migration velocities and invasive behavior of RMS. More specifically, calpains may be considered as markers of tumor aggressiveness since their global activity is linked to migration velocities by a positive linear correlation. Moreover, inhibition of calpain activity in RMS led to migration velocities and invasive behavior similar to those of non-treated LHCN-M2 cells. Because calpain activity is directly involved in migration and invasion of RMS, the proteases would be a good target for the development of novel therapies to control metastasis.

Materials and methods

Cell culture

Cells were grown under 5% CO 2 atmosphere at 37°C in DMEM (Dulbecco's modified Eagle medium) with pyruvate supplemented with 10% FBS (fetal bovine serum) for RMS, and a culture medium containing DMEM without pyruvate (60%), medium 199 (20%) and FBS (20%) for LHCN-M2 cells (human myoblasts cell lines) [START_REF] Zhu | Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies[END_REF]. All cell culture reagents were purchased from Gibco-BRL. Some experiments have been done only with the tumor cell line presenting the most aggressive phenotype, ARMS.

Calpain inhibitor treatments

The chemical calpain inhibitor calpeptin (Z-Leu-Nle-CHO) was purchased from Calbiochem and used at 40 and 60 μM for adhesion assay, 50 μM for migration and invasion assay for immunolocalization and for cytoskeleton organization and 80 μM for Western blot. A second calpain inhibitor, calpain inhibitor III (Z-Val-Phe-CHO, Calbiochem) was used at 50 μM. Antisense oligodeoxynucleotide transient transfections has been realized as previously described [START_REF] Mazères | Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpains[END_REF].

Quantification of calpain activity

All cell lines were cultured in DMEM supplemented with 0.1% FBS during 48 h. The global calpain activity was observed and quantified on living cells using t-BOC-LM-CMAC (7-amino-4chloromethylcoumarin, t-BOC-L-leucyl-L-methionine amide, Molecular Probes) [START_REF] Xu | Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu-and m-calpain in association with increased secretion, cell migration, and invasion[END_REF][START_REF] Carragher | Calpain activity is generally elevated during transformation but has oncogene-specific biological functions[END_REF]. This molecule enters passively in cells and becomes fluorescent after calpain cleavage. Culture medium was replaced by DMEM without serum containing 50 μM of t-BOC-LM-CMAC. Cells were incubated during 30 min in darkness. The μcalpain activity was observed and quantified on fixed and permeabilized cells using Calpain 1 substrate (Chemicon) which become fluorescent after μ-calpain cleavage. After PAF fixation (15 min) and permeabilization with 1% Triton X-100 (3 min), cells were incubated with Calpain 1 substrate 50 μM during 40 min in darkness. The global calpain or the μ-calpain activities were then observed using an inverted epifluorescence microscope (Leica AF DMI6000). The results were quantified using the Metamorph software (Molecular Device).

Immunolocalization of calpains and calpastatin

All cell lines were cultured in DMEM supplemented with 0.1% FBS for 48 h. Next, cells were fixed and permeabilized. A specificity was disrupted by incubation (1 h) with PBS/BSA (3%). Then, m-calpain, μ-calpain and calpastatin were detected using corresponding antibodies (N-19 sc-7533 raised in goat (1/50), Santa Cruz Biotechnology; MAB3104, raised in mouse (1/500), Chemicon; H-300 sc-20779, raised in rabbit (1/50), Santa Cruz Biotechnology respectively, for 3 h). Then, cells were incubated with an appropriate secondary Alexa fluor antibody (anti-goat-Alexa594conjugated (1/1000), anti-mouse-Alexa594-conjugated (1/3000), and anti-rabbit-Alexa488-conjugated (1/1000) antibodies respectively for 90 min). Next cells were observed using an epifluorescence microscope (Leica AF DMI6000).

Adhesion assay

Adhesion assays were performed as described previously [START_REF] Mazères | Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpains[END_REF]. These assays were carried out with (40 μM or 60 μM) or without calpeptin treatment.

Migration assay

All cell lines were seeded in DMEM containing 0.1% FBS at a density of 4.10 3 cells/cm 2 in 35 mm diameter glass-bottom plate. Eight hours after seeding, time-lapse microscopy experiments were performed on an inverted Leica AF DMI6000 microscope equipped with an environmental chamber with phase-contrast optic (images taken every 15 min). Cell velocities of migration, defined as the average of 73 subsequent cell centroid displacements/one time interval between two successive images (15 min), were evaluated using the tracking object of the Metamorph software (Molecular Device). The cell trajectories were recorded for 18 h. These assays have been made in presence vs. absence of calpeptin (50 μM) added at the beginning of the time-lapse.

