Laetitia Bourgeade

Cédric Chauve

Julien Allali
email: julien.allali@labri.frcedric.chauve@sfu.ca

Chaining Sequence/Structure Seeds for Computing RNA Similarity

We describe a new method to compare a query RNA with a static set of target RNAs. Our method is based on (i) a static indexing of the sequence/structure seeds of the target RNAs, (ii) searching the target RNAs by detecting seeds of the query present in the target, chaining these seeds in promising candidate homologs, then (iii) completing the alignment using an anchor-based exact alignment algorithm. We apply our method on the benchmark Bralibase2.1 and compare its accuracy and efficiency with the exact method LocaRNA and its recent seeds-based speed-up ExpLoc-P. Our pipeline RNA-unchained greatly improves computation time of LocaRNA and is comparable to the one of ExpLoc-P, while improving the overall accuracy of the final alignments.

Introduction

A major advance in molecular biology of the last decade has been the discovery that RNA molecules, especially non-coding RNAs (ncRNAs), are involved in many cellular processes such as the regulation of gene expression, splicing, signaling, . . . [START_REF] Thomas | The molecule of the year[END_REF]. This is well illustrated by the growth of the Rfam database [START_REF] Griffiths-Jones | Rfam: an RNA family database[END_REF], whose content went from 15,255 RNAs, in 2002 (date of its creation) to 6,125,803 RNAs, in 2012 (last release). Moreover, several recent studies of the RNA structurome at the whole genome level have lead to the discovery of new families of ncRNAs and to a better understanding of the role of RNAs in the cell [START_REF] Will | Inferring non-coding RNA families and classes by means of genome-scale structure-based clustering[END_REF][START_REF] Kertesz | Genome-wide measurement of RNA secondary structure in yeast[END_REF][START_REF] Wan | Understanding the transcriptome through RNA structure[END_REF].

The general problem of annotating, classifying or clustering of RNA sequences is thus an important problem in computational biology, that relies on solving efficiently and accurately the following computational question: given an RNA query Q and a set of RNA sequence targets D, what are the members of D whose similarity with Q is large enough to indicate a potential relationship, either evolutionary and/or functional? For RNA genes, due to the importance of the structure in terms of biological function, it is natural to consider both the sequence and secondary structure when comparing genes. Most RNA comparison methods can be classified in two families: (i) tools requiring the knowledge of an RNA secondary structure, such as RNAforester [START_REF] Hochsmann | Local similarity in RNA secondary structures[END_REF] or Gardenia [START_REF] Blin | Alignment of RNA structures[END_REF] to cite only two (see [START_REF] Allali | Benchmarking RNA secondary structure comparison algorithms[END_REF] for a thorough evaluation of such methods), and (ii) tools taking only RNA sequences as input and using covariance models or base pairing probabilities such as LocaRNA [START_REF] Will | Inferring non-coding RNA families and classes by means of genome-scale structure-based clustering[END_REF] or Infernal [START_REF] Nawrocki | Infernal 1.1: 100-fold faster RNA homology searches[END_REF]. The first family of approaches relies on the classical notions of edit distance and alignment. RNAs are modeled using tree-like structures and algorithms look either for a set of edit operations of optimal score that transforms the first RNA into the second one, or for an alignment maximizing the similarity. A cubic time complexity is the current reference for pairwise RNA structure comparison (see for example [START_REF] Zhong | Efficient alignment of RNA secondary structures using sparse dynamic programming[END_REF]), underlining the issue of using such approach directly when a large number of pairwise comparison is required. The second family of RNA pairwise comparison methods works directly on RNA sequences. The current reference method LocaRNA aligns RNA sequences based on the pairing probabilities for each sequence, computed from the partition function of the ensemble of all possible foldings into secondary structures, under the assumption of a free-energy based Boltzmann distribution on this ensemble and has a quartic time complexity. Approaches have been introduced to speed-up the alignment, at the expense of guaranteed optimality, such as ExpaRNA-P/Exploc-P [START_REF] Schmiedl | Exact pattern matching for RNA structure ensembles[END_REF]; these methods rely on conserved sequence/structure motifs, called Exact Pattern Matches (EPM), that can be detected in quadratic time and are provided to LocaRNA as alignment constraints, thus breaking the alignment computation into smaller independent problems and reducing the overall computation time. Finally, a last set of tools aims at solving the classification problem, that asks to assign a given query RNA sequence to a set of predefined families, such as the Rfam. The Rfam classification engine Infernal starts by computing covariance models for families based on RNA sequences that are known to belong to them. Again, despite recent improvements this approach remains time consuming, which has motivated the development of filters such as RNA sifter [START_REF] Janssen | Shape based indexing for faster search of RNA family databases[END_REF], based on the abstract shape approach (see [START_REF] Steffen | RNAshapes: an integrated RNA analysis package based on abstract shapes[END_REF]).

In the present work, we address the general problem of the one-against-all RNA pairwise comparison, where a given query RNA Q is compared to unstructured set D of target RNAs. We introduce a new method, RNA-unchained, aimed at computing efficiently high quality alignments between Q and the members of D. Our method is based on a classical principle in sequence comparison following four steps: seed indexing for the target set, seeds look-up in the index for the given query, seeds chaining between the query and the targets sharing common seeds, and finally exact anchor-based alignments. To evaluate RNA-unchained, we followed the approach of [START_REF] Schmiedl | Exact pattern matching for RNA structure ensembles[END_REF] and used the benchmark BRAliBase2.1, which is composed of set of reference pairwise alignments between ncRNA sequences. We measured the accuracy of the alignments obtained by RNA-unchained, using the Sum of Pair Scores statistics (SPS) [START_REF] J D Thompson | A comprehensive comparison of multiple sequence alignment programs[END_REF]. We observe that we obtain alignments of quality comparable or better than LocaRNA, and consistently better than ExpLoc-P, in a time comparable to the time taken by ExpLoc-P.

Methods

The pipeline we describe takes as input a set D of RNA sequences and an RNA sequence query Q, and aims at computing quickly candidate sequences of D that are similar to Q, together with alignments between Q and these candidates. Our pipeline applies to the case where secondary structures are provided or not. It is composed of two elements: a static preprocessing stage for D and, for a given query Q, a dynamic search in D for RNA similar to Q.

Preprocessing D. This stage is static, i.e. is performed once for all. It consists in folding the sequences of D, each into one or several candidate RNA secondary structures, followed by extracting and indexing a set of seeds, defined as sequence/structure motifs of a given length.

Querying D. For a given query Q, its sequence is first folded into an RNA secondary structure and all the seeds it contains are generated. Then the index of seeds from D is searched to identify candidate sequences in D sharing motifs with Q. Next for each candidate from D, an optimal set of seeds that is compatible with the secondary structures of both Q and the candidate is extracted using a fast seeds chaining algorithm, and these seeds are used as anchors for a constrained alignment between the candidate and Q.

We now describe the details of our pipeline, starting with the modeling of RNA secondary structures and of seeds. Next we detail how to index these seeds and look-up for existing seeds. Finally we describe the seeds chaining and anchor-based alignments stages.

Modeling RNA Secondary Structures and Seeds

RNA sequence and secondary structure. An RNA is a molecule composed of four nucleic acids usually symbolized by the alphabet {A, C, G, U }. Pairs of bases in an RNA molecule can form hydrogen links, thus generating a spatial folding of the molecule forming its secondary structure. Here we consider pseudoknot-free RNA secondary structures, i.e. we assume that each base is involved in at most one base pair and that base pairs define a crossing-free planar structure. An RNA secondary structure can be encoded by an arc-annotated sequence (aa-sequence for short) [START_REF] Evans | Finding Common Subsequences with Arcs and Pseudoknots, Combinatorial Pattern Matching[END_REF], and we rely on this modeling to describe our method. An aasequence A = (S, P ) representing a pseudoknot-free RNA structure is composed of a sequence S of length |A| = n on the alphabet {A, C, G, U }, representing the RNA primary structure (sequence) and of a well-parenthesized sequence P of length n on the alphabet {., (, )}, representing the paired bases defining the secondary structure. For a sequence S, we denote by S[i] the i + 1 th symbol of S and by

S[i, j] = S[i]S[i + 1] . . . S[j]
the factor of S of length j -i + 1 starting at position i, for any i ≤ j in {0, . . . , n -1}. Similar definitions hold for P .

Seeds. Seeds, defined as sequence/structure motifs, are used for two purposes in our pipeline. They are first aimed at detecting quickly candidates RNAs from D that share enough seeds with Q. In a second time, an optimal set of seeds that are compatible with the secondary structures of Q and the candidate is computed, and serves as anchors for the final alignment. So the definition of seeds should (1) allow a fast look-up in the indexing structure and (2) satisfy some compactness condition that makes them compatible with the chaining algorithm we use [START_REF] Allali | Efficient chaining of seeds in ordered trees[END_REF].