Invasion assay

The invasive capacity of RMS and LHCN-M2 cells was measured using BD Biocoat Growth Factor Reduced Matrigel Invasion Chambers (BD Biosciences). Cells were seeded at a density of 10 5 cells/cm 2 on the membrane in DMEM with 0.1% FBS. Quiescent medium was also added under the membrane. After 48 h of incubation, cells staying on the membrane were scraped off. Cells lying under the membrane were fixed with PAF (4%), and dehydrated overnight using ethanol. Cells were then stained with Hansen's hemalun and evaluated. The same experiment was realized with culture insert whose PET membrane was recovered by BD Matrigel matrix. Cells crossing the membrane were stained and evaluated. The invasive capacity of all cell lines was expressed by the percentage of cells going through the membrane with Matrigel matrix as compared to the percentage of cells going through the membrane without any Matrigel matrix. The implication of calpains in this phenomenon was evaluated using the same procedures after calpeptin treatment (50 μM).

Cytoskeleton analysis and vinculin localization

Cell actin fibers were stained as previously described [START_REF] Leloup | Involvement of calpains in growth factor-mediated migration[END_REF], with vs. without calpeptin or inhibitor III, and observed using an epifluorescence microscope (Leica AF DMI6000). Vinculin was detected after fixation and permeabilization, using primary antibody (V-9131, Sigma-Aldrich; 1/50) (3 h). Next, cells were incubated with a secondary Alexa fluor antibody from Molecular Probes (anti-mouse-Alexa594-conjugated 1/1000) (90 min).

Western blots

Western blots were performed as described previously [START_REF] Leloup | Involvement of calpains in growth factor-mediated migration[END_REF]. The bicinchoninic acid protein assay kit was purchased from Pierce, polyvinylidene fluoride membrane from Millipore and nitroblue tetrazolium/5-bromo-4chloro-3-indoyl-1-phosphate from Promega. The membranes were incubated with specific antibodies against m-calpain (raised in sheep as previously described ( [START_REF] Brustis | Rat myoblast fusion requires exteriorized m-calpain activity[END_REF]; 1/1000), β-tubulin (Santa Cruz Biotechnology (H-235 sc-9104); 1/500), μ-calpain (Sigma-Aldrich (C-0355); 1/2500) and calpastatin (Santa Cruz Biotechnology (H-300 sc-20779); 1/250). The secondary antibody conjugated to alkaline phosphatase (Sigma-Aldrich; 1/10,000) was used for detection. The apparent density of the band on the membranes was quantified with Totalab (Progenesis) and corrected according to β-tubulin expression.

Extraction of total RNA, reverse transcription and quantitative PCR

Extraction of total RNA, reverse transcription and quantitative PCR were performed as previously described [START_REF] Leloup | Involvement of the ERK/MAP kinase signaling pathway in milli-calpain activation and myogenic cell migration[END_REF]. Forward and reverse primer sequences for μand m-calpain amplification were agtcgtgccgcagcatggtga, acttgtccaggtcaaacttccg and gccatcaagtacctcaaccagg, gctccttggcagatgtctgtg, respectively. Transcript concentration of both isoforms was determined from the standard curve and compared to the control LHCN-M2. GAPDH (glyceraldehyde phosphate dehydrogenase) was used as control; its forward and reverse primer sequences were aaggtcatcccagagctgaacgc and acaacctggtcctcagtgtagcc.

Statistical analysis

The statistical significance of the differences between multiple groups was determined using ANOVA. When F was associated with a probability p < 0.05, intergroup comparisons were conducted using Student's t-test. Unless indicated, all data were expressed as means ± SEM and were representative of an average of at least three independent experiments.

Results

Characterization of the calpain system in rhabdomyosarcoma cells

In RMS cells, the expression of μ-calpain and m-calpain was significantly reduced compared to that of LHCN-M2 cells (Fig. 1A1-2). Moreover, the expression of calpastatin was reduced by 85 ± 1% in ARMS cells and 74 ± 3% in ERMS cells, compared to that of LHCN-M2 cells (Fig. 1A3).

Specific mRNA levels for μand m-calpains were quantified by real time RT-PCR and compared to that of LHCN-M2 control cells (Fig. 1B). The mRNA level of μ-calpain was similar in all cell lines (Fig. 1B1) whereas the expression level of m-calpain mRNA was reduced by more than 40% and 50% for ARMS and ERMS cells, respectively (Fig. 1B2).