Definition ( (l, d)-centered-seed (short name (l, d)-cs) Let A = (S, P ) be an aa-sequence of length n . Let d and l be two integers such that 2d ≤ l. For a given i in {0, . . . , n -l}, the (l, d)-cs of A in position i, denoted by cs i , is the pair (s, p) defined by

p = P [i, i + l -1] and s = S[i + d, i + l -d -1].
Note that s is a sequence of length l -2d and p a sequence of length l, so a (l, d)-cs is not an aa-sequence as both sequences do not have the same length (see Fig. 1). It follows immediately that the maximal number of distinct (l, d)-cs is 3 l 4 l-2d . Furthermore, such seeds can be seen as spaced seeds [START_REF] Brown | Bioinformatics Algorithms: Techniques and Applications, chapter A survey of seeding for sequence alignment[END_REF] with no structural mismatch and possible nucleotide mismatches in a prefix and a suffix of length d of the seed. Next, we define the notion of seed common to two aa-sequences, that we call a hit.

Definition ( hit)

Let A 1 = (S 1 , P 1 ) and A 2 = (S 2 , P 2 ) be two aa-sequences. Let d and l be two integers such that 2d ≤ l. A hit is an (l, d)-cs common to A 1 and A 2 , i.e. a pair (i, j) of integers such that:

-0 ≤ i ≤ |A 1 | -l -1 and 0 ≤ j ≤ |A 2 | -l -1 , -P 1 [i, i + l -1] = P 2 [j, j + l -1], -S 1 [i + d, i + l -d -1] = S 2 [j + d, j + l -d -1].
The score of a hit S between two aa-sequences S 1 and S 2 , composed of a conserved (l, d)-cs located at positions i in S 1 and j in S 2 is defined by:

score(S) = l-1 k=0 f (S 1 [i + k], S 2 [j + k]) wheref (a, a) = 1 and f (a, b) = 0 if a = b
It follows that the score s of S satisfies l -2d ≤ s ≤ l. For example on Fig. 2 the score of {"AA"; "((..))"} is 3.

Fig. 1. Example of hits between two RNAs Q and T . We can notice that hq8 / hq9 and ht8 / ht9 are overlapping hits and hq8 / hq23 and ht8 / ht22 are crossing hits. So there is 6 hits {(hq0;ht0),(hq8;ht8),(hq8;ht23),(hq22;ht8),(hq9;ht9),(hq22;ht23)}.

Seed indexing and hits lookup

The first key element in the method we present consists in indexing in a hash table all (l, d)-cs present in the RNA target set D of interest, for given parameters l and d. We denote by I d l this index. Comparing Q with the RNAs from D starts by searching in I d l the RNAs of D having seeds present in Q.

Indexing seeds. Given an aa-sequence A = (S, P ) of size n and cs parameters l and d, all k = n -l + 1 seeds of A are indexed in I d l . To do this all computed (l, d)cs are converted in integers as follows: the cs encoding for a (l, d)cs on A at i th position is defined by

S V alue (A, i, l, d) = 4 l-2d × i+l-1 j=i (encode(P j ) × 3 i+l-1-j ) + i+l-d-1 j=i+d (encode(S j ) × 4 i+l-d-1-j ) with encode : A = 0; C = 1; G = 2; U = 3; . = 0; (= 1; ) = 2 Given an integer x, I d l [x]
will contain all occurrences of the cs which S V alue is x, that is :

I d l [x] = {(A, i) | S V alue (A, i, l, d) = x}
For example, with the aa-sequences of Fig. 1, the integer associated to the (l, d)cs {"AA", "((..))"} is 5312, and

I 2 6 [5312] = {{(Q, 8), (Q, 22)}, {(T, 8
), (T, 23)}}. Inserting all cs of an RNA is done in linear time using a sliding window of length l. From a practical point of view, this index can easily be modified by adding the seeds of a new RNA sequence and it can compute and use simultaneously indexes for different values of l and d.

Index look-up. Given a query Q, the search for aligning it with the target RNAs from D starts by computing all the seeds of Q, for a given pair (l, d) of parameters, then searching I d l for all RNAs from D that have seeds present in Q. For a given RNA T from D, let LU d l be the set of all (l, d) -cs common to both Q and T , i.e hits:

LU d l (Q, T ) = {(i, j) | S V alue (Q, i, l, d) = S V alue (T, j, l, d)}
For example, using the same aa-sequences as in Fig. 1, LU 2 6 (Q, T ) = {(0, 0), [START_REF] Heyne | Lightweight comparison of RNAs based on exact sequence-structure matches[END_REF][START_REF] Heyne | Lightweight comparison of RNAs based on exact sequence-structure matches[END_REF], [START_REF] Heyne | Lightweight comparison of RNAs based on exact sequence-structure matches[END_REF]23), [START_REF] Zuker | Optimal computer folding of large RNA sequences using thermodynamic and auxiliary information[END_REF][START_REF] Heyne | Lightweight comparison of RNAs based on exact sequence-structure matches[END_REF], [START_REF] Hochsmann | Local similarity in RNA secondary structures[END_REF][START_REF] Hochsmann | Local similarity in RNA secondary structures[END_REF], [START_REF] Zuker | Optimal computer folding of large RNA sequences using thermodynamic and auxiliary information[END_REF]23)}. This phase is done using a standard hashtable look-up using the integer associated to each seed as key. The time required to compute the LU d l is linear in the size of the query and the overall number of hits. RNA-unchained offers the option to reduce the set of candidates based on the number of hits.

Hits/seeds optimization. Preliminary experiments showed that hits that do not contain both structural signal were more likely to be false positive. So in order to obtain more stringent seeds, the hits in LU d l (Q, T ) can be filtered to keep only the ones with two types of RNA structural symbols structure (right base of base pair and left base of base pair) which correspond to seeds that comprise the hairpin-loops: ( ) and stems junction: ) ( motifs, that are known to be well preserved and important structural patterns to detect secondary structure similarity [START_REF] Allali | A multiple layer model to compare RNA secondary structures[END_REF].

Chaining Algorithm and Anchors

The core of our approach to align Q with the target set D is to first compare Q and a member T of D using solely their hits. To do so, we use a recent efficient algorithm for chaining seeds developed in [START_REF] Allali | Efficient chaining of seeds in ordered trees[END_REF], followed by a stage where gaps between seeds are completed using the LocaRNA algorithm. The first steps consists in extending seeds defining hits to account for base-pairing given by the considered RNA secondary structures.

For example on Fig. 3, the last parenthesis of h q9 leads to the extension with the opening parenthesis of Q at position 6.

Note that the corresponding seeds might not be contiguous along the sequences (as illustrated in Fig. 3). It follows that they do not satisfy the definition of EPM; however they satisfy the definition of seeds introduced in [START_REF] Allali | Efficient chaining of seeds in ordered trees[END_REF]. Given a set of k extended hits for two RNAs Q and T , an anchor is a subset of hits such that, first the corresponding seeds are non-overlapping in both Q and T , second the seeds in both RNAs are compatible in terms of secondary structure (see chain definition in [START_REF] Allali | Efficient chaining of seeds in ordered trees[END_REF]). The score of an anchor C is the sum of the scores of the seeds defining the hits it contains, and the chaining score between Q and T is the maximum score of an anchor, taken among all anchors between Q and T (called an optimal anchor ). The algorithm we use computes an optimal anchor in time O(k 2 log k), where k is the number of hits. In our running example, an anchor is composed of the hits {{(Q, 9), (T, 9)}, {(Q, 22), (T, 23)}} (see Fig. 2). At the end of the chaining stage, we thus have, for each RNA T from D a set of hits between Q and T that forms an optimal anchor A(Q, T ). We call gaps the segments of the RNAs Q and T that are not involved in the anchor.

Anchor extension

Prior to aligning the gaps of Q and T with an exact but more costly algorithm, we perform a phase of seeds extension aimed at reducing the gap size and compensating the fact that initial seeds are of bounded length. First, each hit of the anchor between the two RNAs Q and T is extended on both sides based on exact sequence similarity. As an example, on Fig. 3 the hit (h q22 , h t23 ) is extended to the left by two nucleotides.

Next, still prior to the gaps alignment, we fill the remaining gaps using an adapted Longest Common Subsequence (LCS) considering triplet of letters (see Fig. 3 for an example) to avoid irrelevant constraints. This improvement has meaning only if the cover of the anchor is large enough. So we fix a threshold of 4l bases matches.

Anchor-constrained alignment

Finally, for each candidate homolog T , the gaps defined by the anchor between Q and T are aligned using the exact algorithm LocaRNA where the anchor is provided as a set of constraints (see Fig. 4).

Results

In order to assess the ability of RNA-unchained to provide accurate alignments, we applied on the set of reference RNA alignment provided by the benchmark Bralibase2.1 [START_REF] Wilm | An enhanced RNA alignment benchmark for sequence alignment programs[END_REF], composed of 8,976 pairwise alignments, classified into 36 families.

We analyzed this benchmark with RNA-unchained using several sets of options, LocaRNA used as a reference exact alignment tool and ExpLoc-P, a seedbased speed-up of LocaRNA. In order to reproduce the results on Bralibase2.1

shown in [START_REF] Schmiedl | Exact pattern matching for RNA structure ensembles[END_REF], we obtained from the authors of ExpLoc-P the corresponding code and parameters, and we ran ExpLoc-P with these optimized parameters.

In addition to these existing methods, we ran RNA-unchained with the following default parameters: (1) for each RNA sequence, its MFE secondary structure was obtained using RNAfold [START_REF] Lorenz | ViennaRNA Package 2.0[END_REF][START_REF] Zuker | Optimal computer folding of large RNA sequences using thermodynamic and auxiliary information[END_REF], (2) the parameters l and d for seeds were chosen to be l = 9 and d = 1, after exploring a wide range of possible values for these parameters (see discussion below). We denote this default RNA-unchained version 91M F E. In order to assess the impact of adding stringency criterion to the seed selection process, as well as the impact of the anchor extensions methods described in the 2 section, we ran RNA-unchained with additional options. (1) 91r2: only (9, 1)cs containing two-types parenthesis are conserved. (2) 91r2f b91epcLCS36 : 91r2 with seeds optimization, i.e. if there is no 91r2 seed consider 91M F E seeds, and anchor optimization. So all together, we show the results of two reference programs (LocaRNA, ExpLocPOpt) and three versions of RNA-unchained (91M F E, 91r2 and 91r2f b91epcLCS36).

To compare the obtained alignments with the reference alignments from Bral-ibase2.1, we use the SPS statistics (Fig. 5). Given a reference alignment r of length l r and a computed alignment e of length l e , the SPS is defined by the ratio SP e /l r where SP e is the number of pairs (i, j) where position i and j of the aligned sequences form a match in both r and e. In addition, we also consider the coverage in percent of the input RNA by the anchors, defined as the ratio between the number of bases belonging to anchors by the length of the RNAs (Fig. 6). This statistic is important to evaluate the impact of anchors, both in terms of computation time and of accuracy, as a high coverage by wrong hits will mechanically result in a low SPS, while a very low coverage by high confidence hits might not result in a significant gain of computation time. Finally, we display our result according to the similarity between the pairs of compared RNAs, where the similarity value Sim(Q, T ) between a query Q and a target T is defined, from the reference alignments, as follows:

Sim(Q, T ) = l-1 i=0 f (Q [i], T [i]) |Q |
where Q , T are the aligned sequence derived from Q, T and f (a, a) = 1 and f (a, b) = 0 if a = b. Both Fig. 5 and 6 present the number of alignments per similarity level (right scale). We also show the computation time of the different methods that we considered. Note that the difference between the running time of LocaRNA and ExpLocPOpt is not as important as shown in [START_REF] Schmiedl | Exact pattern matching for RNA structure ensembles[END_REF], but the computation time of ExpLocPOpt is comparable between our experiments and [START_REF] Schmiedl | Exact pattern matching for RNA structure ensembles[END_REF]. A first point we can notice is that all methods perform well and with relatively similar behaviour, but for reference alignment between pairs of RNAs that exhibit a similarity in the range [0.6,0.8] (i.e. 60%-80%), where RNA-unchained used with stringent grains composed of at least two kinds of structural elements, performs better, and even obtains better results than LocaRNA, although LocaRNA is guaranteed to obtain alignment with better alignment scores. This shows that adding high quality anchors, even if they cover a smaller part of the considered RNA sequences (see Fig. 6, 91r2), can improve significantly the alignment quality. However, being too stringent in defining hits results in a coverage that is relatively low, which has for consequence that many alignments are constraint-free and rely purely on LocaRNA. Note however, that the low covering hits selected with this method still results in a significant accuracy improvement over LocaRNA in the range [0.6,0.8]. On the opposite, 91M F E has a good coverage meaning that LocaRNA takes great advantage on alignment computation as its computation time is divided by two. However, the accuracy of the alignments can be significantly lower than with the other methods, as some hits with low structural information are false positive that can be selected by the chaining, thus misleading LocaRNA in the final anchor-based exact alignment phase. Finally, the two optimizations we propose, the seed optimization (see paragraph 2.2) and the LCS-based anchor extension (see section 2.4 improves significantly the coverage by the anchor (see method 91r2f b91LCS36) without impacting too much the alignments accuracy. This is reflected by the SPS values for 91r2f b91LCS36 that are accuracy results while the coverage is the best one achieved by our versions. As a result, the gain in terms of alignment time compared to LocaRNA is maximal (divided by two).

Regarding seeds parameters, various values of l and d from (5, 1) to (10, 2) were tested before settling for the combination l = 9 and d = 1. The experiments we carried to explore these parameters (results not shown) indicated clearly that seeds with a small conserved sequence lead to many false positive seeds, while large conserved structures, especially with the additional requirement of two types of structural elements, lead to a very low coverage, and so a higher computational time in the gap alignment phase. In general, these experiments show a relatively consistent pattern of correlated increasing coverage / decreased SPS.

Discussion

Summary. The main contribution we presented in this paper is a complete pipeline for the one-against-all RNA pairwise comparison, based on the notions of seeds, seeds index and seeds chaining. The key points are: a seed model describing both primary and secondary structure elements and a fast (sub-cubic) seeds chaining algorithm. The ability to index quickly and retrieve efficiently common seeds between a query and a set of targets is an important point of our method, that scales well as the main memory consumption of the index is determined by the number of keys in the hash table, which depends only on the seeds parameters (l, d), as the data itself grows linearly with the cumulated size of the target RNAs. Our experiments using the benchmark Bralibase2.1 show clearly that RNA-unchained obtains results that are more accurate than current stateof-the art methods, with comparable computation times (and probably better computation times in the C++ next release).

Seeds model and parameters. The seed model we introduce differ significantly from the ExpaRNA model while EPMs are designed as connected subgraphs of the RNA secondary structure. It is interesting to notice that our seeds model (with the seeds and anchors extensions) provide a coverage of the RNA sequences that is comparable to the one obtained with EPMs (Supplementary Fig. ??). This shows that both models probably are able to capture important conserved structural features. However, we can notice that in the similarity range 60%-80%, where RNA-unchained outperforms ExpLoc-P and LocaRNA in terms of SPS, we observe a significant difference. More generally, our work and the line of work centered on EPM suggest that the general seeds chaining approach deserves to be explored, both in terms of seed models and chaining algorithms. In particular, unlike sequence seeds, that have been deeply studied, formal studies of RNA seeds, including statistical aspects, are lacking. Secondary structure. A major difference between our approach and the Lo-caRNA/ ExpLoc-P lies in the way the secondary structure of RNA is accounted for. We explored several intermediate approaches, based on sampling RNA secondary structures using RNAsubopt and RNAshapes or based on keeping alignment with best score when using several suboptimal structures, but found that the MFE still provided the best accuracy results while minimizing the computation time (results not shown). This apparent concordance between two very different approaches suggests again that the notion of RNA structural seeds still deserves to be further studied.

Gap filling and chaining. An important aspect of RNA-unchained concerns the use of LocaRNA for the anchor-based gaps alignment. As gaps are segments where conserved structural motifs are absent, this allows to reduce the impact of the choice of the MFE, that is used only to detect seeds and compute the anchor, and is likely to be one of the reasons that explains the concordance between both approaches. However, this part of our pipeline is still the most costly in terms of computation time. As suggested by the results obtained with our LCS anchor-extension, a hierarchical/iterative approach, that would consider less conserved sequence and/or structure motifs detected within a given gap, and thus help again to reduce the segments on which an exact alignment algorithm is used, could be an efficient approach. Taking a somewhat extreme point of view, one could even ask if, in applications where exact alignments are not needed, the approach described above, limited to the computation of extended anchors, possibly completed by a quick way to evaluate the similarity between short gaps in RNA sequences, would not be sufficient.

Fig. 2 .

 2 Fig. 2. Compared to Fig. 1 3 hits are lost because of structural composition, crossing and overlapping. So this example show the final hits between Q and T .

Fig. 3 .

 3 Fig. 3. The matches between Q and T are left extended only for hq22 and ht23 and gaps are filled thanks to LCS computation.

Fig. 4 .

 4 Fig. 4. The anchor of our example, seen as sequence constraints.

Fig. 5 .

 5 Fig. 5. SPS value for all 5 methods applied to the Bralibase2.1 benchmark.

Fig. 6 .

 6 Fig. 6. Coverage of RNA sequences by anchors in percent for all 4 seeds-based methods (LocaRNA alone does not use seeds) applied to the Bralibase2.1 benchmark.

Table 1 .

 1 Computation times (in seconds). The time required to build the index is not included but takes less than 1 minute. Experiments were performed on a server with double Intel Xeon 3.3GHz processor. The seeds indexing, hits look-up and chaining are implemented in Java.