Using t-BOC-LM-CMAC, the global calpain activity of RMS cells was measured and compared to that of LHCN-M2 cells (Fig. 1C1-3). Calpain activity was significantly higher in both RMS cell lines than in LHCN-M2 cells (2.6 ± 0.6 and 2.1 ± 0.1 fold for ARMS and ERMS cells respectively) (Fig. 1C4). Furthermore, μ-calpain activity (calpain 1 substrate) was higher in RMS than in LHCN-M2 cells (2.3 ± 0.4 and 2.7 ± 0.4 fold for ARMS and ERMS cells respectively) (Fig. 1C5).

In LHCN-M2 myoblasts, μ-calpain was concentrated around the nucleus with a weak decreasing gradient towards the cell periphery presenting a linear structure parallel to the main axis of the cell (Fig. 1D1). In these cells, m-calpain was more particularly localized around the nucleus in a spot-shaped fashion (Fig. 1D2) and calpastatin presented a higher concentration around the nucleus, decreasing towards the cell periphery (Fig. 1D3). In RMS cells, μ-calpain presented a higher concentration around the nucleus, decreasing towards the cell periphery. m-Calpain and calpastatin localizations were not different between all cell lines (Fig. 1D4-6 for ARMS, data not shown for ERMS cells).

Implication of calpains in adhesion

As shown in Fig. 2A, RMS cells presented a delay and a decrease in their adhesion rates. Eight hours after seeding, the adhesion rates of ARMS and ERMS were 37 ± 3 % and 35 ± 4% respectively, whereas LHCN-M2 myoblasts adhered completely (p < 0.05).

Calpain inhibition drastically decreased the adhesion rate of LHCN-M2 (Fig. 2B1). The presence of calpeptin led to a decrease in the adhesion rate of ARMS. However, this decrease was significant only 2 and 4 h after seeding at 40 μM of calpeptin and after 2 h at 60 μM of calpeptin (Fig. 2B2). The adhesion rates of ERMS were not affected by the presence of calpeptin neither at 40 μM of calpeptin nor at 60 μM (data not shown).

In order to understand the difference of adhesiveness between RMS and LHCN-M2 cells, vinculin (one of the main focal adhesion components) localization was investigated in all cell lines (Fig. 2). In LHCN-M2 cells, vinculin was regularly localized at the ventral surface of the cell (Fig. 2C1). On the contrary, in both RMS cell lines, vinculin was principally localized at the periphery of the cell (Fig. 2C2 for ARMS, data not shown for ERMS). The inhibition of calpain activity led to a reorganization of vinculin. In all cell lines presenting a rounded shape, vinculin was localized predominantly at the periphery of the ventral surface of the cell (Fig. 2C3 andC4 for LHCN-M2 and ARMS cells, respectively, data not shown for ERMS). Contrary to LHCN-M2 myoblasts, in which the presence of calpeptin led to a 107 ± 14% increase in vinculin expression, the presence of calpeptin in ARMS led to a 14 ± 3% decrease in vinculin expression (Fig. 2C5). Antisense oligodeoxynucleotide treatments against μ-calpain led to an increase in LHCN-M2 vinculin expression (14 ± 4%) but did not affect the vinculin expression levels of ARMS (p = 0.30) (Fig. 2C6-7). Antisense oligodeoxynucleotide treatments against m-calpain led to an increase in vinculin expression in LHCN-M2 cells (Fig. 2C6). Conversely, this treatment in ARMS led to a decrease in vinculin expression (Fig. 2C7).

Morphological characteristics and actin cytoskeleton organization of migrating rhabdomyosarcoma cells

Eight hours after seeding, control cells exhibited an elongated shape and membrane protrusions. Migrating RMS cell lines exhibited an elongated shape with a reduced cytoplasm and large nuclei. In addition, membrane protrusions were smaller and thinner than those of LHCN-M2 myoblasts (Fig. 3A1 and3A2 for LHCN-M2 and ARMS cells respectively; data not shown for ERMS). The inhibition of calpain activity impaired cell morphology, all cell lines presenting a rounded shape with very short membrane protrusions (Fig. 3B1 and B2 for LHCN-M2 and ARMS cells, respectively; data not shown for ERMS).

Since the actin cytoskeleton is responsible for cell morphology and considered to be the motor of cell migration [START_REF] Lambrechts | The actin cytoskeleton in normal and pathological cell motility[END_REF], we studied its organization in the different cell lines. In elongated LHCN-M2 cells, the actin cytoskeleton formed stress fibers parallel to the main axis of the cell (Fig. 3C1). On the contrary, in both RMS cell lines, actin cytoskeleton failed to form parallel stress fibers (Fig. 3C2 for ARMS; data not shown for ERMS). The actin cytoskeleton was disorganized and mainly concentrated at the periphery of the cell.