	Computation time Hits/chaining Gaps alignment Total
	LocaRNA	0	9,022	9,022
	ExpLocPOpt	1,492	6,070	7,562
	91M F E	3,386	4,563	7,949
	91r2	3,157	6,242	9,399
	91r2f b91epcLCS36 3,283	4,510	7,793


Florent Pittet
email: author.florent.pittet@univ-rennes1.fr

Océane Le Bot

Cécilia Houdelier

Marie-Annick Richard-Yris

Sophie Lumineau

Motherless quail mothers display impaired maternal behaviour and produce more fearful and less socially motivated offspring

Keywords: Fearfulness, Japanese quail, maternal behaviour, maternal deprivation, maternal effects, precocial bird, sociality, spatial skills

Early maternal deprivation impairs the behavioural development of young individuals.

Recently, strong differences between mothered and maternally-deprived chicks have been reported concerning their emotionality, sociality, and spatial skills. Here we investigated longterm and cross-generational impacts of maternal deprivation by comparing the characteristics of the non-reproductive and the maternal behaviour of 22 mothered and 22 non-mothered adult female Japanese quail (Coturnix c. japonica) and by comparing the behaviour of their respective fostered chicks. We reveal that non-brooded mothers were more fearful and less competent in spatial tasks and expressed impaired maternal care, characterized by more aggression towards chicks, higher activity rates and more abnormal pacing during the first days of the care period. Chicks' behaviour was clearly affected by maternal care inducing strong differences in their fearfulness and social motivation. Our results show both long-term and cross-generational impacts of early maternal deprivation in precocial birds.

The behavioural development of animals is strongly influenced by early post-natal living conditions, particularly interactions with the first care-giver between birth and emancipation [START_REF] Champagne | Like mother, like daughter: evidence for non-genomic transmission of parental behavior and stress responsivity[END_REF][START_REF] Fleming | Neurobiology of mother-infant interactions: experience and central nervous system plasticity across development and generations[END_REF]. During this period, a mother's behaviour affects her offspring's neuronal and physiological development with often long-term consequences on their social and emotional behaviour [START_REF] Berman | Group size, infant development and social networks in free-ranging rhesus monkeys[END_REF][START_REF] Caldji | The effects of early rearing environment on the development of GABAA and central benzodiazepine receptor levels and novelty-induced fearfulness in the rat[END_REF][START_REF] Francis | Maternal care and the development of stress responses[END_REF] and their cognitive skills [START_REF] Bredy | Maternal care influences neuronal survival in the hippocampus of the rat[END_REF][START_REF] Liu | Maternal care, hippocampal synaptogenesis and cognitive development in rats[END_REF]. These influences can occur through various social learning processes [START_REF] Holmes | How mothers influence the development of litter-mate preferences in Belding's ground squirrels* 1[END_REF] or via more passive mechanisms through which mothers directly modify the neurophysiologic development of offspring by tactile stimulations [START_REF] Champagne | Epigenetic mechanisms mediating the long-term effects of maternal care on development[END_REF]. Maternal deprivation paradigms have been widely used to investigate the influence of mothers on the subsequent behaviour of their offspring (see [START_REF] Fleming | Mothering begets mothering: The transmission of behavior and its neurobiology across generations[END_REF]. Early deprivation can be either partial, caused by precocious weaning (see [START_REF] Latham | Maternal deprivation and the development of stereotypic behaviour[END_REF][START_REF] Terranova | Individual differences in mouse behavioural development: effects of precocious weaning and ongomg sexual segregation[END_REF] or repeated separations [START_REF] Dettling | Repeated parental deprivation in the infant common marmoset (callithrix jacchus, primates) and analysis of its effects on early development[END_REF][START_REF] Millstein | Effects of repeated maternal separation on prepulse inhibition of startle across inbred mouse strains[END_REF], or complete (Albers, Timmermans, & Vossen, 1999a;Bertin & Richard-Yris, 2005;[START_REF] Gonzalez | Intergenerational effects of complete maternal deprivation and replacement stimulation on maternal behavior and emotionality in female rats[END_REF][START_REF] Melo | Maternal and littermate deprivation disrupts maternal behavior and social-learning of food preference in adulthood: Tactile stimulation, nest odor, and social rearing prevent these effects[END_REF]. Early repeated separations of young mammals from their care-giver affect their emotional behaviour [START_REF] Ogawa | Periodic maternal deprivation alters stress response in adult offspring: Potentiates the negative feedback regulation of restraint stress-induced adrenocortical response and reduces the frequencies of open field-induced behaviors[END_REF][START_REF] Wigger | Periodic Maternal Deprivation Induces Gender-Dependent Alterations in Behavioral and Neuroendocrine Responses to Emotional Stress in Adult Rats[END_REF], sociality (Livia [START_REF] Terranova | Individual differences in mouse behavioural development: effects of precocious weaning and ongomg sexual segregation[END_REF][START_REF] Seay | Maternal separation in the rhesus monkey[END_REF][START_REF] Tsuda | Long-Lasting Consequences of Neonatal Maternal Separation on Social Behaviors in Ovariectomized Female Mice[END_REF], sexual behaviour [START_REF] Rhees | Effects of maternal separation during early postnatal development on male sexual behavior and female reproductive function[END_REF] and cognitive skills [START_REF] Aisa | Effects of maternal separation on hypothalamic-pituitary-adrenal responses, cognition and vulnerability to stress in adult female rats[END_REF][START_REF] Aisa | Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats[END_REF][START_REF] Garner | Early maternal deprivation reduces prepulse inhibition and impairs spatial learning ability in adulthood: No further effect of postpubertal chronic corticosterone treatment[END_REF].

Complete maternal deprivation of mammals has been comparatively less used. Nevertheless, several studies report that subsequent adult behaviour can be strongly impacted by maternal deprivation, such as reproduction (see [START_REF] Fleming | Mothering begets mothering: The transmission of behavior and its neurobiology across generations[END_REF][START_REF] Gonzalez | Intergenerational effects of complete maternal deprivation and replacement stimulation on maternal behavior and emotionality in female rats[END_REF].

Most specifically, early social environment has strong effects on the quality and intensity of rodents' and primates' parenting behaviour expressed in adulthood: maternal deprivation impairs the expression of maternal behaviour. Harlow's famous studies showed that most of the macaque "motherless mothers" either appeared ignorant or were abusive to their infant [START_REF] Arling | Effects of social deprivation on maternal behavior of rhesus monkeys[END_REF][START_REF] Harlow | Maternal behavior of rhesus monkeys deprived of mothering and peer associations in infancy[END_REF][START_REF] Harlow | Social recovery by isolation-reared monkeys[END_REF]. Similarly, only 14% of the female chimpanzees reared by humans became competent mothers [START_REF] Brent | Evaluation of the chimpanzee breeding program at the Southwest Foundation for Biomedical Research[END_REF] and maternal deprivation led to impoverishment of rats' maternal behaviour (see A. S. [START_REF] Fleming | Mothering begets mothering: The transmission of behavior and its neurobiology across generations[END_REF][START_REF] Gonzalez | Intergenerational effects of complete maternal deprivation and replacement stimulation on maternal behavior and emotionality in female rats[END_REF][START_REF] Melo | Maternal and littermate deprivation disrupts maternal behavior and social-learning of food preference in adulthood: Tactile stimulation, nest odor, and social rearing prevent these effects[END_REF] even towards fostered pups [START_REF] Palombo | Motherless rats show deficits in maternal behavior towards fostered pups[END_REF] confirming the direct effects of maternal deprivation on maternal care.

Without entirely reconsidering these results, several authors pointed out that mammals do not offer the possibility to measure rigorously the impact of complete maternal deprivation. Indeed, some care must be artificially provided by human interventions and the characteristics of this artificial care is likely to influence pups' development [START_REF] Fleming | Mothering begets mothering: The transmission of behavior and its neurobiology across generations[END_REF].

Comparatively, maternally deprived precocial birds can be reared correctly with human intervention limited to providing heat, and consequently they constitute perfect models to investigate the influences of complete maternal deprivation. Complete deprivation procedures have shown that fearfulness and social motivation (Bertin & Richard-Yris, 2005;[START_REF] Fält | Differences in aggressiveness between brooded and non-brooded domestic chicks[END_REF]Perré, Wauters, & Richard-Yris, 2002;[START_REF] Roden | A comparison of the behaviour of domestic chicks reared with or without a hen in enriched pens[END_REF][START_REF] Shimmura | Effect of broody hens on behaviour of chicks[END_REF]), spatial skills (De Margerie et al., 2012) and rhythmicity [START_REF] Formanek | Epigenetic Maternal Effects on Endogenous Rhythms in Precocial Birds[END_REF][START_REF] Wauters | Mothering influences the distribution of activity in young domestic chicks[END_REF] differ strongly between artificially reared and mothered chicks. Nevertheless, these differences have been evidenced only during the first weeks after hatching and comparatively far less is known about long-term consequences of maternal deprivation on precocial birds. The influence of maternal deprivation on the expression of precocial birds' maternal care remains comparatively unknown whereas other aspects of their reproduction behaviour are known to be affected by early experience [START_REF] Immelman | Sexual and other long-term aspects of imprinting in birds and other species[END_REF].

This study aimed to evaluate the long-term influences of total maternal deprivation on adult female precocial birds' emotional reactivity, sociality and spatial skills and on the way they subsequently care for their own chicks. Our model is Japanese quail (Coturnix coturnix japonica), a domestic precocial bird. Mothers are the only care givers in this species, and care lasts only for a short 11-day period. We first evaluated the behavioural characteristics of mothered and non-mothered females using well-established ethological tests [START_REF] Bertin | Mothers' fear of human affects the emotional reactivity of young in domestic Japanese quail[END_REF][START_REF] Forkman | A critical review of fear tests used on cattle, pigs, sheep, poultry and horses[END_REF][START_REF] Formanek | Maternal epigenetic transmission of social motivation in birds[END_REF]. We then induced maternal behaviour in these females and evaluated the maternal care expressed by mothered and non-mothered females. As differences in maternal behaviour are known to influence the behavioural development of Japanese quail offspring [START_REF] Pittet | Age Affects the Expression of Maternal Care and Subsequent Behavioural Development of Offspring in a Precocial Bird[END_REF], our last goal was to determine whether these potential maternal care differences impacted the way chicks subsequently behaved by comparing the behavioural characteristics of chicks fostered by females that developed either with or without a mothering hen. We hypothesized that, as for mammals, effects of maternal deprivation would still be observable in adult precocial birds, that their maternal behaviour would be impaired by this early deprivation and that this would subsequently affect chicks' behaviour.

Methods

Ethic Statement

All experiments were approved by the departmental direction of veterinary services (Ille-et-Vilaine, France, permit number 005283) and were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Our breeding procedure and our ethological tests were approved by the regional ethics committee (agreement number: R-2011-SLU-02).

Animals and Housing

All the subjects were from a broiler line and were provided by an industrial farm (Les Cailles de Chanteloup, Ille et Villaine, France). Food was available ad libitum and conditions were LD (light/dark) 12/12 and 20±1°C.

Grandmothers: 8-week-old adult females (n=22) were acquired by the laboratory, individually marked by a numbered ring on a wing and placed individually in wire mesh breeding cages (51x40x35cm) with a drinker and a feeder. They were left in their cage for 3 weeks before being given chicks for adoption, to become habituated to their environmental conditions. The breeding room contained 44 cages and the females were distributed so that two females were never in neighbouring cages. The other cages were equipped with a heater (38 ± 1°C) for rearing non-brooded chicks. Mothers: They were 176 chicks that had been either adopted by "grandmothers" (brooded) or artificially reared (non-brooded). Male and female chicks were acquired by the laboratory when they were 1 day old. They were immediately placed in groups of 44 in 4 large plastic cages (98x35x42) equipped with a feeder, a drinker and a heater (38±1°C). In the evening of their arrival they were divided into two groups: 22 sets of 4 chicks (brooded chicks) were given to the 22 "grandmothers" and 22 sets of 4 chicks (non-brooded chicks) were placed in similar cages equipped with a heater. During this first breeding period, although cages with mothers needed more care interventions, we systematically intervened in all the cages to avoid differences between B and NB related to interactions with humans.

Adoptive mothers and warming lamps were removed when chicks were 11 days old, when they disperse naturally [START_REF] Orcutt | Nesting and parental behavior in domestic common quail[END_REF]. Sex was determined when they were 3 weeks old and one female was chosen randomly from each cage, individually marked with a numbered ring on a wing and left in her breeding cage until she was 3 months old. When a brood did not include a female (5 broods), we took a female from another brood that included two or more females. Brooded females are noted B (n=22) and females that developed without mothers are noted NB (n=22). Emotional reactivity, sociality and spatial abilities of B and NB were assessed using several well-established poultry behavioural tests during the three weeks before they adopted chicks.

Chicks: 200 one-day-old newly acquired chicks were placed in groups of 40 in 5 large plastic cages (98x35x42) equipped with a feeder, a drinker and a heater (38±1°C). In the evening, 176 of these chicks were randomly selected to be adopted either by B (22 sets of 4 chicks: Bc) or by NB (22 sets of 4 chicks: NB-c). After they were separated from their mothers, the emotional reactivity, sociality and spatial skills of B-c and NB-c were evaluated.

Fostering procedure and observation of maternal Behaviour

Fostering procedure

The same fostering procedure was used to induce maternal behaviour in both grandmothers and mothers.

Maternal behaviour was induced using the induction procedure described by [START_REF] Richard-Yris | Comportement parental chez les gallinacées: Importance du facteur émotivité dans la vitesse d'émergence des réponses parentales. Apport du modèle caille japonaise[END_REF]. At the beginning of the dark phase (20:00), 4 one-day old chicks were placed gently underneath each female who had been shut up in her nest box (18x18x18cm) one hour before.

Boxes were shut up again for the whole night (12 hours) during which the chicks' vocal and physical solicitations induced rapid expression of maternal behaviour by the adult females.

The next morning, all the boxes were opened and removed from the cages at 08:00. During the first day that the mothers spent with this first brood, their maternal behaviour was monitored and females that did not express any warming behaviour of the young chicks or that expressed aggression resulting in injuries of chicks were excluded from the experiment: none of the 22 females had to be excluded from the initial breeding period (grandmothers breeding mothers), but 4 B and 2 NB were excluded from the second breeding (B and NB breeding respectively B-c and NB-c) period.

Chicks that showed signs of hypothermia (motionless, eyes closed and trembling) when leaving the boxes or later during the breeding period were discarded from the experiment.

They were immediately placed under heaters where they recovered swiftly. These chicks were replaced by chicks of the same age, identified by a leg ring, so that they were not tested later.

At the end of the breeding period, 60 NB-c and 57 B-c had spent the whole breeding period with their mother. Numbers of chicks replaced did not differ significantly between NB and B (NB: 1.15±0.33, B: 0.67±0.21, U=206, p=0.4) and as many NB as B broods involved at least one replacement during the breeding period (NB: 13/20, B: 11/18, χ²=0.06, p=0.8).

Mothers were removed from the cages on post-hatching day (PHD) 11. Chicks then remained with their siblings for two more weeks during which the fearfulness, the social motivation and the spatial skills of two chicks from each cage were evaluated (when two chicks that spent the whole breeding period with the mother were available: n NB-c = 32; B-c = 33). As morphological sexual dimorphism appears only at 3 weeks [START_REF] Mills | The behavior of the Japanese or domestic quail Coturnix japonica[END_REF], chicks were chosen randomly, but sex ratios did not differ significantly between B-c and NB-c (sex ratios: B-c=0.88, NB-c=0.89; χ²=0.00, df = 1, p=0.99). During this test period, chicks were weighed when they were 14 and 28 days old.

Observations of maternal behaviour

Maternal behaviour was recorded on PHD 3, 5, 7 and 9. During the breeding period when B and NB females adopted chicks, we evaluated maternal behaviour using instantaneous scan sampling to establish mothers' time-budgets and, simultaneously, focal animal sampling to note complete behavioural sequences (see below for details). The observer stood behind a one-way mirror.

Instantaneous scan sampling: Each day we recorded 60 scans at 5-minute intervals: 30 scans in the morning and 30 in the afternoon. Each scan recorded whether the mother was warming chicks and if she was, we recorded her posture and how many chicks were being warmed. We also recorded the mother's activity and the distance between each chick and its mother. An index of distance was calculated as the average score of all chicks that were not being warmed. The five classes of distances and associated scores were: on opposite sides of the cage (1), more than half a cage away (0.75), less than half a cage away (0.5), one chick length away (0.25) and in contact (0). The higher the index was, the further from the mother the chicks were when not being warmed. We also recorded whether chicks were warming one another. Data were sampled using an ipod Touch (Apple©) and the application "scan sampling" (Vincent Richard ©).

Focal sampling: Each cage was observed for two 5-minute sessions when the mother's entire behavioural sequence was recorded. The experimenter noted the frequency of interactive behaviours (aggression: the mother attacked chicks while producing threat vocalizations followed by the chick moving away, pecking: the mother pecked at the chick with her beak closed, trampling chicks and contact breaks) and non-interactive behaviours (locomotion, exploration, observation and maintenance).

Separation test

To assess the strength of the bond between mothers and chicks, we removed chicks from each cage for 5 minutes on PHD 6 and placed them in similar cages without their mother. Mothers' reactions to this separation were observed behind a one-way mirror and the observer recorded vocalizations and latencies to resume comfort behaviours (eating and resting). At the same time, the reactions of 2 chicks a cage were videotaped to assess latency and frequency of their distress calls and steps.

Behavioural characteristics of foster mothers and chicks

Procedures assessing fearfulness

Behavioural tests assessed the levels of fearfulness of both B and NB mothers before they adopted chicks and of their chicks after separation. As fearfulness is a multidimensional trait [START_REF] Roussot | Factorial correspondence analysis of fear-related behaviour traits in Japanese quail[END_REF] that cannot be estimated by a single procedure, several tests presenting different environmental conditions and various fear-inducing stimuli were realized.

We investigated fearfulness using the procedures described below to assess shyness, neophobia and reactions to humans.

All observations except reactions to humans were recorded behind a one-way mirror.