In presence of calpeptin, the LHCN-M2 F-actin was no longer at the center of the cell. However it still remained organized at the periphery of the cell where it was concentrated (Fig. 3C3). In both RMS cell lines, the inhibition of calpains induced an increase in the concentration of F-actin at the periphery of the cell (Fig. 3C4, data not shown for ERMS).

In all cell lines, the presence of calpeptin led to an increase in the quantity of F-actin (Fig. 3D1). This result was confirmed by the use of calpain inhibitor III leading to an increase in the level of Factin by 18 ± 3% in ARMS (Fig. 3D2).

Implication of calpain in the arrangement of actin cytoskeleton

In LHCN-M2 cells, F-actin and μ-calpain colocalized by 93% (F-actin overlapping μ-calpain: 93.2 ± 0.5% and μ-calpain overlapping Factin: 93.2 ± 1.0%) (Fig. 4A1). In these cells, stress fibers were associated with a peak of μ-calpain concentration (Fig. 4A2). In addition, F-actin in ARMS cells colocalized with μ-calpain at a lower extent than in LHCN-M2 myoblasts (F-actin overlapping μcalpain: 66.5 ± 3.5% and μ-calpain overlapping F-actin: 46.7 ± 2.9) (Fig. 4A3-4). In both cell lines, the presence of calpeptin led to a relocalization of F-actin as well as μ-calpain at the periphery of the cell (Fig. 4A5-8).

In order to precise the role of each calpain on α-actin and βactin expression (components of F-actin), the expression of each isoform of actin monomer was determined in the presence vs. absence of calpeptin. In LHCN-M2 cells, the inhibition of calpains induced an increase of both α-actin and β-actin (α-actin increased by 51 ± 14% and β-actin increased by 33 ± 7) (Fig. 4B1-2). In ARMS conversely, this inhibition induced a decrease of both αactin and β-actin (α-actin decreased by 38 ± 5% and β-actin decreased by 15 ± 3%) (Fig. 4B1-2).

Moreover, in LHCN-M2 cells, the inhibition of μ-calpain (by antisense oligodeoxynucleotide treatments) up-regulated the expression of α-actin but did not affect the expression level of β-actin (Fig. 4B3 and5) while the inhibition of m-calpain downregulated the expression of α-actin and up-regulated the expression of β-actin (Fig. 4B4 and6). It appeared that the inhibition of μcalpain as well as m-calpain in ARMS down-regulated the expression of both α-actin and β-actin (Fig. 4B4 and6).

Kinetic characteristics of rhabdomyosarcoma cell lines

As presented in Fig. 5A, the migration velocities of cancer cells were significantly higher (about 2.3 and 1.6 fold for ARMS and ERMS respectively) than those measured in controls. The inhibition of calpain activity drastically impaired migration velocities of all cell lines (Fig. 5A). The velocities decreased by 11% for LHCN-M2, 60% for ARMS and 28% for ERMS cells. Similar results were obtained using the calpain inhibitor III (data not shown).

To complete this study, the different cell trajectories were analyzed and are presented in Fig. 5B. The ARMS dispersion area was higher than that of LHCN-M2 cells (Fig. 5B2 vs. B1, ERMS dispersion area was intermediate, data not shown). As presented in Fig. 5C1, Δ (vector between the initial point of each trajectory and the final localization of the cell) confirmed that the dispersive capacity was higher for the ARMS cell line. The inhibition of calpain activity significantly impaired the dispersion area of all cell lines (Fig. 5B3 vs. B1 and B4 vs. B2) as well as the dispersive behavior (Fig. 5C2-3; data not shown for ERMS). The dispersive capacity decreased by 35%, 44% and 49% for LHCN-M2, ARMS and ERMS cells respectively. Moreover, the dispersive capacity of treated ARMS cells was similar to the dispersive capacity of LHCN-M2 in standard conditions (p = 0.07).

These results highlight the role of calpains in migration and dispersive behavior of all cell lines. Moreover, the global activity of calpains was directly correlated with the velocity of each cell Fig. 1 -Characterization of calpain system components in rhabdomyosarcoma cells. Expression of μ-calpain, m-calpain and calpastatin in all cell lines (A1, A2, and A3, respectively). Quantification of μ-calpain and m-calpain mRNA expression, in all cell lines (B1 and B2, respectively). Global calpain activity in LHCN-M2, ARMS and ERMS cells (C1, C2, and C3, respectively). Quantification of global calpain activity and μ-calpain activity (C4 and C5, respectively). Localization of μ-calpain, m-calpain and calpastatin in LHCN-M2 cells (D1, D2, and D3, respectively) and in ARMS cells (D4, D5, and D6, respectively). Results are mean values ± SEM from at least 3 independent experiments. ⁎Significantly different from the control LHCN-M2 cells (p < 0.05). Pictures shown are representative of at least three separated sets of culture. line (Fig. 5D). Indeed, calpain activity and migration velocity showed a strong positive linear correlation: y = 13.71x -0.395 (R 2 = 0.9401).