Emergence test: This test followed a protocol similar to that described by Jones et al. (1991).

Quail were individually transported in a wooden box (18x18x18cm) with a removable wall. This box was placed against the apparatus: a large and well-lit wooden box (62x60x33cm) with wood-shavings covering the floor and an observation window. The transport box was kept closed for 1 minute before the door was opened to allow access to the apparatus. The experimenter noted the latency between raising of the door and the emergence of the subject's head out of the box, and its total emergence.

Novel object test: This test assesses neophobia by the reactions subjects express in the presence of an unfamiliar object (R. B. Jones, 1996). Mothers were tested in their home cage, but chicks had to be socially isolated and were consequently tested in a polyhedral openfield (1m², h=60cm) after a 5-minute habituation. The novel stimulus was an unfamiliar plastic Tshaped object. During a 10-minute focal sampling the experimenter recorded latency to approach the object, frequency of locomotion (walks, runs) and frequency of fear behaviours expressed towards the novel object including escape, withdrawal (slowly walk away from the object keeping it in sight), jumps, fear postures (crouching) and freezing (Jones, 1996;[START_REF] Mills | The behavior of the Japanese or domestic quail Coturnix japonica[END_REF]. The experimenter also recorded the frequency of defecation, observations and explorations of the object (pecking at the object), of the apparatus (pecking at a wall or at the floor). Scan samples, at 10-second intervals, recorded at the same time the position of the subject in relation to the object. During observations of mothers, the cages were virtually divided into two zones and the experimenter noted whether the female was in the half of the cage containing the object or not. During observations of chicks, the openfield was divided into three equal zones and the experimenter noted whether the chick was in the object zone, in a middle zone or in the zone opposite to the object.

Human observer test: This test assesses reactions to humans [START_REF] Jones | Reduction of the domestic chick's fear of human beings by regular handling and related treatments[END_REF]. Subjects were tested in their familiar environment. The experimenter, using instantaneous scan sampling, passed in front of each cage at 5-minute intervals recording a total of 32 scans for each cage.

Each time he passed in front of a cage, he stopped for few seconds and recorded the instantaneous activity of all subjects: fear reactions (subject interrupts its ongoing activity and moves away from the observer), observation of the observer, explorations, feeding, resting or maintenance behaviours (resting, self-preening).

Procedures to assess sociality

Inter-individual distances in home-cage: When they were 21 days old, inter-individual distances between each chick and its nearest conspecific were recorded by 32 scans of each cage, made at 4-minute intervals. We used the same distance classes and scores as those used to describe distances between mothers and chicks during the brooding period.

Runway test: This test is an adaptation of the treadmill test [START_REF] Mills | The treadmill test for the measurement of social motivation in Phasianidae chicks[END_REF]) that evaluates subjects' motivation to reach a social stimulus [START_REF] Formanek | Maternal epigenetic transmission of social motivation in birds[END_REF]. The apparatus is a 100cm-long wire-netting tunnel. Test subjects were transported individually in a wooden box (18x18x18cm), which was then placed at the tunnel entrance. At the other end of the tunnel was a cage (20x35x20cm) containing three unfamiliar conspecifics of the same age as the tested individual, representing a social stimulus. The corridor was divided into four zones: the closest zone to the social stimulus, "1 bird long" (zone P) and 3 equal 32cm-long zones called, from the entrance to zone P: zones A (beginning of the tunnel), B (middle) and C (end of the tunnel). One minute after the transport box had been put in place, the door was opened and the subject was observed for 5 minutes. The experimenter noted latency to emerge completely from the box, to reach zone P, number of crossed zones and time spent in each zone. An index of sociality was calculated using the following formula: Index of sociality = Time (s) in zone P + 0.66*time in zone C + 0.33*time in zone B.

The higher the index is, the closer the individual remained to the social stimulus. The experimenter also recorded latency to emit a distress call, frequency of distress calls, exploration of the cage containing conspecifics (number of soft pecking against the conspecifics' cage), aggressive behaviour (number of violent pecking against the conspecific's cage, associated with threat vocalizations), fear postures, and jumps.

Reaction to a stuffed conspecific: Only adult females were tested to assess their reactions to a model adult Japanese quail. The social stimulus was a stuffed female quail. Test subjects were first placed in the centre of a plastic arena (Ø120x60cm) with a linoleum floor, for a 5 minutes habituation period. Then the light was switched off for one minute when the lure was placed in the centre of the apparatus. When the light had been switched on again they were observed for a 5-minute focal sequence with the lure and, after a one minute without light, for another 5-minute focal sequence without the lure. During each sequence, the experimenter recorded latency of first distress call, number of distress calls, latency of first step, number of steps and frequencies of observation, exploration and maintenance activities. When exposed to the lure, latencies to approach and to contact the stuffed female were also recorded.

Spatial skills test

Detour task: The apparatus was a cross between [START_REF] Zucca | Animal lateralization and social recognition: quails use their left visual hemifield when approaching a companion and their right visual hemifield when approaching a stranger[END_REF] and [START_REF] Zucca | Detour behaviour in three species of birds: quails (Coturnix sp.), herring gulls (Larus cachinnans) and canaries (Serinus canaria)[END_REF] detour apparatuses (Fig. 1). Test mothers and chicks were food deprived for 12h by presenting food for only 2 minutes at middle of the restriction period but water was available continuously. The apparatus was a rectangular arena with an obstacle through which a feeder placed just behind it could be seen. Test subjects were placed in the dark in front of the obstacle and had to go round it to reach the feeder. The experimenter noted latency to take first step, to get round the obstacle, to reach the feeder, which side of the obstacle the subject went and all the subject's behavioural activities (vocalizations, locomotion, jumps, observations, fear posture and maintenance).

*Figure 1*

Statistical analyses

As most of our data were not normally distributed, we used non-parametric tests to compare NB's and B's, as well as, NB-c's and B-c's behavioural data, separately per each behavioural variable. Mann-Whitney tests were performed to compare frequencies, latencies and proportions of time (spent in a particular area or performing a particular behaviour) between sets, and Chi-square tests were performed to compare proportions of quail of each set that expressed or did not express a behaviour.

For bivariate data (repeated measurements), we computed ANOVAs on repeated measurements after checking response variable and residual normalities by Kolmogorov-Smirnov tests and the homogeneity of variances by Mauchly's sphericity test. Data analyses were computed using Statistica® and XLStat®.

Results

Long-term effects of mother deprivation on adult behaviour

Effects on emotional reactivity

During the emergence test, NB put their head out of the shelter earlier than did B (NB: 5.68 ± 2.09s, B: 20.81 ± 8.10s; Mann-Whitney U-test: U = 132, P = 0.01), but latencies to emerge completely from the shelter did not differ between the two sets of females (P > 0.05). When facing a novel object, latencies to approach and to explore the object did not differ significantly between NB and B (P > 0.05). Nevertheless, NB moved more frequently (NB: 35.0 ± 2.7, B: 26.7 ± 3.0; U = 153, P = 0.04), avoided the object more (U = 120, P = 0.004; Fig. 2a) and defecated more than did B (NB: 0.77 ± 0.11, B: 0.45 ± 0.13; U = 168.5, P = 0.05). The frequencies of other behaviours did not differ significantly between B and NB (P > 0.05). More B than NB spent a significantly higher proportions of time near the object (NB: 8/18, B: 13/17; χ² 1 = 3.73, P = 0.05). NB females expressed more fear postures in reaction to humans (U = 159, P = 0.04; Fig. 2b). The proportions of scans spent in other activities did not differ significantly between B and NB (P > 0.05).

*Figure 2* Effects on sociality NB took longer to approach the lure than did B (NB: 79.41±13.47s, B: 48.18±13.05s, U = 159, P = 0.05). After removal of the lure, NB took longer to take their first step (NB: 85.77 ± 13.03s, B: 27.59 ± 9.75s; U=111.5, P=0.002) and tended to take less steps (NB: 25.5 ± 7.68, B: 38 ± 8.74; U = 162, P = 0.06). The other behavioural traits measured in this test did not reveal significant differences between NB and B (P > 0.05).The runway test revealed no significant differences between females' behavioural expressions or the times they spent in the apparatus' different zones (P > 0.05).

Effects on spatial skills

Fewer NB than B were successful in the detour task (NB: 4/22, B: 13/22; χ² 1 = 7.76, P = 0.005). None of the other behavioural traits measured in this test, including latency to take first step and frequency of locomotor acts differed significantly (P > 0.05).

Effects of mother deprivation on maternal behaviour

Reaction to induction

The first interactions with chicks (aggressive behaviour, warming parameters) of females that were maternal after the induction procedure and the number of chicks showing signs of hypothermia during the first day following induction did not differ significantly between B and NB (Mann-Whitney U-test: P > 0.05).

Maternal traits

Brooding parameters (time spent warming chicks, number of chicks warmed and posture preferences), contact breaks or distance to chicks did not differ significantly between B and NB during the whole breeding period (Mann-Whitney; P > 0.05).

Nevertheless, aggressive behaviours towards chicks and time-budgets showed several differences between the two sets of females. Indeed, NB were more aggressive towards chicks than B at the beginning of the brooding period (PHD3: NB: 2.15 ± 0.71; B: 0.39 ± 0.23; Mann-Whitney U test: U = 119.5, P = 0.04) and pecked them more on PHD 7 (NB: 1.15 ± 0.45, B: 0.11 ± 0.08; U = 132, P = 0.03).

Frequencies of chick trampling did not differ between B and NB females on any day of the breeding period (P > 0.05). NB's and B's time-budgets differed. NB spent more time active than B (see Fig. 3 for details), but their activity was not affected by chicks' age and we found no significant interaction between set of females and age of chicks (Fig. 3). More NB than B expressed stereotypic pacing (abnormally repeated flight attempts with exaggerated locomotion and pecking against the cage walls) on PHD3, PHD5 and tended to on PHD7 (Fig. 4), and NB spent higher proportions of time pacing on these days (PHD3: NB: 4.16 ± 1.56%, B: 2.50 ± 2.50%; Mann-Whitney U-test: U = 96.5, P = 0.003; PHD5: NB: 2.16 ± 0.46%, B: Reactions (latencies and frequencies of behaviours and vocalizations) to separation from chicks did not differ significantly between B and NB on PHD6 (P > 0.05). Similarly, reactions to separation from their mother did not differ significantly between NB-c and B-c (P > 0.05)

Chicks' growth and behaviour

Weights of NB-c and B-c did not differ significantly on PHD 14 or on PHD 28, but we found an interaction between age and set due to B-c's faster weight gain (Fig. 5).

*Figure 5* Emotional reactivity

Emergence test data indicated that NB-c's mean latencies were more than twice as long as Bc's for both emergence of the head (NB-c: 18.5 ± 6.54 s, B-c: 5.53 ± 2.26 s; U = 354.5, p = 0.005) and full emergence (NB-c: 21.76 ± 6.78 s, B-c: 8.47 ± 3.34 s; U = 307, P = 0.0007).

When exposed to the observer, NB-c spent less scans self-preening (NB: 0.76±0.33 scans; B: 2.16 ± 0.63 scans; U = 462, P = 0.04). We could find no other significant differences between NB-c's and B-c's behavioural expressions during the emergence test and the reaction to the observer test (P > 0.05). NB-c's and B-c's behaviours did not differ significantly in the novelobject test (P > 0.05).

Sociality

Observations of NB-c in their cages indicated that they tended to be more frequently in parts of their cage opposite to their nearer conspecific (NB-c: 4.1±0.7%, B-c: 2.9±0.6%, U = 490, P = 0.09). Distance scores did not differ significantly between NB-c and B-c (P > 0.05).

Our distance index indicated that NB-c were further from the social stimulus in the runway test (NB: 160.9 ± 16.0, B: 204.

Spatial skills

As many NB-c as B-c were successful in the detour task (NB-c: 24/32, B-c: 24/33; χ² 1 = 0.04, P > 0.05) and the latencies of the successful subjects to reach to the feeder did not differ between the two sets (Mann-Whitney U-test: P > 0.05).

Discussion

This study evaluated the modifications induced by maternal deprivation on females' subsequent non-reproductive and maternal behaviour in adulthood. We found that maternally deprived females displayed higher fearfulness and impaired spatial skills. When maternal, these motherless mothers were more aggressive towards chicks and more active during the first half of the care period when they also expressed more stereotypic behaviours than did mothered females. When assessing consequences of these maternal care differences on chick development, we found strong differences between NB-c and B-c as NB-c's fearfulness was higher and their social motivation was lower.

Effects of maternal deprivation on adult behaviour

First we showed that maternal deprivation impacts the non-reproductive behaviour and spatial skills of females when adult. Although previous reports have shown differences between mothered and non-mothered precocial birds, these reports assessed their behaviour only during the first weeks after hatching and no conclusions could be drawn concerning the longterm influences of this early experience (Bertin & Richard-Yris, 2005;[START_REF] De Margerie | Effect of mothering on the spatial exploratory behavior of quail chicks[END_REF][START_REF] Formanek | Epigenetic Maternal Effects on Endogenous Rhythms in Precocial Birds[END_REF][START_REF] Perré | Influence of mothering on emotional and social reactivity of domestic pullets[END_REF][START_REF] Roden | A comparison of the behaviour of domestic chicks reared with or without a hen in enriched pens[END_REF][START_REF] Shimmura | Effect of broody hens on behaviour of chicks[END_REF].

In the present study, NB were clearly more fearful than B, they were more neophobic in the presence of a novel object and more fearful in the presence of humans. These results are consistent with Roden's (1998) and Perré's (2002) reports concerning domestic chicks, but contradict reports concerning Japanese quail chicks (Bertin & Richard-Yris, 2005). This last inconsistency might be related to the age or sex of test subjects (adult females vs. mixed-sex chicks), but we think they result from the adoptive mothers. Indeed, the females Bertin & Richard-Yris (2005) used as adoptive mothers for the brooded chicks had been selected for a particular level of emotionality [START_REF] Mills | Divergent selection for duration of tonic immobility and social reinstatement behavior in Japanese quail (Coturnix coturnix japonica) chicks[END_REF]. As maternal emotional reactivity is transmitted to fostered chicks [START_REF] Houdelier | Development of Fearfulness in Birds: Genetic Factors Modulate Non-Genetic Maternal Influences[END_REF][START_REF] Richard-Yris | Nongenomic inheritance of emotional reactivity in Japanese quail[END_REF], using mothers from different lines can affect chicks in such a way that they appear either more or less fearful than non-brooded chicks.

Another reported consequence of maternal deprivation is impairment of development of sociality [START_REF] Dettling | Repeated parental deprivation in the infant common marmoset (callithrix jacchus, primates) and analysis of its effects on early development[END_REF][START_REF] Terranova | Individual differences in mouse behavioural development: effects of precocious weaning and ongomg sexual segregation[END_REF][START_REF] Seay | Maternal separation in the rhesus monkey[END_REF][START_REF] Tsuda | Long-Lasting Consequences of Neonatal Maternal Separation on Social Behaviors in Ovariectomized Female Mice[END_REF]. We found that NB reacted more fearfully to a stuffed conspecific than did B. This reaction could be considered either as a lesser social competence or as a neophobic reaction because they had never been exposed to an adult. The fact that the runway test results revealed no differences in proximity with unfamiliar conspecifics between the two sets, led us to favour the second explanation. Non-brooded chicks were reported to behave less socially than chicks that developed with a mother (Bertin & Richard-Yris, 2005;Perré et al., 2002), but we did not find a similar difference when they were adult. We do not suggest that this result indicates no differences in sociality between B and NB, but rather that characteristics of this species are involved [START_REF] Guyomarc'h | La reproduction chez la caille des blés (Coturnix C. coturnix). II: Croissance et développement sexuel des jeunes[END_REF], as adult females display solitary phases whereas chicks are highly gregarious, making differences in sociality much harder to highlight in adults than in chicks.

NB's detour results suggest that their spatial skills have been impaired. Exploratory behaviour of these subjects was assessed when they were chicks (in mixed-sex flocks of 4 mothered or non-mothered chicks), and the exploratory skills of non-mothered chicks already showed a deficit [START_REF] De Margerie | Effect of mothering on the spatial exploratory behavior of quail chicks[END_REF]) that seems to be still observable in adulthood. This impairment of spatial skills is consistent with the literature reporting the necessity of maternal stimulation to promote mammals' hippocampal synaptogenesis and spatial learning [START_REF] Liu | Maternal care, hippocampal synaptogenesis and cognitive development in rats[END_REF]. Early maternal deprivation experiments highlighted delayed or impaired spatial learning [START_REF] Aisa | Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats[END_REF][START_REF] Garner | Early maternal deprivation reduces prepulse inhibition and impairs spatial learning ability in adulthood: No further effect of postpubertal chronic corticosterone treatment[END_REF]. As suggested by de Margerie (2012), NB's spatial skills could also be a by-product of their higher fearfulness as fear-related behaviour can inhibit exploration [START_REF] Murphy | The practical problems of recognizing and measuring fear and exploration behaviour in the domestic fowl[END_REF], and NB behaved more fearfully in other tests.

Nevertheless inhibition of NB's exploration is not supported by our results since latencies to move and frequencies of moving, freezing or flight attempts did not differ significantly between NB and B. We consequently consider that the impoverishment of spatial stimulations due to maternal deprivation during NB's early life induced their poorer spatial ability.