Invasive behavior of rhabdomyosarcoma cells

All cell lines were capable of breaching the Matrigel. Nevertheless, the invasive capacities of both RMS were significantly higher than that of control cells (Fig. 6A). The invasive index was 11.23 ± 0.45 for ARMS and 12.32 ± 0.20 for ERMS cell lines (Fig. 6B).

The inhibition of calpain activity led to a significant decrease in the invasive behavior of all cell lines. The invasive behavior of LHCN-M2 myoblasts decreased by about 2 fold. In the case of RMS cell lines, it decreased by more than 10 fold. Consequently, the invasive behavior of RMS treated with calpeptin as well as their invasive factor were equivalent to that of non-treated LHCN-M2 Fig. 2 -Implication of calpains in cell adhesion. Adhesion rates of all cell lines (A) and adhesion rates of LHCN-M2 (B1) and ARMS cells (B2) in calpeptin presence (40 μM or 60 μM) or not. Localization of vinculin in LHCN-M2 and ARMS cells (C1 and C2, respectively) or in LHCN-M2 and ARMS treated with calpeptin (50 μM) (C3 and C4), respectively. Expression of vinculin in LHCN-M2 and ARMS cells treated or not with calpeptin (80 μM) (C5). Expression of vinculin in LHCN-M2 and ARMS (C6 and C7, respectively) transfected with antisense oligodeoxynucleotides directed against μ-calpain or m-calpain. Results are mean values ± SEM from at least 3 independent experiments. ⁎Significantly different from the control (LHCN-M2 cells (A), non-treated cells (B1, B2, and C5) or cells transfected with random (C6, C7)) (p < 0.05). Pictures shown are representative of at least three separated sets of culture. cells (Fig. 6A-B). Similar results were obtained using the calpain inhibitor III (data not shown).

Discussion

RMS cells can acquire invasive behavior and can form metastases in lung, bone, marrow, and lymphatic nodes decreasing the chances of patients' healing to less than 20% [START_REF] Dagher | Rhabdomyosarcoma: an overview[END_REF]. The comprehension of the mechanisms that regulate cancer cells migration and invasion may be a key for the development of new therapies for limiting metastases.

The determination of the mRNA expression level of μ-calpain and m-calpain, in all cell lines showed that it was similar for μcalpain and it was reduced for m-calpain, in RMS cells, compared to control. However, Western blots showed that μ-calpain as well as m-calpain were reduced in RMS when compared to control. These results could be explained by a potential post-transcriptional regulation for RMS μ-calpain and by an absence of such regulation for m-calpain. In RMS cell lines, even though the expression of the calpain system components was decreased compared to LHCN-M2 myoblasts, both global and μ-calpain activities were higher than in LHCN-M2 cells. This deregulation of RMS calpain system is in agreement with the high activity or expression of calpains detected in human renal cell carcinoma [START_REF] Braun | Expression of calpain I messenger RNA in human renal cell carcinoma: correlation with lymph node metastasis and histological type[END_REF], in squamous cell carcinoma [START_REF] Reichrath | Different expression patterns of calpain isozymes 1 and 2 (CAPN1 and 2) in squamous cell carcinomas (SCC) and basal cell carcinomas (BCC) of human skin[END_REF], and in human colorectal adenocarcinoma [START_REF] Lakshmikuttyamma | Overexpression of m-calpain in human colorectal adenocarcinomas[END_REF]. In RMS cells, calpastatin expression is highly down-regulated. Thereby, the increase in calpain activity could be due to an accumulation of the non-calpastatin-inhibited proteolyzed active forms of the proteases. The immunolocalization of calpain system components was different between the cell lines. In RMS cells, μ-calpain presented a gradient of concentration decreasing toward the periphery of the cell. Conversely, in LHCN-M2 cells, μ-calpain presented linear structures parallel to the main axis of the cell. This difference of μ-calpain organization between LHCN-M2 and RMS cells presumes a different role for this protease as a function of the cell type. The localization of m-calpain was identical in all cell types and this observation confirms the results obtained by Samanta et al. showing the localization of mcalpain at the endoplasmic reticulum level [START_REF] Samanta | Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum[END_REF].

In RMS cells, the weak adhesion rate compared to LHCN-M2 is characteristic of the metastatic phenotype of these cells. In both RMS cell lines, the quantity of focal adhesions, marked by vinculin, was weaker than that of LHCN-M2 cells. Moreover, in RMS, vinculin was concentrated essentially at the periphery of the cell. This vinculin distribution may be responsible for the weak adhesion rate of RMS. These results are in agreement with the decreased adhesiveness observed in circulating tumor cells in prostate cancer [START_REF] Howard | Decreased adhesiveness, resistance to anoikis and suppression of GRP94 are integral to the survival of circulating tumor cells in prostate cancer[END_REF]. A reduction in the adhesiveness may increase the motility of RMS cells which is a key step of invasion [START_REF] Kassis | Motility is rate-limiting for invasion of bladder carcinoma cell lines[END_REF]. Inhibition of calpain activity impaired the adhesion of LHCN-M2 and ARMS cells involving calpains in this process. Moreover, the inhibition of μ-calpain as well as m-calpain led to an overexpression of vinculin in LHCN-M2 cells. These results indicate that vinculin is a calpain substrate in human myoblasts, in agreement with the results obtained by Taylor et al. in Z-disc [START_REF] Taylor | Is Z-disk degradation responsible for postmortem tenderization?[END_REF]. In ARMS, the expression of vinculin was regulated only by m-calpain that stimulated its expression. Together, these results show the role of calpain in adhesion of LHCN-M2 and ARMS. They also support a differential role for μ-calpain and m-calpain in the regulation of vinculin depending on the cell type. These differences at the adhesive capacity level may be responsible for some differences at the migration level.

Indeed, in all cell lines, the determination of migration velocities showed that RMS migrated faster and further away than LHCN-M2. The high migration velocities of RMS cells linked to the high invasive behavior of these cell lines are both characteristics of tumor and metastatic cells. In LHCN-M2 as well as in RMS cell lines, the inhibition of calpain activity led to a decrease in cell migration velocities. This decrease is in agreement with the decrease of migration observed in fibroblasts capn 4 -/- [START_REF] Dourdin | Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts[END_REF]. Moreover, in presence of calpeptin, the migration velocities of ARMS were similar to the migration velocities of LHCN-M2 cells in standard conditions (p = 0.52, NS). Similarly, the dispersion behavior of all cell lines was reduced and in presence of calpeptin, the dispersive capacity of ARMS was similar to the dispersive capacity of LHCN-M2 cells in standard conditions. Together, these results indicate that calpains are involved in migration velocities and dispersive capacity of migrating cells.

To summarize, RMS showed a high calpain activity and high migration velocities. One of the main results was the determination of a positive linear correlation between migration velocities and calpain activity. Indeed, calpain activity was directly linked to migration velocities pointing these proteases as markers of tumor aggressiveness.

The reorganization of the actin cytoskeleton is a fundamental step in cell adhesion, migration and invasion [START_REF] Lambrechts | The actin cytoskeleton in normal and pathological cell motility[END_REF]. In LHCN-M2 cells, the actin cytoskeleton was organized in stress fibers that were parallel to the main axis of the cell. This organization confers a strong adhesiveness to these cells. Moreover, the F-actin Fig. 4 -Implication of calpain in the actin cytoskeleton arrangement. Immunolocalization of F-actin (green) and μ-calpain (red) in LHCN-M2 cells, in standard conditions (A1). Representation of colocalization of F-actin (green) and μ-calpain (red), at the level of the line drawn on A1 (A2). A width at the middle of the peak overlapping zone higher than 2 pixels illustrated colocalization of both F-actin and μ-calpain (white line). Numbers indicate the amount of pixel between the limit of the overlapping zone (A2). Immunolocalization and representation of colocalization of F-actin and μ-calpain in ARMS cells, in standard conditions (A3 and A4, respectively). Immunolocalization and representation of colocalization of F-actin and μ-calpain in LHCN-M2 and in ARMS, in the presence of calpeptin (50 μM) (A5 and A6, respectively, for LHCN-M2 cells and A7 and A8, respectively, for ARMS cells). Quantification of α-actin and β-actin expression in LHCN-M2 and ARMS cells with or without calpeptin (80 μM) (B1 and B2, respectively). Quantification of α-actin expression in LHCN-M2 and ARMS cells after antisense oligonucleotides treatment (B3 and B4, respectively) and quantification of β-actin expression in LHCN-M2 and ARMS cells, in the same conditions (B5 and B6). Results are mean values ± SEM from at least 3 independent experiments. ⁎Significantly different from the control (non-treated cells or cells transfected with random) (p < 0.05). Pictures shown are representative of at least three separated sets of culture.