Effects of maternal deprivation on maternal behaviour

Our results indicate that the maternal behaviour of maternally deprived females was impaired, at least at the beginning of the breeding period. This impairment is characterized by more aggression, more activity and more frequent pacing stereotypies. The negative impact of maternal deprivation has already been reported in mammals. The maternal behaviour of handreared female primates is greatly deficient. [START_REF] Brent | Evaluation of the chimpanzee breeding program at the Southwest Foundation for Biomedical Research[END_REF] reported that only 14% of handreared chimpanzee females were able to provide adequate care and ensure the survival of their infants. Maternally-deprived female primates also behave aggressively towards their offspring [START_REF] Harlow | Social recovery by isolation-reared monkeys[END_REF]. Artificially reared rats retrieved fewer pups during a retrieval test and exhibited reduced pup licking and crouching behaviours [START_REF] Gonzalez | Intergenerational effects of complete maternal deprivation and replacement stimulation on maternal behavior and emotionality in female rats[END_REF].

Currently, we cannot draw any conclusion about a direct influence of early experience on aggression of chicks as this could also be the expression of NB's higher fearfulness. High levels of activity when facing stressful situations and expression of stereotypic behaviours are also a known particularity of maternally-deprived mammals [START_REF] Gonzalez | Intergenerational effects of complete maternal deprivation and replacement stimulation on maternal behavior and emotionality in female rats[END_REF][START_REF] Latham | Maternal deprivation and the development of stereotypic behaviour[END_REF]. Interestingly, differences between NB and B females were only measurable during the first half of the breeding period. Contrary to all expectations, primate motherless mothers' brutality or indifference finally decreases in response to infants' persistent solicitations [START_REF] Harlow | Social recovery by isolation-reared monkeys[END_REF]. We suggest that, similarly, stimulation of their mother by chicks eventually induces NB mothers to express a maternal behaviour comparable to that of brooded females.

Nevertheless, maternal deprivation did not appear to modify several fundamental traits of maternal behaviour, including time spent warming, warming posture preference or the strength of bond with chicks (as reactions to separation from chicks did not differ between NB and B). This result could suggest low plasticity of these traits to experiential factors since they were not clearly influenced by the presence of a mother during the first weeks of the female's life or by the female's breeding experience (Pittet, Coignard, Houdelier, Richard-Yris, & Lumineau, in press). This hypothesis subsequently implies the existence of an important individual determinant that could be highlighted by analysis of mothering styles in our species as reported for many mammals (P. [START_REF] Albers | Evidence for the existence of mothering styles in guinea pigs (Cavia aperea f. porcellus)[END_REF][START_REF] De Lathouwers | Comparing maternal styles in bonobos (Pan paniscus) and chimpanzees (Pan troglodytes)[END_REF][START_REF] Dwyer | Maternal behaviour in domestic sheep (Ovis aries): constancy and change with maternal experience[END_REF][START_REF] Hill | All Mothers are Not the Same: Maternal Styles in Bottlenose Dolphins (Tursiops truncatus)[END_REF][START_REF] Maestripieri | Social structure, infant handling, and mothering styles in group-living old world monkeys[END_REF], but never investigated in birds. We can also consider that chicks' rearing conditions and particularly the fact that they were reared in social groups strongly influenced this result.

Several studies dissociate the confused effects of early social isolation and maternal deprivation in mammals and show that deprived individuals reared with social peers had reduced social and maternal deficits [START_REF] Melo | Maternal and littermate deprivation disrupts maternal behavior and social-learning of food preference in adulthood: Tactile stimulation, nest odor, and social rearing prevent these effects[END_REF]. Similarly, social conditions may have limited the impact of maternal deprivation in our study. Another possibility is that the expression of precocial animals' maternal care is less sensitive to early maternal stimulations than that of altricial species. The maternal care of precocial guinea pigs reared in social isolation, with social partners or with a mother and social partners did not differ (P. Albers et al., 1999a;[START_REF] Stern | Effects of social isolation until adulthood on maternal behavior in guinea pigs[END_REF].

Development of chicks brooded by NB and B mothers

The fact that our results indicate that the early rearing conditions of mothers did not influence chicks' survival rates appears logical as most of the fundamental maternal behaviour traits did not differ between B and NB mothers and our laboratory conditions were non-restrictive. We nevertheless found that chicks brooded by B mothers presented a higher weight gain after separation from their mothers. We could consider here that the slightly harsher conditions in which NB-c developed as their mothers were more aggressive and more active probably impaired their weight gains slightly from the first days although differences between NB-c and B-c became significant only a few weeks after separation. Another possibility is that slight maternal behaviour differences did not affect weight directly, but as NB-c were more reactive in several tests, the testing period could have been a more stressful for these chicks and led to a lesser weight gain after separation from the mother since fearfulness and fear behaviour are negatively associated with growth [START_REF] Jones | Fear-related behaviour in Japanese quail divergently selected for body weight[END_REF].

Ability to resolve the detour task did not differ between the two sets of chicks and chicks' success was higher than that of their mothers. Our results indicate that the presence of a mother during early development clearly influences the future ability to resolve spatial tasks.

Results from mammals indicate that the behavior of mothers can affect the spatial behavior of their offspring [START_REF] Albers | Is maternal behavior correlated with later explorative behavior of young guinea pigs ( Cavia aperea f. porcellus )?[END_REF]. Thus we expected the differences between NB's and B's maternal behaviour to induce differences in chicks' detour abilities. Indeed, precocial chicks brooded by a hen tend to follow their mother and hence explore larger areas than non-brooded chicks [START_REF] Wauters | Mothering influences the distribution of activity in young domestic chicks[END_REF] and show greater ability to explore new environments [START_REF] De Margerie | Effect of mothering on the spatial exploratory behavior of quail chicks[END_REF]. NB's higher frequencies of maternal aggression and pacing could have led NB-c to follow their mother less and to be, consequently, less stimulated to explore their cage. The absence of effects of these maternal behaviour differences on NB-c's and B-c's spatial abilities could be related to physical characteristics of their housing environment where distances are limited and individuals never lose sight of one another. We suggest that such maternal stimulations differences could have stronger impacts on chicks' later spatial abilities if the breeding period took place in a larger and structured environment.

We found that the emotional and social behaviour differed between chicks brooded by mothered and non-mothered females. This result is in accordance with our previous reports and confirms an influence of maternal care on chicks' subsequent behavioural development.

Interestingly, we highlighted maternal behaviour differences between NB and B only during the first half of the care period, but nevertheless chicks' behaviour differed greatly between sets, suggesting that they were more sensitive during this early period. NB-c were more paralleled the differences observed between their mothers. NB-c were also less socially motivated, as they were further from both familiar and unfamiliar conspecifics. We did not find evidence of any differences between their mothers concerning their sociality when adult, but, as chicks, B and NB presented similar differences (Bertin & M. A. Richard-Yris, 2005).

Altogether, these results describe a clear case of non-genomic transmission of behavioural characteristics from mothers to chicks, similar to that already reported for both emotionality (M. A. [START_REF] Richard-Yris | Nongenomic inheritance of emotional reactivity in Japanese quail[END_REF] and sociality [START_REF] Formanek | Maternal epigenetic transmission of social motivation in birds[END_REF] in quail.

Figures and legends

  8 ± 16.1, U = 378, P = 0.034). Moreover in this test, NB-c crossed more zones (NB-c: 17.8+79 ± 1.77, B-c: 11.31 ± 1.25; U = 321.5, P = 0.004) and emitted distress calls earlier than did B-c (NB-c: 188 ± 21.27s, B-c: 254.81 ± 15.43s; U = 392, P = 0.032).

Figure

  Figure 1

  Figure 1

Figure 1 .

 1 Figure 2

Figure 2

 2 Figure 2. a: Novel object test. Mean (±SEM) frequencies of avoidance of the novel object by NB (grey bar) and B (black bar). b: Reaction to humans test. Mean (±SEM) proportions of scans (in % of total number of scans) in fear postures in the presence of a human by NB (grey bar) and B (black bar). Mann-Whitney U-test: **p<0.005, *p<0.05. (nNB=22, nB=22).

Figure 3 .

 3 Figure3. Mothers' activity in relation to chick age. Mean (±SEM) proportions of scans (in % of total scans) when B and NB were active (including locomotion, exploration, feeding, stereotypic pacing, jumps, alert observations, pecking and aggression). PHD: Days of the breeding period post hatching. Analysis of variance indicated that NB's (n=20) activity level was higher than B's (n=18), but no effect of chicks' age, nor an interaction between set and age of chicks. Grey bar: NB; black bar: B.

Figure 4 .

 4 Figure 4. Mothers' stereotypic pacing in relation to chick age. Proportions of subjects that expressed stereotypic pacing during the breeding period. Chi-square tests indicated that more NB (n=20) than B (n=18) expressed stereotypic pacing at that beginning of the breeding period (PHD3 and PHD5). Grey bar: NB; black bar: B.

Figure 5 .

 5 Figure 5. Chicks' weights in relation to their age. Mean (±SEM) weight of NB-c and B-c on PHD14 and PHD28. Analysis of variance indicated an effect of age and an interaction between set and age. (nNB-c=32, nB-c=33). Grey bar: NB-c; black bar: B-c.

Acknowledgements

The authors would like to thank C. Petton for his help in rearing and maintaining the animals used in this study. We are grateful to Dr Ann Cloarec for improving the writing of the manuscript and to the referees for their interesting and helpful comments on the manuscript.