constituting the stress fibers colocalized with μ-calpain. The same colocalization was previously reported in dendritic cells [START_REF] Calle | Inhibition of calpain stabilises podosomes and impairs dendritic cell motility[END_REF] and may implicate μ-calpain in the organization and rearrangement of the actin cytoskeleton. Even if μ-calpain seems to play a role at the time of cell adhesion [START_REF] Glading | Cutting to the chase: calpain proteases in cell motility[END_REF], its association with the stress fibers presumes of a singular role in migration. On the contrary, the actin cytoskeleton of RMS was disorganized and concentrated at the periphery of the cell. This lack of stress fibers has already been described in v-Src transformed cells in which calpain activity simulates the loss of actin stress fibers and induces some morphological modifications [START_REF] Carragher | Cleavage of focal adhesion kinase by different proteases during SRC-regulated transformation and apoptosis. Distinct roles for calpain and caspases[END_REF]. This disorganization may be responsible for the weak adhesion rate and the high migration velocities of RMS cells [START_REF] Perrin | Proteolysis of cortactin by calpain regulates membrane protrusion during cell migration[END_REF]. In all cell lines, the inhibition of calpain activity induced morphological modifications: cells became rounded, with small protrusions. These changes contrast with the modifications observed in CHO cells treated with calpain inhibitor 1 which present longer shape than controls [START_REF] Huttenlocher | Regulation of cell migration by the calcium-dependent protease calpain[END_REF]. Indeed, fibroblasts over-expressing calpastatin show a rounded morphology as observed in treated LHCN-M2 and RMS cells [START_REF] Potter | Calpain regulates actin remodelling during cell spreading[END_REF]. In LHCN-M2 cells, calpain inhibition induced a disorganization of the central actin cytoskeleton and the F-actin was concentrated at the periphery of the cell. These results are in agreement with those obtained in C2C12 cells over-expressing calpastatin [START_REF] Dedieu | Myoblasts migration is regulated by calpain through its involvement in cell attachment and cytoskeletal organization[END_REF]. This rearrangement of actin cytoskeleton was coupled with the relocalization of μ-calpain. This result is in agreement with the changes in μ-calpain localization coordinated with the actinrelated cytoskeleton rearrangements observed during early embryonic development of Drosophila [START_REF] Emori | Calpain localization changes in coordination with actin-related cytoskeletal changes during early embryonic development of Drosophila[END_REF]. In LHCN-M2 cells, the persistence of peripheral actin stress fibers, coupled with the disorganization of central fibers could be explained by studies showing that two different pathways are involved in stress fiber regulation: one regulates peripheral stress fibers, depending on calcium and involving calmodulin while the other implies Rho and Rho-kinase [START_REF] Katoh | Rho-kinase dependent organization of stress fibers and focal adhesions in cultured fibroblasts[END_REF]. The same concentration of F-actin at the Fig. 5 -Kinetic characteristics of rhabdomyosarcoma cell lines. Average of migration velocities of all cell lines, with or without calpeptin (A). Individual trajectories of cell displayed in diagrams drown with the initial point of each trajectory placed at the origin of the plot (B). LHCN-M2 and ARMS cells trajectories in standard conditions (B1 and B2, respectively) and in calpeptin presence (B3 and B4, respectively). Distance Δ between the initial point of each trajectory and the final localization of cell, after 18 h of time-lapse (C). Δ for LHCN-M2, ARMS, and ERMS cells, in standard conditions (C1). Δ for LHCN-M2 and ARMS cells with or without calpeptin (C2 for LHCN-M2 and C3 for ARMS cells). Positive linear correlation between calpain activity and migration velocity (D). Results are mean values ± SEM from at least 3 independent experiments. ⁎Significantly different from the control LHCN-M2 cells (p < 0.05). periphery of the cell was observed in RMS cell lines. These results may involve calpains in the Rho and Rho-kinase-dependant stress fiber regulation.

Although the F-actin was disorganized when calpain activity was inhibited, its quantity was higher in all treated cell lines than in controls. Such increase has already been observed in dendritic cells [START_REF] Calle | Inhibition of calpain stabilises podosomes and impairs dendritic cell motility[END_REF]. In these cells, the accumulation of vinculin, that binds the Arp 2/3 complex, may stimulate the actin polymerization and then increase F-actin quantity. In LHCN-M2 cells, same interactions may be responsible for F-actin increase since inhibition of calpain induced an increase in vinculin expression. On the contrary, in ARMS cells, the increase in F-actin quantity observed when calpain activity was inhibited may result from another process given the fact that calpain inhibition induced a decrease in vinculin expression.

F-actin is formed by α-actin and β-actin. As a consequence, the increase in F-actin caused by the inhibition of calpain may be due to either a stimulation of the polymerization or to an inhibition of the depolymerization. In LHCN-M2 cells, the inhibition of calpains induced an increase of both actin isoforms. Indeed, the increase in F-actin may be due to a stimulation of actin polymerization. These results are in agreement with those of Calle et al. proposing a vinculin-Arp2/3 stimulation of actin polymerization [START_REF] Calle | Inhibition of calpain stabilises podosomes and impairs dendritic cell motility[END_REF]. On the contrary, in the same conditions in ARMS, both actin isoforms decreased. The increase in F-actin observed in ARMS may result from an inhibition of F-actin depolymerization. Moreover, in LHCN-M2 cells, μ-calpain seemed to play a preponderant role in the down-regulation of α-actin expression while m-calpain downregulated β-actin expression. In ARMS cells, both μ-calpain and mcalpain had the same effect on α-actin and β-actin. Both calpains stimulated the expression of the two actin isoforms.

To summarize, regulation of actin cytoskeleton organization and arrangement was dependent on calpain activity and on the specific cell type.

The invasive tests realized with all cell lines showed the higher invasive capacities of RMS compared to that of LHCN-M2 cells. This result confirms the invasive phenotype of RMS.

The inhibition of calpain activity induced a decrease in the invasive capacity of all cell lines. These results showed that calpains play a significant role in the invasive process and are in agreement with those obtained in other different cell types such as cancer prostate cells [START_REF] Mamoune | Calpain-2 as a target for limiting prostate cancer invasion[END_REF], lung cancer cells [START_REF] Xu | Suppression of cancer cell migration and invasion by protein phosphatase 2A through dephosphorylation of mu-and m-calpains[END_REF] or osteoclastoma cells [START_REF] Fan | Silencing of calpain expression reduces the metastatic potential of human osteosarcoma cells[END_REF]. More specifically, in the presence of calpeptin, the invasive capacities of both RMS cell lines were similar to that of LHCN-M2 cells in standard conditions. This result is the second major point of this study directly involving calpain activity in tumor aggressiveness.

The different implications of calpains in adhesion, migration and invasion which are all key steps of metastatic dissemination indicate that μ-calpain and m-calpain are potential therapeutic targets in the aim of stemming tumor invasion and development of metastases. Moreover, the direct relationship between calpain activity and migration velocities or invasive behavior indicates that calpains can serve as useful markers of tumor aggressiveness.

Fig. 3 -

 3 Fig. 3 -Morphological characteristics and actin cytoskeleton organization of migrating rhabdomyosarcoma cells. LHCN-M2 and ARMS cell morphology in standard conditions (A1 and A2, respectively) and in the presence of calpeptin (50 μM) (B1 and B2, respectively). Cytoskeleton organization of LHCN-M2 and ARMS, in standard condition (C1 and C2, respectively) and in the presence of calpeptin (50 μM) (C3 and C4, respectively). Quantification of F-actin quantity, in all cell lines, with or without calpeptin treatment (50 μM) (D1). Quantification of ARMS F-actin with or without calpain inhibitor III treatment (D2). Results are mean values ± SEM from at least 3 independent experiments. Pictures shown are representative of at least three separated sets of culture. ⁎Significantly different from the control (p < 0.05).

Fig. 6 -

 6 Fig. 6 -Invasive behavior of rhabdomyosarcoma cells. Invasive capacity of LHCN-M2, ARMS and ERMS cells with or without calpeptin (50 μM) (A). Invasive index of LHCN-M2, ARMS and ERMS cells with or without calpeptin (50 μM) (B). Results are mean values ± SEM from at least 3 independent experiments. ⁎Significantly different from the control LHCN-M2 cells; ⁎⁎significantly different from data without calpeptin (p < 0.05).

E X P E R I M E N T A L C E L L R E S E A R C H 3 1 6 ( 2 0 1 0 ) 1 5 8 7 -1 5 9 9

Acknowledgments

We thank very much Dr V. Mouly and Dr G. S. Butler-Browne for the gift of LHCN-M2 myoblasts and Dr A. Bonnieu for the gift of RMS. We also thank Dr M. Godfroy for reading the manuscript and A. Pires-Alves for helping with additional experiments.

The authors appreciate the assistance provided by Dr J.-L. Morel for confocal imagery on confocal microscope Leica SP5.

This work was supported by grants from the Association Française contre les Myopathies (AFM), the Ligue National Contre le Cancer, Comités Aquitaine Charente, and from the Institut National de Recherche Agronomique (INRA-France, PHASE Department).

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